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Development, Optimization and Characterization of a Surface Enhanced Raman Spectroscopic Method for Detection of Dipicol...


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1 DEVELOPMENT, OPTIMIZATION AND CHARACTERIZATION OF A SURFACE ENHANCED RAMAN SPECTROSCOPIC METHOD FOR DETECTION OF DIPICOLINIC ACID By JOY D. GUINGAB A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 Copyright 2007 by Joy D. Guingab

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3 To my parentsm y strength and foundation

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4 ACKNOWLEDGMENTS I would like to express my sincere gratitude to my research adviser Prof. James D.Winefordner for his continued guidance and encour agement. I am especially grateful to have had the opportunity to work with a mentor w ho encourages personal gr owth through research and teaching. I would like to acknowledge Dr. Nicolo Omenetto for his valuable research guidance. His admirable love for science is truly inspiring. I am very grateful to Dr. Benjamin Smith for all his advice in building my experimental se t up. He has set a very good example for all the students in the Winefordner group. I would like to extend my a ppreciation to my committee members for their valuable advice on the completion of this research dissertation. The Winefordner-Omenetto group is worthy of th anks for the camaraderie and friendship. I am especially grateful to Dr. Jamshid Temi rov, Benoit Lauly and Nicholas Taylor for all Raman discussions and the assistance extended du ring the setting up of the Raman system. I am thankful that I have had the opportunity to be in the same group as Dr.Xihong Wu for the past 3 years and have developed a great friendship. Many thanks go to Dr. Kirby Amponsah-Manager, Ron Whiddon and Pam Monterola for their constant encouragement and friendship. I would like to thank Dr. David Hahn and Dr Timothy Anderson for allowing me to use their Raman systems in the preliminary stages of this research. I am grateful to Young Seok Kim for all the assistance in the use of their Raman system. All the UV-Vis measurements were conducted in laboratory of Dr. Nigel Richard, Dr. Charles Martin and Dr. Kathryn Williams. Thanks go to the SEM laboratory staff for all training and assistance in the use of the SEM microscope.

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5 I would like to extend my appr eciation to all support sta ff in chemistry, including the glass, machine and electronics shop for all the work their contributions in the instrument development. It has been great having Ms. Lori Clark all these years as the most helpful person to every Chemistry graduate st udent. Special thanks go to Ms. Jeanne Karably for all her assistance and great ad vice on almost anything. My life in Gainesville had not been this me morable without the company of good friends. I acknowledge all my friends in chemistry for all the good times shared. I am so fortunate to have a very good support group from my fellow Filipino graduate students and their families. My heartfelt gratitude goes to the Javelosas for being so accommodating, to Jemy and Fair for their friendship and constant encouragement, to Jhoana and Dodge for their good company and editorial and computer-related assistance, a nd to John for his thoughtfulness. A special appreciation is owed to my d ear friend Rina for being the best roommate and more importantly my best friend. Those years of friendship will always be treasured and cherished. I am grateful to my relatives for all their suppo rt. I am very grateful to the Flores family in Montreal for all the support extended during my graduate studies and for being my family away from home. I would like to thank the Lamar family for graciously accommodating me in their home in Jacksonville and for treating me as a family member. I am blessed with a family who endlessly s upports and believes in me. I would like to acknowledge my brothers, Jaymar and Jun for thei r love and support. My brothers and I were surrounded by a tremendous wealth of love and s upport from my grandparent s while growing up. I am forever grateful to my mother for her in credible strength and unconditional love. Without her believing in my potential, I would never have reached this point. I owe my father gratitude for instilling in me the strength th at I need to face the challenges in life. He has been my driving

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6 force in all my accomplishments. Much apprec iation is extended to my husband, Emil for all the support and encouragement and most importan tly for his unwavering friendship and love.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES................................................................................................................ .......10 CHAPTERS 1 INTRODUCTION..................................................................................................................15 Background and Significance.................................................................................................15 Scope of Research Dissertation..............................................................................................18 2 THEORETICAL OVERVIEW..............................................................................................20 Raman Scattering............................................................................................................... .....20 Historical Background.....................................................................................................20 Spontaneous Raman Effect..............................................................................................21 Raman Signal Enhancement Techniques........................................................................26 Surface Enhanced Raman Spectroscopy (SERS)...................................................................27 Historical Background.....................................................................................................27 Enhancement Mechanisms..............................................................................................27 Substrates for SERS........................................................................................................31 Advantages and Limitations of SERS.............................................................................35 3 DESIGN, OPTIMIZATION AND CHARAC TERIZATION OF A RAMAN SYSTEM FOR SERS APPLICATION...................................................................................................40 Introduction................................................................................................................... ..........40 Design, Optimization and Characterization............................................................................41 Excitation Source.............................................................................................................41 Sampling Mode and Collection Optics............................................................................43 Monochromator...............................................................................................................44 Optical Filters................................................................................................................ ..47 Detector....................................................................................................................... ....49 Data Collection................................................................................................................53 Evaluation of Performance of the Raman System..................................................................54 4 STABILITY OF SILVER COLLOID AS SUBSTRATE FOR SURFACE ENHANCED RAMAN DETECTION OF DIPICOLINIC ACID................................................................83 Introduction................................................................................................................... ..........83 Experiments and Methods......................................................................................................84

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8 Results and Discussion......................................................................................................... ..85 Conclusions.................................................................................................................... .........88 5 EVALUATION OF EXPERIMENTAL CONDITIONS FOR THE SURFACE ENHANCED RAMAN DETECTION OF DIPI COLINIC ACID (DPA) ON SILVER COLLOIDS GENERATED BY FLOW INJECTION ANALYSIS (FIA)..........................100 Introduction................................................................................................................... ........100 Experimental Design and Methods.......................................................................................102 Results and Discussion.........................................................................................................104 Factors Affecting SERS of DPA...................................................................................104 FIA-SERS of DPA........................................................................................................109 Conclusion..................................................................................................................... .......109 6 FEASIBILITY OF SERS DETECT ION OF BACTERIAL SPORES.................................122 Introduction................................................................................................................... ........122 Experimental Methods and Procedures................................................................................124 Results and Discussion.........................................................................................................124 Conclusions.................................................................................................................... .......127 7 CONCLUSIONS AND FUTURE WORK...........................................................................133 Conclusions.................................................................................................................... .......133 Future Research Directions...................................................................................................134 LIST OF REFERENCES.............................................................................................................138 BIOGRAPHICAL SKETCH.......................................................................................................145

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9 LIST OF TABLES Table page 3-1 Common lasers used in Raman Spectroscopy...................................................................57 3-2 Nominal output power of the visi ble lines of the Argon ion laser ....................................58 3-3 A summary of the important char acteristics of the Spex 1680B double monochromator..................................................................................................................66 3-4 The monochromator scan speed at different increments...................................................67 3-5 A summary of the characteristic s of the Hammamatsu R-928 PMT.................................73 4-1 A summary of normalized signal intensities and calculated enhancement factors from SERS of DPA on silver colloids........................................................................................99 6-1 A survey of SERS methods for detection of dipicolinic acid and bacterial spores.........132

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10 LIST OF FIGURES Figure page 2-1 Energy diagram illustrating the Raman effect...................................................................37 2-2 A schematic diagram of surface enhanced Raman process...............................................38 2-3 A simplified schematic diagram illustrating the SERS electromagnetic enhancement mechanism...................................................................................................................... ...39 3-1 The Raman experimental set-up........................................................................................56 3-2 The Argon ion visible lines................................................................................................59 3-3 The experimental set-up used for measur ing Argon ion laser pr ofile and laser short and long term fluctuations.................................................................................................60 3-4 The short-term fluctuation of th e 514.5 nm line of the Argon ion laser............................61 3-5 The long-term fluctuation of th e 514.5 nm line of the Argon ion laser.............................62 3-6 A schematic diagram of the 90o sampling geometry adapted for Raman system..............63 3-7 A diagram illustrating (a) the position of the dove prism in the Raman system and (b) the image rotation by a dove .............................................................................................64 3-8 A comparison of normal Raman spectr a of saturated DPA illustrating the improvement in signal intensity due to the image rotation using a dove prism................65 3-9 Transmission spectra of different types of optical filters used in Stokes Raman Spectroscopy................................................................................................................... ...68 3-10 A comparison of the optical density plot of a long wave pass filter vs. a notch filter.......69 3-11 A spectrum of Ar+ laser with a lser transmitting filter.......................................................70 3-12 The decrease in the intensity of Rayl eigh scatter peak using a high pass filter.................71 3-13 A comparison of the Rayleigh peak a ttenuation by two diffe rent long wave pass filters........................................................................................................................ ..........72 3-14 A typical pulsed height di stribution. Disc 1 a nd Disc 2 are the discriminators of the photon counter................................................................................................................. ..74 3-15 The pulse height distribution for the photon counting system for determining the discrimination level........................................................................................................... .75

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11 3-16 Benzene spectra obtained using anal og and photon counting mode illustrating the difference in signal to noise...............................................................................................76 3-17 A timing diagram used for the photon counter and monochromator interfacing and synchronization................................................................................................................ ..77 3-18 Mercury lamp spectra obtained with (a) analog and (b) photon counting mode showing the narrow mercury lines.....................................................................................78 3-19 Raman spectra of several pure solven ts measured using the analog mode........................79 3-20 Raman spectra of 1.0 x 10-4 M beta-carotene in (a) THF and (b) ethanol.........................80 3-21 A normal Raman spectrum of saturated DPA in 1 M KOH..............................................81 3-22 Raman spectra of an empty cuve tte, deionized water and 1 M KOH................................82 4-1 Comparison of the normal Raman and SE RS spectra of dipicolinic acid (DPA)..............91 4-2 SERS signal of DPA on silv er colloids aged over 1 h ......................................................93 4-3 Absorption spectra of silver hydrosol befo re and after addition of dipicolinic acid (DPA).......................................................................................................................... .......94 4-4 Absorbance of silver colloids at the p eak maximum 401 nm as a function of sodium borohydride.................................................................................................................... ....95 4-5 SERS signal of DPA on silver colloid with and without sodi um sulfite as oxygen scavenger...................................................................................................................... ......96 4-6 Absorbance of the silver collo ids at the peak maximum 402 nm......................................97 4-7 SEM images of silver colloids prepar ed using sodium boro hydride treated with sodium sulfite................................................................................................................. ....98 5-1 The Flow Injection Analysis (FIA)-SERS set up............................................................110 5-2 Absorbance readout record ed from the FIA system........................................................111 5-3 SERS signal of DPA at 1013 cm-1 on silver colloid prepared at different volume ratios......................................................................................................................... ........112 5-4 Absorption spectra of silver colloids prepared at diffe rent volume ratios of sodium borohydride to silver nitrate.............................................................................................113 5-5 SEM images of silver colloid prepared by batch addition with different volume ratios......................................................................................................................... ........114 5-6 SERS signal of DPA at 1013 cm-1 on silver colloids generate d at different flow rates..115

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12 5-7 Scanning electron micrographs of silver colloid prepared by batch addition and generated by FIA system at different total flow rates......................................................116 5-8 The effect of addition of different con centrations of nitric acid and potassium hydroxide on the SERS signal of DPA at 1013 cm-1.......................................................117 5-9. The effect of aggregation of silver colloi d upon addition of differe nt concentrations of sodium sulfate on the SERS of DPA...............................................................................118 5-10 The effect of aggregation of silver co lloid upon addition of different concentrations of sodium chloride on the SERS of DPA.........................................................................119 5-11 A typical FIA peak profile of DPA measured at 1013 cm-1 using the FIA-SERS system......................................................................................................................... .....120 5-12 Comparison of percent relative standard deviation of SERS of DPA under static condition and FIA-mode..................................................................................................121 6-1 The SERS spectrum of 25 ppm DPA used for estimating the limit of detection 6-2 SERS spectra of different concentrations of DPA...........................................................129 6-3 A plot of DPA signal (1013 cm-1) vs concentration (0-78 ppm) using the unknown peak (966 cm-1) as an internal standard............................................................................130 6-4 SERS spectra of 78 ppm DPA measured us ing freshly prepared and 2-day old silver colloids....................................................................................................................... ......131

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENT, OPTIMIZATION AND CHARACTERIZATION OF A SURFACE ENHANCED RAMAN SPECTROSCOPIC METHOD FOR DETECTION OF DIPICOLINIC ACID By Joy D. Guingab May 2007 Chair: James D. Winefordner Major Department: Chemistry Surface Enhanced Raman Spectroscopy (SERS) ha s been explored as a tool to study biological agents. The anthrax di stribution in the US postal in Oc tober 2001 intensified the need for a rapid and accurate detection of bacterial spores. Dipicolinic acid (DPA) is a unique component of bacterial spores and has been used as a signature molecule for detection schemes. The feasibility of using SERS as a detection technique for bacterial spores is evaluated in this research. A conventional Raman system was designed, optimized and characterized. The labconstructed Raman system was used for the SERS studies of DPA on silver colloid dispersions. Experimental conditions required to obtain re producible and intense SERS signal from DPA molecule were evaluated and optimized. A conti nuous flow system was c onstructed for SERS of DPA studied under flow mode. Improvement in SERS signal reproducibility was demonstrated with the use of controlled conditions of a custom -built Flow Injection An alysis (FIA) system. The developed FIA-SERS system allowed silv er colloid generation, sample introduction and

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14 DPA detection. Characterization of the silv er colloids was done by absorption method and Scanning Electron Microscopy (SEM). The feasibility of using SERS as a detection technique for bacterial spores was evaluated. The absolute limit of detection was estimated to be 40 ng or 106 spores which is 2 orders of magnitude higher than the infect ious dose set by the Centers for Diseases Control and Prevention and required limit of detection for bacterial s pores. The signal enhancement factor of the prominent DPA peak at 1013 cm-1 was approximated to be 2 orde rs of magnitude increase from the normal Raman signal of saturated DPA. A lthough, quantitative analysis of DPA was not demonstrated in this research, Raman peaks at tributed to DPA molecules were observed in concentration as low as 25 ppm.

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15 CHAPTER 1 INTRODUCTION Background and Significance Airborne microorganisms, which ex ist in respirable size of 1 m, can cause adverse effects on humans and can potentially cause fatal diso rders. One of these biological agents is the bacterial spore. Bacteria are known to thrive almost everywhere. There is a wide array of bacterial species ranging from the benign to the infectious such as Bacillus anthracis, the causative agent for anthrax. Bacteria are micros copic but complex organisms. Aside from their ability to multiply exponentially, th ey can adapt to any kind of environmental stress by triggering some response mechanisms.1 When exposed to unfavorable gr owth conditions such as depleting food and water supply or extreme temperatures, physiological changes and cellular responses are triggered to combat starvation a nd other harsh conditions. Failure of these immediate responses is the onset of a period of metabolic dormancy and resistance to stress or sporulation. Bacterial spores can stay dormant for a few hours to m illions of years until conditions are favorable for germination. Metabolic dormancy and resistance to chemical agents, radiation and heat are the main reasons for their long-term stability.2 Endurance to all possible harsh environmental factors makes bacterial spores a potential hazard to human health. The recent anthrax attack in 2001 shows the potential danger that spores may pose. Substantial progress towards the development of rapid detection methods for bioaerosols, particularly bacterial spores, ha s been made over the past few years. The need for rapid and accurate detection of these airbor ne contaminants has been intensified by the anthrax distribution in the US postal system in October 2001. Detec tion of these biological agents is extremely important in defense against bioterrorism, preven tion of disease outbreak as well as monitoring in industrial or clinical setting. Time consuming and tedious microbiological assays based on the

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16 morphology of the microorganism are commonly used for analysis of biological agents. Routine identification of spores include s nucleic acid sequencing using the Polymerase Chain Reaction (PCR) technique.3-5 Immunoassays are commonly em ployed as spore identification techniques.6,7 Although these assays are nondestructiv e and sensitive, they require sample preparation and do not allow automation of anal ysis. Fluorescence spectroscopy, which plays an important role in the bioanalyti cal field, has also been used.8-10 Laser Induced Breakdown Spectroscopy (LIBS) has also been explored fo r its feasibility to study bacterial spores.11-13 However, these methods are not capable of distinguishing bacterial spores from other interference such as pollens and molds. Dipicolinic acid (2, 6-pyridin edicarboxylic acid or DPA) in the form of calcium dipicolinate (CaDPA) is known to be a uni que constituent of bacterial spores.2 Techniques geared towards the detection of CaDPA and its derivatives as Bacillus anthracis signature are now being developed. Sensitive and highly selective detection methods, which involve extraction of DPA from spores such as mass spectrometry.14-16 A DPA triggered terbium luminescence has been employed as a method fo r rapid detection of DPA extracted from bacterial spores.17,18 The drawback of these methods is that sample preparation is time consuming and often tedious, making the analysis time longer. The field of vibrational spectroscopy has been explored as a tool for w hole organism fingerprinting; the most promising is Raman spectroscopy.19 As a versatile, information-rich technique Raman spectroscopy plays an important role in the identification and characterization of micr oorganisms. The advent of Raman microscopy even makes it possible to probe and ch aracterize an individual bacterium20 as well as a bacterial spore.21 Detection of a single bact erial spore using Raman spectro scopy coupled with optical

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17 trapping has been investigated.22 Although Raman spectroscopy can provide rich information about a molecule, its major disadva ntage is that of having a very weak signal due to the small cross section of the inte raction process. In fact, the Rama n cross section is approximately 12 orders of magnitude lower than the competitive effect, fluorescence.23 Not until the 1970s, when an unexpectedly high signal enhancement was observed from pyridine on a roughened silver electrode24, had Surface Enhanced Raman Spectroscopy (SERS) shown promise to overcome the traditionally low sensitivity of normal Raman spectroscopy. In the past decades, studies have been conducted to understand the signal enhancement observed when a molecule is attached to variou s metals such as silver, gold and copper. The signal enhancement has now been increased to 14 orders of magnitude making single molecule detection possible.24-26 This signal enhancement would tran slate into a Raman cross section of approximately 10-16 cm2/molecule. There are two operative mechanisms responsible for the SERS phenomenon, namely electromagnetic field e nhancement and chemical enhancement. The former contributes to the enhanc ed Raman signal when scatteri ng takes place in the enhanced local optical fields of the metal and the latter is due to metal-molecule interaction. With the inherent selectivity of Raman spectroscopy co mbined with signal enhancement, SERS has become a valuable analytical tool with low det ection limits and short analysis time. Moreover, unlike with infrared (IR) measurements, water doe s not interfere, making SERS very useful in bioanalysis. Substrates used for SERS evolved from r oughened silver electrodes to a variety of substrates prepared in various ways.27 Both chemical reduction28-33 and photo reduction34-36 have been used to generate metal hydrosols. Laser ablation of metal films has also been used for preparation of colloidal sols.37-41 Metal vapor evaporation42,43 and surface etching44-46 have been

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18 employed to prepare solid support-based SERS subs trates. Metal colloids as SERS substrate is of particular interest due to straightforward preparation and simplicity of characterization and manipulation.47 However, it has been shown that extr eme dependence of the SERS effect on the physico-chemical properties of the colloids impose s a rather severe restriction on the usefulness of SERS on colloids as an analytical technique.30 It has been shown that a continuous flow configuration SERS can improve the reproducibili ty of the colloid prep aration, thus improving sensitivity.28,48-50 Bacterial spores studies usi ng SERS has been reported recen tly. It has been used as a tool for probing specific biochemical com ponents and for discrimination of bacteria.51,52 DPA extracted from spores53,54, as well as in whole bacterial spores has also been detected.19,55 SERS of a single bacterial spore has been made possible by optical trapping.56 The long term goal of this research is to de tect DPA in bacterial spores. In the present study, a SERS method was developed for detection of DPA. Controlled c onditions of a custombuilt flow injection system allowed precise preparation of the colloid, controlled aggregation and thorough mixing of the colloid and th e bacterial spores. This appr oach addressed the problem of poor reproducibility common in silver colloid prep aration that leads to n on-linearity of the SERS signal with the analyte concentration while prov iding a sensitive detec tion method for bacterial spores. The feasibility of SERS detection of 10000 spores, which is the infectious dose for Bacillus anthracis57, was evaluated. Scope of Research Dissertation The focus of this dissertation is on the deve lopment, optimization and characterization of a Surface Enhanced Raman Spectroscopy system fo r the detection of ba cterial spores. The research involved SERS of DPA studied both in static conditions and continuous flow mode.

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19 Static condition SERS studies of DPA were vital in the deve lopment and optimization of the Flow Injection Analysis (FIA)-SERS system. Theoretical considerations of Raman spectroscopy are important before focusing on SERS and are addressed in Chapter 2. This includes both classical a nd quantum mechanical description of Raman Spectroscopy. An ove rview of SERS including the enhancement mechanisms involved is discussed in Chapter 2. A variety of SERS substr ates are available but the use of metal colloids is investigated in this dissertation and briefly covered in Chapter 2. The design, optimization and characterization of a c onventional Raman system for the SERS study are presented in Chapter 3. In Chapter 4, the stability of silver colloids as SERS substrate for DPA detection is evaluated. Op timization of conditions for SE RS of DPA in FIA mode is presented in Chapter 5. The capability of SERS detection of DPA in spores is evaluated in Chapter 6. General conclusions and recommendations for future directions are described in Chapter 7.

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20 CHAPTER 2 THEORETICAL OVERVIEW Raman Scattering A brief overview of Raman scattering is n ecessary in order to understand the theory behind surface enhanced Raman spectroscopy. Se veral Raman reference books were consulted in writing this section of the dissertation chapter.23,58-61 Historical Background In the 1920s, researchers were interested in the scattering of light by charged particles. The Compton effect which explained the cha nges in the wavelength of X-ray photons when scattered by electrons was first documented.62 There was also a related study predicting this process by Kramers and Heisenberg in 1925.62 Theoretical studies c onducted by Smekal in 1923 led to the first predicti on of the Raman effect.62 Chandrasekhar Venkata (C.V) Raman in India first experimentally observed Raman scattering in 1928.63 The investigation involved focusing sunlight and using filters while observing color changes in the scat tered light. Simultaneous to the discovery by C.V Raman, a study about Rama n scattering was reported by Landsberg and Mandelstam in Moscow.62 C.V Raman initially referred to the scattering process as Feeble Fluorescence and later termed the phenomenon New Radiation. The importance of this discovery was recognized by the awarding of the Nobel Prize in Physics to C.V Raman in 1930. Ever since, it has been known as Raman Spectroscopy, Raman effect or Raman scattering. Following these pioneer investigations, more fundamental studies and applications of Raman spectroscopy had been conducted. Until the mid 1980s, the Raman literature had limited reports on the chemical applic ations of the technique. Th e limiting factors were both

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21 fundamental and technical. Fundamentally, Raman scatter is inherently weak and can be easily overwhelmed by fluorescence. Technical issues were addressed by th e introduction of PMTphoton counting detection systems a nd the laser. The feasibility of routine chemical analysis became possible after the recent introduction of Fourier-transform (FT)-Raman, charged coupled devices (CCD), small powerful computers and th e availability of near infrared lasers. Spontaneous Raman Effect Raman and Brillouin scattering are two types of inelastic scattering. Raman scattering is caused by rotational and vibrational transitions in molecules. Rotational transitions are lower in energy and slower than vibrational transitions. The molecule may have collisions with other molecules during rotational transition causing a cha nge in the rotational state of the molecule. Rotational Raman spectroscopy is carri ed out in gas phase at low pre ssure to ensure that the time for collisions is greater than the time for a transi tion. Relatively large frequency shifts which are independent of scattering angle are observed in Raman scattering. Brill ouin scattering, which is caused by thermal fluctuations of the medium, yiel ds small frequency shifts that are dependent on the scattering angle. Raman spectroscopy is an inelastic light scatteri ng effect due to the interaction of light with a sample. When a sample is illuminated with intense monochromatic light, photons are reflected, absorbed and scattered. It is the sc attering process that gives information about the structure of a molecule. The light can be scattered in three ways. Most of the scattered radiation has the same energy as the incident light result ing to elastic scatter which is referred as the Rayleigh scatter. A small fracti on of these photons is scattered inelastically by either losing energy to the molecule or gain ing energy from the molecule. Photons which lose energy give rise to Stokes Raman scattering a nd photons which gain energy give rise to anti-Stokes Raman scattering. The energy gained by the molecule in Stokes Raman scattering appears as vibrational

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22 energy. When a molecule has excess vibrational energy above the ground st ate, it is this energy that is lost to the anti-Stokes Raman scattering. A Raman spectrum is a plot of the scattered light intensity as a function of the Raman shift from the incident light in cm-1. The Raman shift is calculated with respect to the frequency of the excitation light us ing a general equation 1 10 0 (2-1) where 0 is the wavenumber of the excitation source, is the Raman wavenumber of the sample, o is the wavelength of the laser and is the wavelength co rresponding to the Raman data of the sample. This equation illustrate s that the Raman shift is independent of the wavelength of the excitation source. Both elas tic and inelastic scatter can be observed in a Raman spectrum and are symmetrically positione d on each side of the Rayleigh scatter. Although the Stokes and anti-Stokes lines are sy mmetrically positioned about the Rayleigh peak, their intensities are different w ith the Stokes being more intense. The Rayleigh peak appearing at 0 cm-1 can be greatly attenuated by a band rejection filter. The Raman effect has been described using both classical and quantum mechanical model. The discussion of the two models in this dissertation serves as a general overview. Classical model When an incoming radiation with an osci llating electric field impinges on a molecule, this induces a change in the dipole moment of the molecule. When a molecule is in the presence of an oscillating electric fiel d, its electron cloud is distorted resulting in an induced dipole moment. The induced polarization is proportional to the stre ngth of the electric field, E as given in the equation E (2-2)

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23 where the proportionality constant, corresponds to the polarizab ility of the molecule. Polarizability is related to the ease with wh ich the electron cloud can be distorted when a molecule is subjected to an elec tric field. The electromagnetic radiation generates a fluctuating dipole moment of the same fre quency and can be expressed as ) 2 cos( cos0 0 0t E t E Eo (2-3) whereoE is the electric field st rength at equilibrium and 0 is the angular frequency of the electromagnetic radiation. Equa tion 2-2 is a generalization and only describes the induced dipole that has a vibrational frequency j equal to the frequency of the incident lighto This induced dipole will scatter light at the sa me frequency as the laser light which is the Rayleigh scatter. Molecular vibrations are considered to be composed of normal modes,jQ .t Q Qj o j j2 cos (2-4) where j is the characteristic harmonic frequency of the jth normal mode. For a molecule with N atoms jQ is equal to 3N-6 and for linear molecules jQ is equal to 3N-5. During vibration, the changes in polarizability and the amplitude of the change is given by ...0 j jQ Q (2-5) When a molecule interacts with the incident radi ation with an electroma gnetic field strength E, equation 2-2 becomes t E Eo o 2 cos (2-6) Substituting equations 2-4 and 2-5 into 2-6 yields: j o o j o o ot E Q Q t Ej 2 cos 2 cos 2 cos0 (2-7)

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24 Using the identity 2 cos cos cos cos y x y x y x (2-8) equation 2-7 can be expressed as: j o j o j o j o o oQ Q E t E 2 cos 2 cos 2 2 cos0 (2-9) This equation summarizes that the induced dipole w ill radiate light at three frequencies. The first term in the equation is the Rayleigh scatter wh ich has the same frequency as the incident radiation and magnitude propor tional to the inherent polar izability of the molecule, o. The second term describes the anti -Stokes Raman scatter occurrin g at a frequency equal to j o and the third term is the Stokes Raman scatter occurring at j o The classical description of Raman scattering a lthough incomplete, provides some us eful insights. It indicates a linear dependence of polarization and both Ra yleigh and Raman scattering intensities on the intensity of the incident radi ation. At higher values ofoE nonlinear Raman scattering can occur. Another insight is that only molecular vibrations that cause a change in polarizability of the molecule yield Raman scattering 0jQ This has been the basis of the primary selection rule of Raman in contrast to IR absorption whic h requires a change in the dipole moment. The signs in the equation indicate that Raman scatter can be positive or negative. The intensities of Stokes and anti-Stokes are determined mainly by the Boltzmann distribution. Anti-Stokes scattering is dependent on the population of the firs t vibrational excited stat e. The population of the ground state is always higher th an that of the excited state re sulting in a more intense Stokes

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25 scattering intensity. The difference in intensity of Stokes and anti-Stokes scattering is given by the ratio predicted based on the Boltzmann distri bution indicating the dependence on temperature kT h I Ij o j o j o R j o R exp4 (2-10) where h is the Plancks constant, k is the Bo ltzmann constant and T is the temperature. Another important insight from the classical desc ription is that the change in polarizability jQ may vary significantly for different mol ecules and for different modes in a given molecule. Lastly, Rayleigh scatter is always more intense than the Raman scatter so jQ is expected to be much sma ller than the inherent polarizability of the moleculeo. Quantum mechanical model The quantum mechanical treatment of Rama n scattering recognizes that the vibrational energy of a molecule is quantized according to the relationship j oh E h E (2-11) where E is the net energy change of the molecule and h is the net energy change of the photons. The mechanism of the Raman process can be illustrated simplest by an energy level diagram. As depicted in Figure 2-1, when radiation interacts with a molecule, it is excited to a virtual state. The virtual state is not a true quan tum state of a molecule but can be considered as a short lived distortion of the electron cloud caused by the light oscillating electric field. Relaxation of the molecule from the virtual state to the ground state leads to Rayleigh scattering. The energy of Rayleigh scatter is the same as th e energy of the incident radiation. Raman scattering is shown in which light scattered is lower (Stokes) or higher (anti-Stokes) in energy by

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26 the amount equal to the vibrational transition h1. Stokes Raman scattering results from molecules relaxing to an energy level higher than the original energy level and yields a positive E. On the other hand, anti-Stokes Raman scattering yields a negative E since the molecule relaxes to an energy level lower than the original As discussed in the classical description of Raman scattering, Stokes has higher in intensit y than anti-Stokes. The energy population is governed by the Boltzmann distribution. Molecu les at room temperature are in the lowest vibrational level in the electroni c ground state. The intensit y difference of Stokes and antiStokes are discussed in the previous section. Raman Signal Enhancement Techniques Although Raman spectroscopy can provide rich information about a molecule, it is inherently a weak process. Ha ving a weak signal is a major disa dvantage of the technique due to a rather small cross section of the interaction process. In fact, the Raman cross section is approximately 6-8 orders of ma gnitude lower than fluorescence. Several processes can be used to enhance the sensitivity of Raman spectroscopy such as resonance Raman and surface enhanced Raman sp ectroscopy (SERS). Resonance Raman effect is observed when the lasers wavelength is tune d to the absorption wavelength of the molecule. The intensity of the vibrational modes is affect ed by the associated electronic transition. Resonance Raman spectroscopy is very useful in th e analysis of biological molecules as it is both selective and sensitive. Its sensitivity is 26 orders of magnitude higher than normal Raman spectroscopy. The weak Raman pro cess has a cross-section between 10-31 and 10-29 cm2 sr-1 molecule-1. Moreover, this enhancement technique is limited by the availability of lasers that can be tuned to be resonant with the electroni c transitions of the molecule. Surface enhanced Raman spectroscopy has shown promise to overcome the traditionally low sensitivity of

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27 spontaneous Raman spectroscopy. A remarkab le signal enhancement is observed when a molecule is attached to various metals such as silver, gold and copper (Fig. 2-2). Twenty years after the discovery of SERS, the signal enhancem ent factor approximated to be 3-4 orders of magnitude improved to about 14 orders of magnitude.26 This would translate into a Raman cross section of approximately 10-16 cm2/ sr-1 molecule-1. The succeeding section of this chapter of the dissertation provides a general ove rview of the theory of SERS based on several theoretical discussions.26,64-70 Surface Enhanced Raman Spectroscopy Historical Background An intense Raman signal from pyridine adso rbed on electrochemically roughened silver electrodes was observed by Fleishma nn, Hendra and McQuillan in 1974.24 This remarkably strong signal was attributed to the presence of a large number of pyridin e adsorbed on a greater surface area provided by the surface of the roughened electrode. Th ree years later, two papers published separately reported an increase in Raman signal from pyridine adsorbed on silver surfaces and attributed the signal enhancement to an intrinsic enhancement mechanism. In contrast to the conclusion made earlier, the strong Raman signal was caused by an increase in Raman cross section in the presence of an adjace nt rough metal structure and not to the increase in number of pyridine molecules adsorbed on th e surface of the electrode. These independent investigations of J eanmaire and Van Duyne71 and Albrecht and Creighton72 led to the emergence of what is known as surface enhanced Raman spectroscopy (SERS). SERS Enhancement Mechanisms After the discovery of the SERS effect, many researchers have conducted fundamental studies on the mechanism behi nd the signal enhancement. Understanding of the SERS mechanisms has been a subject of many ongoing research projects. Electromagnetic field

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28 enhancement and chemical enhancement are th e two operative mechanisms that have been proposed to explain the increase in the Raman cr oss section of molecules adsorbed on metallic nanostructures. It has been observed that some form of roughness is required for SERS. Equation 2-9 shows that the Raman Stokes compone nt of the polarizatio n contains both the polarizability and the electromagnetic field intens ity. The local field intensity relates to the electromagnetic field enhancement mechanism while the inherent polarizability of the molecule relates to the chemical enhancement mechan ism. The observed signal enhancement is contributed by the combination of these two m echanisms. The contribution by each mechanism depends on the molecule and the optical and chem ical characteristic of the SERS substrate. In normal Raman, the total Stokes Rama n signal is proportional to the Raman cross section as well as the laser intensity. This relationship is given by L R free s NRI N P (2-12) where s NRP is the total Stokes Raman signal in W sr-1, N is the number of molecules present in the probe volume, R free is the Raman cross section in cm2 sr-1 molecule-1and LI is the laser intensity in W. This expression can be modified to describe the total SERS Stokes signalS SERSP Based on the proposed SERS mechanis ms, equation 2-12 can be expressed as L S L R ads S SERSI A A N P 2 2' (2-13) where 'Nis the number of molecules in volved in the SERS process, R ads is the increased Raman cross section. The terms LA and SA are enhancement factors for the laser and for the Raman scattered field, respectively.

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29 Electromagnetic field enhancement mechanism In understanding the electromagnetic en hancement mechanism, the size, shape and material of the surface have to be taken into co nsideration since these characteristics determine the resonant frequency of the conduction electron s in the metallic partic le. When a metallic nanoparticle is exposed to electromagnetic field of the same frequency, electrons on the surface oscillate. Metals have many free electrons which vibrate or oscill ate when excited by an electromagnetic wave at a particular frequency. This collective resonant oscillation known as plasmons generates an additional electric fi eld on the surface of the metallic particle. Electromagnetic field enhancement mechanism is based on the idea that scattering takes place within the enhanced local optical fields of the nanoparticle. A simplified schematic diagram (Fig. 2-3) aids in understanding the elect romagnetic field enhancement mechanism.26 It shows a spherical metallic nanoparticl e with a dielectric constant, o, present in a medium with a dielectric constant, The particle has a diameter of 2r which is smaller than the wavelength of the incident laser. A molecule positioned at a distance, d, from the su rface of the spherical particle experiences an electric field, ME. This field is the superposition of the incident electric field oE and the induced field on the surface of the particle, spE sp o ME E E (2-14) The field induced on the metal sphere is depend ent on the dielectric constants of the medium and the particle, the size of the particle as well as th e distance of the molecule to the particle. This relationship is given by 3 31 2 d r E r Eo o o sp (2-15)

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30 The field enhancement factor A can be expressed as the ratio of the field at the position of the molecule and the incident field. 32 ~ d r r E E Ao o o M (2-16) Based on this equation, the conditions of the re sonant excitation of the surface plasmon can be described. Maximum field enhancement factor can be achieved when the real part of the dielectric constant of the particle is equal too 2 This can be achieved for silver and gold particles using wavelengths within the visible and near infrared region. The imaginary part of the dielectric constant needs to be small. If the enhancing factors for the laser and the Stokes field enhancement are taken into account, an expression for the electromagnetic e nhancement factor for the Stokes signal can be written as 12 2 2 2 22 2 ~ d r r A A Go S o S o L o L S L S em (2-17) Several useful insights describing the electrom agnetic enhancement mechanism can be deduced from equation 2-17. It indicates th at the enhancement scales as the 4th power of the local field of the metallic particle. This can be maximized wh en the excitation and the scattered field are in resonance with the localized surface plasmon of the metal. Electromagnetic enhancement mechanism does not require the molecule to be adsorbed directly on th e metallic surface but the distance dependence is extremely strong scaling as a 12th factor. An enhancement factor of 104 1012 has been attributed to the electroma gnetic enhancement mechanism

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31 Chemical enhancement mechanism Chemical enhancement mechanism requires direct adsorption of a molecule on the surface of the metallic nanostructure. The co ntribution to the total enhancement has been estimated to be a factor of about 100. This m echanism is associated with the overlapping of the electronic states of the metal and the adsorbed molecule. A char ge transfer state is created between the metal and the adsorbed molecule. Th e conduction band of the metal is divided by the Fermi level. This is the boundary between the filled and unfilled orbitals within the conduction band which moves up or down depending on the electric potential of the metal. An adsorbate molecule that is widely separated from the metal has discrete energy levels. When a molecule is adsorbed on the metal, the orbitals overlap to form a bond between the metal and the adsorbate. This orbital interact ion permits electrons to tunnel from the adsorbate molecule to the metal. This process is referred to as charge tr ansfer. The charge transfer state increases the probability of a Raman transition to o ccur by providing resonant excitation. The chemical enhancement mechanism via the charge transfer theory has several characteristics that distinguish it from elect romagnetic field enhancement mechanism. The charge transfer theory indicates that the enhanc ement is due to the interaction of the metal and the molecule and not the optical properties of the metallic nanostructure. A site specific interaction between the metal and the adsorbate is required for chemical enhancement. The more prominent characteristic is that chemical enhanc ement is short range in nature and limited to the first layer of adsorbed molecules. SERS substrates Since the discovery of SERS in 1970, the SE RS-active substrates have evolved from the electrochemically roughened silver electrode to a variety of forms. The pioneer works on both electrodes and colloidal systems have provided the foundations of SERS theory. Substrates for

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32 SERS, commonly noble metals such as silver, go ld or copper are used in colloidal form or immobilized on surface.27 For these substrates to be SERS-ac tive, attention must be given to the type and preparation procedure. Based on the discussion of the electromagnetic enhancement and chemical enhancement mechanisms, factors that can affect the SERS signal include the dielectric constants of both the metal and its medium, the morphology of the metallic surface, the distance between the adsorbate molecule and th e metal surface and the excitation frequency. According to the electromagnetic enhancement m echanism, SERS intensity is also affected by excitation of the local field of the metal surface. Th erefore, it is critical to control the factors that influence the excitation of the localized surface plasmon resonance (LSPR) of the metal in order to achieve the maximum SERS signal. Metal colloids Formation of metal colloids is very simp le and easy; not requiring sophisticated instrumentation. Colloidal systems are well su ited for solution phase SERS measurements. They can be easily characterized using absorption spectroscopy. There is definite dependence of SERS signal on the size and shape of the nanopartic les. The use of metal colloids for analytical detection has several limitations. SERS has been shown to require a certain degree of colloid aggregation. The aggreg ative state of the metal can easily be affected by the introduction of the analyte. Not only does SERS depend on the manner of introduction of the analyte but also depend on the exact preparation procedure of the meta l colloids. It is not surprising that SERS on metal colloids have been found to be irreproducible. Metal colloids or hydrosols can be prepared by chemical reduction of simple salts of silver or gold. Reducing agents include borohydride28-32,48,49 and citrate.33 Turkevich et al used citrate as a reducing agent to produce gold colloids of uniform size and spherical shape.73 By controlling the concentration of citrate, gold coll oids of different sizes were produced. Gold

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33 colloid preparation normally involve s heating and vigorous stirring while that of silver colloids is simple. However, methods for preparation of silver particles of uniform size and spherical shapes are less developed. Silver colloids prepared from chemical reduction of silver nitrate and sodium borohydride are usually a mixt ure of irregular sizes and shapes.74 Photo-reduction has also been used to obtain metal colloids.75-77 Laser ablation of thin metal films submerged in deionized water has been an alternative way to generate metal colloids that are free of impurities.78-82 Characterization of metal colloids normally invo lves their absorption (extinction) spectra. Silver colloid solutions are yellow and have a single extinction band at 385 nm while gold colloids are wine red with an extinction band at 520 nm.74 These extinction bands result from the localized surface plasmon resonance (LSPR) excitation of the metallic nanoparticles and depend on their size and shape. The extinction sp ectrum of a metal colloid is a contribution of both absorption and scattering of the light incident to the particles. When visible light illuminates a small particle, the particle can absorb and scatter the light. The absorption contribution (absC ) to the extinction of small particles of sizes within the Rayleigh scattering regime (a < 20 nm) is given by 2 1 83 2 M absI a C (2-18) where a is the radius of the particle, = 1 + i 2 is the dielectric constant of the particle at the optical frequency relative to that of the surrounding medium and is the wavelength of the light in the medium. For a colloidal solution with N particles per unit volume, the absorption is given by l NC Aabs (2-19)

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34 where l is the path length. The scattering contribution (scaC ) to the extinction is expressed as 2 4 6 52 1 3 128 a Csca (2-20) In the Rayleigh scattering regime, both absorp tion and scattering cont ribution are at their maxima when the real part of the dielectric cons tant is equal to -2. Absorption predominates over scattering when the particle size is within this regime. On the other hand, as the radius increases (a> 20 nm), scattering is the ma in contributor of the extinction. In the context of SERS using colloidal particles, the shift of the extinction band to longer wavelength is of particular interest rather than that of an isolated particle at the Rayleigh regime. The shift in excitation resonan ce can be achieved by using bigger particles or aspherical or particles in aggregates. Theo retical calculations of the LSPR of triangular, aggregated and irregularly shaped particles were conducted to provide insight into the electromagnetic enhancement mechanism by nanoparticles.83 Surface based SERS substrates Different methods have been used to pr epare surface based SERS substrates. The following are the methods that are being used by different groups:27 Vapor or vacuum deposition Corrosive etching or el ectrochemical roughening Nanoparticle deposition/ordering Sputter coating Aqueous sol deposition Subsequent assembly Sol-gel polymerization Noble metal embedding Metal colloid monolayer assembly Colloid multilayer formation

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35 The wide range of surface SERS substrates a llows researchers to study SERS in diverse environment. A great amount of work has been focused on the development of more stable, novel SERS substrates that can pr omote optimum SERS enhancement. Advantages and Limitations of SERS SERS as an analytical technique has many advantages. The advantages of normal Raman Spectroscopy also apply for SERS. As a vibr ational spectroscopy technique, a SERS spectrum provides information about the molecular structure. It addresses the tradit ionally poor sensitivity of normal Raman spectroscopy. SERS requires the mo lecule to be adhered to or near the metal surface thus providing non-radiative pathways for th e decay of excited states and thus, quenching fluorescence. The abrupt decay of the electrom agnetic fields ensures that only the molecules adsorbed or close to the SERS substrate are being probed making SERS a selective technique. SERS has been demonstrated to be very useful in the analysis of biol ogical samples which are normally in aqueous environment since wate r does not give a strong SERS signal. The limitations of SERS have to be considered as well. A major limitation is linked to the poor reproducibility of SERS substrates preparation making co rrelation of theoretical and experimental SERS challenging. The dynamic cha nges in metal colloids due to aggregation of the particles lead to irreproducib le signal intensities. Production of assembled nanoparticles still exhibits some degree of irreproducibility. The rou ghness feature that is required for SERS needs to be characterized properly in order to understa nd the signal enhancement. Quantitative SERS analysis has been restricted by the poor linearity of the technique. The limited choice of active SERS substrates that exhibit surface plasmon re sonance is another major constraint. Other substrates aside from gold, silver and copper have b een explored. In order for them to be used as SERS substrates, a thin coating of an active substrate is necessary which complicates the preparation procedure. SERS applicability is limited to molecules that naturally adsorb

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36 chemically or physically onto the surface of th e SERS substrate. Manipulation of these molecules must be done in order for them to be with in the enhanced optical field of the substrate. High background continuum is often observed in SE RS spectra. Although the source of the high background is still under debate, the background can overwhelm the SERS signal from the molecule.

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37 ___________________________________Excited electronic state --------------------------------------------------V irtual State ___________________________________ ___________________________________ Ground state Figure 2-1. Energy diagram illustrating the Raman effect. Rayleigh Scattering Stokes Raman Scattering Anti-Stokes Raman Scattering h o h oh 1 h o+ h 1 1

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38 Figure 2-2. A schematic diagram of surface enhanced Raman process showing molecules adsorbed on the surface of an aggregate of gol d particles. The part icle size is between 10-100 nm. Laser SERS Normal Raman

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39 ' i Figure 2-3. A simplified schematic diagram illustrating the SERS electromagnetic enhancement mechanism. The metal is spherical and its size is smaller than the wavelength of the laser (adapted from reference # 26). r d Laser Eo Metal Molecule

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40 CHAPTER 3 DESIGN, OPTIMIZATION AND CHARACTE RIZATION OF A RAMAN SYSTEM FOR SERS APPLICATION Introduction High-end commercial Raman instruments which meet different set of needs are currently available. Instrument choice depends on the intended application particularly the sample requirement and the research objectives. In the effo rt to develop a system that can be used in a wide range of applications, the design of commercial instruments becomes complex which results in difficulty of operation and increased syst em costs. The long-term goal of this research is to detect bacterial spores using Surf ace Enhanced Raman Spectroscopy (SERS). The application requirement is a simple, conventiona l Raman system with the basic components to study the SERS of bacterial spores. A Raman inst rument consists of five basic components: an excitation source, a sample illuminating system a sample holder, a wavelength selector, a detector and a data acquisition system.84,85 Although modifications such as computer interfacing for instrument control and data collection is common, the basic components remain unchanged in the majority of applications. A schematic of the lab-constructed Raman instrument is depicted in Figure 3-1. It consists of a Spectra-Physics BeamLok 2060 Ar+ laser (Mountain View,CA) and a SPEX 1680B double monochromator (Horiba JobinYvon; Edison NJ). A thermoelectrically cooled Hammamatsu R-928 (Bridgewater, NJ) photomultip lier tube coupled with a SR 400 gated photon counter (Stanford Research; Sunnyvale,CA) was used as a detector. A dove prism (Edmund Optics; Barrington, NJ) tilted at a 45o angle was used to rotate the image making it parallel to the entrance slit of the double monochromator. A LabVIEW program was used for

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41 photon counter control and data acquisition. All measurements were done using a standard 1 cm x 1 cm quartz cuvette. SERS spectra were recorded in 90o collection geometry. Characterization and optimization of the Raman system are covered in the succeeding sections in this Chapter. Since selection of components ba sed on different criter ion was not the objective of the research, thorough discussion of all existing Raman instru ment components is not included in this dissertation. This chapter is dedicated specifi cally to describe the characteristics and the performance of the Raman system designed for the particular SERS application. Design, Optimization and Characterization Excitation Source Background A variety of lasers are currently available for Raman spectroscopy. The discovery of the laser has been one of the technological advanc es behind the renaissance of Raman spectroscopy as a useful analytical techni que. Table 3-1 shows the wavelengths and output power ranges of the most common commercial lasers used in Raman Spectroscopy.86 The most common of these lasers are the Ar+ and Kr+ lasers. Their popularity in Raman spectroscopy has been attributed to their high output power, variety of output wavelengths, and relatively long lifetime. As shown in Table 3-1, different alternatives are availabl e depending on the application requirement. Source specifications Spectra-Physics BeamLok 2060 Ar+ laser with a maximum mu lti-line output power of 7 watts was used as an excitation laser. The lase r is equipped with a broadband optic that allows output of multi lines. Single line operation is done by using a prism instead of a broad band optic. The prism disperses the laser beam se parating the lines based on their wavelengths. Adjustment procedures must be followed to ge t the desired wavelength which can be confirmed by their relative power. Maximum output power specifications of the visible lines of the Ar+

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42 laser are summarized in Table 3-2.87 Visible single line outpu t power specified for 488 and 514.5 nm are 1.8 W and 2.4 W, respectively. Th e laser power that reaches the sample was approximately half of the original power due to some losses in the beam alignment and focusing. All spectra obtained in this re search were acquired using a la ser power range of 100-800 mW. Experimental characterization The laser profile was obtained using a fibe r optic and CCD spectrometer (Ocean Optics; Dunedin, Fl). Figure 3-2 shows the laser profil e of the Argon ion laser. Specifications of the Argon ion laser provided a power stability of 0.3 % and % over 2 h and 8 h period, respectively.87 Depending on the lifetime of the laser, the performance may change over time. While lasers are good sources of monochromatic li ght, fluctuations are in herent in the output power and relatively old lasers ma y be more susceptible to these fluctuations. For this reason, short-term and long-term fluctuations for the Ar+ laser were considered in system characterization. The same experimental set up used to record the laser profile was used to perform the measurement (Fig.3-3). For short term fluctuation measurement, the laser signal was monitored for 1000 s. For long-term fluctu ation analysis, the laser signal was measured over 5000 s. The integration time was set at 100 ms. The percent relative standard deviation (%RSD) was calculated for each measurement. The short-term laser fluctuation, which yielded 7% RSD, indicated that for shor ter analysis times, the noise cont ributed by the laser was minimal (Fig.3-4). This is important for determining th e systems limiting noise source. The calculated %RSD for the long-term fluctuation analysis was 8% (Fig.3-5). This can provide an insight about possible laser drift dur ing long measurement time such as the DPA SERS spectra measurement.

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43 Sampling Mode and Collection Optics Background The sampling geometry and collection optics ar e very important in the design of a Raman system. The alignment of the laser, the samp le and collection aperture has a huge influence on the magnitude, reproducibility and S/N of the Raman signal. Since the Raman signal is inherently weak, it is best to have an e fficient collection of the scattered photons. Raman sampling modes are divided into thr ee categories: conventional, remote and microscopy modes.86 Conventional sampling involves liquid samples in cuvettes or solids such as powders and pellets. Remote sampling involves the use of fibe r optic cables which can be any usable length for remote Raman detection with in meters or kilometers away from the spectrometer. The third mode of sampling uses a microscope. With the use of a microscope objective, Raman microscopy allows probing of small regions usually less than 1 m in diameter and a few m in depth. For this dissertation, th e conventional mode was used because the samples were in liquid form. Experimental characterization Common conventional sampli ng geometries are the 90o and 180o geometries. The 90o geometry was chosen for this research. With th is geometry it is easy to align the laser focal cylinder with the monochromator entrance slit. Figure 3-6 depicts the arrangement of the optics used. A transparent sample is placed in a cuve tte and the laser is directed by a mirror on the sample cuvette. The laser focal cylinder is imaged on the entrance slit of the monochromator with a slit width of 3 m. With the two lenses, the image is perpendicular to the slit, making the image area entering the monochromator too small. A dove prism was used to rotate the image on the entrance slit. A dove prism rotates an image tw ice as much as the angle that it is tilted

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44 (Fig. 3-7).84,88 By positioning the dove prism between the two lenses at a 45 degree tilt, the image was rotated by 90o. The improvement in signal collection by using a dove prism is illustrated in Figure 3-8. Monochromator Background The Raman signal is inherently weak due to a small Raman cross section in the order of 10-29 cm2 sr-1. It is difficult to detect a Raman signal amidst intense stray light, which causes an overwhelming background. For this reason, many Raman instruments use double or triple monochromators, which provide stray light rejection without sacrificing throughput. Monochromators are dispersive instruments that isolate a sm all wavelength band from a polychromatic source.84 Most monochromator configuratio ns are based on the Czerny-Turner design.84 Light passes through an entrance slit an d reflects off a collimating mirror to a diffraction grating. The grating separates the light according to wavelength. A focusing mirror reflects the diffracted light onto an ex it slit to the detector. It is important to take note of several characteristics of the monochromator such as line ar dispersion, resolution and spectral bandpass. Linear dispersion shows the capability of the in strument to disperse light. It is how far apart two wavelengths are in the focal plan e. Linear dispersion is expressed as DL = dx/d = fDa (3-2) where f is the focal length of the focusing element in mm and Da is the angular dispersion in rad nm-1. The unit for linear dispersion is mm nm-1 Manufacturers of these instruments provide a more common term, the reciprocal linear dispersion, RD which is given by RD = (DL)-1 = d /dx = (FDa)-1 (3-3) The reciprocal linear dispersion represents the wavelength range within a unit distance in the focal plane and is conveniently expressed in units of nm mm-1.

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45 The size of the diffraction grating plays a key role in determining the solid angle, the fnumber, the throughput and the resolution. With a limiting aperture diameter of L in mm, a projected area A and a focal length F in mm, the f-number (f/#) is expressed as f/# = f/L (3-4) The solid angle in sr can now be defined as = A/f2 = ( /4) / (f/#)2 (3-5) The spectral bandpass, sg, is the half-width of the wa velength distribution that passes across the exit slit. The spectral bandpass is expr essed in units of nm. With the exception of very small slit widths where diffraction effects a nd aberrations occur, the spectral bandpass for a given slit width, W in mm is given by sg = RDW (3-6) Generally, monochromators have equal entrance and exit slit widths. The slit widths determine the spectral profile of the imag e at the exit slit. In the case of equal slit widths, when monochromatic light is passed th rough the entrance slit, a monochromatic image of the entrance slit is formed on the exit slit. As the grating is rotated, the entrance slit image is scanned across the exit slit. The monochromators resolution is closely rela ted to its spectral di spersion. Dispersion indicates the distance of 2 wave lengths in the focal plane while resolution specifies the distinguishable separation of the two wavelengths. For larger slit widths where aberrations and diffraction effects are negligible, spectral reso lution is expressed as the slit width limited resolution, s in units of nm s = 2sg = 2RDW (3-7)

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46 Resolving power is another way to expre ss how well a monochroma tor distinguishes two adjacent wavelengths. Experimentally, this can be determined using the expression Rexp = ave/smin (3-8) where smin is the spectral bandpass at the minimum slit width. The theoretical resolving power is calculated using the expression Rth = ave/ d (3-9) Stray light in a monochromator is considered to be any light that passes that is outside os where o is the wavelength setting and sg is the spectral bandpass. The intensity of the light of other wavelengths is referred to as the stray light level. Stray light level is one of the most important specifications of a monoc hromator for many applications. Experimental characterization A Spex 1680B double monochromator with a fo cal length of 0.220 m is used in this research. It is equipped with a 1200 gr ooves/mm grating. The double monochromators dispersion is 1.8 nm mm-1, the resolution is 0.2 nm at 500 nm a nd the numerical aperture is f/4. It can be scanned over a spectral range of 185-900 nm The most important characteristics of the double monochromator are summarized in Table 3-3. The next criteria to consider are the monochromators accur acy and speed in acquiring a spectrum. The grating motor drive dictates the accuracy and speed of measuring a spectrum. The double monochromator used has an accuracy of .4 nm and a repeatability of .2 nm. Experimentally, the monochromator scan speed in the continuous mode was determined at by scanning from 500 to 600 nm at different wavelengt h increments (Table 34). The results of these measurements also provided the approximate rotation time of the grating at specific increments which was considered in the sync hronization of the scanning with the photon counting detection. This is covered in the next section of this Chapter. Monochromators involve

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47 scanning the spectral features of the optical sign al in both continuous and step modes. As a result, the measurement process is slower than th at of a spectrograph with a multi-array detector such as charged coupled detect ors (CCD). These types of sp ectrometers operate in a fixed grating position and directly acquire spectrum according to their dispersion as opposed to scanning the grating in the case of monochrom ators. The Spex 1680B double monochromator has an internal drive ci rcuitry that permits coupling with a scan controller. The scan can be programmed to be in continuous or step mode The continuous scan mode can easily be achieved by using an external control unit th at commonly comes with the monochromator. However, these types of scan controllers cannot be used in step mode, which is necessary for PMT-photon counting detection. An altern ative and more convenient approach to monochromator scan cont rol can be achieved using a PC peri pheral and software. Interfacing of the monochromator to a computer was done usin g a SPEX232/488 spectrometer control interface (JobinYvon; Edison, NJ), which has a dual co mpatibility. It can communicate with the spectrometer via an RS232 serial port or the IEEE 488. A Wi ndows-based spectrometer control software (JobinYvon; Edison, NJ) was used to control the monochromator. The continuous scan mode was used for direct current measurements while the step scan mode was used for photon counting mode. Optical Filters Background A Raman spectrometer must be able to meas ure weak Raman signal in the presence of a stronger Rayleigh or diffuse reflections at the la ser frequency. Raman spectrometers must have an outstanding stray light rejection, which can be achieved by using double or triple monochromators. The stray light cut-off of a typical double m onochromator sometimes is not enough to address this issue. T hus, optical filters are used to minimize detection of undesired

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48 radiation entering the spectrometer, which can overwhelm the weak Raman signal. There are two general types of filters that are used in Raman spectroscopy: laser transmitting and laser blocking filter.89 Laser blocking filters are further groupe d into two types: notch filter and long wavelength pass filter or cut-off filter. Thes e filters are characterized by their transmission spectra. The transmission spectra illustrated in Figure 3-9 show th e specific applications of the three different types of filters.89 Laser transmitting filter positioned between the laser and the sample limits undesired light from the laser such as broadband spontaneous emission or plasma lines from being detected. Such filters clean up the laser lines allowing only a single laser line to reach the sample. The thin-film laser blocking f ilter is positioned between the spectrometer and the sample to suppress the Rayleigh scatter so th at the weak Raman signals can be accurately measured. In systems where both laser transmitti ng and notch filters are in use, both Stokes and anti-Stokes Raman scattering can be measured simultaneously. The long wave pass filter on the other hand, only measures the Stokes Raman sca tter. The advantage of using a long wave pass filter over a notch filter is illu strated by their transmission spectra Long wave pass filters have the ability to transmit light close to the laser li ne. A graph comparing th e optical density [-log (Transmittance)] of the two types of filters is shown in Fig. 3-10,89 which illustrates the increase in the edge steepness of a long wa ve pass filter relative to a no tch filter. The steepness of the edge results in a narrow transition region sepa rating the laser line and the transmitting region of the filter spectrum, allowing the observation of St okes Raman shifts very close to the laser line. Experimental characterization The Ar+ laser used was equipped with a broadband optic instead of a prism so the output was a mixture of all the lines of an Argon i on laser. A laser transmitting filter (MaxLineTM, Semrock, NY) was used to clean up the laser light before reaching the sample. This filter only allowed 514.5 nm laser line to pass while bl ocking the other laser lines (Fig. 3-11). A

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49 transmission efficiency of more than 90% prevented significant loss in laser power. The Rayleigh scatter has higher intensity than the Raman scatter and can overwhelm the weak Raman signal. A laser blocking filter (Chroma Technology Corp; Rockingham, VT), positioned between the monochromator and the sample blocks the Rayleigh scatter. This research only involved Stokes Raman scatter measurement so a long wave pass filter was chosen over a notch filter. A steep 514.5 nm long wave pass filter was used to block the Rayleigh scatter and improve Stokes Raman measurement. The position of the filter was optimized and the Rayleigh scatter in both configur ations was compared (Fig. 3-12). Ra yleigh rejection was observed to be more efficient when the filter was positioned ve ry close to the monochromator entrance slit. Two different high pass filters were compared fo r their effectiveness in blocking the Rayleigh scatter (Fig.3-13). The Razor E dge filter (Semrock; Rochester, NY) proved to be more effective than the H-filter (Chroma Technol ogy Corp; Rockingham, VT). Comparison of the properties of these filters is presented in Table 3-5.89,90 Detector Background Optical detectors are grouped into three categories: thermal, photon and multichannel detectors.84 Photon detectors and multichannel de tectors are commonly used in Raman spectroscopy. Photon detectors are based on the rate of photon arrival an d the spectral response varies with wavelength while mutichannel dete ctors can provide simultaneous detection of dispersed wavelength.84 Several types of photon and multic hannel detectors are available. Photon detectors are grouped into different types of devices as photoemissive devices (photomultipliers and phototubes), pn -junction devices (photodiode s and phototransistors), photoconductive cells, a nd photovoltaic cells.91

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50 Photomultiplier (PMT) tubes fall under the photon detector category and are commonly used in Raman measurements. Photomultiplier tubes are low noise light detectors with high gain. Single photon detection can be achieve d over a spectral range of 180-900 nm. Photons striking the photocathode of a PM T cause ejection of an electron by the photoelectric effect. The electron directed to a series of dynode s or high voltage steps gets amplified. These electrons are collected at th e anode with the signal out put in the form of a pulse. Photomultiplier detecti on has two main types: analog and photon counting. Analog mode looks at the DC portion of the pul se. The DC current is the sum of all pulse s regardless of their source. For this reason, noise is added to th e signal and difficult to eliminate. Analog measurement is useful for measurements of high intensity signals because the noise contribution is negligible. For low intens ity signal measurements such as Raman spectroscopy, photon counting is preferred. In photon co unting, the pulses are directed to an amplifier/ current-voltage converter and then to a discriminator. The disc riminator sets the cut-off voltage to filter low voltage noise pulses, thus higher signal-to-noise ratios (S/N) are obt ained. Most of the noise is generated after the cathode and within the dynodes. These low voltage pulses are filtered by the discriminator unlike in analog where noise is tightly bound to the sign al. PMTs have high voltage requirements and changes in the supplied voltage do not a ffect the pulse count while the analog signal is affected. Anot her advantage is that photon counting is not affected by the RC time constant that is inherent in the electronics of analog systems Experimental characterization A side-on R-928 HammamatsuTM photomultiplier tube cooled to about -20o C was used as a detector. Important characteristics of th is PMT are summarized in Table 3-6. Pulse amplitude from the PMT was estimated based on gain and rise time provided by the manufacturer using the formula92

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51 Amplitude (mV) = Q x G x / tR (3-10) where Q is the elementary charge in C/e, G is the gain, is the impedance of the preamplifier in ohms and tR is the rise time in ns. The output from a PMT is a current pulse, which travels down a 50 ohm cable that is terminated by a 50 ohm input impedance of a pre-amplifier. For an R-928 PMT, the gain is 1 x 107 and the rise time is 2.2 ns. Usi ng the above formula, the calculated amplitude of the pulse from the PMT is 36.4 mV. A snubber was connected to the PMT to improve the shape of the pulse for photon counting.92 This is a network consisting of a shor t piece of 50 ohm coaxial cable which is terminated with a resistor of less than 50 ohms. Ringing, which can cause multiple counts from a single photon, is very common when using a PM T. This can be minimized by connecting a snubber to the PMT prior to photon counting. Th e snubber network is a 10 inch coax cable connected to a small 50 ohm potentiometer with terminating impedance ranging from 0 to 50 ohms. The other end of the coaxial cable was connected to the PMT together with the output signal cable that was connected to the preamplifie r. The output current from the PMT is divided into the signal cable and into the snubber. Adjusting the snubber to 50 ohms attenuates the signal by a factor of 2. No signa l is reflected back to the anode unless the pot is set to less than 50. Reflections are delayed by the cable roundtr ip time and sent out the signal cable. The round trip time in the snubber cable can be adjusted so that the reflections can cel the signal ringing. With the correct snubber cable length, the round tr ip time of the cable can approximately match the period of the signal ringing and thus, the sign al ringing is cancelled. For a 10-inch snubber cable, the round trip time is abou t 5 s. The snubber network conn ected to the PMT improved the shape of the pulse. However, si gnal ringing was not completely can celled. In addition to the 50 ohms impedance of the amplifier, the snubber ha d a terminating impedance of 50 ohms. Taking

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52 into account these two 50 ohm loads, the pulse amplitude was estimated to be 18.2 mV and a preamplifier gain of 25 yielded about 455 mV. Small amplitude noise pulses result from thermal emissions of the PMT dynodes and amplifier. A discriminator is used to block these low amplitude noise. Setting the correct discrimination level is important in photon counting. To achieve a good S/N, the discriminator level should be set above the noise amplitude and below the signal amplitude. Another reason for setting the correct discriminator level is to reduce drift. Slight change in the PMT gain may lead to drastic change in the photon counts if the discriminator le vel is within the center of a signal pulse height distribution. The discrimination level can be determined by plotting a pulse height distribution as de picted in Figure 3-14.92 Generally, the optimum discriminator level for a photon counter is within the valley of the pulse height distribution. There is no concrete rule in setting the discriminator value as it depends on th e nature of the measurement. With high dark counts, the threshold should be set at a lower va lue. For a cooled PMT where dark counts are not high, a higher threshold shoul d work. A pulse height distri bution presented in Figure 3-15 was plotted for the PMT. A narrow discriminator window is set by adjusti ng discriminator 1 to 0 V and discriminator 2 to -5 mV. The two discrimi nators were connected to the same input. The narrow window between the two discriminators was scanned across all pulse height and the photon counts were recorded. The discriminator level was set to 70 mV which is slightly higher than the lowest value. This setting was chosen in order to eliminate the signal ringing, which the snubber failed to cancel. The SR-400 gated photon counter (Stanfor d Research; Sunnyvale, CA) was operated using continuous gating and so the photon count er was enabled througho ut the entire count period. Computer control of the photon count er was done through the GPIB interface. The

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53 photon counter was controlled by a LabVIEW program. All the parameters such as discriminator level, integration time and count period were set using the LabVIEW software. For all photon counting measurements, the inte gration time was set between 1 s to 30 s. The spectrum of benzene recorded in analog wa s compared with that obtained with photon counting. Figure 3-16 shows that photon countin g gave a better S/N than the analog mode. Data Collection The LabVIEW program was also used to acquire spectra. Two different computer interfacing methods were employed to drive the grating at a stepping mode and control the photon counter. Synchronization of the monochr omator control with the photon counter was necessary to achieve accurate collection of Raman spectrum. In contrast to scanning continuously, the step mode permitted longer inte gration time while scanning when recording a Raman spectrum. A timing diagram (Fig.3 -17) was followed to achieve smooth and synchronized measurement and data collection. For the monochromator, the total time for each measurement is not only the time set for the grat ing to stop at a certain wavelength but also the time required for the grating to rotate from one wavelength to anothe r during scanning. The rotation time was calibrated at different wavele ngth increment (Table 3-4). The photon counter has an integration time, which is the length of time the counter is set to count photons, and a dwell time which is the time between count pe riods. Total measurement time set for the photon counter should be equal to the total measuremen t time that the monochromator stops in between scans. The data acquired by the LabVIEW program were the counts corresponding to each count period. Each data count corresponds to a specific wavelength. Data processing was done using Origin 7.5 (Northampton, MA). The height of the peaks was determined by taking the two lowest points on each side of the peak of interest and fitting a linear function.

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54 All spectra were recorded usi ng wavelength increments of 0.1 nm to 0.2 nm. In order to confirm that no peaks were skipped using thes e increments during photon counting, a spectrum of a mercury lamp was recorded using both analog and photon counting mode. Figure 3-18 indicates that none of the narrow mercury line s were skipped during the scanning in the photon counting mode. Evaluation of Performance of the Raman System The lab-built Raman system was designed spec ifically for measurements of liquids in cuvettes. Several solvent spectra were obtained as part of the system characterization. Using both analog and photon counting modes, spectra of pure solvents including benzene, methanol, ethanol, tetrahydrofuran (THF) and acetone were r ecorded. The spectra of the pure solvents in analog mode are presented in Figure 3-19. For all solvents, the spectra showed Raman peaks matching those in literature. For the purpose of S/N consideration, the signal was defined as the average intensity value and noise as standard deviation of the p eak intensity. A comparison of the benzene spectra indicated an improvement in the S/N was observed from analog mode to photon counting mode (Fig. 3-16). The ability of the system to observe Raman scattering from analytes in dilute solution was demonstrated by the spectra obtained from be ta-carotene in different solvents (Fig. 3-20). Raman spectra of dilute samples of beta -carotene in THF were also measured. The first step toward the SERS of DPA on silver colloids was to obtain a normal Raman spectrum of saturated DPA as part of the sy stem characterization (Fig. 3-21). A huge background was observed from the saturated DPA. To determine the source of this background, the spectra of an empty cuvette, deionized wa ter and 1 M KOH were recorded and compared (Fig. 3-22). The cuvette gave no signal while the deionized water and 1 M KOH showed the

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55 same shape of background. Deionized water wa s present in both samples. A spectrum of deionized water in literature confirmed the same Raman spectrum.93 After the design, optimization and characte rization of the Raman system, a broader and deeper understanding of the system has been attained. It is cl ear that the lab designed Raman system is capable of measuring Raman scatter from a variety of liquid samples. The next step is to assess its usefulness in SERS det ection of DPA in bacterial spores.

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56 Figure 3-1. The Raman experimental set-up. ARGON ION LASER Cooled PMT PHOTON COUNTER DOUBLE MONOCHROMATOR AMPLIFIER MONOCHROMATOR DRIVER INTERFACE 0 100 200 300 400 500 600 700 800 4006008001000120014001600 Wavenumber (cm-1)Raman Signal GPIB-USB 514.5 nm

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57 Table 3-1. Common lasers us ed in Raman Spectroscopy (Adapted from Reference #3). Laser Type Wavelength (nm) Typical average Power (mW) Ar+ CW 244, 247 200 Ar+ CW 457, 488, 514.5 100-2000 Kr+ CW 406, 647, 752 100-1000 He-Ne CW 632.8 5-50 Ti sapphire CW 690-1000 500-2000 diode CW 690-900 5-500 Nd:YAG Pulsed or quasiCW 200-400 10-500 Nd:YAG doubled CW and pulsed 535 50-500

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58 Table 3-2. Nominal output power of the visible lines of the Argon ion laser (adapted from reference #87) Wavelength (nm) Output Power (W) Visible Multiline 454.5-514.5 7.00 454.5 0.14 457.9 0.42 465.8 0.18 472.7 0.24 476.5 0.72 488.0 1.80 496.5 0.72 501.7 0.48 514.5 2.40 Visible Single Line 528.7 0.42

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59 Figure 3-2. The Argon ion visible lin es measured using an OceanOpticsTM CCD spectrometer. 0 1000 2000 3000 4000 5000 6000 450460470480490500510520530540550 Wavelength (nm)Signal (Counts/s)457.9 476.5 488 496.5 514.5

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60 Figure 3-3. The experimental se t-up used for measuring Argon i on laser profile and laser short and long term fluctuations. Argon ion Laser Spectrometer Laser Transmitting filter Fiber optic White cardboard 02004006008001000 0 500 1000 1500 2000 Signal (Counts)Time (s)

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61 02004006008001000 0 500 1000 1500 2000 Signal (Counts)Time (s) Figure 3-4. The short-term fluctu ation of the 514.5 nm line of the Ar+ laser (% RSD=7%).

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62 0100020003000400050006000 0 500 1000 1500 2000 Signal (Counts)Time (s) Figure 3-5. The long-term fluctuation of the 514.5 nm line of the Arg+ laser (% RSD = 8%).

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63 Figure 3-6. A schematic diagram of the 90o sampling geometry adapted for Raman system. Monochromator slit H filter lens lens Diaphragm

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64 Figure 3-7. A diagram illustrating (a) the positio n of the dove prism in the Raman system and (b) the image rotation by a dove pris m (adapted from reference # 88). H-filter f/4 4 4 f/4 4 Adjustable diaphragm slit a b

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65 4006008001000120014001600 4000 6000 8000 10000 12000 14000 16000 SERS Signal (Counts)Raman Shift (cm-1) no dove prism with dove prism Figure 3-8. A comparison of normal Raman spectra of saturated DPA illustrating the improvement in signal intensity due to the image rotation using a dove prism.

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66 Table 3-3. A summary of the important characteristics of the Spex 1680B double monochromator. Grating 1200 grooves/mm Focal length 0.220 m Reciprocal Linear Dispersion 1.8 nm/mm Resolution 0.2 nm at 500 nm Repeatability .2 nm Aperture f/4 Spectral Range 185-900 nm Accuracy .4 nm

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67 Table 3-4. The monochromator scan speed at different increments. Increment (nm) rotation time (s) scan speed (nm/s) 0.1 0.240 0.417 0.2 0.243 0.823 0.5 0.256 1.953 1.0 0.355 2.817 2.0 0.469 4.264 5.0 0.829 6.031 10.0 1.499 6.671

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68 Figure 3-9. Transmission spectra of different types of SemrockTM optical filters used in Stokes Raman Spectroscopy: (a) laser transmitting filter (b) long wave pass filter, and (c) notch filter (adapted from reference 89 ). a c b

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69 Figure 3-10. A comparison of the optical density pl ot of a long wave pass filter vs. a notch filter (adapted from reference #89).

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70 Figure 3-11. A spectrum of Ar+ laser showing the different output wavelengths (blue). Inserting a 514.5 nm MaxlineTM Semrock laser line filter between the laser and the sample all wavelengths except 514.5 nm. 0 500 1000 1500 2000 2500 3000 3500 4000 4500 450460470480490500510520530540550 Wavelength (nm)Signal (Counts/s) no Maxline laser filter with Maxline laser filter

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71 Figure 3-12. The decrease in th e intensity of Rayleigh scatter peak (514.5 nm) using a high pass filter that blocks light from 500-515 with a tr ansmission efficiency of >90% placed at different positions. 0 50000 100000 150000 200000 250000 300000 512513514515516517518519520 Wavelength (nm)Signal (Counts/s) position a position b Monochromator slit H filter lens lens Diaphragm Monochromator slit H filter lens lens Diaphragm Position a Position b

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72 Figure 3-13. A comparison of the Rayleigh peak attenuation by two di fferent long wave pass filters. acetone 0 2000 4000 6000 8000 10000 12000 14000 16000 513513.5514514.5515515.5516 Wavelength (nm)Counts/s Chroma H filter Semrock RazorEdge filter

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73 Table 3-5. A summary of the characte ristics of the Hammamatsu R-928 PMT. Photocathode material Multi alkali Window Material UV glass Peak Wavelength 400 nm Spectral Range 185-900 nm Dynode Stages 9 Gain 1 x 107 Anode to cathode voltage (max) 1250 V Average anode current (max) 0.1 mA Anode dark current (after 30 min) typical 3 nA Anode dark current (after 30 min) max 50 nA Rise time 2.2 ns Transit time 22 ns

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74 Figure 3-14. A typical pulsed hei ght distribution. Disc 1 and Disc 2 are the discriminators of the photon counter. A window discriminator (Disc 2-Disc 1) is scanned with a td interval to generate a pulse height distribution in order to determine the discrimination level for the photon counter. Single photon peak Noise Pulse Height (mV) C o u n t s Disc 1 Disc 2 td

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75 Figure 3-15. The pulse height distribution for the photon counting system for determining the discrimination level. Inset is the blown up portion of the plot to clearly show the data points. -50000000 0 50000000 100000000 150000000 200000000 250000000 300000000 350000000 400000000 450000000 -200-180-160-140-120-100-80-60-40-200 Pulse Height (mV)Counts 1 s integration time 2s integration time 5s integration time 10s integration time 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 -150-140-130-120-110-100-90-80-70-60-50-40 Pulse HeightCounts 1s integration time 2s integration time 5s integratio time 10s integration time

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76 0 1000 2000 3000 4000 5000 6000 500100015002000250030003500 Raman Shift (cm-1)Raman Signal (mV) Figure 3-16. Benzene spectra obtained using analog and photon counting mode illustrating the difference in signal to noise. 0 5000 10000 15000 20000 25000 30000 500100015002000250030003500 Raman shift (cm-1)Raman Signal (Counts) Analog Photon counting

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77 Figure 3-17. A timing diagram used for the photon counter and monochromator interfacing and synchronization. The integration time a nd dwell time of the photon counter were set using the LabVIEW program. The monochr omator stop time and rotation time were set using the monochromotor controller. Stop Time Integration Time Stop Time Rotation Time Integration time Dwell Time Monochromator Photon Counter Start Stop Dwell Time Rotation Time Count period

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78 Figure 3-18. Mercury lamp spectra obtained with (a) analog and (b ) photon counting mode showing the narrow mercury lines at 546.08, 576.96 and 579.07 nm. This demonstrates that the monochromator scanni ng does not skip any lines even when set to 0.2 nm increments. 0 500 1000 1500 2000 2500 3000 3500 542544546548550552554556 Wavelength (nm)Signal (mV) 0.2 nm increment 0.1 nm increment 0 500 1000 1500 2000 2500 3000 3500 4000 540545550555560565570575580585 Wavelength (nm)Counts/s 0.2 nm increment 0.1 nm increment a b

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79 0 100 200 300 400 500 600 700 6007008009001000110012001300140015001600 Raman Shift (cm-1)Raman Signal (mV) 0 100 200 300 400 500 600 700 800 6007008009001000110012001300140015001600 Raman Shift (cm-1)Ram an Signal (m V) 0 50 100 150 200 250 300 350 400 6007008009001000110012001300140015001600 Raman Shift (cm-1)Raman Signal (mV) Figure 3-19. Raman spectra of several pure solvents measured using the analog mode. acetone ethanol THF

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80 0 200 400 600 800 1000 1200 6007008009001000110012001300140015001600 Raman Shift (cm-1)Raman Signal (mV) beta-carotene + ethanol ethanol 0 200 400 600 800 1000 1200 1400 1600 1800 2000 6007008009001000110012001300140015001600 Raman Shift (cm-1)Ram an Signal (m V) beta-carotene+tetrahydrofuran tetrahydrofuran Figure 3-20. Raman spectra of 1.0 x 10-4 M beta-carotene in (a) etha nol and (b) tetrahydrofuran. The spectra of the solvents are shown and can be superimposed on the beta carotene spectrum. The arrows represent the peaks attribute to beta-carotene. a b

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81 40060080010001200140016001800 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Signal (Counts)Raman Shift (cm-1) Figure 3-21. A normal Raman spectrum of saturated DPA in 1 M KOH.

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82 40060080010001200140016001800 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 Raman Signal (Counts)Raman Shift (cm-1) empty quartz cuvette deionized water 1 M KOH Figure 3-22. Raman spectra of an empty cuvette, deionized water, and 1 M KOH.

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83 CHAPTER 4 STABILITY OF SILVER COLLOID AS SUBSTRATE FOR SURFACE ENHANCED RAMAN DETECTION OF DIPICOLINIC ACID Introduction Metal colloids (hydrosols) ha ve been commonly used as SE RS substrates. Creighton et al were the first to report SERS of pyridine on silver and gold colloids.47,72 Their measurements have been significant in demonstrating that SERS is associated with surface plasmon excitation on the metal surface.72 Subsequent experiments which followe d the first observation of SERS of pyridine on roughened electrodes invol ved the use of these types of substrates. However, they are less favorable for fundamental studies of th e SERS phenomenon. Researchers shifted their attention to the use of metal colloids since it is much easier to ch aracterize their optical properties particularly their absorption spectra, and to account for these properties in terms of roughness in a precise way. The main adva ntages of using metal colloids for SERS measurements are their ease of formation and manipulation for analytical studies. Chemical reduction of simple salts of silver or gold with either sodium borohydride or sodium citrate is most commonly used as a method of preparing these metal hydrosols. These colloidal systems include particles w hose diameters range from 1 nm to 100 nm.94 These metallic particles are usually negatively charged due to the adsorbed anions. Neutral species added to the hydrosol can be adsorbed on the particles replacing the negative charges. This disrupts the aggregative stability of the system and larger aggregates can form.68 It has been shown many times that SERS requires partial aggregation and adsorption of the analyte.28,30,32,48,49,68,95,96 Although, analytical applications of SERS have been reported, the use for quantitative analysis has been problematic be cause of the poor precis ion of the SERS signal due to irreproducible colloid preparation. In order to understand the factors affecting the

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84 irreproducibility of the SERS response, it is impor tant to study SERS under static conditions. This present study evaluates important factors su ch as silver colloid aging, analyte-induced aggregation and the stability of the sodium bor ohydride. Dipicolinic acid (DPA) which is a signature of Bacillus spores, is used in this study. Experiments and Methods Reagents and Procedures Aqueous solutions of analytical reag ent grade sodium borohydride (2 x10-3 M) and silver nitrate (1 x 10-3 M) were used for generation of silver colloids. The chemical reaction is:28 Ag + + BH4 +3H2O Ag + H3BO3 + 7/2 H2 (g) (4-1) Hydrogen gas is produced by the reduction of silver ions as well as the sl ow reduction of water by the sodium borohydride at room temperature. BH4 + 3H2O H2BO3 + 4H2 (g) (4-2) The silver hydrosols were obtai ned by mixing sodium borohydride a nd silver nitrate solutions at 6/1 (v/v) volume ratio. Aqueous solu tion of dipicolinic acid (1.5 x 10-3M) was mixed to the resulting silver colloid at a 1:1 ratio. A list of reagents is provided in Table 4-1. Silver Colloid Characterization Characterization of the silver colloids by absorption spectroscopy was performed using an Agilent UV-Vis double beam spectrophotometer. A 1 mL sample from each batch of silver colloids was used for all absorption measurements Deionized water was used as the blank. Scanning electron microscopy (SEM) was used to study the surface morphology of the silver colloids. 50 L aliquots were spotted on sample st ubs and air-dried overnight in a desiccator. The SEM samples were coated thin ly with gold by sputtering at 50 mA for 50 s. SERS Instrumentation SERS measurements were conducted using th e Raman system described in Chapter 3

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85 (Fig.3-1). Briefly, it consisted of a Spectra Physics BeamLok 2060 Ar+ laser and a SPEX 1680B double monochromator. A thermoelectrically cooled Hamamatsu R-928 photomultiplier tube coupled with a Stanford research photon counting system was used as a detector. A dove prism tilted at a 45o angle is used to rotate the image making it parallel to the entrance slit of the double monochromator. A LabVIEW program was used for photon counter control and data acquisition. All measurements were done using a standard 1 cm x 1 cm qu artz cuvette. SERS spectra were recorded in 90-degree collection ge ometry. All SERS signals were normalized to the measured laser power after obtaining the peak. Results and Discussion The most commonly used method of generati ng silver hydrosols for SERS measurements is the chemical reduction of silver nitrate by sodium borohydride solution. The quality of the SERS signal depends on a variety of factors such as reagent concentration, mixing procedures as well as the incorporation of the sample.28,48,49 Because of the dependenc y of the silver colloid quality on several factors, it is not surprising that SERS precision is poor. This study evaluates these important factors in order to improve the reproducibility of SERS measurement of dipicolinic acid. Farquharson and co-workers observed the fo llowing band in the SERS spectrum of DPA in water: 657, 815, 1008, 1382, 1445, and 1567 cm-1.97 Normal Raman and SERS spectra of dipicolinic acid were recorded to observe these bands (Fig. 4-1). The peaks in the normal Raman and SERS spectra of DPA are summarized in Tabl e 4-2. The most prominent peak observed at 1013 cm-1 was barely visible in the normal Raman of 7.5 x 10-3 M DPA in water. The hydrosols obtained in this study were ch aracterized by their ab sorption spectra. Provided that the silver particles are smaller than the wavelength of light and approximately spherical in shape and monodisperse silver colloids are yellow with a single extinction band at

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86 385 nm. This band is due to the resonant exc itation of plasma oscilla tions in the confined electron gas of the particles.68 Figure 4-2 shows the effect of aging the silver hydrosols on the absorption spectra. A sample hydrosol was obtaine d at different times after preparation for UVVis absorption measurement. All the spectra showed a single maximum peak at about 400 nm which is characteristic of the surface plasmon res onance of particles that are roughly spherical in shape. An increase in the absorbance due to an increase in the concentration of the silver particles over time was observed. Th is indicated that more silver particles form in a span of 1 hour after the reaction started. It is also evident that ag ing the silver colloid affects the SERS signal of DPA. Figure 4-2 illustrates that as a consequence of the increase in the concentration of silver particles through ag ing of the hydrosol, the SERS re sponse of DPA increased for the first 30 min and was constant for longer times. Silver colloids usually have a negatively char ged surface due to adsorbed anions from the reagents used in the reaction and electrostatic re pulsion keeps the colloid stable from aggregation provided that there is sufficient charge.68 Introduction of molecules such as DPA can replace these negative charges leading to ag gregation of the silver particles.68 Before addition of DPA, the silver colloids were yellow in color with a sharp extinc tion band centered at 400 nm. Addition of DPA changed the color of the silver colloid solution from yellow to pink indicating the formation of aggregates. Aggregate formati on was evident from the change in the shape of the silver hydrosol absorpti on spectrum with the appearan ce of another peak at longer wavelengths (Fig.4-3). No drastic changes in the SERS signal of DPA were observed when the DPA and the silver colloid mixture was allowed to stand for 1 h. Therefore, further silver colloid aggregation induced by the DPA was mi nimal which could also be deduced from the observed absorption curves of the mixt ure monitored within the 1 h period.

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87 Sodium borohydride has been commonly used as a reducing agent for the generation of silver hydrosols. Equation 4-2 shows that furthe r oxidation of the borohydride ions occur even at room temperature. To avoid such concen tration changes over time, sodium borohydride solutions must be freshly prepared. Figure 44 demonstrates the depl etion of borohydride ions available to reduce silver nitrat e over a period of 1 h. Assuming that all silver nitrate reacted with sodium borohydride, a decrease in the absorbance at the maximum peak ca. 400 nm indicates a decrease in the resu lting silver colloid concentratio n. The absorbance observed after 1 h of standing the borohydride solution decr eased two-fold as shown in Figure 4-4a. Deoxygenation of the deionized water was done pr ior to preparation of sodium borohydride solution to avoid further oxidation to occur. Purging the soluti on with an inert gas such as nitrogen is the most commonly used method of minimizing oxygen in the solution. However, this method did not completely stop the fu rther oxidation of borohydride based on the absorbance at the peak maximum spectra of th e silver hydrosols (Fig. 4-4b). Another way to address this problem is to introduce another re ducing agent such as sodium sulfite. Sodium sulfite is used as an oxygen scavenging agent in wa ter treatment. This method has been used to prevent quenching of phosphorescence by oxygen.98 This method is based on the reaction 2SO3 -2 + O2 2SO4 -2 (4-3) This deoxygenation method showed better results as no drastic decr ease in the absorbance at the maximum peak was observed (Fig. 4-4c). The resu lting silver concentrations remained constant with the addition of sodium sulfite. In Fi gure 4-5, an improvement in the SERS signal reproducibility was obtained largely a result of limiting borohydride concentration changes with the aid of the sodium sulfite addition.

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88 It was also observed that the formation of the yellow silver colloid solution, upon addition of sodium borohydride to silver nitrate occurred at a slower rate when sodium sulfite was added. In the absence of sodium sulfite, th e color change is instantaneous. A kinetic study of the silver colloid formation was carried out ove r a period of 3 h. A co mparison of 2 different concentrations of sodium sulfite is presented in Figure 4-6. The abso rbance reading reached a plateau after 50 min and 75 min for the silver colloid treated with 0.005 M and 0.01 M Na2SO3, respectively. The stability of SERS signal observe d from DPA using silver colloids treated with 0.050 M Na2SO3 after 50 min (Fig. 4-5a) was consistent with the results obtained from the absorption measurements of the silv er. SEM images of the silver colloids collected at different times are shown in Figure 4-7. No change in the particle size was observed from each silver colloid. Comparison of the SERS signal obtained using silver colloids treated with 0.005 M and 0.010 M Na2SO3 yielded different signal enhancement f actors (Table 4-3). Two orders of magnitude enhancement was achieved using 0.005 M Na2SO3 and one order of magnitude with 0.01 M Na2SO3. Sulfate aggregation has been shown to promote SERS of DPA.99 Based on the equation 4-3, sulfate induced aggregation may o ccur after the oxidation of sulfite. Addition of 0.005 M Na2SO3 was sufficient to form aggregation re sulting in a 100-fold DPA SERS signal enhancement while 0.010 M resulted in excess ag gregation, hence yielding only an order of magnitude enhancement. It was also observed that sulfite addition ga ve a higher DPA SERS signal as compared to sulfate additi on as summarized in Table 4-3. Conclusions This preliminary study of the SERS of DPA is crucial in improving the reproducibility of SERS as an analytical technique. It is im portant to understand the SERS of DPA on silver colloids in static conditions to address the problem of poor precisi on. It was shown that the age of the silver colloid affects the observed SERS signal of DPA with th e optimum signal achieved

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89 using silver colloids aged for at least 30 min. The DPA-induced aggregation study shows that the SERS signal measured over 1 h was stable al lowing longer measurement time, as in the case of scanning PMT-photon counting detection. Co ntrol over the deple tion of borohydride ions available for reduction of silver ions was achie ved by using sodium sulfite for deoxygenation. All these findings are useful in the developmen t of a Flow Injection Analysis (FIA)-SERS system for the detection of DPA which is the subject of the Chapter 5.

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90 Table 4-1. List of reagents used. Reagent Systematic name Formula Molecular weight (g/mol) Physical state Company Dipicolinic acid Pyridine-2,6dicarboxylic acid (C5H3N)(COOH)2167.12 White powder SigmaAldrich Sodium borohydride Sodium tetrahydroborate NaBH4 37.83 White powder Fisher Chemicals Silver nitrate Silver nitrate AgNO3 169.8731 White crystalline solid Fisher Chemicals Sodium sulfite Sodium sulfite Na2SO3 126.04 White crystalline solid J.T Baker, NJ Sodium chloride Sodium chloride NaCl 58.44 White crystalline solid Fisher Chemicals, Sodium sulfate Sodium sulfate decahydrate Na2SO4.10 H2O 322.19 White crystalline solid Fisher Chemicals Nitric Acid Nitric acid HNO3 63.01 Liquid Fisher Chemicals Potassium hydroxide Potassium hydroxide KOH 56.11 White pellets Fisher Chemicals

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91 40060080010001200140016001800 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Signal (Counts)Raman Shift (cm-1) Figure 4-1. Comparison of the normal Raman spect ra of (a) saturated DPA in KOH and (c) 7.5 x 10-3 M DPA in water; and (b) SERS spectrum of 7.5 x 10-3 M DPA in water. The silver colloids used for the SERS measur ement were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1 (NaBH4 to AgNO3). a b c

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92 Table 4-2. A summary of observed normal Raman and SERS peaks of DPA. Literature100 Observed Normal Raman SERS Normal Raman SERS Peak Assignments 647 657 674 674 817 815 835 835 998 1008 1013 1013 Symmetric ring stretch 1384 1382 1393 1393 OCO symmetric stretch 1434 1428 1425 1425 CH bend 1569 1567 1574 1574 OCO symmetric stretch

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93 Figure 4-2. SERS signal of DP A on silver colloids aged over 1 h and the absorbance of the silver colloid at 402 nm measured from 0 to 60 min. The silver colloids used were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1 (NaBH4 to AgNO3). 0102030405060 0 5000 10000 15000 20000 25000 30000 35000 Time (min)Normalized SERS Signal at 1013 cm-1 (Counts/W)0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 Absorbance

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94 300400500600700800 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Absorbance (A.U)Wavenumber (cm-1) Silver Colloid Silver Colloid + DPA Figure 4-3. Absorption spectra of silver hydrosol before (bl ack) and after (red) addition of dipicolinic acid (DPA). The silver colloid s were prepared by ch emical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1 (NaBH4 to AgNO3).

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95 0102030405060 0.0 0.2 0.4 0.6 0.8 1.0 Absorbance at 402 nmTime (min) no treatment with nitrogen purging with sodium sulfite c b a Figure 4-4. Absorbance of silver colloids at the peak maximum 401 nm as a function of sodium borohydride aging over a period of 1 h: (a) no treatment, (b) with nitrogen purging and (c) with sodium sulfite. The silver co lloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1 (NaBH4 to AgNO3).

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96 Figure 4-5. SERS signal of DPA on silver colloid with (a) and without sodium sulfite as oxygen scavenger (b The silver colloids used in the SERS measurements were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1 (NaBH4 to AgNO3). 020406080100120 0 10000 20000 30000 40000 50000 60000 70000 80000 Normalized SERS Signal (Counts/W)Time (min)a b020406080100120 0 5000 10000 15000 20000 25000 Normalized SERS Signal (Counts/W)Tim e (min)

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97 020004000600080001000012000 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 0.005 M Na2SO3 0.010 M Na2SO3Absorbance at 402 nmTime (s) Figure 4-6. Absorbance of the s ilver colloids at the peak maximu m 402 nm. The silver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4 stabilized with 0.005 M and 0.010 M Na2SO3. The volume ratio used was 6 to 1 (NaBH4 to AgNO3).

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98 Figure 4-7. SEM images of silver colloids pr epared using sodium bor ohydride treated with sodium sulfite. Silver colloid morphol ogy was observed (a) 15 min and (b) 30 min after preparation. The silver colloids we re prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4 stabilized with 0.005 M Na2SO3. The volume ratio used was 6 to 1 (NaBH4 to AgNO3). a b

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99 Table 4-3. A summary of normaliz ed signal intensities and calculated enhancement factors from SERS of DPA on silver colloids. Normalized Intensity (Counts/W) DPA Concentration Silver Colloid Treatment Normal Raman SERS Enhancement Factor 1.50E-02 1074 7.50E-03 0.005 M Na2SO4 38253 71 7.50E-03 0.010 M Na2SO4 44039 82 7.50E-03 0.005 M Na2SO3 54062 101 7.50E-03 0.010 M Na2SO3 5857 11

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100 CHAPTER 5 EVALUATION OF EXPERIMENTAL CONDITIONS FOR THE SURFACE ENHANCED RAMAN DETECTION OF DIPICO LINIC ACID ON SILVER COLLOIDS GENERATED BY FLOW INJECTION ANALYSIS Introduction Flow injection analysis (FIA) is based on the injection of a plug of sample into a moving and continuous carrier liquid. The injected sample forms a zone and is transported to a detector which continuously records a signal as the disper sed sample passes through a flow cell. The simplest FIA system is composed of a pump to push the carrier liquid through the small diameter tubing, an injection port for repr oducible injection of th e sample to the carrier stream, a reaction coil for sample dispersion and reaction with the car rier liquid, and a detection flow cell. An FIA output shows that peak height is related to the analyte concentration. A well designed FIA system has an extremely rapid response in the ra nge of 5-20 s with injected volumes between 1 L to 200 L.101 This makes FIA a high throughput techni que with minimum reagent and sample requirement. FIA systems are designed for the purpose of analyzing the maximum number of samples at short time and minimu m reagent and sample volumes. FIA is governed by three basic principles as described by Ruzicka and Hansen.102 These are reproducible timing, sample injection and cont rolled dispersion. The purpose of transporting sample solution using an FIA is to treat the sample material in such a way that it will yield not only a sensitive output but also reproducible. In an FIA system, physical mixing of the reagent and sample is carried out reproducibly. The use of an injection valve prevents variation in the sample volume. In such systems, band broadening or sample zone dispersion is controlled to suit the detection and chemistry associated with it. The process of dispersion, as the sample pl ug is transported through FIA system, can be quantitated using the disp ersion coefficient. Dispersion is defined as the amount by which the

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101 original concentration has decreased as a result of sample dilution during the transport within the FIA manifold. Mathematically, dispersion can be expressed as: D = Co/Cmax (5-1) where D is the dispersion coefficient, Co is the original concentration of the injected sample and Cmax is the maximum concentration of the sample at the peak of the FIA signal. Experimentally, this can be measured by pumping a dye soluti on (i.e. Rhodamine-6G) of known concentration into the FIA system and recording the absorban ce as the dye passes through the detection flow cell. The signal is evaluated and th e concentration of the pure dye, Co, is determined. The dye solution is replaced by a carrier solution and the dye solution is injected through the injection valve. Cmax is calculated from the observed signal and the dispersion coefficient can be calculated. The dispersion coefficient can be used to compare different manifolds when developing an FIA system. It is used to verify the extent of sample dilution due to changes in the manifold during system development. The degree of disp ersion in FIA systems have been conveniently classified into three groups: limited (D=1-2), medi um (D=2-10), and large (D=10-10000). Depending on the application requirement, the di spersion coefficient can be controlled by altering the manifold design allowing a large degr ee of flexibility. Factors such as flow rate, tubing dimensions, and types of manifold (i.e. simple coiled t ubing or mixing chambers) can be adjusted to yield a desired degree of dispersion. Controlled conditions of a flow injection syst em allow precise preparation of the colloid, controlled aggregation and thor ough mixing of the colloid and the sample. Laserna and coworkers pioneered the application of SERS to FIA.28,48,49 It has been shown that a continuous flow configuration SERS can improve the repr oducibility of the colloid preparation, thus

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102 improving sensitivity.28,48,49 In the present study, the objec tive was to develop, optimize and characterize a SERS system in a continuous flow configuration for detection dipicolinic acid (DPA). Although FIA-SERS has been evaluated fo r detection of differen t analytes, a continuous flow configuration SERS of bacterial spores has not been reported in literature. This research addressed the problem of poor reproducibility common in silver colloid preparation that leads to non-linearity of the SERS signal w ith analyte concentration. This work evaluated experimental conditions for SERS detection of DPA in an FIA system. Experimental Design and Methods Raman Experimental Setup All SERS measurements were conducted using a conventio nal Raman system described in Chapter 3. Static and FIA SERS measurem ents were done using a standard 1 cm x 1 cm quartz cuvette and a 0.440 ml flow cell, respectiv ely. All SERS signal were normalized to the power of the laser to correct for possible laser power drift. Flow Injection Analysis (FIA) set up A custom-built flow injection system was designed and constructed as described in literature.28 A schematic diagram of the FIA system is shown in Figure 5-1. Peristaltic pumps (Rainin, Model Rabbit) were se t at desired flow rates to deliver sodium borohydride (NaBH4) and silver nitrate (AgNO3) solutions and transfer the resulting hydrosol formed in the mixing cell through the 6-port injection valve. A custom-m ade degasser cell with approximate volume of 1.2 mL prevented the entrapment of hydrogen gas formed by the reduction of silver ions and water. Constant stirring in the cell ensured thorough mixing and complete reduction. A six-port low pressure valve with an injection loop (250 L) was used to introduce the sample and to control the volume of sample injected. The sample and the silver colloid were thoroughly mixed a 1 mL mixing cell. A micro-flow cell (0.440 mL) was used as the detect ion cell. Peristaltic

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103 pump tubing of desired inner diameter was used to obtain the necessary flow rate for each pump. For the peristaltic pumps to achieve smooth flow the flow rate range should be within 0.3-5 ml/min101. Chromatographic Teflon tubing (0.5 mm ID) and Valco Cheminert connectors designed with flangeless fittings were used to connect all the components of the continuous flow system. Dispersion coefficients were calculate d for the system following the simple procedure described in the previous s ection of this Chapter. Reagents and Procedures Silver colloids were prepared by reduction of AgNO3 by NaBH4 under different conditions. The chemical reaction i nvolved in the colloid formation is: Ag+ + BH4 + 3H2O Ag + H3BO3 + 7/2 H2 (g) (5-2) Incorporation of a bubble remover in the FIA system removes hydrogen gas formed by the reduction of silver ions and th e slow reduction of water taking place at room temperature: BH4 + 3H2O H2BO3 + 4 H2 (g) (5-3) Freshly prepared aqueous solutions of analytical grade reagents were used for colloid generation and deionized water was used throughout. NaBH4 solution was prepared immediately prior to use to minimize concentration changes cau sed by further reduction of water by NaBH4. Dipicolinic acid (2, 6-pyridinedica rboxylic acid or DPA) (Fisher Sc ientific, USA) was used as a model compound without further puri fication. Silver deposition was removed by periodically washing the flow system with 50% (v/v) nitric acid followed by filling w ith deionized water to prevent contamination. Characterization of the silver particles obtained at different conditions was done by measurement of absorption spectra using an Ag ilent double beam spectrophotometer. For the FIA generated colloids, a sample of the silver hydrosol was collected after the bubble remover cell. All surface characterization by SEM wa s done using a Hitachi S-400 Scanning Electron

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104 Microscope of magnification range of 18xx and a maximu m resolution of 1.5 nm. Sample preparation was done as described in Chapter 4. Results and Discussion The design of the FIA system was ba sed on the work of Laserna et al.28,49 The purpose of using an FIA system was to have reproducib le mixing, sample introduction and detection because smooth undisrupted flow must be maintained during analysis. The system flow rate was chosen to characterize the system in terms of its dispersion coefficient since it dramatically affects the quality and ability of the FIA-generate d silver colloids to promote SERS of DPA. The flow rate of the sample as it passes through the flow cell was varied and dispersion coefficients were calculated from the output signal. Figure 5-2 shows the FIA output of rhodamine dye that was injected into th e FIA system and dete cted using a UV-Vis spectrophotometer. The type of manifold used was a mixing cell to thoroughly mix the silver colloids and the sample before reaching the detection cell. Factors Affecting SERS of DPA Silver colloids prepared by reduc tion of silver nitrate with sodium borohydride have been commonly used as a SERS substrate. The characte ristics, stability and ability to enhance Raman signals depend on the relative concentration and volume ratios of the reactants, temperature and even the manner of mixing the reagents. With al l these factors affecting the quality of silver colloids, it is important to develop a reproducible procedure of silver colloid preparation that will lead to better SERS measuremen t. The flow injection system has been shown to address this poor precision problem common in SERS when silver colloids are used.28,48,49 Previous studies established that SERS require partial aggregation of silver colloids by studying the SERS activity of p-aminobenzoic aci d on silver colloids obtained from various preparation procedures.28,30,31,48,49 In order to understand the SERS activity of DPA in bacterial

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105 spores, SERS of pure DPA was studied. Prior to the application of the FIA-SERS detection of DPA, all measurements were done under static conditions. Relative concentration and volume ratios of reagents One of the factors that affect the composition of silver colloids for SERS is the relative concentration of the AgNO3 and NaBH4 solutions. Winefordner and co-workers used 0.001 M AgNO3 and 0.002 M NaBH4 to prepare silver colloids for SERS.28,30,32,48,49,74 The physicochemical property of the silver colloids is influenced by the volume ratio of the AgNO3 to NaBH4 solution. In the present stud y, the same concentrations used in the previous studies were implemented while different volume ratios of the reactants (NaBH4:AgNO3) were used for preparation of the silver colloids. For all volume ratios studied, NaBH4 was added drop wise to AgNO3 with constant stirring. DP A solution was added to the resu lting silver hydrosol at a 1:1 volume ratio. The mixture was agitated to ensure uniform adsorption of the DPA on the silver clusters. In Figure 5-3, the SE RS signal of DPA on silver colloi ds prepared at different volume ratios is shown. The highest SERS intensity was observed on silver colloid prepared with a volume ratio of 6:1 (NaBH4:AgNO3). One advantage of using silver colloids as the SERS substrate is the simplicity of characterization of the colloids by absorption spectrometry. It has been shown that an absorption spectrum can give an indication of the size and shape of the silver particles.68 The absorption spectra of the silver hydros ols collected at different volume ratio are shown in Figure 5-4. The spectra showed a si ngle maximum peak at about 400 nm which is characteristic of the surface plasmon resonance of particles that are roughly spherical in shape and about 50 nm size. Figure 5-5 shows SEM images of silver colloids prepared at different volume ratio.

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106 Effect of Flow Rate To study the extent of mixing, the peristaltic pump of the FIA system was set at different flow rates while keeping the volume ratio optimi zed at 6:1 as the preceding study. Aqueous solutions of 2 x10-3M NaBH4 and 1 x 10-3 M AgNO3 were used. A sample of the resulting hydrosol from the flow rate combinations was collected after the bubble remover cell. This ensured completeness of the reduction and elimination of bubbles due to hydrogen gas formation. Aqueous DPA solution was mixed t horoughly with the hydrosol sample at a 50/50 proportion. SERS activity of the DPA on these hydr osols was measured. The optimum flow rate determined was based on the SERS intensity of the 1013 cm-1 band which is attributed to SERS of DPA. The observed SERS intensities of DPA w ith silver colloids prepared at different flow rates are summarized in Figure 5-6. The SERS activity has been shown to depend on the extent of silver colloid aggregation.29 The continual change over time in the stat e of aggregation of the colloids at room temperature is indicative of a relationship of SERS and the age of the h ydrosol. The age of the hydrosol can be defined as the residence time of the hydrosol in the mixing chamber that opens an opportunity to form aggregates. In the FIA sy stem, the residence time of the hydrosol in the mixing chamber depended solely on the system flow rate. A study of the morphology of the silver colloids by SEM sugge sted that the FIA generated silver particles generally have similar shape as those prepared by batch addition (Fig. 5-7) By controlling the flow rate of the reagents, the shape and size of the silver colloid can be varied as indicated in Figures 5-7b and c. Faster flow rates tend to form silver particles of more uniform spherical shape as compared to those generated at slower flow ra tes, which produced a mixture of rods and spheres.

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107 Effect of pH and aggregating agents A dramatic increase in sensitivity has been observed by injecting acidified samples compared to samples at neutral pH. The signal e nhancement has been related to the activation of the silver particles by compression of the electrical double layer by protons.30 Measurement of pH in the spore coat indicated a pH of 6.3-6.5, which is more acidic than in a vegetative cell.2 Elevation of the spore core pH to about 8 has be en shown to have no effect on the dormancy of the spores and the same is true with decreasing pH.2 Extraction of DPA from the spores has been done by using surfact ants such as dodecylamine103 or simply by adding HNO3.54 Based on these accounts, the effect of pH on SERS of DPA was studied. Preliminary studies on the pH effect were conducted by simply mixing equal vo lumes of the FIA-generated silver colloids and acidified samples of DPA. DPA samples prepared in HNO3 at different concentrations were added to silver colloids in a 1: 1 volume ratio. SERS response of DPA at different concentrations was evaluated as a function of n itric acid concentration to determine the optimum concentration that promoted the greatest signal enhancement (F ig. 5-8a). Although DPA has higher solubility in strong base than in water, addition of KOH re sulted in a loss in signal enhancement as shown in Figure 5-8b. DPA dissolved in KOH is fully disso ciated resulting to more negative charge on DPA, leading to repulsion between the negatively charged silver particle and DPA. The SERS signal is quenched probably due to the decreas e in the adsorption of DPA on the silver aggregates thus, SERS is not observed. The use of aggregating agents such as poly (L-lysine) has been shown to improve the poor binding of anions to the surface of the silver particles.99 The mechanism is that the positively charged polymer molecules interact with the negatively charged silver surface providing sites of adsorption for anions. Althou gh, it has been shown that addition of poly(Llysine lead to aggregation of silver colloids, no detectable SERS signal was observed from very

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108 low concentration of DPA.99 In contrast, addition of Na2SO4 has been shown to promote remarkable DPA signal enhancement.99 The effect of aggregati on of the silver colloid by addition of different concentrations of Na2SO4 was investigated (Fig.5-9). The results showed that sulfate aggregation aids in improving the SERS of DPA at lower concentr ations but the signal decreased at higher concentrations of Na2SO4. Figure 5-10 shows the effect using NaCl as aggregating agent. This result is consistent with the results obtained by Bell and coworkers99. Addition of a singly charge anion completely quenched the SERS signal of DPA. The chloride ions replaced the negative charge on the silv er colloid and decreasing the adsorbed negatively charged DPA on the nanoaggregates. The effectiveness of Na2SO4 as an aggregating agent has been attributed to the relatively large solubility product of Ag2SO4. The Ksp of Ag2SO4 (1.2 x 10-5 mol3 dm-9) is much larger than AgCl (1.77 x 10-10 mol2 dm-6) so that even after addition of sulfate ions, there are no strongly bound anions on the surface of the s ilver to prevent the adsorption of DPA on the enhancing silver surfaces. It has been shown that Na2SO3 prevents the further oxidation of NaBH4 solution. It is used as an oxygen scavenging agent in water treat ment and has been used to prevent quenching of phosphorescence by oxygen.98 This method is based on the reaction 2SO3 -2 + O2 2SO4 -2 (5-4) The addition of Na2SO3 aided in the stability of NaBH4 as well as promoted a significant DPA signal enhancement. Interestingl y, the DPA SERS signal observed on Na2SO3 treated silver colloid was higher compared to that observed on Na2SO4 treated silver collo ids. In the present work, the signal enhancement achieved with sulf ite-treated silver collo ids was 100 times while about 70 times enhancement was observed with sulfate-aggregated silver colloids.

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109 FIASERS of DPA To improve the reproducibility of the colloid preparation, it has been shown that SERS can be conveniently studied in a flow inje ction configuration resulting in an excellent reproducibility (~3% RSD).28 The improvement has been linke d to the on-line preparation and delivery of the sample to the detection cell. An improvement in the reproducibility of the SERS signal from DPA was achieved by measuring SERS in a continuous flow mode. A remarkable improvement in the percent relative standard deviation (%RSD) obtained with the FIA-SERS system (7%) compared to SERS measured under static conditions. Figure 5-11 shows a SERS signal of DPA measured in a cont inuous flow mode at a flow rate of 1.0 ml/min. The shape of the peak generally follows the shape of a sa mple zone passing through a detection cell as described by Ruzicka.102 The incorporation of a mixing cham ber in the FIA manifold creates a broad peak.101,102 The peak response of 10 repeated injections of DPA sample yielded a remarkable improvement in the signal reproducibility. Figure 5-12 shows a comparison of calculated % RSD of SERS of DPA meas ured under static and FIA conditions. Conclusion The results of the study demonstrated the f easibility of measuri ng SERS of DPA in a continuous flow mode. Experi mental conditions required in achieving a reproducible SERS measurement were evaluated and optimized. By controlling these experimental conditions, a remarkable signal enhancement and precision were obtained in the SERS of DPA. With sulfite treated silver colloids, two orders of magnitude signal enhancement was achieved for DPA. A decrease in the percent relativ e standard deviation from 28% to 7% was observed by measuring SERS in the FIA mode.

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110 Figure 5-1. The Flow Injection Analysis (FIA)-SERS set up Silver Nitrate Sodium Borohydride peristaltic pumps Bubble remover 6-port injection valve Mixing cell Magnetic stirrer Raman System

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111 Figure 5-2. Absorbance readout recorded from the FIA system. The system flow rates were varied and dispersion coeffi cients were calculated. The sample injected was 100 L of 1 X 10-6 M Rhodamine-6G dye solution. The volum e of the flow cell was 3.5 mL. 020040060080010001200 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 AbsorbanceTime (s) 1.0 mL min-1D=177020040060080010001200 0.00 0.02 0.04 0.06 0.08 0.10 AbsorbanceTime (s) 0.50 mL min-1D=196 020040060080010001200 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 AbsorbanceTime (s) 2.00 mL min-1D=120020040060080010001200 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 AbsorbanceTime (s) 3.00 mL min-1D=100020040060080010001200 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Absorbance TIme (s) 4.00 mL min-1D=21

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112 1234567891011 1000 2000 3000 4000 5000 6000 7000 SERS Signal (Counts) Volume Ratio (NaBH4 to AgNO3) Figure 5-3. SERS signal of DPA at 1013 cm-1 on silver colloid prepared at different volume ratios (NaBH4 to AgNO3). The silver coll oids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The x-axis represents the different volumes of NaBH4 solution with constant volume of silver nitrate solution (1 ml). The silver colloid was mixed with 1.5 x 10-2 M DPA in a 1:1 volume ratio.

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113 300400500600700800 0.00 0.25 0.50 Absorbance (A.U)Wavelength (nm) a b c d Figure 5-4. Absorption spectra of silver colloids prepared at different volume ratios of NaBH4 solution (2 x 10-3 M) to AgNO3 solution (1 x 10-3 M): (a) 5/1, (b) 2.5/1, (c) 1/5 and (d) 1/2.5.

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114 Figure 5-5. SEM images of silver colloid pr epared by batch addition with different volume ratios (NaBH4:AgNO3): (a) 3:1 and (b) 6:1. The s ilver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. a b

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115 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 5000 10000 15000 20000 25000 30000 35000 40000 0.10 0.15 0.20 0.25 0.30 0.35 N o r m a l i z e d S E R S S i g n a l ( C o u n t s / W )F l o w R a t e A g N O3 ( m l / m i n )F l o w R a t e N a B H4 ( m l / m i n ) Figure 5-6. SERS signal of 1.5 x 10-2 M DPA at 1013 cm-1 on silver colloids generated at different flow rates. The s ilver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4.

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116 Figure 5-7. Scanning electron micrographs of si lver colloid prepared by (a) batch addition and generated by FIA system at different total flow rates: (b) 0.70 ml min-1 and 1.75 ml min-1. Total flow rate is the sum of the NaBH4 (2 x 10-3 M) flow rate and AgNO3 (1 x 10-3 M ) delivery flow rate. a c b

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117 Figure 5-8. The effect of addition of different concentrations of nitric acid (a) and potassium hydroxide (b) on the SERS signal of 1.5 x 10-2 M DPA at 1013 cm-1. The silver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. 0.000.020.040.060.080.100.120.14 10000 20000 30000 40000 50000 60000 70000 Normalized SERS Signal (Counts/W)Concentration (M) 0.000.020.040.060.080.100.120.14 0 5000 10000 15000 20000 Normalized SERS Signal (Counts/W)Concentration (M)a b

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118 0.000.020.040.060.080.10 25000 30000 35000 40000 45000 Normalized SERS Signal (Counts/W)Concentration (M) Figure 5-9. The effect of aggr egation of silver colloid upon addi tion of different concentrations of sodium sulfate on the SERS of DPA. The silver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4.

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119 0.000.020.040.060.080.10 0 5000 10000 15000 20000 Normalized SERS Signal (Counts/W)Concentration (M) Figure 5-10. The effect of aggregation of silver colloid upon addition of different concentrations of sodium chloride on the SERS of 1.5 x 10-2 M DPA. The silver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4.

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120 Figure 5-11. A typical FIA peak profile of 1.5 x 10-2 M DPA measured at 1013 cm-1 using the FIA-SERS system. The sample flow rate was 1ml min-1 through a 0.440 ml flow cell. The silver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. 050100150200250300 SERS Signal At 10 13cm-1Time (s)

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121 Figure 5-12. Comparison of per cent relative standard deviation of SERS of DP A under static condition and FIA-mode. Static 1=same sample, static =2different samples, FIA1=sample aspirated and FIA2= sample injected. The silver colloids were prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. static 1static 2FIA 1FIA 2-0 5 10 15 20 25 30 35 40 % relative standard deviationMode of SERS Measurement

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122 CHAPTER 6 FEASIBILITY OF SERS DETECTION OF BACTERIAL SPORES Introduction The threat of biological and chemical weapons aimed at the military and civilian population presents a challenging task for resear chers to develop fast and reliable detection methods. In the military scenario, the detection re quirement is generally to warn the soldiers of the possible threat. The soldiers are highly trained and prepared for hazardous exposure. In the event of a terroristic attack, these agents become a tool to havoc fear among the civilian population. Therefore, detection of these agents in civilian scen ario is much more challenging than in the military scenario. The need for a ra pid and accurate detection technique for bacterial spores has been intensified by the anthrax di stribution in the US mail in October 2001. Detection of low concentrations of bacterial spores in any une xpected setting has become the objective of many techniques being developed such as SERS. Dipicolinic acid (DPA), a unique component of bacterial spores, has been the mo st widely used signature molecule for SERS detection of bacterial spores. Since all spore-forming bacter ia contain DPA, SERS cannot differentiate harmless from infectious or even fatal bacteria. Therefore, detection of DPA does not necessarily indicate that the sample is a bioha zard. However, detection of bacterial spores in places that are unexpected is the first step to further investigation and identification during an emergency situation. In the present study, the objective is to esta blish whether it is possible for the developed SERS system to detect DPA in the concentration range required for bacterial spore detection. DPA which is linked to the dormancy of bacteria l spores has been found to be 15% of the dry weight of a spore.1,2 The concentration of DPA present in spores can infer the concentration of

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123 spores in counts per unit volume. This is based on the approximation that 2 x 108 DPA molecules or 4 x 10-16 moles of DPA can be extracted from a single spore through germination or physical lysis 104. Several characteristics of biological agents that are relevant for proper emergency include the infectious dose incubation time and transmissibility.105 SERS has the sensitivity needed for detecti on but lack selectivity for iden tification. In evaluating the sensitivity of the developed SERS technique for bacterial spore detection, the infectious dose must be taken into account. Infectious dose is the minimum number of vi able cells of bioagents that enter the human body that can initiate an infection or disease.105 Therefore, a lower infectious dose indicates more dangerous mi crobes, and so a single cell can be dangerous because microorganisms multiply easily as long as th ey have a suitable environment for growth. The dose level depends on the species of b acteria and the manner they are prepared. Environmental factors during the ev ent of dissemination also affect the level of an infectious dose as well as the susceptibility of the individua l to infection. Anthrax spores can enter the body by ingestion, inhalation and cutaneous exposure.57 Exposure to infected animals and their products is the usual pathway of anthrax spores in the exposure for humans. Many workers are exposed to significant levels of anthrax spores but not sufficient to in itiate symptoms. The Centers for Disease Control (CDC) has estimate d that inhalation of 10000 anthrax spores or 100 ng is lethal to 50% of an exposed population.57 Taking into account the lethal dose for Bacillus anthracis the concentration range for SERS detection at this level can be approximated. Suspensi ons of spores which serve as biological indicators for sterili zation procedures can be obtaine d in concentrations up to 108 spores/0.1 ml. For the purpose of calculating th e DPA concentration that can be released from spores, the spore concentration can be expressed in units of spores/0.1 ml suspension. All SERS

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124 detection in this study was done using aqueous so lutions of DPA standards. Based on the DPA concentration inside a singl e spore, a suspension of 104/0.1 ml spores contains about 0.7 ppb or 4 nM DPA. This means that the detection limit should be below this concentration in order to detect anthrax spores before reaching the lethal dos e. In this study, the feasibility of detecting spores was evaluated. The limit of detection was determined and compared with 10000 spores. Experimental Methods and Procedures Silver colloids were generated from the FIA system at the optimum volume ratio and flow rate, and collected after the bubble remover (Fig.5-1). The sodium borohydride solution was stabilized with 0.005 M s odium sulfite. Aqueous solution s of DPA were prepared (0-2500 ppm) for SERS measurement. In the quantitative measurements of DPA, 1 ml of silver colloid was mixed with 1 ml of DPA solution. SERS meas urements were done in cuvettes under static conditions using the conventional Raman system described in Chapter 3 (Fig.3-1). Results and Discussion In the previous chapters of this dissertation, it has been shown that it is possible to obtain acceptable SERS spectra of a high c oncentration of DPA. To evalua te the feasibility of detecting spores, low concentrations of DPA were measur ed. The lowest concentration that gave detectable DPA peaks was 25 ppm. Based on the S/N of the DPA peak at 1013 cm-1 for 25 ppm, the relative limit of detection is 5 ppm or 30 M DPA (Fig.6-1). Taking into account the probe volume used in the SERS measurement, the abso lute limit of detection is 40 ng DPA. In the context of spore detection, assuming the calculations are correct, then at least 106 spores are needed to observe an acceptable SERS signal for DPA using the existing SERS method. The enhancement factor using 25 ppm DPA is about 2 orders of magnitude. Several groups are developing SERS methods us ing a variety of SERS substrates fo r detection of bacterial spores.

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125 Table 6-1 presents a summary of the results of di fferent SERS investigation of DPA in bacterial spores. The SERS spectra of different concentrati ons of DPA measured under static conditions are shown in Fig. 6-2. The spectra show that although the SERS response is not linear over the concentration range, all the spectra above 50 ppm gave good signal to noise. All these concentrations showed fingerprint spectra of DP A. Clearly, quantitativ e SERS measurement of DPA under static conditions ha s poor reproducibility. In normal Raman measurements, quantitative analysis is done by introducing an internal standard which is presumably inert and yields a detectable Raman signal. The internal standard accounts for variations in the laser intensity and the sample that l eads to irreproducibility. For SE RS measurements, an internal standard plays the same role however, its interact ion with the SERS substr ate is very important. The introduction of an internal standard in SE RS measurements takes into account not only the factors affecting Raman measuremen ts but also changes in the SERS substrate. KSCN has been used as an internal standard in SERS quantit ative analysis of DPA (0-50 ppm) with silver colloids99. It has been shown to play a role in elim inating false negatives since a deactivation of the silver colloids lead to a decrease in both DPA and thiocyanate peak intensities. The symmetric stretch vibration of NO3 at 1050 cm-1 has been observed in the SERS spectra of Bacillus subtilis spores in HNO3 and has been used as an internal standard to account for variations in the sample.106 In this study, 0.005 M Na2SO3 was used as a stabilizer for NaBH4 solution. This was used as an oxygen scavenger that follows the equation 2SO3 2+ O2 2SO4 2-

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126 Assuming that all the SO3 2ions were converted to SO4 2-, an observable peak at 980 cm-1 can be assigned as the SO4 2peak and can be used as an internal standard. A prominent unknown peak was observed at about 966 cm-1 in the spectra of 50 to 625 ppm DPA solution (Fig. 6-2c-g). The unknown peak which was suspected to be SO4 2peak was not observed in the blank sample (Fig. 6-2a) or in the 25 ppm DPA (Fig.6-2b) as well as concentrations higher than 625 ppm (Fig.6-2hi). The blank contained 0 ppm of DPA and silver colloids. Fo r the concentration range between 50-625 ppm, the ratio of the intensit ies of the DPA peak at 1013 cm-1 to the intensity of the suspected SO4 2peak was plotted against the DPA con centration to obtain a calibration curve (Fig.6-3). Linear response was observed at rela tively higher concentratio ns. For concentrations lower than 78 ppm, the sulfate peak in tensity relative to the DPA peak at 1013 cm-1 is not reproducible. The unknown peak at 980 cm-1 seems to behave differently at very low concentrations and relatively high concentrations of DPA. The results show that DPA and SO4 2responses are linear only within a particular conc entration range. At high concentrations, the disappearance of the SO4 2peak can be explained by the satu ration of the SERS substrate active sites by DPA molecules. With the assumption that SO3 2ions were completely converted to SO4 2ions, all samples contained the same concentration of SO4 2which was much lower compared to DPA. On the other hand, at DPA concentrations below 78 ppm, few DPA molecules compete with the SO4 2ions. The erratic behavior of the SO4 2peak indicates the presence of other factors affecting the SERS activity of SO4 2ions. Finally, it should also be noted that the silver colloids are not stable after a day. The spectrum 78 ppm was measured and presented in Fig.6-4. The quality of the spectrum is not comparable to that measured the same da y the silver colloids were prepared

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127 Conclusions The results have demonstrated that SERS of low concentrations of DPA can be observed using silver colloids as the SERS substrat e. The S/N of the SERS peak at 1006 cm-1 of the 25 ppm DPA solution of 14 suggests a relative lim it of detection (S/N=3) of 5 ppm or 30 M. The absolute limit of detection is 40 ng or 106 spores. The absolute limit of detection is 2 orders of magnitude above the lethal dose of 10000 spores set by CDC. The SERS enhancement factor relative to the normal Raman signal intensity of saturated DPA is estimated to be 2 orders of magnitude. In order to achieve th is limit of detection, a signal enhancement of at least 5 orders of magnitude has to be observed from the silver co lloids. This indicates that further investigation has to be conducted to fully achieve a quantitativ e method for the detection of bacterial spores.

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128 Figure 6-1. The SERS spectrum of 25 ppm DPA used for estimating the limit of detection. Inset shows the DPA peak at 1013 cm-1. 9801000102010401060108011001120114011601180 -50 0 50 100 150 SERS Signal (Counts/s)Raman Shift (cm-1)400600800100012001400160018002000 0 1000 2000 3000 4000 5000 6000 SERS Signal (Counts/s)Raman Shift (cm-1)

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129 Figure 6-2. SERS spectra of diffe rent concentrations of DPA. The silver colloids were prepared using:2 x 10-3 M NaBH4 and 1 x 10-3 M AgNO3. A peak at 966 cm-1, suspected to be a SO4 -2 peak, appeared in the spectra of 50-625 ppm DPA. 400600800100012001400160018002000 0.0 0.2 0.4 0.6 0.8 1.0 SERS SignalRaman Shift (cm-1) 25 ppm DPA400600800100012001400160018002000 0.0 0.2 0.4 0.6 0.8 1.0 0 2 4 6 8 10 SERS SignalRaman Shift (cm-1) 0 ppm400600800100012001400160018002000 0.0 0.2 0.4 0.6 0.8 1.0 SERS Signal Raman Shift (cm-1) 50 ppm400600800100012001400160018002000 0.2 0.4 0.6 0.8 1.0 SERS Signal Raman Shift (cm-1) 78 ppm DPA400600800100012001400160018002000 0.2 0.4 0.6 0.8 1.0 SERS Signal Raman Shift (cm-1) 156 ppm DPA400600800100012001400160018002000 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 SERS Signal Raman Shift (cm-1) 312 ppm DPA400600800100012001400160018002000 0.2 0.4 0.6 0.8 1.0 SERS SignalRaman Shift (cm-1) 625 ppm DPA400600800100012001400160018002000 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 SERS SignalRaman Shift (cm-1) 1250 ppm400600800100012001400160018002000 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 SERS SignalRaman Shift (cm-1) 2500 ppm DPA a b c d e f g h i

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130 0100200300400500600700 0 1 2 3 4 5 6 7 8 IDPA/Iunknown peakDPA concentration (ppm) Figure 6-3. A plot of DPA signal (1013 cm-1) vs concentration (0-78 ppm) using the unknown peak (966 cm-1) as an internal standard.

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131 Figure 6-4. SERS spectra of 78 ppm DPA measured using freshly prepared and 2-day old silver colloids. 400600800100012001400160018002000 0.2 0.4 0.6 0.8 1.0 SERS SignalRaman Shift (cm-1) same day after 2 days

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132 Table 6-1. A survey of SERS methods for dete ction of dipicolinic acid and bacterial spores Type of SERS Substrate Borohydride reduced silver colloid* Silver film over nanospheres (AgFON)54 Silver doped sol gel107 Citrate reduced silver colloid99 Absolute limit of detection 8 x 105 spores (~106spores) 2.3 x 103 spore (~103 spores) 2.5 x 104 spores (~104 spores) 1 ppm (105 spores) Enhancement factor 100 Total sample volume 2 ml 0.2 L 0.1 mL 200 L *The authors research results

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133 CHAPTER 7 CONCLUSIONS AND FUTURE WORK Conclusions The research described in this dissertation involved the development of a SERS detection method for DPA, a signature molecule of bacter ial spores. The work was divided into three stages: (1) Raman instrument design, optimi zation and characterizat ion; (2) SERS method development and characterization, and (3) feasib ility of SERS detection of bacterial spores. In the first stage of the research, a conve ntional Raman system was designed for SERS application. The Raman system consisted of an Ar+ laser, a double monochromator and a PMTphoton counting detector. Instrument contro l was done through computer interfacing. Characterization and optimization studies were conducted. The perf ormance of the Raman system was evaluated. Several spectra of pure so lvents and dilute solutions were measured and corresponding S/N were calculated. The nor mal Raman spectrum of saturated DPA was measured in order to calculate the signal enha ncement factor achieved using SERS on silver colloids. The second research stage involved the SERS study of DPA on silver colloids. This phase of the research included SERS of DPA studied under static and flow condi tions. Static measurements of DPA were done to study the stab ility of silver colloids as SERS substrate which included silver colloid aging and silver co lloid aggregation due to analyte addition. The stability of NaBH4 against further oxidation was also studie d. It was observed that addition of Na2SO3 to the NaBH4 solution prevented depletion of NaBH4 available for reduction of AgNO3. The effect of addition of HNO3, KOH, NaCl and Na2SO4 on the SERS signal of DPA was studied. It was conclu ded that addition of Na2SO4 proved to enhance the DPA signal linearly to a limit in Na2SO4 concentration. A Flow Injectio n Analysis system was designed and

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134 characterized for the flow mode SERS studies of DPA. Evaluation and optimization of experimental conditions which in cluded the volume ratio of NaBH4 and AgNO3 and flow rates were conducted. The FIA-SERS system improved the precision of SERS on silver colloid from an RSD of 28% to 7% by measuring in the continuous flow mode. The third stage was the evaluation of the feas ibility of SERS detection of DPA. SERS spectra of several concen trations of DPA solutions were measured. Quantitative analysis of DPA was not demonstrated in this research. In order to achieve qua ntitative analysis, an internal standard must be used to correct for the po ssible deactivation of th e silver colloid. The lowest concentration which showed DPA peaks was 25 ppm. The enhancement factor was calculated to be 100. The ab solute limit of detection was calculated to be 40 ng or 106 spores. The current limit of detection was 2 orders of ma gnitude above that required for bacterial spores based on the infectious dose set by the Center for Disease Control and Prevention. It was concluded that for this method, an enhancement f actor of about 4 orders of magnitude has to be achieved to observe acceptable SERS signal from 10000 spores. The SERS studies of DPA conducted in this re search provide valuable preliminary results which can be useful in developing a de tection method for bacterial spores. Future Research Directions Based on the results presented in this disse rtation, several paths can be followed for future research studies. The future work can be grouped into (1) sensitivity improvement and (2) instrument field adaptability. The more important and apparent direction is the improvement in sensitivity of the technique for detection of b acterial spores. Remarkable SERS enhancement has been linked to the quality of the SERS substrat e. In this research, silver colloid prepared by chemical reduction of AgNO3 by NaBH4 was presented. The silver particles produced from the reaction are negatively charged due to the adsorbed residual anion (e.g.NO3 -). Partial

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135 dissociation of DPA yields a ne gative charge. DPA molecules replace this anions the on the surface of the silver nanoparticles. It has been shown that addition of SO4 -2 ions induces aggregation without competing with th e DPA ions for the SERS active sites.108,109 Competitive binding by anions have been studied us ing citrate reduced silver colloids.108 The binding of DPA on borohydride reduced silver colloid can be studied in the presence of different anions (i.e. Cl-, NO3 -,SO3 -2 and SO4 -2) added simultaneously. A similar study can also be done using impurityfree silver colloid dispersions. Silver colloids fr ee of residual ions from chemical reduction can be obtained commercially (Ted Pella, Inc.). La ser ablation of thin silver films in submerged deionized has been demonstrated to be a good source of silver particles for SERS that are free of impurities.110-112 Although low concentrations of DPA gave good SERS spectra, quantita tive analysis of DPA was not demonstrated in this research. Bell and coworkers have demonstrated the possibility of quantitative SERS measurement of DPA in the concentration range (0-50 ppm).109 It is important to be able to quantify the con centration of DPA in spore samples in order to pinpoint a possible threat. For static SERS measurements, the reproducibility problem can be corrected using an internal standard. An internal standard must have the same interaction with the SERS substrate as the analyte. The cha nges in the SERS signal of the analyte and the internal standard must be parallel in order to correct for any possible changes in the SERS environment. Previous study has shown the use of KSCN as an internal standard to take into account any possible deactivation of the silv er colloids during the SERS measurement.99 Small anions can be used as internal standard as long as they easily give accep table SERS signal. In this research, an attempt to lot a calibration curve for DPA wa s done using an unknown peak that appeared at 966 cm-1. Aside from the ions (Na+, NO3 and DPA-) present in the silver colloid

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136 analyzed for SERS, SO4 -2 (produced from the SO3 -2 by addition to the NaBH4) was also present. The Raman band at 980 cm-1 has been attributed to SO4 -2. The unknown peak was suspected to be the SO4 -2 peak. However, the appearance of the peak in the SERS spectrum was not consistent at all concentrations of DPA measur ed. Further confirmatory experiments should be done to characterize SERS of SO4 -2 on silver colloids. After achieving the sensitivity required for the detection, the s econd direction to be considered is improving toward field adaptability of the detection system. A portable system is ideal for spore detection in unexpected places. For the developed system, the first step is miniaturization of the FIA system. The custom-made FIA system has been shown to improve the reproducibility of the DPA SERS signal. Du ring the development of the FIA system, it is desirable that the manifold desi gn be simple and easy to modify. The manifold is where physical mixing of the reagents takes place. The manifo ld design for the FIA-SERS system consisted of flexible tubing and mixing chambers. Since th e method has been developed and optimized for SERS detection, a fixed and rigi d structure can now be designed for routine use of the system. Ruzicka and Hansen introduced the use of micr o-conduits made from a block of plexiglass.101,102 Instead of using flexible tubing that can be da maged easily, channels can be engraved on the plexiglass block. The mixing chambers can be replaced by engraved wells containing small magnetic stirrers. There are many advantages of using integrated micro-conduits. They are rigid and stable making routine analysis possible. They are small and compact and easy to connect to other components of an FIA system (i .e. peristaltic pumps and injection valve). The channels are protected preventing disturbance in the flow as in the case of exposed flexible tubing. Miniaturization of the manifold block reduces reagent consumption and sample volume. The conventional Raman system used for the qual itative studies of DPA can be replaced with a

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137 portable system. The bulky Ar+ laser can be replaced with small diode lasers. Simple fiber optic probe can be used instead of the complex optical system. An air-cooled CCD spectrometer can be used a detector.

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138 LIST OF REFERENCES 1. Fundamental and Applied Aspect s of Bacterial Spores, Academic Press: London, 1985. 2. Setlow, P. Fundamental and Applied Asp ects of Bacterial Spores Gould, G. W.; Russel, A. D.Stewart-Tull, D. E. S., Eds.; Blackwell Scientific PUblications: Oxford, 1994. 3. Makino, S.; Cheun, H. Journal of Microbiological Methods 2003, 53 141-47. 4. Reif, T. C.; Johns, M.; Pillai, S. D.; Carl, M. Applied and Environm ental Microbiology 1994, 60 1622-25. 5. Sperveslage, J.; Stackebrandt, E.; Lembke, F. W.; Koch, C. Journal of Microbiological Methods 1996, 26 219-24. 6. McBride, M. T.; Gammon, S.; Pitesky, M.; O'Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Analytical Chemistry 2003, 75 192430. 7. Stratis, D. N.; Griffin, G. D; Mobley, J; Vass, A. A; Vo-Dihn, T Analytical Chemistry 2003, 75 275-80. 8. Ho, J. Analytica Chimica Acta 2002, 457 125-48. 9. Kunnil, J.; Swartz, B.; Reinisch, L. Applied Optics 2004, 43 5404-09. 10. Kunnil, J.; Sarasanandarajah, S.; Chacko, E.; Swartz, B.; Reinisch, L. Aerosol Science and Technology 2005, 39 842-48. 11. Dixon, P. B; Hahn, D. W Analytical Chemistry 2005, 77 631-38. 12. Gibb-Snyder, E; Gullet, B; Ryan, S; Oudejans, L; Touati, A Applied Spectroscopy 2006, 60 860-70. 13. Kim, T.; Specht, Z. G.; Vary, P. S.; Lin, C. T. Journal of Physical Chemistry B 2004, 108 5477-82. 14. Beverly, M. B.; Basile, F.; Voorhees, K. J.; Hadfield, T. L. Rapid Communications in Mass Spectrometry 1996, 10 455-58. 15. Snyder, A. P.; Thornton, S. N.; Dworzanski, J. P.; Meuzelaar, H. L. C. Field Analytical Chemistry and Technology 1996, 1 49-59. 16. White, D. C.; Lytle, C. A.; Gan, Y. D. M.; Piceno, Y. M.; Wimpee, M. H.; Peacock, A. D.; Smith, C. A. Journal of Microbiological Methods 2002, 48 139-47. 17. Ponce, A.; Lester, E.; Kirby, J. P. Abstracts of Papers of th e American Chemical Society 2003, 225 U169.

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139 18. Lester, E. D; Bearman, G; Ponce, A IEEE Engineering in Medicine and Biology 2004, 23 130-35. 19. Grow, A. E.; Wood, L. L.; Claycomb, J. L.; Thompson, P. A. Journal of Microbiological Methods 2003, 53 221-33. 20. Schuster, K. C; Urlaub, E; Gapes, J. R Journal of Microbiological Methods 2000, 42 2938. 21. Esposito, A. P.; Talley, C. E.; Huser, T.; Hollars, C. W.; Schaldach, C. M.; Lane, S. M. Applied Spectroscopy 2003, 57 868-71. 22. Chan, J. W.; Esposito, A. P.; Talley, C. E.; Hollars, C. W.; Lane, S. M.; Huser, T. Analytical Chemistry 2004, 76 599-603. 23. McCreery.R Raman Spectroscopy for Chemical Analysis, John Wiley and Sons, Inc: New York, 2000. 24. Fleishman, M; Hendra, P. J.; McQuillan, A. J Chemical Physics Letters 1974, 26 163-66. 25. Kneipp, K.; Kneipp, H.; Kartha, V. B.; Manoharan, R.; Deinum, G.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Physical Review e 1998, 57 R6281-R6284. 26. Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Journal of PhysicsCondensed Matter 2002, 14 R597-R624. 27. Baker, G. A.; Moore, D. S. Analytical and Bioanalytical Chemistry 2005, 382 1751-70. 28. Berthod, A.; Laserna, J. J.; Winefordner, J. D. Applied Spectroscopy 1987, 41 1137-41. 29. Berthod, A.; Laserna, J. J.; Winefordner, J. D. Journal of Pharmaceu tical and Biomedical Analysis 1988, 6 599-608. 30. Laserna, J. J.; Torres, E. L.; Winefordner, J. D. Analytica Chimica Acta 1987, 200 46980. 31. Laserna, J. J.; Campiglia, A. D.; Winefordner, J. D. Analytica Chimica Acta 1988, 208 21-30. 32. Laserna, J. J.; Sutherland, W. S.; Winefordner, J. D. Analytica Chimica Acta 1990, 237 439-50. 33. Lee, P. C.; Meisel, D. Journal of Physical Chemistry 1982, 86 3391-95. 34. Muniz-Miranda, M. Journal of Raman Spectroscopy 2002, 33 295-97. 35. Muniz-Miranda, M. Colloids and Surfaces A-Physicoc hemical and Engineering Aspects 2003, 217 185-89.

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140 36. Muniz-Miranda, M. Journal of Raman Spectroscopy 2004, 35 839-42. 37. Lee, I.; Han, S. W.; Kim, K. Journal of Raman Spectroscopy 2001, 32 947-52. 38. Prochazka, M.; Mojzes, P.; Step anek, J.; Vlckova, B.; Turpin, P. Y. Analytical Chemistry 1997, 69 5103-08. 39. Prochazka, M.; Stepanek, J. ; Vlckova, B.; Srnova, I.; Maly, P. Journal of Molecular Structure 1997, 410 213-16. 40. Smejkal, P.; Siskova, K.; Vlckova, B.; Pf leger, J.; Sloufova, I.; Slouf, M.; Mojzes, P. Spectrochimica Acta Part A-Mol ecular and Biomolecular Spectroscopy 2003, 59 232129. 41. Zhang, J. B.; Fang, Y. Colloids and Surfaces A-Phys icochemical and Engineering Aspects 2005, 266 38-43. 42. Saito, Y.; Wang, J. J.; Smith, D. A.; Batchelder, D. N. Langmuir 2002, 18 2959-61. 43. Sutherland, W. S.; Winefordner, J. D. Journal of Raman Spectroscopy 1991, 22 541-49. 44. Ruperez, A.; Laserna, J. J. Analytica Chimica Acta 1994, 291 147-53. 45. Ruperez, A.; Laserna, J. J. Talanta 1997, 44 213-20. 46. Perez, R.; Ruperez, A.; Rodri guez-Castellon, E.; Laserna, J. J. Surface and Interface Analysis 2000, 30 592-96. 47. Creighton, J. A. Surface Enhanced Raman Scattering Chang, R.; Furtak, T., Eds.; Plenum Press: New York, 198. 48. Laserna, J. J.; Berthod, A.; Winefordner, J. D. Microchemical Journal 1988, 38 125-36. 49. Laserna, J. J.; Berthod, A.; Winefordner, J. D. Talanta 1987, 34 745-47. 50. Angebranndt, M. J.; Winefordner, J. D. Talanta 1992, 39 569-72. 51. Jarvis, R. M.; Goodacre, R. Analytical Chemistry 2004, 76 40-47. 52. Jarvis, R. M.; Brooker, A.; Goodacre, R. Faraday Discussions 2006, 132 281-92. 53. Farquharson, S.; Gift, A. D.; Maksymiuk, P.; Inscore, F. E. Applied Spectroscopy 2004, 58 351-54. 54. Zhang, X. Y.; Young, M. A.; Lyandres, O.; Van Duyne, R. P. Journal of the American Chemical Society 2005, 127 4484-89. 55. Daniels, J. K.; Caldwell, T. P.; Christensen, K. A.; Chumanov, G. Analytical Chemistry 2006, 78 1724-29.

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141 56. Alexander, T. A.; Pellegrino, P. M.; Gillespie, J. B. Applied Spectroscopy 2003, 57 1340-45. 57. Basic Diagnostic tes ting protocols for level A laboratiories for the presumptive identification of Bacillus anthracis. Centers for Disease Control and Prevention, American Society of Microbiology 2004. Ref Type: Electronic Citation 58. Stevenson, C. L.; Vo-Dinh, T. Modern Techniques in Raman Spectroscopy Laserna, J. J., Ed.; John Wiley: Chichester, 1996; Chapter 1. 59. Ingle, J. D.; Crouch, S. R. Spectrochemical Analysis Prentice-Hall, Inc: New Jersey, 1988; Chapter 16. 60. Gardiner, D. J. Practical Raman Spectroscopy Gardiner, D. J.; Graves, P. R., Eds.; 1989; Chapter 1. 61. Bulkin, B. Analytical Raman Spectroscopy Grasselli, J.; Bulkin, B., Eds.; John Wiley: New York, 1991; Chapter 1. 62. Bulkin, B. Analytical Raman Spectroscopy Grasselli, J.; Bulkin, B., Eds.; John Wiley: New York, 1991; Chapter 1. 63. Raman, C. V; Krishnan, K. S Nature 1928, 121 64. Wang, Z.; Rothberg, L. J. Applied Physics B-Lasers and Optics 2006, 84 289-93. 65. Pustovit, V. N.; Shahbazyan, T. V. Chemical Physics Letters 2006, 420 469-73. 66. Persson, B. N. J.; Zhao, K.; Zhang, Z. Y. Physical Review Letters 2006, 96 67. Kerker, M. Accounts of Chemical Research 1984, 17 271-77. 68. Creighton, J. A. Surface Enhanced Raman Scattering Chang, R.; Furtak, T., Eds.; Plenum Press: New York, 198. 69. Centeno, S. P.; Lopez-Tocon, I.; Arenas, J. F.; Soto, J.; Otero, J. C. Journal of Physical Chemistry B 2006, 110 14916-22. 70. Brown, R. J. C.; Wang, J.; Tantra, R.; Yardley, R. E.; Milton, M. J. T. Faraday Discussions 2006, 132 201-13. 71. Jeanmarie, D. L; Van Duyne, R. P. Journal of Electroanalytical Chemistry 1977, 84 72. Creighton, J. A; Blatch ford, C. G; Albrecht, M. G J.Chem.Soc.Faraday Trans II 1979, 75 73. Turkevich, J; Stevenson, P. C; Hillier, J Disc.Farady Soc. 1951, 11

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142 74. Creighton, J. A. Surface Enhanced Raman Scattering Chang, R.; Furtak, T., Eds.; Plenum Press: New York, 1982. 75. Muniz-Miranda, M. Journal of Raman Spectroscopy 2004, 35 839-42. 76. Muniz-Miranda, M. Colloids and Surfaces A-Physicoc hemical and Engineering Aspects 2003, 217 185-89. 77. Muniz-Miranda, M. Journal of Raman Spectroscopy 2002, 33 295-97. 78. Zhang, J. B.; Fang, Y. Colloids and Surfaces A-Phys icochemical and Engineering Aspects 2005, 266 38-43. 79. Smejkal, P.; Siskova, K.; Vlckova, B.; Pf leger, J.; Sloufova, I.; Slouf, M.; Mojzes, P. Spectrochimica Acta Part A-Mol ecular and Biomolecular Spectroscopy 2003, 59 232129. 80. Lee, I.; Han, S. W.; Kim, K. Journal of Raman Spectroscopy 2001, 32 947-52. 81. Prochazka, M.; Mojzes, P.; Step anek, J.; Vlckova, B.; Turpin, P. Y. Analytical Chemistry 1997, 69 5103-08. 82. Prochazka, M.; Stepanek, J. ; Vlckova, B.; Srnova, I.; Maly, P. Journal of Molecular Structure 1997, 410 213-16. 83. Hao, E; Scahtz, G. C Journal of Chemical Physics 2004, 120 357-66. 84. Ingle, J. D.; Crouch, S. R. Spectrochemical Analysis Prentice-Hall, Inc: New Jersey, 1988; Chapter 16. 85. Strommen, D. P.; Nakamoto, K. Laboratory Raman Spectroscopy, John Wiley & Sons, Inc: New York, 1984. 86. Chase, B. Chemical Analysis: A Series of M onographs on Analytical Chemistry and Applications Winefordner, J. D.; Kolthoff, I. M., Eds.; John Wiley & Sons,Inc: New York, 1991; Chapter 2. 87. Spectra-Physics Lasers. BeamLok 2060 and 208-0 Ion Lase rs: User's Manual. 1992. Ref Type: Catalog 88. Edmund Optics Worldwide. http://www.edmundoptics.com/onlinecatalog 2006. Ref Type: Electronic Citation 89. Semrock High-Performance Optical Filters. 2005. Ref Type: Catalog 90. Chroma Technology Corp. Handbook of Opti cal Filters for Fluorescence Microscopy. 2000. Ref Type: Catalog

PAGE 143

143 91. Spectrochemical Analysis Ingle, J. D.; Crouch, S. R., Eds.; Prentice Hall: NJ, 1988; Chapter 4. 92. Standard Research Systems. Model SR-400 Gated Photon Counter Manual. 1987. Ref Type: Catalog 93. Vallee, P. H; Lafait, J; Ghomi, M; Jouanne, M; Morhange, J. F Journal of Molecular Structure 2003, 652 371-79. 94. Voyutsky, s. Colloid Chemistry Mir Publications: Mosc ow, 1978; Chapter 2. 95. Angebranndt, M. J.; Winefordner, J. D. Talanta 1992, 39 569-72. 96. Sutherland, W. S.; Winefordner, J. D. Journal of Raman Spectroscopy 1991, 22 541-49. 97. Farquharson, S.; Gift, A. D.; Maksymiuk, P.; Inscore, F. E. Applied Spectroscopy 2004, 58 351-54. 98. Phosphorimetry:Theory, Instru mentation, and Applications Hurtubise, R., Ed.; VCH Publishers: New York, 1990; Chapter 10. 99. Bell, S. E. J.; Mackle, J. N.; Sirimuthu, N. M. S. Analyst 2005, 130 545-49. 100. Farquharson, S.; Gift, A. D.; Maksymiuk, P.; Inscore, F. E. Applied Spectroscopy 2004, 58 351-54. 101. Karlberg, B.; Pacey, G. Flow Injection Analysis : A Practical Guide, Amsterdam, 1989. 102. Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; John Wiley & Sons, Inc: New York, 1988. 103. Farquharson, S.; Gift, A. D.; Maksymiuk, P.; Inscore, F. E. Applied Spectroscopy 2004, 58 351-54. 104. Hindle, A. A; Hall, A. H Analyst 1999, 124 1599-604. 105. Graham, T. W; Sabelnikov, A. G Journal of Homeland Security and Emergency Management 2004, 1 1-13. 106. Yonzon, C. R.; Stuart, D. A.; Zhang, X. Y.; McFarland, A. D.; Haynes, C. L.; Van Duyne, R. P. Talanta 2005, 67 438-48. 107. Farquharson, S.; Gift, A. D.; Maksymiuk, P.; Inscore, F. E. Applied Spectroscopy 2004, 58 351-54. 108. Bell, S. E. J.; Sirimuthu, N. M. S. Journal of Physical Chemistry A 2005, 109 7405-10. 109. Bell, S. E. J.; Sirimuthu, N. M. S. Journal of Physical Chemistry A 2005, 109 7405-10.

PAGE 144

144 110. Prochazka, M.; Mojzes, P.; Step anek, J.; Vlckova, B.; Turpin, P. Y. Analytical Chemistry 1997, 69 5103-08. 111. Prochazka, M.; Stepanek, J. ; Vlckova, B.; Srnova, I.; Maly, P. Journal of Molecular Structure 1997, 410 213-16. 112. Smejkal, P.; Siskova, K.; Vlckova, B.; Pf leger, J.; Sloufova, I.; Slouf, M.; Mojzes, P. Spectrochimica Acta Part A-Mol ecular and Biomolecular Spectroscopy 2003, 59 232129.

PAGE 145

145 BIOGRAPHICAL SKETCH Joy D. Guingab was born on September 17, 1975 in the province of Isabela, Philippines to Elpidio C. Guingab and Thelma G. Deray. She has two brother, Javier 4 years her senior and Elpidio Jr 9 years her junior. She attended th e prestigious Philippine Science High School in Quezon City, Metro Manila and graduated in 199 2. She moved to Los Baos, Laguna to pursue her Bachelors degree in Chemistry at the University of the Philippines. After her graduation in 1996, she stayed in Los Baos to work as a Research Associate in the National Institutes of Molecular Biology and Biotechnology and then as a Researcher in the International Rice Research Institute. She came to Florida to pursue her PhD in Analytical Chemistry in 2001 under the research supervis ion of Prof. James D. Winefordner.


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DEVELOPMENT, OPTIMIZATION AND CHARACTERIZATION OF
A SURFACE ENHANCED RAMAN SPECTROSCOPIC METHOD
FOR DETECTION OF DIPICOLINIC ACID














By

JOY D. GUINGAB


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007


























Copyright 2007

by

Joy D. Guingab




























To my parents--my \roenlgth and foundation









ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my research adviser Prof. James

D.Winefordner for his continued guidance and encouragement. I am especially grateful to have

had the opportunity to work with a mentor who encourages personal growth through research

and teaching.

I would like to acknowledge Dr. Nicolo Omenetto for his valuable research guidance.

His admirable love for science is truly inspiring. I am very grateful to Dr. Benjamin Smith for

all his advice in building my experimental set up. He has set a very good example for all the

students in the Winefordner group.

I would like to extend my appreciation to my committee members for their valuable

advice on the completion of this research dissertation.

The Winefordner-Omenetto group is worthy of thanks for the camaraderie and friendship.

I am especially grateful to Dr. Jamshid Temirov, Benoit Lauly and Nicholas Taylor for all

Raman discussions and the assistance extended during the setting up of the Raman system. I am

thankful that I have had the opportunity to be in the same group as Dr.Xihong Wu for the past 3

years and have developed a great friendship. Many thanks go to Dr. Kirby Amponsah-Manager,

Ron Whiddon and Pam Monterola for their constant encouragement and friendship.

I would like to thank Dr. David Hahn and Dr. Timothy Anderson for allowing me to use

their Raman systems in the preliminary stages of this research. I am grateful to Young Seok Kim

for all the assistance in the use of their Raman system. All the UV-Vis measurements were

conducted in laboratory of Dr. Nigel Richard, Dr. Charles Martin and Dr. Kathryn Williams.

Thanks go to the SEM laboratory staff for all training and assistance in the use of the SEM

microscope.









I would like to extend my appreciation to all support staff in chemistry, including the

glass, machine and electronics shop for all the work their contributions in the instrument

development. It has been great having Ms. Lori Clark all these years as the most helpful person

to every Chemistry graduate student. Special thanks go to Ms. Jeanne Karably for all her

assistance and great advice on almost anything.

My life in Gainesville had not been this memorable without the company of good friends.

I acknowledge all my friends in chemistry for all the good times shared. I am so fortunate to

have a very good support group from my fellow Filipino graduate students and their families.

My heartfelt gratitude goes to the Javelosas for being so accommodating, to Jemy and Fair for

their friendship and constant encouragement, to Jhoana and Dodge for their good company and

editorial and computer-related assistance, and to John for his thoughtfulness. A special

appreciation is owed to my dear friend Rina for being the best roommate and more importantly

my best friend. Those years of friendship will always be treasured and cherished.

I am grateful to my relatives for all their support. I am very grateful to the Flores family

in Montreal for all the support extended during my graduate studies and for being my family

away from home. I would like to thank the Lamar family for graciously accommodating me in

their home in Jacksonville and for treating me as a family member.

I am blessed with a family who endlessly supports and believes in me. I would like to

acknowledge my brothers, Jaymar and Jun for their love and support. My brothers and I were

surrounded by a tremendous wealth of love and support from my grandparents while growing up.

I am forever grateful to my mother for her incredible strength and unconditional love. Without

her believing in my potential, I would never have reached this point. I owe my father gratitude

for instilling in me the strength that I need to face the challenges in life. He has been my driving









force in all my accomplishments. Much appreciation is extended to my husband, Emil for all the

support and encouragement and most importantly for his unwavering friendship and love.












TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. ...............4.....


LI ST OF T ABLE S ................. ...............9................


LIST OF FIGURES .............. ...............10....


CHAPTERS



1 INTRODUCTION ................. ...............15.......... ......


Back ground and Si gnifi chance ................. ................. 15......... ....
Scope of Research Dissertation ................. ...............18.......... ....

2 THEORETICAL OVERVIEW .............. ...............20....


Raman Scattering............... ...............2
Historical Background............... ...............2
Spontaneous Raman Effect ................. ...............21........... ....
Raman Signal Enhancement Techniques .............. ...............26....
Surface Enhanced Raman Spectroscopy (SERS) .............. ...............27....
Historical Back ground ............... ...............2
Enhancement Mechanisms .............. ...............27....
Substrates for SERS ................ ......... ...............3
Advantages and Limitations of SERS .............. ...............35....


3 DESIGN, OPTIMIZATION AND CHARACTERIZATION OF A RAMAN SYSTEM
FOR SERS APPLICATION................ ..............4


Introducti on .............. .. ......__ ..... ...............40

Design, Optimization and Characterization............... ............4
Excitation Source................ ......... ..........4

Sampling Mode and Collection Optics............... ...............43.
M onochromator ................. ...............44.......... ......

Optical Filters .............. ...............47....
D etector .............. ...............49....
Data Collection ................. .............. ....... ........ ..........5
Evaluation of Performance of the Raman System ................. ...............54........... ..

4 STABILITY OF SILVER COLLOID AS SUBSTRATE FOR SURFACE ENHANCED
RAMAN DETECTION OF DIPICOLINIC ACID .............. ...............83....


Introducti on ................... ......... ...............83.......

Experiments and Methods .............. ...............84....












Results and Discussion .............. ...............85....
Conclusions............... ..............8


5 EVALUATION OF EXPERIMENTAL CONDITIONS FOR THE SURFACE
ENHANCED RAMAN DETECTION OF DIPICOLINIC ACID (DPA) ON SILVER
COLLOIDS GENERATED BY FLOW INJECTION ANALYSIS (FIA) ..........................100


Introducti on ................ .. ........ ..... ...............100......

Experimental Design and Methods............... ...............102
Results and Discussion ................... ........... ...............104......
Factors Affecting SERS of DPA ............ .....__ ...............104
FIA-SERS of DPA .............. ...............109....
Conclusion ............ ..... ._ ...............109...


6 FEASIBILITY OF SERS DETECTION OF BACTERIAL SPORES .............. ...............122


Introducti on ................. ............. .. ...............122.....

Experimental Methods and Procedures .............. ...............124....
Results and Discussion ................ ...............124................
Conclusions............... ..............12


7 CONCLUSIONS AND FUTURE WORK ................. ...............133........... ...


Conclusions............... .. .............13
Future Research Directions............... ..............13


LIST OF REFERENCES ................. ...............138................


BIOGRAPHICAL SKETCH ................. ...............145......... ......










LIST OF TABLES


Table page

3-1 Common lasers used in Raman Spectroscopy. ............. ...............57.....

3-2 Nominal output power of the visible lines of the Argon ion laser ................. ................58

3-3 A summary of the important characteristics of the Spex 1680B double
monochromator. .............. ...............66....

3-4 The monochromator scan speed at different increments. ............. .....................6

3-5 A summary of the characteristics of the Hammamatsu R-928 PMT. ............. ..............73

4-1 A summary of normalized signal intensities and calculated enhancement factors from
SERS of DPA on silver colloids. ........... ..... .__ ...............99.

6-1 A survey of SERS methods for detection of dipicolinic acid and bacterial spores .........132










LIST OF FIGURES


Figure page

2-1 Energy diagram illustrating the Raman effect. ............. ...............37.....

2-2 A schematic diagram of surface enhanced Raman process .............. .....................3

2-3 A simplified schematic diagram illustrating the SERS electromagnetic enhancement
m echanism .............. ...............39....

3-1 The Raman experimental set-up .............. ...............56....

3-2 The Argon ion visible lines ..........._ ..... ..__ ...............59.

3-3 The experimental set-up used for measuring Argon ion laser profile and laser short
and long term fluctuations. ............. ...............60.....

3-4 The short-term fluctuation of the 5 14.5 nm line of the Argon ion laser ..........................61

3-5 The long-term fluctuation of the 5 14.5 nm line of the Argon ion laser. ..........................62

3-6 A schematic diagram of the 900 sampling geometry adapted for Raman system ..............63

3-7 A diagram illustrating (a) the position of the dove prism in the Raman system and (b)
the image rotation by a dove ............. ...............64.....

3-8 A comparison of normal Raman spectra of saturated DPA illustrating the
improvement in signal intensity due to the image rotation using a dove prism. ...............65

3-9 Transmission spectra of different types of optical filters used in Stokes Raman
Spectroscopy ................. ...............68.......... .....

3-10 A comparison of the optical density plot of a long wave pass filter vs. a notch filter.......69

3-11 A spectrum of Ar' laser with a Iser transmitting filter ................. .......... ...............70

3-12 The decrease in the intensity of Rayleigh scatter peak using a high pass filter .................71

3-13 A comparison of the Rayleigh peak attenuation by two different long wave pass
filters. ............. ...............72.....

3-14 A typical pulsed height distribution. Disc 1 and Disc 2 are the discriminators of the
photon counter .......... ............... .............. .................. ...............74

3-15 The pulse height distribution for the photon counting system for determining the
discrimination l evel ................. ...............75......___ ...










3-16 Benzene spectra obtained using analog and photon counting mode illustrating the
difference in signal to noise. ............. ...............76.....

3-17 A timing diagram used for the photon counter and monochromator interfacing and
synchronization ........... ..... .._ ._ ...............77....

3-18 Mercury lamp spectra obtained with (a) analog and (b) photon counting mode
showing the narrow mercury lines ................. ...............78........... ...

3-19 Raman spectra of several pure solvents measured using the analog mode........................79

3-20 Raman spectra of 1.0 x 10-4 M beta-carotene in (a) THF and (b) ethanol ................... ......80

3-21 A normal Raman spectrum of saturated DPA in 1 M KOH. ............. .....................8

3 -22 Raman spectra of an empty cuvette, deionized water and 1 M KOH ........._..... ..............82

4-1 Comparison of the normal Raman and SERS spectra of dipicolinic acid (DPA).............. 91

4-2 SERS signal of DPA on silver colloids aged over 1 h .............. ...............93....

4-3 Absorption spectra of silver hydrosol before and after addition of dipicolinic acid
(D PA ). .............. ...............94....

4-4 Absorbance of silver colloids at the peak maximum 401 nm as a function of sodium
b orohy dri de. .............. ...............9 5....

4-5 SERS signal of DPA on silver colloid with and without sodium sulfite as oxygen
scavenger. ........... ..... ._ ...............96...

4-6 Absorbance of the silver colloids at the peak maximum 402 nm. ............. ...............97

4-7 SEM images of silver colloids prepared using sodium borohydride treated with
sodium sulfite. ........... ........... ...............98....

5-1 The Flow Inj section Analysis (FIA)-SERS set up ................. ...............110........... .

5-2 Absorbance readout recorded from the FIA system ................. .......... ................1 11

5-3 SERS signal of DPA at 1013 cml on silver colloid prepared at different volume
ratios............... ...............112

5-4 Absorption spectra of silver colloids prepared at different volume ratios of sodium
borohydride to silver nitrate ........._.__...... ..__. ...............113..

5-5 SEM images of silver colloid prepared by batch addition with different volume
ratios ................. ...............114................

5-6 SERS signal of DPA at 1013 cml on silver colloids generated at different flow rates ..1 15










5-7 Scanning electron micrographs of silver colloid prepared by batch addition and
generated by FIA system at different total flow rates ................. .......... ...............116

5-8 The effect of addition of different concentrations of nitric acid and potassium
hydroxide on the SERS signal of DPA at 1013 cm~l ................ ....__ ................1 17

5-9. The effect of aggregation of silver colloid upon addition of different concentrations of
sodium sulfate on the SERS of DPA. ................ .....__ ....................1

5-10 The effect of aggregation of silver colloid upon addition of different concentrations
of sodium chloride on the SERS of DPA. .....__.....___ ..........__ .........19

5-11 A typical FIA peak profile of DPA measured at 1013 cm-l using the FIA-SERS
system .............. ...............120....

5-12 Comparison of percent relative standard deviation of SERS of DPA under static
condition and FIA-mode ................. ...............121....... ......

6-1 The SERS spectrum of 25 ppm DPA used for estimating the limit of detection

6-2 SERS spectra of different concentrations of DPA. ......____ ........__ ................129

6-3 A plot of DPA signal (1013 cm l) vs concentration (0-78 ppm) using the unknown
peak (966 cm-0) as an internal standard. .......................... ........130

6-4 SERS spectra of 78 ppm DPA measured using freshly prepared and 2-day old silver
colloids. ........... ..... ._ ...............131...












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DEVELOPMENT, OPTIMIZATION AND CHARACTERIZATION OF A
SURFACE ENHANCED RAMAN SPECTROSCOPIC METHOD
FOR DETECTION OF DIPICOLINIC ACID

By

Joy D. Guingab

May 2007

Chair: James D. Winefordner
Major Department: Chemistry

Surface Enhanced Raman Spectroscopy (SERS) has been explored as a tool to study

biological agents. The anthrax distribution in the US postal in October 2001 intensified the need

for a rapid and accurate detection of bacterial spores. Dipicolinic acid (DPA) is a unique

component of bacterial spores and has been used as a signature molecule for detection schemes.

The feasibility of using SERS as a detection technique for bacterial spores is evaluated in this

research.

A conventional Raman system was designed, optimized and characterized. The lab-

constructed Raman system was used for the SERS studies of DPA on silver colloid dispersions.

Experimental conditions required to obtain reproducible and intense SERS signal from DPA

molecule were evaluated and optimized. A continuous flow system was constructed for SERS of

DPA studied under flow mode. Improvement in SERS signal reproducibility was demonstrated

with the use of controlled conditions of a custom-built Flow Injection Analysis (FIA) system.

The developed FIA-SERS system allowed silver colloid generation, sample introduction and









DPA detection. Characterization of the silver colloids was done by absorption method and

Scanning Electron Microscopy (SEM).

The feasibility of using SERS as a detection technique for bacterial spores was evaluated.

The absolute limit of detection was estimated to be 40 ng or 106 Spores which is 2 orders of

magnitude higher than the infectious dose set by the Centers for Diseases Control and Prevention

and required limit of detection for bacterial spores. The signal enhancement factor of the

prominent DPA peak at 1013 cml was approximated to be 2 orders of magnitude increase from

the normal Raman signal of saturated DPA. Although, quantitative analysis of DPA was not

demonstrated in this research, Raman peaks attributed to DPA molecules were observed in

concentration as low as 25 ppm.










CHAPTER 1
INTTRODUCTION

Background and Significance

Airborne microorganisms, which exist in respirable size of 1-10 Clm, can cause adverse

effects on humans and can potentially cause fatal disorders. One of these biological agents is the

bacterial spore. Bacteria are known to thrive almost everywhere. There is a wide array of

bacterial species ranging from the benign to the infectious such as Bacillus anthracis, the

causative agent for anthrax. Bacteria are microscopic but complex organisms. Aside from their

ability to multiply exponentially, they can adapt to any kind of environmental stress by triggering

some response mechanisms.l When exposed to unfavorable growth conditions such as depleting

food and water supply or extreme temperatures, physiological changes and cellular responses are

triggered to combat starvation and other harsh conditions. Failure of these immediate responses

is the onset of a period of metabolic dormancy and resistance to stress or sporulation. Bacterial

spores can stay dormant for a few hours to millions of years until conditions are favorable for

germination. Metabolic dormancy and resistance to chemical agents, radiation and heat are the

main reasons for their long-term stability.2 Endurance to all possible harsh environmental factors

makes bacterial spores a potential hazard to human health. The recent anthrax attack in 2001

shows the potential danger that spores may pose.

Substantial progress towards the development of rapid detection methods for bioaerosols,

particularly bacterial spores, has been made over the past few years. The need for rapid and

accurate detection of these airborne contaminants has been intensified by the anthrax distribution

in the US postal system in October 2001. Detection of these biological agents is extremely

important in defense against bioterrorism, prevention of disease outbreak as well as monitoring

in industrial or clinical setting. Time consuming and tedious microbiological assays based on the









morphology of the microorganism are commonly used for analysis of biological agents. Routine

identification of spores includes nucleic acid sequencing using the Polymerase Chain Reaction

(PCR) technique.3-5 Immunoassays are commonly employed as spore identification

techniques.6,7 Although these assays are nondestructive and sensitive, they require sample

preparation and do not allow automation of analysis. Fluorescence spectroscopy, which plays an

important role in the bioanalytical Hield, has also been used.s-10 Laser Induced Breakdown

Spectroscopy (LIBS) has also been explored for its feasibility to study bacterial spores.11-1

However, these methods are not capable of distinguishing bacterial spores from other

interference such as pollens and molds.

Dipicolinic acid (2, 6-pyridinedicarboxylic acid or DPA) in the form of calcium

dipicolinate (CaDPA) is known to be a unique constituent of bacterial spores.2 Techniques

geared towards the detection of CaDPA and its derivatives as Bacillus utin acltl i\ signature are

now being developed. Sensitive and highly selective detection methods, which involve

extraction of DPA from spores such as mass spectrometry.14-16 A DPA triggered terbium

luminescence has been employed as a method for rapid detection of DPA extracted from

bacterial spores.l7l The drawback of these methods is that sample preparation is time

consuming and often tedious, making the analysis time longer. The Hield of vibrational

spectroscopy has been explored as a tool for whole organism Eingerprinting; the most promising

is Raman spectroscopy.l

As a versatile, information-rich technique, Raman spectroscopy plays an important role

in the identification and characterization of microorganisms. The advent of Raman microscopy

even makes it possible to probe and characterize an individual bacterium20 as well as a bacterial

spore.21 Detection of a single bacterial spore using Raman spectroscopy coupled with optical









trapping has been investigated.22 Although Raman spectroscopy can provide rich information

about a molecule, its major disadvantage is that of having a very weak signal due to the small

cross section of the interaction process. In fact, the Raman cross section is approximately 12-14

orders of magnitude lower than the competitive effect, fluorescence.23 Not until the 1970s, when

an unexpectedly high signal enhancement was observed from pyridine on a roughened silver

electrode24, had Surface Enhanced Raman Spectroscopy (SERS) shown promise to overcome the

traditionally low sensitivity of normal Raman spectroscopy.

In the past decades, studies have been conducted to understand the signal enhancement

observed when a molecule is attached to various metals such as silver, gold and copper. The

signal enhancement has now been increased to 14 orders of magnitude making single molecule

detection possible.24-26 This signal enhancement would translate into a Raman cross section of

approximately 10-16 CM2/mOlecule. There are two operative mechanisms responsible for the

SERS phenomenon, namely electromagnetic field enhancement and chemical enhancement. The

former contributes to the enhanced Raman signal when scattering takes place in the enhanced

local optical fields of the metal and the latter is due to metal-molecule interaction. With the

inherent selectivity of Raman spectroscopy combined with signal enhancement, SERS has

become a valuable analytical tool with low detection limits and short analysis time. Moreover,

unlike with infrared (IR) measurements, water does not interfere, making SERS very useful in

bioanalysis.

Substrates used for SERS evolved from roughened silver electrodes to a variety of

substrates prepared in various ways.27 Both chemical reduction28-3 and photo reduction34-36 have

been used to generate metal hydrosols. Laser ablation of metal films has also been used for

preparation of colloidal sols.37-41 Metal vapor evaporation42,4 and surface etching44-4 have been










employed to prepare solid support-based SERS substrates. Metal colloids as SERS substrate is

of particular interest due to straightforward preparation and simplicity of characterization and

manipulation.47 However, it has been shown that extreme dependence of the SERS effect on the

physico-chemical properties of the colloids imposes a rather severe restriction on the usefulness

of SERS on colloids as an analytical technique.30 It has been shown that a continuous flow

configuration SERS can improve the reproducibility of the colloid preparation, thus improving

senitiity28,48-50

Bacterial spores studies using SERS has been reported recently. It has been used as a

tool for probing specific biochemical components and for discrimination of bacteria.51,52 DPA

extracted from spores53,54, as well as in whole bacterial spores has also been detected.19,55 SERS

of a single bacterial spore has been made possible by optical trapping.56

The long term goal of this research is to detect DPA in bacterial spores. In the present

study, a SERS method was developed for detection of DPA. Controlled conditions of a custom-

built flow injection system allowed precise preparation of the colloid, controlled aggregation and

thorough mixing of the colloid and the bacterial spores. This approach addressed the problem of

poor reproducibility common in silver colloid preparation that leads to non-linearity of the SERS

signal with the analyte concentration while providing a sensitive detection method for bacterial

spores. The feasibility of SERS detection of 10000 spores, which is the infectious dose for

Bacillus untinut~l i\5 was evaluated.

Scope of Research Dissertation

The focus of this dissertation is on the development, optimization and characterization of

a Surface Enhanced Raman Spectroscopy system for the detection of bacterial spores. The

research involved SERS of DPA studied both in static conditions and continuous flow mode.









Static condition SERS studies of DPA were vital in the development and optimization of the

Flow Inj section Analysis (FIA)-SERS system.

Theoretical considerations of Raman spectroscopy are important before focusing on

SERS and are addressed in Chapter 2. This includes both classical and quantum mechanical

description of Raman Spectroscopy. An overview of SERS including the enhancement

mechanisms involved is discussed in Chapter 2. A variety of SERS substrates are available but

the use of metal colloids is investigated in this dissertation and briefly covered in Chapter 2. The

design, optimization and characterization of a conventional Raman system for the SERS study

are presented in Chapter 3. In Chapter 4, the stability of silver colloids as SERS substrate for

DPA detection is evaluated. Optimization of conditions for SERS of DPA in FIA mode is

presented in Chapter 5. The capability of SERS detection of DPA in spores is evaluated in

Chapter 6. General conclusions and recommendations for future directions are described in

Chapter 7.










CHAPTER 2
THEORETICAL OVERVIEW



Raman Scattering


A brief overview of Raman scattering is necessary in order to understand the theory

behind surface enhanced Raman spectroscopy. Several Raman reference books were consulted

in writing this section of the dissertation chapter.23,58-61

Historical Background

In the 1920s, researchers were interested in the scattering of light by charged particles.

The Compton effect which explained the changes in the wavelength of X-ray photons when

scattered by electrons was first documented.62 There was also a related study predicting this

process by Kramers and Heisenberg in 1925.62 Theoretical studies conducted by Smekal in 1923

led to the first prediction of the Raman effect.62 Chandrasekhar Venkata (C.V) Raman in India

first experimentally observed Raman scattering in 1928.63 The investigation involved focusing

sunlight and using filters while observing color changes in the scattered light. Simultaneous to

the discovery by C.V Raman, a study about Raman scattering was reported by Landsberg and

Mandelstam in Moscow.62 C.V Raman initially referred to the scattering process as "Feeble

Fluorescence" and later termed the phenomenon "New Radiation." The importance of this

discovery was recognized by the awarding of the Nobel Prize in Physics to C.V Raman in 1930.

Ever since, it has been known as Raman Spectroscopy, Raman effect, or Raman scattering.

Following these pioneer investigations, more fundamental studies and applications of

Raman spectroscopy had been conducted. Until the mid 1980s, the Raman literature had limited

reports on the chemical applications of the technique. The limiting factors were both









fundamental and technical. Fundamentally, Raman scatter is inherently weak and can be easily

overwhelmed by fluorescence. Technical issues were addressed by the introduction of PMT-

photon counting detection systems and the laser. The feasibility of routine chemical analysis

became possible after the recent introduction of Fourier-transform (FT)-Raman, charged coupled

devices (CCD), small powerful computers and the availability of near infrared lasers.

Spontaneous Raman Effect

Raman and Brillouin scattering are two types of inelastic scattering. Raman scattering is

caused by rotational and vibrational transitions in molecules. Rotational transitions are lower in

energy and slower than vibrational transitions. The molecule may have collisions with other

molecules during rotational transition causing a change in the rotational state of the molecule.

Rotational Raman spectroscopy is carried out in gas phase at low pressure to ensure that the time

for collisions is greater than the time for a transition. Relatively large frequency shifts which are

independent of scattering angle are observed in Raman scattering. Brillouin scattering, which is

caused by thermal fluctuations of the medium, yields small frequency shifts that are dependent

on the scattering angle.

Raman spectroscopy is an inelastic light scattering effect due to the interaction of light

with a sample. When a sample is illuminated with intense monochromatic light, photons are

reflected, absorbed and scattered. It is the scattering process that gives information about the

structure of a molecule. The light can be scattered in three ways. Most of the scattered radiation

has the same energy as the incident light resulting to elastic scatter which is referred as the

Rayleigh scatter. A small fraction of these photons is scattered inelastically by either losing

energy to the molecule or gaining energy from the molecule. Photons which lose energy give

rise to Stokes Raman scattering and photons which gain energy give rise to anti-Stokes Raman

scattering. The energy gained by the molecule in Stokes Raman scattering appears as vibrational










energy. When a molecule has excess vibrational energy above the ground state, it is this energy

that is lost to the anti-Stokes Raman scattering. A Raman spectrum is a plot of the scattered light

intensity as a function of the Raman shift from the incident light in cm- The Raman shift is

calculated with respect to the frequency of the excitation light using a general equation


Av=vo (2-1)


where vo is the wavenumber of the excitation source, v is the Raman wavenumber of the

sample, h0 is the wavelength of the laser and h is the wavelength corresponding to the Raman

data of the sample. This equation illustrates that the Raman shift is independent of the

wavelength of the excitation source. Both elastic and inelastic scatter can be observed in a

Raman spectrum and are symmetrically positioned on each side of the Rayleigh scatter.

Although the Stokes and anti-Stokes lines are symmetrically positioned about the Rayleigh peak,

their intensities are different with the Stokes being more intense. The Rayleigh peak appearing

at 0 cm-l can be greatly attenuated by a band rej section filter. The Raman effect has been

described using both classical and quantum mechanical model. The discussion of the two

models in this dissertation serves as a general overview.

Classical model

When an incoming radiation with an oscillating electric field impinges on a molecule,

this induces a change in the dipole moment of the molecule. When a molecule is in the presence

of an oscillating electric field, its electron cloud is distorted resulting in an induced dipole

moment. The induced polarization pu is proportional to the strength of the electric field, E as

given in the equation

p = aE (2-2)









where the proportionality constant, a, corresponds to the polarizability of the molecule.

Polarizability is related to the ease with which the electron cloud can be distorted when a

molecule is subj ected to an electric field. The electromagnetic radiation generates a fluctuating

dipole moment of the same frequency and can be expressed as

E = E, cos mi,t = E, cos(2xivot) (2-3)

where E, is the electric field strength at equilibrium and mi, is the angular frequency of the

electromagnetic radiation. Equation 2-2 is a generalization and only describes the induced dipole

that has a vibrational frequency v, equal to the frequency of the incident light vo. This induced

dipole will scatter light at the same frequency as the laser light which is the Rayleigh scatter.

Molecular vibrations are considered to be composed of normal modes, Q1


.Q, = Qocos2xyrVt (2-4)

where vl is the characteristic harmonic frequency of the jth normal mode. For a molecule with N

atoms Q, is equal to 3N-6 and for linear molecules Q, is equal to 3N-5. During vibration, the

changes in polarizability and the amplitude of the change is given by


a ~ a + 6~2,a : +...(2-1


When a molecule interacts with the incident radiation with an electromagnetic field strength E,

equation 2-2 becomes

pu = aE = aE, cos 2xivot (2-6)

Substituting equations 2-4 and 2-5 into 2-6 yields:


p~~ = coE coxot OE, cos 2xyr,t cos 2xyv (2-7)









Using the identity


[cos(x + y) + costx y)l
cos xcosy = (2-8)


equation 2-7 can be expressed as:


pu = aoE, cos 2xivot + Eg acs@oV)cs@o- 29


This equation summarizes that the induced dipole will radiate light at three frequencies. The first

term in the equation is the Rayleigh scatter which has the same frequency as the incident

radiation and magnitude proportional to the inherent polarizability of the molecule, ao The

second term describes the anti-Stokes Raman scatter occurring at a frequency equal to

(vo + v, and the third term is the Stokes Raman scatter occurring at (vo v,). The classical

description of Raman scattering although incomplete, provides some useful insights. It indicates

a linear dependence of polarization and both Rayleigh and Raman scattering intensities on the

intensity of the incident radiation. At higher values ofEo nonlinear Raman scattering can occur.

Another insight is that only molecular vibrations that cause a change in polarizability of the


moleule ieldRama scatein s 0 This has been the basis of the primary selection



rule of Raman in contrast to IR absorption which requires a change in the dipole moment. The

signs in the equation indicate that Raman scatter can be positive or negative. The intensities of

Stokes and anti-Stokes are determined mainly by the Boltzmann distribution. Anti-Stokes

scattering is dependent on the population of the first vibrational excited state. The population of

the ground state is always higher than that of the excited state resulting in a more intense Stokes









scattering intensity. The difference in intensity of Stokes and anti-Stokes scattering is given by

the ratio predicted based on the Boltzmann distribution indicating the dependence on temperature


I,=, exp ,ih (2-10)
IR oV o, I\,-V kT

where h is the Planck' s constant, k is the Boltzmann constant and T is the temperature.

Another important insight from the classical description is that the change in polarizability


Smay vary significantly for different molecules and for different modes in a given



molecule. Lastly, Rayleigh scatter is always more intense than the Raman scatter so is


expected to be much smaller than the inherent polarizability of the molecule a"

Quantum mechanical model

The quantum mechanical treatment of Raman scattering recognizes that the vibrational

energy of a molecule is quantized according to the relationship

AE +hA v= AE+ h(vo F,) (2-11)

where AE is the net energy change of the molecule and h~v is the net energy change of the

photons. The mechanism of the Raman process can be illustrated simplest by an energy level

diagram. As depicted in Figure 2-1, when radiation interacts with a molecule, it is excited to a

virtual state. The virtual state is not a true quantum state of a molecule but can be considered as

a short lived distortion of the electron cloud caused by the light oscillating electric field.

Relaxation of the molecule from the virtual state to the ground state leads to Rayleigh scattering.

The energy of Rayleigh scatter is the same as the energy of the incident radiation. Raman

scattering is shown in which light scattered is lower (Stokes) or higher (anti-Stokes) in energy by










the amount equal to the vibrational transition hot. Stokes Raman scattering results from

molecules relaxing to an energy level higher than the original energy level and yields a positive

AE. On the other hand, anti-Stokes Raman scattering yields a negative AE since the molecule

relaxes to an energy level lower than the original. As discussed in the classical description of

Raman scattering, Stokes has higher in intensity than anti-Stokes. The energy population is

governed by the Boltzmann distribution. Molecules at room temperature are in the lowest

vibrational level in the electronic ground state. The intensity difference of Stokes and anti-

Stokes are discussed in the previous section.

Raman Signal Enhancement Techniques

Although Raman spectroscopy can provide rich information about a molecule, it is

inherently a weak process. Having a weak signal is a maj or disadvantage of the technique due to

a rather small cross section of the interaction process. In fact, the Raman cross section is

approximately 6-8 orders of magnitude lower than fluorescence.

Several processes can be used to enhance the sensitivity of Raman spectroscopy such as

resonance Raman and surface enhanced Raman spectroscopy (SERS). Resonance Raman effect

is observed when the laser' s wavelength is tuned to the absorption wavelength of the molecule.

The intensity of the vibrational modes is affected by the associated electronic transition.

Resonance Raman spectroscopy is very useful in the analysis of biological molecules as it is both

selective and sensitive. Its sensitivity is 2-6 orders of magnitude higher than normal Raman

spectroscopy. The weak Raman process has a cross-section between 10-31 and 10-29 CM2 ST-1

molecule- Moreover, this enhancement technique is limited by the availability of lasers that

can be tuned to be resonant with the electronic transitions of the molecule. Surface enhanced

Raman spectroscopy has shown promise to overcome the traditionally low sensitivity of










spontaneous Raman spectroscopy. A remarkable signal enhancement is observed when a

molecule is attached to various metals such as silver, gold and copper (Fig. 2-2). Twenty years

after the discovery of SERS, the signal enhancement factor approximated to be 3-4 orders of

magnitude improved to about 14 orders of magnitude.26 This would translate into a Raman cross

section of approximately 10-16 CM2/ ST-1 moleCUle-1. The succeeding section of this chapter of the

dissertation provides a general overview of the theory of SERS based on several theoretical

discussions.26,64-70

Surface Enhanced Raman Spectroscopy

Historical Background

An intense Raman signal from pyridine adsorbed on electrochemically roughened silver

electrodes was observed by Fleishmann, Hendra and McQuillan in 1974.24 This remarkably

strong signal was attributed to the presence of a large number of pyridine adsorbed on a greater

surface area provided by the surface of the roughened electrode. Three years later, two papers

published separately reported an increase in Raman signal from pyridine adsorbed on silver

surfaces and attributed the signal enhancement to an intrinsic enhancement mechanism. In

contrast to the conclusion made earlier, the strong Raman signal was caused by an increase in

Raman cross section in the presence of an adj acent rough metal structure and not to the increase

in number of pyridine molecules adsorbed on the surface of the electrode. These independent

investigations of Jeanmaire and Van Duyne7 and Albrecht and Creighton72 led to the emergence

of what is known as surface enhanced Raman spectroscopy (SERS).

SERS Enhancement Mechanisms

After the discovery of the SERS effect, many researchers have conducted fundamental

studies on the mechanism behind the signal enhancement. Understanding of the SERS

mechanisms has been a subject of many ongoing research projects. Electromagnetic field









enhancement and chemical enhancement are the two operative mechanisms that have been

proposed to explain the increase in the Raman cross section of molecules adsorbed on metallic

nanostr-uctures. It has been observed that some form of roughness is required for SERS.

Equation 2-9 shows that the Raman Stokes component of the polarization contains both the

polarizability and the electromagnetic field intensity. The local field intensity relates to the

electromagnetic field enhancement mechanism while the inherent polarizability of the molecule

relates to the chemical enhancement mechanism. The observed signal enhancement is

contributed by the combination of these two mechanisms. The contribution by each mechanism

depends on the molecule and the optical and chemical characteristic of the SERS substrate.

In normal Raman, the total Stokes Raman signal is proportional to the Raman cross

section as well as the laser intensity. This relationship is given by

Ph (Vs) c~ No~ree L) (2- 12)

where Pa (v,)is the total Stokes Raman signal in W sr- N is the number of molecules present

in the probe volume, G~re is the Raman cross section in cm2 ST-1 moleCUle-land I(v, ) is the

laser intensity in W. This expression can be modified to describe the total SERS Stokes

signal Pssn vs) Based on the proposed SERS mechanisms, equation 2-12 can be expressed as

Pssn (s) acx N' oR A(v, ) 2 IA(vs ) 2 ~(L ) (2-13)

where N' is the number of molecules involved in the SERS process, oR, is the increased Raman

cross section. The terms A(v, ) and A(vs ) are enhancement factors for the laser and for the

Raman scattered field, respectively.









Electromagnetic field enhancement mechanism

In understanding the electromagnetic enhancement mechanism, the size, shape and

material of the surface have to be taken into consideration since these characteristics determine

the resonant frequency of the conduction electrons in the metallic particle. When a metallic

nanoparticle is exposed to electromagnetic field of the same frequency, electrons on the surface

oscillate. Metals have many free electrons which vibrate or oscillate when excited by an

electromagnetic wave at a particular frequency. This collective resonant oscillation known as

plasmons generates an additional electric field on the surface of the metallic particle.

Electromagnetic field enhancement mechanism is based on the idea that scattering takes place

within the enhanced local optical fields of the nanoparticle. A simplified schematic diagram

(Fig. 2-3) aids in understanding the electromagnetic field enhancement mechanism.26 It shows a

spherical metallic nanoparticle with a dielectric constant, o,~ present in a medium with a

dielectric constant, e. The particle has a diameter of 2r which is smaller than the wavelength of

the incident laser. A molecule positioned at a distance, d, from the surface of the spherical

particle experiences an electric field, En This field is the superposition of the incident electric

field Eo and the induced field on the surface of the particle, EI

E, = Eo + Es (2-14)

The field induced on the metal sphere is dependent on the dielectric constants of the medium and

the particle, the size of the particle as well as the distance of the molecule to the particle. This

relationship is given by


Es = r 3E (2-15)
S+ 2eo a(r+ d)3









The field enhancement factor A(v) can be expressed as the ratio of the field at the position of the

molecule and the incident field.


A(v)= o(-6
EEv e+ 2E r+d (-6

Based on this equation, the conditions of the resonant excitation of the surface plasmon can be

described. Maximum field enhancement factor can be achieved when the real part of the

dielectric constant of the particle is equal to 2Eo. This can be achieved for silver and gold

particles using wavelengths within the visible and near infrared region. The imaginary part of

the dielectric constant needs to be small.

If the enhancing factors for the laser and the Stokes field enhancement are taken into

account, an expression for the electromagnetic enhancement factor for the Stokes signal can be

written as


G,,,,(vs)= A)vL AFvs) L o S i o2 12 (2-17)
E(VL)+ 2Eo I I(s)+ 2Eo r+d

Several useful insights describing the electromagnetic enhancement mechanism can be deduced

from equation 2-17. It indicates that the enhancement scales as the 4th pOWeT Of the local field of

the metallic particle. This can be maximized when the excitation and the scattered field are in

resonance with the localized surface plasmon of the metal. Electromagnetic enhancement

mechanism does not require the molecule to be adsorbed directly on the metallic surface but the

distance dependence is extremely strong scaling as a 12th factor. An enhancement factor of 104

1012 has been attributed to the electromagnetic enhancement mechanism









Chemical enhancement mechanism

Chemical enhancement mechanism requires direct adsorption of a molecule on the

surface of the metallic nanostructure. The contribution to the total enhancement has been

estimated to be a factor of about 100. This mechanism is associated with the overlapping of the

electronic states of the metal and the adsorbed molecule. A charge transfer state is created

between the metal and the adsorbed molecule. The conduction band of the metal is divided by

the Fermi level. This is the boundary between the filled and unfilled orbitals within the

conduction band which moves up or down depending on the electric potential of the metal. An

adsorbate molecule that is widely separated from the metal has discrete energy levels. When a

molecule is adsorbed on the metal, the orbitals overlap to form a bond between the metal and the

adsorbate. This orbital interaction permits electrons to tunnel from the adsorbate molecule to the

metal. This process is referred to as charge transfer. The charge transfer state increases the

probability of a Raman transition to occur by providing resonant excitation.

The chemical enhancement mechanism via the charge transfer theory has several

characteristics that distinguish it from electromagnetic field enhancement mechanism. The

charge transfer theory indicates that the enhancement is due to the interaction of the metal and

the molecule and not the optical properties of the metallic nanostructure. A site specific

interaction between the metal and the adsorbate is required for chemical enhancement. The more

prominent characteristic is that chemical enhancement is short range in nature and limited to the

first layer of adsorbed molecules.

SERS substrates

Since the discovery of SERS in 1970, the SERS-active substrates have evolved from the

electrochemically roughened silver electrode to a variety of forms. The pioneer works on both

electrodes and colloidal systems have provided the foundations of SERS theory. Substrates for









SERS, commonly noble metals such as silver, gold or copper are used in colloidal form or

immobilized on surface.27 For these substrates to be SERS-active, attention must be given to the

type and preparation procedure. Based on the discussion of the electromagnetic enhancement

and chemical enhancement mechanisms, factors that can affect the SERS signal include the

dielectric constants of both the metal and its medium, the morphology of the metallic surface, the

distance between the adsorbate molecule and the metal surface and the excitation frequency.

According to the electromagnetic enhancement mechanism, SERS intensity is also affected by

excitation of the local field of the metal surface. Therefore, it is critical to control the factors that

influence the excitation of the localized surface plasmon resonance (LSPR) of the metal in order

to achieve the maximum SERS signal.

Metal colloids

Formation of metal colloids is very simple and easy; not requiring sophisticated

instrumentation. Colloidal systems are well suited for solution phase SERS measurements.

They can be easily characterized using absorption spectroscopy. There is definite dependence of

SERS signal on the size and shape of the nanoparticles. The use of metal colloids for analytical

detection has several limitations. SERS has been shown to require a certain degree of colloid

aggregation. The aggregative state of the metal can easily be affected by the introduction of the

analyte. Not only does SERS depend on the manner of introduction of the analyte but also

depend on the exact preparation procedure of the metal colloids. It is not surprising that SERS

on metal colloids have been found to be irreproducible.

Metal colloids or hydrosols can be prepared by chemical reduction of simple salts of

silver or gold. Reducing agents include borohydride28-32,48,49 and citrate.33 Turkevich et al used

citrate as a reducing agent to produce gold colloids of uniform size and spherical shape.73 By

controlling the concentration of citrate, gold colloids of different sizes were produced. Gold









colloid preparation normally involves heating and vigorous stirring while that of silver colloids is

simple. However, methods for preparation of silver particles of uniform size and spherical

shapes are less developed. Silver colloids prepared from chemical reduction of silver nitrate and

sodium borohydride are usually a mixture of irregular sizes and shapes.74 Photo-reduction has

also been used to obtain metal colloids.757 Laser ablation of thin metal films submerged in

deionized water has been an alternative way to generate metal colloids that are free of

impunities.78-8

Characterization of metal colloids normally involves their absorption (extinction) spectra.

Silver colloid solutions are yellow and have a single extinction band at 385 nm while gold

colloids are wine red with an extinction band at 520 nm.74 These extinction bands result from

the localized surface plasmon resonance (LSPR) excitation of the metallic nanoparticles and

depend on their size and shape. The extinction spectrum of a metal colloid is a contribution of

both absorption and scattering of the light incident to the particles. When visible light

illuminates a small particle, the particle can absorb and scatter the light. The absorption

contribution (Cabs) to the extinction of small particles of sizes within the Rayleigh scattering

regime (a < 20 nm) is given by


Cabs M(-8


where a is the radius of the particle, E= 81 + ie2 is the dielectric constant of the particle at the

optical frequency relative to that of the surrounding medium and h is the wavelength of the light

in the medium. For a colloidal solution with N particles per unit volume, the absorption is given

by


(2-19)


A = NCabs1









where 1 is the path length. The scattering contribution ("s= ) to the extinction is expressed as

1287/a6 l_ 12
sea 321 e + 2 (2-20)


In the Rayleigh scattering regime, both absorption and scattering contribution are at their

maxima when the real part of the dielectric constant is equal to -2. Absorption predominates

over scattering when the particle size is within this regime. On the other hand, as the radius

increases (a> 20 nm), scattering is the main contributor of the extinction.

In the context of SERS using colloidal particles, the shift of the extinction band to longer

wavelength is of particular interest rather than that of an isolated particle at the Rayleigh regime.

The shift in excitation resonance can be achieved by using bigger particles or aspherical or

particles in aggregates. Theoretical calculations of the LSPR of triangular, aggregated and

irregularly shaped particles were conducted to provide insight into the electromagnetic

enhancement mechanism by nanoparticles.83

Surface based SERS substrates

Different methods have been used to prepare surface based SERS substrates. The

following are the methods that are being used by different groups:27

Vapor or vacuum deposition
Corrosive etching or electrochemical roughening
Nanoparticle deposition/ordering
Sputter coating
Aqueous sol deposition
Subsequent assembly
Sol-gel polymerization
Noble metal embedding
Metal colloid monolayer assembly
Colloid multilayer formation









The wide range of surface SERS substrates allows researchers to study SERS in diverse

environment. A great amount of work has been focused on the development of more stable,

novel SERS substrates that can promote optimum SERS enhancement.

Advantages and Limitations of SERS

SERS as an analytical technique has many advantages. The advantages of normal Raman

Spectroscopy also apply for SERS. As a vibrational spectroscopy technique, a SERS spectrum

provides information about the molecular structure. It addresses the traditionally poor sensitivity

of normal Raman spectroscopy. SERS requires the molecule to be adhered to or near the metal

surface thus providing non-radiative pathways for the decay of excited states and thus, quenching

fluorescence. The abrupt decay of the electromagnetic fields ensures that only the molecules

adsorbed or close to the SERS substrate are being probed making SERS a selective technique.

SERS has been demonstrated to be very useful in the analysis of biological samples which are

normally in aqueous environment since water does not give a strong SERS signal.

The limitations of SERS have to be considered as well. A maj or limitation is linked to

the poor reproducibility of SERS substrates preparation making correlation of theoretical and

experimental SERS challenging. The dynamic changes in metal colloids due to aggregation of

the particles lead to irreproducible signal intensities. Production of assembled nanoparticles still

exhibits some degree of irreproducibility. The roughness feature that is required for SERS needs

to be characterized properly in order to understand the signal enhancement. Quantitative SERS

analysis has been restricted by the poor linearity of the technique. The limited choice of active

SERS substrates that exhibit surface plasmon resonance is another major constraint. Other

substrates aside from gold, silver and copper have been explored. In order for them to be used as

SERS substrates, a thin coating of an active substrate is necessary which complicates the

preparation procedure. SERS applicability is limited to molecules that naturally adsorb










chemically or physically onto the surface of the SERS substrate. Manipulation of these

molecules must be done in order for them to be within the enhanced optical field of the substrate.

High background continuum is often observed in SERS spectra. Although the source of the high

background is still under debate, the background can overwhelm the SERS signal from the

molecule.



















































Ground state

Anti-Stokes Rayleigh Stokes Raman
Raman Scattering Scattering Scattering


Figure 2-1. Energy diagram illustrating the Raman effect.


Excited electronic state


---------------1--------;-r------4-----Vita State


hoo+ hot


hoo- hot
















SERS


Laser







Normal Raman








Figure 2-2. A schematic diagram of surface enhanced Raman process showing molecules
adsorbed on the surface of an aggregate of gold particles. The particle size is between
10-100 nm.






























Laser a aa

Eo
Molecule





Metal

E = E + lS
Figure 2-3. A simplified schematic diagram illustrating the SERS electromagnetic enhancement
mechanism. The metal is spherical and its size is smaller than the wavelength of the
laser (adapted from reference # 26).










CHAPTER 3
DESIGN, OPTIMIZATION AND CHARACTERIZATION OF A RAMAN SYSTEM FOR
SERS APPLICATION



Introduction

High-end commercial Raman instruments which meet different set of needs are currently

available. Instrument choice depends on the intended application particularly the sample

requirement and the research obj ectives. In the effort to develop a system that can be used in a

wide range of applications, the design of commercial instruments becomes complex which

results in difficulty of operation and increased system costs. The long-term goal of this research

is to detect bacterial spores using Surface Enhanced Raman Spectroscopy (SERS). The

application requirement is a simple, conventional Raman system with the basic components to

study the SERS of bacterial spores. A Raman instrument consists of Hyve basic components: an

excitation source, a sample illuminating system, a sample holder, a wavelength selector, a

detector and a data acquisition system.84,85 Although modifications such as computer interfacing

for instrument control and data collection is common, the basic components remain unchanged in

the maj ority of applications.

A schematic of the lab-constructed Raman instrument is depicted in Figure 3-1. It

consists of a Spectra-Physics BeamLok 2060 Ar+ laser (Mountain View,CA) and a SPEX 1680B

double monochromator (Horiba JobinYvon; Edison NJ). A thermoelectrically cooled

Hammamatsu R-928 (Bridgewater, NJ) photomultiplier tube coupled with a SR 400 gated

photon counter (Stanford Research; Sunnyvale,CA) was used as a detector. A dove prism

(Edmund Optics; Barrington, NJ) tilted at a 450 angle was used to rotate the image making it

parallel to the entrance slit of the double monochromator. A LabVIEW program was used for










photon counter control and data acquisition. All measurements were done using a standard 1 cm

x 1 cm quartz cuvette. SERS spectra were recorded in 900 collection geometry. Characterization

and optimization of the Raman system are covered in the succeeding sections in this Chapter.

Since selection of components based on different criterion was not the obj ective of the research,

thorough discussion of all existing Raman instrument components is not included in this

dissertation. This chapter is dedicated specifically to describe the characteristics and the

performance of the Raman system designed for the particular SERS application.

Design, Optimization and Characterization

Excitation Source

Background

A variety of lasers are currently available for Raman spectroscopy. The discovery of the

laser has been one of the technological advances behind the renaissance of Raman spectroscopy

as a useful analytical technique. Table 3-1 shows the wavelengths and output power ranges of

the most common commercial lasers used in Raman Spectroscopy.86 The most common of these

lasers are the Ar~ and Kr' lasers. Their popularity in Raman spectroscopy has been attributed to

their high output power, variety of output wavelengths, and relatively long lifetime. As shown in

Table 3-1, different alternatives are available depending on the application requirement.

Source specifications

Spectra-Physics BeamLok 2060 Ar+ laser with a maximum multi-line output power of 7

watts was used as an excitation laser. The laser is equipped with a broadband optic that allows

output of multi lines. Single line operation is done by using a prism instead of a broad band

optic. The prism disperses the laser beam separating the lines based on their wavelengths.

Adjustment procedures must be followed to get the desired wavelength which can be confirmed

by their relative power. Maximum output power specifications of the visible lines of the Ar









laser are summarized in Table 3-2.87 Visible single line output power specified for 488 and

514.5 nm are 1.8 W and 2.4 W, respectively. The laser power that reaches the sample was

approximately half of the original power due to some losses in the beam alignment and focusing.

All spectra obtained in this research were acquired using a laser power range of 100-800 mW.

Experimental characterization

The laser profie was obtained using a fiber optic and CCD spectrometer (Ocean Optics;

Dunedin, Fl). Figure 3-2 shows the laser profile of the Argon ion laser. Specifieations of the

Argon ion laser provided a power stability of a 0.3 % and 5 % over 2 h and 8 h period,

respectively." Depending on the lifetime of the laser, the performance may change over time.

While lasers are good sources of monochromatic light, fluctuations are inherent in the output

power and relatively old lasers may be more susceptible to these fluctuations. For this reason,

short-term and long-term fluctuations for the Ar' laser were considered in system

characterization. The same experimental set up used to record the laser profile was used to

perform the measurement (Fig.3-3). For short term fluctuation measurement, the laser signal

was monitored for 1000 s. For long-term fluctuation analysis, the laser signal was measured

over 5000 s. The integration time was set at 100 ms. The percent relative standard deviation

(%RSD) was calculated for each measurement. The short-term laser fluctuation, which yielded

7% RSD, indicated that for shorter analysis times, the noise contributed by the laser was minimal

(Fig.3-4). This is important for determining the system's limiting noise source. The calculated

%RSD for the long-term fluctuation analysis was 8% (Fig.3-5). This can provide an insight

about possible laser drift during long measurement time such as the DPA SERS spectra

measurement.










Sampling Mode and Collection Optics

Background

The sampling geometry and collection optics are very important in the design of a Raman

sy stem. The alignment of the laser, the sample and collection aperture has a huge influence on

the magnitude, reproducibility and S/N of the Raman signal. Since the Raman signal is

inherently weak, it is best to have an efficient collection of the scattered photons.

Raman sampling modes are divided into three categories: conventional, remote and

microscopy modes.86 COnventional sampling involves liquid samples in cuvettes or solids such

as powders and pellets. Remote sampling involves the use of fiber optic cables which can be any

usable length for remote Raman detection within meters or kilometers away from the

spectrometer. The third mode of sampling uses a microscope. With the use of a microscope

obj ective, Raman microscopy allows probing of small regions usually less than 1 Cpm in diameter

and a few Cpm in depth. For this dissertation, the conventional mode was used because the

samples were in liquid form.

Experimental characterization

Common conventional sampling geometries are the 90o and 1800 geometries. The 900

geometry was chosen for this research. With this geometry it is easy to align the laser focal

cylinder with the monochromator entrance slit. Figure 3-6 depicts the arrangement of the optics

used. A transparent sample is placed in a cuvette and the laser is directed by a mirror on the

sample cuvette. The laser focal cylinder is imaged on the entrance slit of the monochromator

with a slit width of 3 pm. With the two lenses, the image is perpendicular to the slit, making the

image area entering the monochromator too small. A dove prism was used to rotate the image on

the entrance slit. A dove prism rotates an image twice as much as the angle that it is tilted









(Fig. 3-7).84,88 By positioning the dove prism between the two lenses at a 45 degree tilt, the

image was rotated by 900. The improvement in signal collection by using a dove prism is

illustrated in Figure 3-8.

Monochromator

Background

The Raman signal is inherently weak due to a small Raman cross section in the order of

10-29 CM12 ST-1. It is difficult to detect a Raman signal amidst intense stray light, which causes an

overwhelming background. For this reason, many Raman instruments use double or triple

monochromators, which provide stray light rej section without sacrificing throughput.

Monochromators are dispersive instruments that isolate a small wavelength band from a

polychromatic source.84 Most monochromator configurations are based on the Czerny-Turner

design.84 Light passes through an entrance slit and reflects off a collimating mirror to a

diffraction grating. The grating separates the light according to wavelength. A focusing mirror

reflects the diffracted light onto an exit slit to the detector. It is important to take note of several

characteristics of the monochromator such as linear dispersion, resolution and spectral bandpass.

Linear dispersion shows the capability of the instrument to disperse light. It is how far

apart two wavelengths are in the focal plane. Linear dispersion is expressed as

DL = dx/dh = fDa (3 -2)

where f is the focal length of the focusing element in mm and Da is the angular dispersion in rad

nm- The unit for linear dispersion is mm nm-l Manufacturers of these instruments provide a

more common term, the reciprocal linear dispersion, RD which is given by

RD = (DL -1 = dh /dx = (FDa)^1 (3-3)

The reciprocal linear dispersion represents the wavelength range within a unit distance in the

focal plane and is conveniently expressed in units of nm mml









The size of the diffraction grating plays a key role in determining the solid angle, the f-

number, the throughput and the resolution. With a limiting aperture diameter of L in mm, a

projected area A and a focal length F in mm, the f-number (f/#) is expressed as

f/# = flL (3-4)

The solid angle 0Z in sr can now be defined as

0z = A/f2 = (2/4) / (f/#)2 (3-5)

The spectral bandpass, s,, is the half-width of the wavelength distribution that passes

across the exit slit. The spectral bandpass is expressed in units of nm. With the exception of

very small slit widths where diffraction effects and aberrations occur, the spectral bandpass for a

given slit width, W in mm is given by

s, = RDW (3 -6)

Generally, monochromators have equal entrance and exit slit widths. The slit widths determine

the spectral profie of the image at the exit slit. In the case of equal slit widths, when

monochromatic light is passed through the entrance slit, a monochromatic image of the entrance

slit is formed on the exit slit. As the grating is rotated, the entrance slit image is scanned across

the exit slit.

The monochromator' s resolution is closely related to its spectral dispersion. Dispersion

indicates the distance of 2 wavelengths in the focal plane while resolution specifies the

distinguishable separation of the two wavelengths. For larger slit widths where aberrations and

diffraction effects are negligible, spectral resolution is expressed as the slit width limited

resolution, ahs in units of nm

Ahs = 2s, = 2RDW (3 -7)









Resolving power is another way to express how well a monochromator distinguishes two

adjacent wavelengths. Experimentally, this can be determined using the expression

Rexp = have/Smin (3-8)

where smin is the spectral bandpass at the minimum slit width. The theoretical resolving power is

calculated using the expression

Rth = have ad (3 -9)

Stray light in a monochromator is considered to be any light that passes that is outside

his where h, is the wavelength setting and s, is the spectral bandpass. The intensity of the light

of other wavelengths is referred to as the stray light level. Stray light level is one of the most

important specifications of a monochromator for many applications.

Experimental characterization

A Spex 1680B double monochromator with a focal length of 0.220 m is used in this

research. It is equipped with a 1200 grooves/mm grating. The double monochromator's

dispersion is 1.8 nm mm l, the resolution is 0.2 nm at 500 nm and the numerical aperture is f/4.

It can be scanned over a spectral range of 185-900 nm. The most important characteristics of the

double monochromator are summarized in Table 3-3.

The next criteria to consider are the monochromator's accuracy and speed in acquiring a

spectrum. The grating motor drive dictates the accuracy and speed of measuring a spectrum.

The double monochromator used has an accuracy of 10.4 nm and a repeatability of 10.2 nm.

Experimentally, the monochromator scan speed in the continuous mode was determined at by

scanning from 500 to 600 nm at different wavelength increments (Table 3-4). The results of

these measurements also provided the approximate rotation time of the grating at specific

increments which was considered in the synchronization of the scanning with the photon

counting detection. This is covered in the next section of this Chapter. Monochromators involve










scanning the spectral features of the optical signal in both continuous and step modes. As a

result, the measurement process is slower than that of a spectrograph with a multi-array detector

such as charged coupled detectors (CCD). These types of spectrometers operate in a fixed

grating position and directly acquire spectrum according to their dispersion as opposed to

scanning the grating in the case of monochromators. The Spex 1680B double monochromator

has an internal drive circuitry that permits coupling with a scan controller. The scan can be

programmed to be in continuous or step mode. The continuous scan mode can easily be

achieved by using an external control unit that commonly comes with the monochromator.

However, these types of scan controllers cannot be used in step mode, which is necessary for

PMT-photon counting detection. An alternative and more convenient approach to

monochromator scan control can be achieved using a PC peripheral and software. Interfacing of

the monochromator to a computer was done using a SPEX232/488 spectrometer control interface

(JobinYvon; Edison, NJ), which has a dual compatibility. It can communicate with the

spectrometer via an RS232 serial port or the IEEE 488. A Windows-based spectrometer control

software (JobinYvon; Edison, NJ) was used to control the monochromator. The continuous scan

mode was used for direct current measurements while the step scan mode was used for photon

counting mode.

Optical Filters

Background

A Raman spectrometer must be able to measure weak Raman signal in the presence of a

stronger Rayleigh or diffuse reflections at the laser frequency. Raman spectrometers must have

an outstanding stray light rej section, which can be achieved by using double or triple

monochromators. The stray light cut-off of a typical double monochromator sometimes is not

enough to address this issue. Thus, optical filters are used to minimize detection of undesired









radiation entering the spectrometer, which can overwhelm the weak Raman signal. There are

two general types of filters that are used in Raman spectroscopy: laser transmitting and laser

blocking filter.89 Laser blocking filters are further grouped into two types: notch filter and long

wavelength pass filter or cut-off filter. These filters are characterized by their transmission

spectra. The transmission spectra illustrated in Figure 3-9 show the specific applications of the

three different types of filters.89 Laser transmitting filter positioned between the laser and the

sample limits undesired light from the laser such as broadband spontaneous emission or plasma

lines from being detected. Such filters clean up the laser lines allowing only a single laser line to

reach the sample. The thin-film laser blocking filter is positioned between the spectrometer and

the sample to suppress the Rayleigh scatter so that the weak Raman signals can be accurately

measured. In systems where both laser transmitting and notch filters are in use, both Stokes and

anti-Stokes Raman scattering can be measured simultaneously. The long wave pass filter on the

other hand, only measures the Stokes Raman scatter. The advantage of using a long wave pass

filter over a notch filter is illustrated by their transmission spectra. Long wave pass filters have

the ability to transmit light close to the laser line. A graph comparing the optical density [-log

(Transmittance)] of the two types of filters is shown in Fig. 3-10,89 which illustrates the increase

in the edge steepness of a long wave pass filter relative to a notch filter. The steepness of the

edge results in a narrow transition region separating the laser line and the transmitting region of

the filter spectrum, allowing the observation of Stokes Raman shifts very close to the laser line.

Experimental characterization

The Ar+ laser used was equipped with a broadband optic instead of a prism so the output

was a mixture of all the lines of an Argon ion laser. A laser transmitting filter (MaxLineTM

Semrock, NY) was used to clean up the laser light before reaching the sample. This filter only

allowed 514.5 nm laser line to pass while blocking the other laser lines (Fig. 3-11). A









transmission efficiency of more than 90% prevented significant loss in laser power. The

Rayleigh scatter has higher intensity than the Raman scatter and can overwhelm the weak Raman

signal. A laser blocking filter (Chroma Technology Corp; Rockingham, VT), positioned

between the monochromator and the sample blocks the Rayleigh scatter. This research only

involved Stokes Raman scatter measurement so a long wave pass filter was chosen over a notch

filter. A steep 514.5 nm long wave pass filter was used to block the Rayleigh scatter and

improve Stokes Raman measurement. The position of the filter was optimized and the Rayleigh

scatter in both configurations was compared (Fig. 3-12). Rayleigh rej section was observed to be

more efficient when the filter was positioned very close to the monochromator entrance slit.

Two different high pass filters were compared for their effectiveness in blocking the Rayleigh

scatter (Fig.3-13). The Razor Edge filter (Semrock; Rochester, NY) proved to be more effective

than the H-filter (Chroma Technology Corp; Rockingham, VT). Comparison of the properties of

these filters is presented in Table 3-5.89,90

Detector

Background

Optical detectors are grouped into three categories: thermal, photon and multichannel

detectors.84 Photon detectors and multichannel detectors are commonly used in Raman

spectroscopy. Photon detectors are based on the rate of photon arrival and the spectral response

varies with wavelength while mutichannel detectors can provide simultaneous detection of

dispersed wavelength.84 Several types of photon and multichannel detectors are available.

Photon detectors are grouped into different types of devices as photoemissive devices

(photomultipliers and phototubes), pn-junction devices (photodiodes and phototransistors),

photoconductive cells, and photovoltaic cells.91









Photomultiplier (PMT) tubes fall under the photon detector category and are commonly

used in Raman measurements. Photomultiplier tubes are low noise light detectors with high

gain. Single photon detection can be achieved over a spectral range of 180-900 nm.

Photons striking the photocathode of a PMT cause ej section of an electron by the

photoelectric effect. The electron directed to a series of dynodes or high voltage steps gets

amplified. These electrons are collected at the anode with the signal output in the form of a

pulse. Photomultiplier detection has two main types: analog and photon counting. Analog mode

looks at the DC portion of the pulse. The DC current is the sum of all pulses regardless of their

source. For this reason, noise is added to the signal and difficult to eliminate. Analog

measurement is useful for measurements of high intensity signals because the noise contribution

is negligible. For low intensity signal measurements such as Raman spectroscopy, photon

counting is preferred. In photon counting, the pulses are directed to an amplifier/current-voltage

converter and then to a discriminator. The discriminator sets the cut-off voltage to filter low

voltage noise pulses, thus higher signal-to-noise ratios (S/N) are obtained. Most of the noise is

generated after the cathode and within the dynodes. These low voltage pulses are filtered by the

discriminator unlike in analog where noise is tightly bound to the signal. PMT's have high

voltage requirements and changes in the supplied voltage do not affect the pulse count while the

analog signal is affected. Another advantage is that photon counting is not affected by the RC

time constant that is inherent in the electronics of analog systems

Experimental characterization

A side-on R-928 HammamatsuThl photomultiplier tube cooled to about -200 C was used

as a detector. Important characteristics of this PMT are summarized in Table 3-6. Pulse

amplitude from the PMT was estimated based on gain and rise time provided by the

manufacturer using the formula92









Amplitude (mV) = Q x G x Z / tR (3-10)

where Q is the elementary charge in C/e, G is the gain, 0Z is the impedance of the preamplifier in

ohms and tR is the rise time in ns. The output from a PMT is a current pulse, which travels down

a 50 ohm cable that is terminated by a 50 ohm input impedance of a pre-amplifier. For an R-928

PMT, the gain is 1 x 107 and the rise time is 2.2 ns. Using the above formula, the calculated

amplitude of the pulse from the PMT is 36.4 mV.

A snubber was connected to the PMT to improve the shape of the pulse for photon

counting.92 This is a network consisting of a short piece of 50 ohm coaxial cable which is

terminated with a resistor of less than 50 ohms. Ringing, which can cause multiple counts from

a single photon, is very common when using a PMT. This can be minimized by connecting a

snubber to the PMT prior to photon counting. The snubber network is a 10 inch coax cable

connected to a small 50 ohm potentiometer with terminating impedance ranging from 0 to 50

ohms. The other end of the coaxial cable was connected to the PMT together with the output

signal cable that was connected to the preamplifier. The output current from the PMT is divided

into the signal cable and into the snubber. Adjusting the snubber to 50 ohms attenuates the

signal by a factor of 2. No signal is reflected back to the anode unless the pot is set to less than

50. Reflections are delayed by the cable roundtrip time and sent out the signal cable. The round

trip time in the snubber cable can be adjusted so that the reflections cancel the signal ringing.

With the correct snubber cable length, the round trip time of the cable can approximately match

the period of the signal ringing and thus, the signal ringing is cancelled. For a 10-inch snubber

cable, the round trip time is about 5 s. The snubber network connected to the PMT improved the

shape of the pulse. However, signal ringing was not completely cancelled. In addition to the 50

ohms impedance of the amplifier, the snubber had a terminating impedance of 50 ohms. Taking









into account these two 50 ohm loads, the pulse amplitude was estimated to be 18.2 mV and a

preamplifier gain of25 yielded about 455 mV.

Small amplitude noise pulses result from thermal emissions of the PMT dynodes and

amplifier. A discriminator is used to block these low amplitude noise. Setting the correct

discrimination level is important in photon counting. To achieve a good S/N, the discriminator

level should be set above the noise amplitude and below the signal amplitude. Another reason

for setting the correct discriminator level is to reduce drift. Slight change in the PMT gain may

lead to drastic change in the photon counts if the discriminator level is within the center of a

signal pulse height distribution. The discrimination level can be determined by plotting a pulse

height distribution as depicted in Figure 3-14.92 Generally, the optimum discriminator level for a

photon counter is within the valley of the pulse height distribution. There is no concrete rule in

setting the discriminator value as it depends on the nature of the measurement. With high dark

counts, the threshold should be set at a lower value. For a cooled PMT where dark counts are

not high, a higher threshold should work. A pulse height distribution presented in Figure 3-15

was plotted for the PMT. A narrow discriminator window is set by adjusting discriminator 1 to 0

V and discriminator 2 to -5 mV. The two discriminators were connected to the same input. The

narrow window between the two discriminators was scanned across all pulse height and the

photon counts were recorded. The discriminator level was set to -70 mV which is slightly higher

than the lowest value. This setting was chosen in order to eliminate the signal ringing, which the

snubber failed to cancel.

The SR-400 gated photon counter (Stanford Research; Sunnyvale, CA) was operated

using continuous gating and so the photon counter was enabled throughout the entire count

period. Computer control of the photon counter was done through the GPIB interface. The










photon counter was controlled by a LabVIEW program. All the parameters such as

discriminator level, integration time and count period were set using the LabVIEW software.

For all photon counting measurements, the integration time was set between 1 s to 30 s.

The spectrum of benzene recorded in analog was compared with that obtained with photon

counting. Figure 3-16 shows that photon counting gave a better S/N than the analog mode.

Data Collection

The LabVIEW program was also used to acquire spectra. Two different computer

interfacing methods were employed to drive the grating at a stepping mode and control the

photon counter. Synchronization of the monochromator control with the photon counter was

necessary to achieve accurate collection of Raman spectrum. In contrast to scanning

continuously, the step mode permitted longer integration time while scanning when recording a

Raman spectrum. A timing diagram (Fig.3-17) was followed to achieve smooth and

synchronized measurement and data collection. For the monochromator, the total time for each

measurement is not only the time set for the grating to stop at a certain wavelength but also the

time required for the grating to rotate from one wavelength to another during scanning. The

rotation time was calibrated at different wavelength increment (Table 3-4). The photon counter

has an integration time, which is the length of time the counter is set to count photons, and a

dwell time which is the time between count periods. Total measurement time set for the photon

counter should be equal to the total measurement time that the monochromator stops in between

scans.

The data acquired by the LabVIEW program were the counts corresponding to each

count period. Each data count corresponds to a specific wavelength. Data processing was done

using Origin 7.5 (Northampton, MA). The height of the peaks was determined by taking the two

lowest points on each side of the peak of interest and fitting a linear function.









All spectra were recorded using wavelength increments of 0. 1 nm to 0.2 nm. In order to

confirm that no peaks were skipped using these increments during photon counting, a spectrum

of a mercury lamp was recorded using both analog and photon counting mode. Figure 3-18

indicates that none of the narrow mercury lines were skipped during the scanning in the photon

counting mode.

Evaluation of Performance of the Raman System

The lab-built Raman system was designed specifically for measurements of liquids in

cuvettes. Several solvent spectra were obtained as part of the system characterization. Using

both analog and photon counting modes, spectra of pure solvents including benzene, methanol,

ethanol, tetrahydrofuran (THF) and acetone were recorded. The spectra of the pure solvents in

analog mode are presented in Figure 3-19. For all solvents, the spectra showed Raman peaks

matching those in literature. For the purpose of S/N consideration, the signal was defined as the

average intensity value and noise as standard deviation of the peak intensity. A comparison of

the benzene spectra indicated an improvement in the S/N was observed from analog mode to

photon counting mode (Fig. 3-16).

The ability of the system to observe Raman scattering from analytes in dilute solution

was demonstrated by the spectra obtained from beta-carotene in different solvents (Fig. 3-20).

Raman spectra of dilute samples of beta-carotene in THF were also measured.

The first step toward the SERS of DPA on silver colloids was to obtain a normal Raman

spectrum of saturated DPA as part of the system characterization (Fig. 3-21). A huge

background was observed from the saturated DPA. To determine the source of this background,

the spectra of an empty cuvette, deionized water and 1 M KOH were recorded and compared

(Fig. 3-22). The cuvette gave no signal while the deionized water and 1 M KOH showed the









same shape of background. Deionized water was present in both samples. A spectrum of

deionized water in literature confirmed the same Raman spectrum.93

After the design, optimization and characterization of the Raman system, a broader and

deeper understanding of the system has been attained. It is clear that the lab designed Raman

system is capable of measuring Raman scatter from a variety of liquid samples. The next step is

to assess its usefulness in SERS detection of DPA in bacterial spores.


















































Figure 3-1. The Raman experimental set-up.





















Table 3-1. Common lasers used in Raman
Laser Type


Spectroscopy (Adapted from Reference #3).
Wavelength (nm) Typical average
Power (mW)


Ar
Ar
Kr
He-Ne

Ti sapphire
diode
Nd:YAG

Nd:YAG doubled


CW
CW
CW
CW
CW

CW

Pulsed or quasi-
CW
CW and pulsed


244, 247
457, 488, 514.5
406, 647, 752
632.8
690-1000
690-900
200-400

535


200
100-2000
100-1000
5-50
500-2000
5-500
10-500


50-500






















Table 3-2. Nominal output power of the visible lines of the Argon ion laser (adapted from
reference #87)


Wavelength (nm)


Output Power (W)


Visible Multiline




Visible Single Line


454.5-514.5
454.5
457.9
465.8
472.7
476.5
488.0
496.5
501.7
514.5
528.7


7.00
0.14
0.42
0.18
0.24
0.72
1.80
0.72
0.48
2.40
0.42





























6ann



5000

488 496.5 514.5

4000



3000 476.5



2000





1000n 47.


520 530 540 550


470 480 490 500 510
Wavelength (nm)


Figure 3-2. The Argon ion visible lines measured using an OceanOpticsTM CCD spectrometer.





Laser
Transmitting
filter


Argn in Lse


White cardboard


Fiber optic


Spectrometer


Figure 3-3. The experimental set-up used for measuring Argon ion laser profile and laser short
and long term fluctuations.





































2000-




1500-



o
1000-




500-





0 200 400 600 800 1000

T im e (s)





Figure 3-4. The short-term fluctuation of the 514.5 nm line of the Ar' laser (%/ RSD=7%).



































2000 -


o





500-





0 1000 2000 3000 4000 5000 6000
T im e (s)




Figure 3-5. The long-term fluctuation of the 514.5 nm line of the Arg' laser (% RSD = 8%).























lens lens

Monochromatorlmi
slit i(

filIter

Diaphragm


Figure 3-6. A schematic diagram of the 900 sampling geometry adapted for Raman system.























Adjustable diaphragm


~ 4 LL~q~~ ~ 4~1~


Figure 3-7. A diagram illustrating (a) the position of the dove prism in the Raman system and
(b) the image rotation by a dove prism (adapted from reference # 88).





























16000-
-no dove prism
14000--withdove prism


12000-


C 10000-


[ 8000-







41 0 600 800 1000 1200 1400 1600
Ram an Shift (cm -1)




Figure 3-8. A comparison of normal Raman spectra of saturated DPA illustrating the
improvement in signal intensity due to the image rotation using a dove prism.

























Table 3-3. A summary of the important characteristics of the Spex 1680B double
monochromator.


Grating
Focal length
Reciprocal Linear Dispersion
Resolution
Repeatability
Aperture
Spectral Range
Accuracy


1200 grooves/mm
0.220 m
1.8 nm/mm
0.2 nm at 500 nm
10.2 nm


185-900 nm
10.4 nm

























n speed (nm/s)
17
23
153
17
.64
131
171


Table 3-4. The monochromator scan speed at different increments.
Increment (nm) rotation time (s) sca
0.1 0.240 0.4
0.2 0.243 0.8
0.5 0.256 1.9
1.0 0.355 2.8
2.0 0.469 4.2
5.0 0.829 6.0
10.0 1.499 6.6











1001
90
80
70
60
50
40
30

-Measured

450j 475 50P0 25 550 575 000
Wavelengthr (mn)


b












210

0
425~ 475


525; 575- 625 675 725
Wavelength (nm)


~2"





5


380


-- Oe~g~


430 480 530 500
Wavelength (nm)


530 650


Figure 3-9. Transmission spectra of different types of SemrockTM Optical filters used in Stokes
Raman Spectroscopy: (a) laser transmitting filter, (b) long wave pass filter, and (c)
notch filter (adapted from reference 89 ).































Ege Design
7T NolBch Design
Labsr Lina
810 615 L20 02?5 6930 635P 180 645 660s 655 060a
Wavelenrgt (nmR)



Figure 3-10. A comparison of the optical density plot of a long wave pass filter vs. a notch filter
(adapted from reference #89).








































S2500

2000

1500

1000

500


450 460 470 480 490 500 510 520 530 540 550
Wavelength (nm)


Figure 3-11. A spectrum of Ar' laser showing the different output wavelengths (blue). Inserting
a 514.5 nm MaxlineTM Semrock laser line filter between the laser and the sample all
wavelengths except 514.5 nm.

























3OOOOO


250000


2OOOOO


100000


50000
















Monochromator
slit


512 513 514 515 516
Wavelength (nm)


517 518 519 520


lens
lens




slit
H filter_ fle


lens









Diaphragm


Diaphragm


Position a


Position b


Figure 3-12. The decrease in the intensity of Rayleigh scatter peak (5 14.5 nm) using a high pass
filter that blocks light from 500-515 with a transmission efficiency of >90% placed at

different positions.









































I ,,--Semrock nRzor~dge 1e



6000


4000


2000



513 513 5 514 514 5 515 5155 516
Wavelength (nm)









Figure 3-13. A comparison of the Rayleigh peak attenuation by two different long wave pass
filters.
























Table 3-5. A summary of the characteristics of the Hammamatsu R-928 PMT.
Photocathode material Multi alkali
Window Material UV glass
Peak Wavelength 400 nm
Spectral Range 185-900 nm
Dynode Stages 9
Gain 1 x107
Anode to cathode voltage (max) 1250 V
Average anode current (max) 0.1 mA
Anode dark current (after 30 min) typical 3 nA
Anode dark current (after 30 min) max 50 nA
Rise time 2.2 ns
Transit time 22 ns




















Single photon peak


Noi se


o Disc 1

Disc 2



tt






Pulse Height (mV)




Figure 3-14. A typical pulsed height distribution. Disc 1 and Disc 2 are the discriminators of the
photon counter. A window discriminator (Disc 2-Disc 1) is scanned with a Atd
interval to generate a pulse height distribution in order to determine the
discrimination level for the photon counter.











































450000000


1s Iegraton ime00
2s ltegrabon bme ,0
5siegratotime






150 140 130 120 110 100 9o so 70 605o4
Pulse night






30 -180 -160 -140 -20 -100 -80 -60 -4


Pulse Height (mV)


Figure 3-15. The pulse height distribution for the photon counting system for determining the

discrimination level. Inset is the blown up portion of the plot to clearly show the data

points.


400000000
-5000


350000000





300000000

250000000


-2

















6000


O


5000


Analog


4000









2000




1000


1000


2000
Raman Shift (cm-1)


2500


3000


3500


500 1000 1500 2000 2500 3000 3500
Raman shift (cm-1)


Figure 3-16. Benzene spectra obtained using analog and photon counting mode illustrating the

difference in signal to noise.































Photon Counter


Monochromator


Count period


Figure 3-17. A timing diagram used for the photon counter and monochromator interfacing and
synchronization. The integration time and dwell time of the photon counter were set
using the LabVIEW program. The monochromator stop time and rotation time were
set using the monochromotor controller.
























3500


30001


2500


S2000


rn 1500


-0 2nm Increm ent
|-01 nm Increm entI


Wavelength (nm)


3500
b
3000


2500


2000


1500


1000


500



540 545 550 555 560 565 570 575 580 585
Wavelength (nm)










Figure 3-18. Mercury lamp spectra obtained with (a) analog and (b) photon counting mode

showing the narrow mercury lines at 546.08, 576.96 and 579.07 nm. This

demonstrates that the monochromator scanning does not skip any lines even when set

to 0.2 nm increments.





























































600 700 800 900 1000 1100 1200 1300 1400 1500 1600

Raman ShiR (cm-1)


800 -



700



600



500



400



300



200



100




600


700 800 900 1000 1100 1200 1300 1400 1500 1600
Raman ShiR (cm-1)


350



300












200

-o


600 700 800 900 1000 1100 1200 1300 1400 1500 1600

Raman Shift (cm-1)


Figure 3-19. Raman spectra of several pure solvents measured using the analog mode.





















a





1000- beta-carotene + ethanol
--ethanol


800








400



200




600 700 800 900 1000 1100 1200 1300 1400 1500 1600
Raman Shift (cm-1)
b



1800
-beta-carotene+tetrahydrofuran
-tetrahydrofuran
1600


1400







S800

600

400


200



600 700 800 900 1000 1100 1200 1300 1400 1500 1600
Raman Shift (cm-1)





Figure 3-20. Raman spectra of 1.0 x 10-4 M beta-carotene in (a) ethanol and (b) tetrahydrofuran.

The spectra of the solvents are shown and can be superimposed on the beta carotene

spectrum. The arrows represent the peaks attribute to beta-carotene.
































45000 -

40000-

35000-

30000 -

25000 -

20000 -

15000-

10000 -

5000 -

0 -


600 800 1000 1200 1400

R am anShift (cm -1)


1600 1800


Figure 3-21. A normal Raman spectrum of saturated DPA in 1 M KOH.































20000-

18000-

16000 -1 -e m pty q u artz c uve tte
9 -deionized water
] 14000 -( -1 M KO H

O 12000-

S 10000-

S 8000-

S 6000-

4000-

2000-


4( 0 600 800 1000 1200 1400 1600 1800

Raman Shift (cm l)






Figure 3-22. Raman spectra of an empty cuvette, deionized water, and 1 M KOH.










CHAPTER 4
STABILITY OF SILVER COLLOID AS SUBSTRATE FOR SURFACE ENHANCED
RAMAN DETECTION OF DIPICOLINIC ACID

Introduction

Metal colloids (hydrosols) have been commonly used as SERS substrates. Creighton et

al were the first to report SERS of pyridine on silver and gold colloids.47,72 Their measurements

have been significant in demonstrating that SERS is associated with surface plasmon excitation

on the metal surface.72 Subsequent experiments which followed the first observation of SERS of

pyridine on roughened electrodes involved the use of these types of substrates. However, they

are less favorable for fundamental studies of the SERS phenomenon. Researchers shifted their

attention to the use of metal colloids since it is much easier to characterize their optical

properties particularly their absorption spectra, and to account for these properties in terms of

roughness in a precise way. The main advantages of using metal colloids for SERS

measurements are their ease of formation and manipulation for analytical studies. Chemical

reduction of simple salts of silver or gold with either sodium borohydride or sodium citrate is

most commonly used as a method of preparing these metal hydrosols.

These colloidal systems include particles whose diameters range from 1 nm to 100 nm.94

These metallic particles are usually negatively charged due to the adsorbed anions. Neutral

species added to the hydrosol can be adsorbed on the particles replacing the negative charges.

This disrupts the aggregative stability of the system and larger aggregates can form.68 It has

been shown many times that SERS requires partial aggregation and adsorption of the

analyte.28,30,32,48,49,68,95,96 Although, analytical applications of SERS have been reported, the use

for quantitative analysis has been problematic because of the poor precision of the SERS signal

due to irreproducible colloid preparation. In order to understand the factors affecting the









irreproducibility of the SERS response, it is important to study SERS under static conditions.

This present study evaluates important factors such as silver colloid aging, analyte-induced

aggregation and the stability of the sodium borohydride. Dipicolinic acid (DPA) which is a

signature of Bacillus spores, is used in this study.

Experiments and Methods

Reagents and Procedures

Aqueous solutions of analytical reagent grade sodium borohydride (2 x10-3 M) and silver

nitrate (1 x 10-3 M) were used for generation of silver colloids. The chemical reaction is:28

Ag' + BH4- +3H20+ Ag +H3BO3 +7/2 H 2(g) (4-1)

Hydrogen gas is produced by the reduction of silver ions as well as the slow reduction of water

by the sodium borohydride at room temperature.

BH4- + 3H20 H2BO3- + 4H 2 (g) (4-2)

The silver hydrosols were obtained by mixing sodium borohydride and silver nitrate solutions at

6/1 (v/v) volume ratio. Aqueous solution of dipicolinic acid (1.5 x 10-3M) was mixed to the

resulting silver colloid at a 1:1 ratio. A list of reagents is provided in Table 4-1.

Silver Colloid Characterization

Characterization of the silver colloids by absorption spectroscopy was performed using

an Agilent UV-Vis double beam spectrophotometer. A 1 mL sample from each batch of silver

colloids was used for all absorption measurements. Deionized water was used as the blank.

Scanning electron microscopy (SEM) was used to study the surface morphology of the

silver colloids. 50 CLL aliquots were spotted on sample stubs and air-dried overnight in a

desiccator. The SEM samples were coated thinly with gold by sputtering at 50 mA for 50 s.

SERS~ Instrumentation

SERS measurements were conducted using the Raman system described in Chapter 3









(Fig.3-1). Briefly, it consisted of a Spectra Physics BeamLok 2060 Ar+ laser and a SPEX 1680B

double monochromator. A thermoelectrically cooled Hamamatsu R-928 photomultiplier tube

coupled with a Stanford research photon counting system was used as a detector. A dove prism

tilted at a 450 angle is used to rotate the image making it parallel to the entrance slit of the double

monochromator. A LabVIEW program was used for photon counter control and data

acquisition. All measurements were done using a standard 1 cm x 1 cm quartz cuvette. SERS

spectra were recorded in 90-degree collection geometry. All SERS signals were normalized to

the measured laser power after obtaining the peak.

Results and Discussion

The most commonly used method of generating silver hydrosols for SERS measurements

is the chemical reduction of silver nitrate by sodium borohydride solution. The quality of the

SERS signal depends on a variety of factors such as reagent concentration, mixing procedures as

well as the incorporation of the sample.28,48,4 Because of the dependency of the silver colloid

quality on several factors, it is not surprising that SERS precision is poor. This study evaluates

these important factors in order to improve the reproducibility of SERS measurement of

dipicolinic acid.

Farquharson and co-workers observed the following band in the SERS spectrum of DPA

in water: 657, 815, 1008, 1382, 1445, and 1567 cm 1.97 Normal Raman and SERS spectra of

dipicolinic acid were recorded to observe these bands (Fig. 4-1). The peaks in the normal Raman

and SERS spectra of DPA are summarized in Table 4-2. The most prominent peak observed at

1013 cm-l was barely visible in the normal Raman of 7.5 x 10-3 M DPA in water.

The hydrosols obtained in this study were characterized by their absorption spectra.

Provided that the silver particles are smaller than the wavelength of light and approximately

spherical in shape and monodisperse, silver colloids are yellow with a single extinction band at









385 nm. This band is due to the resonant excitation of plasma oscillations in the confined

electron gas of the particles.68 Figure 4-2 shows the effect of aging the silver hydrosols on the

absorption spectra. A sample hydrosol was obtained at different times after preparation for UV-

Vis absorption measurement. All the spectra showed a single maximum peak at about 400 nm

which is characteristic of the surface plasmon resonance of particles that are roughly spherical in

shape. An increase in the absorbance due to an increase in the concentration of the silver

particles over time was observed. This indicated that more silver particles form in a span of 1

hour after the reaction started. It is also evident that aging the silver colloid affects the SERS

signal of DPA. Figure 4-2 illustrates that as a consequence of the increase in the concentration

of silver particles through aging of the hydrosol, the SERS response of DPA increased for the

first 30 min and was constant for longer times.

Silver colloids usually have a negatively charged surface due to adsorbed anions from the

reagents used in the reaction and electrostatic repulsion keeps the colloid stable from aggregation

provided that there is sufficient charge.68 Introduction of molecules such as DPA can replace

these negative charges leading to aggregation of the silver particles.68 Before addition of DPA,

the silver colloids were yellow in color with a sharp extinction band centered at 400 nm.

Addition of DPA changed the color of the silver colloid solution from yellow to pink indicating

the formation of aggregates. Aggregate formation was evident from the change in the shape of

the silver hydrosol absorption spectrum with the appearance of another peak at longer

wavelengths (Fig.4-3). No drastic changes in the SERS signal of DPA were observed when the

DPA and the silver colloid mixture was allowed to stand for 1 h. Therefore, further silver

colloid aggregation induced by the DPA was minimal which could also be deduced from the

observed absorption curves of the mixture monitored within the 1 h period.









Sodium borohydride has been commonly used as a reducing agent for the generation of

silver hydrosols. Equation 4-2 shows that further oxidation of the borohydride ions occur even at

room temperature. To avoid such concentration changes over time, sodium borohydride

solutions must be freshly prepared. Figure 4-4 demonstrates the depletion of borohydride ions

available to reduce silver nitrate over a period of 1 h. Assuming that all silver nitrate reacted

with sodium borohydride, a decrease in the absorbance at the maximum peak ca. 400 nm

indicates a decrease in the resulting silver colloid concentration. The absorbance observed after

1 h of standing the borohydride solution decreased two-fold as shown in Figure 4-4a.

Deoxygenation of the deionized water was done prior to preparation of sodium borohydride

solution to avoid further oxidation to occur. Purging the solution with an inert gas such as

nitrogen is the most commonly used method of minimizing oxygen in the solution. However,

this method did not completely stop the further oxidation of borohydride based on the

absorbance at the peak maximum spectra of the silver hydrosols (Fig. 4-4b). Another way to

address this problem is to introduce another reducing agent such as sodium sulfite. Sodium

sulfite is used as an oxygen scavenging agent in water treatment. This method has been used to

prevent quenching of phosphorescence by oxygen.98 This method is based on the reaction

2SO3-2 + 02 + 2SO4-2 (4-3)

This deoxygenation method showed better results as no drastic decrease in the absorbance at the

maximum peak was observed (Fig. 4-4c). The resulting silver concentrations remained constant

with the addition of sodium sulfite. In Figure 4-5, an improvement in the SERS signal

reproducibility was obtained largely a result of limiting borohydride concentration changes with

the aid of the sodium sulfite addition.









It was also observed that the formation of the yellow silver colloid solution, upon

addition of sodium borohydride to silver nitrate occurred at a slower rate when sodium sulfite

was added. In the absence of sodium sulfite, the color change is instantaneous. A kinetic study

of the silver colloid formation was carried out over a period of 3 h. A comparison of 2 different

concentrations of sodium sulfite is presented in Figure 4-6. The absorbance reading reached a

plateau after 50 min and 75 min for the silver colloid treated with 0.005 M and 0.01 M Na2SO3,

respectively. The stability of SERS signal observed from DPA using silver colloids treated with

0.050 M Na2SO3 after 50 min (Fig. 4-5a) was consistent with the results obtained from the

absorption measurements of the silver. SEM images of the silver colloids collected at different

times are shown in Figure 4-7. No change in the particle size was observed from each silver

colloid. Comparison of the SERS signal obtained using silver colloids treated with 0.005 M and

0.010 M Na2SO3 yielded different signal enhancement factors (Table 4-3). Two orders of

magnitude enhancement was achieved using 0.005 M Na2SO3 and one order of magnitude with

0.01 M Na2SO3. Sulfate aggregation has been shown to promote SERS of DPA.99 Based on the

equation 4-3, sulfate induced aggregation may occur after the oxidation of sulfite. Addition of

0.005 M Na2SO3 WAS sufficient to form aggregation resulting in a 100-fold DPA SERS signal

enhancement while 0.010 M resulted in excess aggregation, hence yielding only an order of

magnitude enhancement. It was also observed that sulfite addition gave a higher DPA SERS

signal as compared to sulfate addition as summarized in Table 4-3.

Conclusions

This preliminary study of the SERS of DPA is crucial in improving the reproducibility of

SERS as an analytical technique. It is important to understand the SERS of DPA on silver

colloids in static conditions to address the problem of poor precision. It was shown that the age

of the silver colloid affects the observed SERS signal of DPA with the optimum signal achieved









using silver colloids aged for at least 30 min. The DPA-induced aggregation study shows that

the SERS signal measured over 1 h was stable allowing longer measurement time, as in the case

of scanning PMT-photon counting detection. Control over the depletion of borohydride ions

available for reduction of silver ions was achieved by using sodium sulfite for deoxygenation.

All these findings are useful in the development of a Flow Inj section Analysis (FIA)-SERS

system for the detection of DPA which is the subj ect of the Chapter 5.





















Table 4-1. List of reagents used.
Reagent Systematic Formula Molecular Physical Company
name weight state
(g/mol)
Dipicolinic Pyridine-2,6- (C5H3N)(COOH)2 167.12 White Sigma-
acid dicarboxylic powder Aldrich
acid
Sodium Sodium NaBH4 37.83 White Fisher
borohydride tetrahydroborate powder Chemicals
Silver nitrate Silver nitrate AgNO3 169.8731 White Fisher
crystalline Chemicals
solid
Sodium Sodium sulfite Na2SO3 126.04 White J.T Baker,
sulfite crystalline NJ
solid
Sodium Sodium NaCl 58.44 White Fisher
chloride chloride cry stalline Chemi cal s,
solid
Sodium Sodium sulfate Na2SO4. 10 H20 322.19 White Fisher
sulfate decahydrate crystalline Chemicals
solid
Nitric Acid Nitric acid HNO3 63.01 Liquid Fisher
Chemicals
Potassium Potassium KOH 56.11 White Fisher
hydroxide hydroxide pellets Chemicals






























45000, -

40000-

35000-

30000 b

S25000-

20000-

15000-

10000-

5000-


4* 0 600 800 1000 1200 1400 1600 1800
Raman Shift (cm-1)




Figure 4-1. Comparison of the normal Raman spectra of (a) saturated DPA in KOH and (c) 7.5 x
10-3 M DPA in water; and (b) SERS spectrum of 7.5 x 10-3 M DPA in water. The
silver colloids used for the SERS measurement were prepared by chemical reduction
of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1
(NaBH4 to AgNO3).


























Table 4-2. A summary of observed normal Raman and SERS peaks of DPA.
Literatureloo Observed
Normal Raman SERS Normal Raman SERS Peak Assignments
647 657 674 674


815
1008
1382

1428
1567


817
998
1384

1434
1569


835
1013
1393

1425
1574


835
1013
1393

1425
1574


Symmetric ring stretch
O-C-O symmetric
stretch
C-Hbend
O-C-O symmetric
stretch



























l i l ii l II I .20
35000 .- -- 0.18


O 30000-
0 0.16

o 25000- -I 0.14


20000- ----- m 01
m 0.10 0
S15000-


O'' 10000 -0.06

-a 0.04
5000-
I 0.02

0 0 ~~....0.00
2 0 10 20 30 40 50 60

Tim e (m in)



Figure 4-2. SERS signal of DPA on silver colloids aged over 1 h and the absorbance of the
silver colloid at 402 nm measured from 0 to 60 min. The silver colloids used were
prepared by chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The
volume ratio used was 6 to 1 (NaBH4 to AgNO3 -



























0.5 -I | liver loniola + v ey


0.4-


0.3-


0.2-


0.1-


0.0 ggg
300 400 500 600 700 800

Wavenum ber (cm l)


Figure 4-3. Absorption spectra of silver hydrosol before (black) and after (red) addition of
dipicolinic acid (DPA). The silver colloids were prepared by chemical reduction of 1
x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1 (NaBH4 to
AgNO3).






















1.0 -



0.8-



0.6-



S0.4-
o I I ~no treatment
Q ~ --ith n itrog en p urgin g
0.2-c with sodium sulfite a -



0.0 ggggg .
O 10 20 30 40 50 60
Time (min)




Figure 4-4. Absorbance of silver colloids at the peak maximum 401 nm as a function of sodium
borohydride aging over a period of 1 h: (a) no treatment, (b) with nitrogen purging
and (c) with sodium sulfite. The silver colloids were prepared by chemical reduction
of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio used was 6 to 1
(NaBH4 to AgNO3).






































SI I I I I


0 2D AD O ED 1D ~120

lirm(nin)


ilIII)-

Y
~ Bm]D
o
O
ICIII)-
m

v, ~D00D-
v,
ct
w JIII)-
v,

N ~mxr
m
E
L 10m)-
o
z


25000-


Y
~ awoo-
o
o
m
~ 15000-
cn
v,
v,

W 10000-
v,
-cr
.N



z


line(nin)


Figure 4-5.


SERS signal of DPA on silver colloid with (a) and without sodium sulfite as oxygen

scavenger (b The silver colloids used in the SERS measurements were prepared by

chemical reduction of 1 x 10-3 M AgNO3 by 2 x 10-3 M NaBH4. The volume ratio

used was 6 to 1 (NaBH4 to AgNO3).




























I I


0.9 -mg

0.8- *

(11 0 7-

W 0.6

c 0.5-



< 0.3-
4I m 0.005 M Na2SO3
0.2-
a 0.010 M Na2SO3
0.1-

0 2000 4000 6000 8000 10000 12(000

Time (s)


Figure 4-6. Absorbance of the silver colloids at the peak maximum 402 nm. The silver colloids
were prepared by chemical reduction of 1 x 10-3 M A~gNO3 by 2 x 10-3 M NaBH4
stabilized with 0.005 M and 0.010 M Na2SO3. The volume ratio used was 6 to 1

(NaBH4 to AgNO3).
















































Figure 4-7. SEM images of silver colloids prepared using sodium borohydride treated with
sodium sulfite. Silver colloid morphology was observed (a) 15 min and (b) 30 min
after preparation. The silver colloids were prepared by chemical reduction of 1 x 10-3
M AgNO3 by 2 x 10-3 M NaBH4 Stabilized with 0.005 M Na2SO3. The volume ratio
used was 6 to 1 (NaBH4 to AgNO3).





















Table 4-3. A summary of normalized signal intensities and calculated enhancement factors from
SERS of DPA on silver colloids.
Normalized Intensity (Counts/W)
DPA Silver Colloid
Concentration Treatment Enhancement
Normal Raman SERS Factor
1.50E-02 1074
7.50E-03 0.005 M Na2SO4 38253 71
7.50E-03 0.010 M Na2SO4 44039 82
7.50E-03 0.005 M Na2SO3 54062 101
7.50E-03 0.010 M Na2SO3 5857 11










CHAPTER 5
EVALUATION OF EXPERIMENTAL CONDITIONS FOR THE SURFACE ENHANCED
RAMAN DETECTION OF DIPICOLINIC ACID ON SILVER COLLOIDS GENERATED BY
FLOW INJECTION ANALYSIS

Introduction

Flow inj section analysis (FIA) is based on the inj section of a plug of sample into a moving

and continuous carrier liquid. The injected sample forms a zone and is transported to a detector

which continuously records a signal as the dispersed sample passes through a flow cell. The

simplest FIA system is composed of a pump to push the carrier liquid through the small diameter

tubing, an injection port for reproducible injection of the sample to the carrier stream, a reaction

coil for sample dispersion and reaction with the carrier liquid, and a detection flow cell. An FIA

output shows that peak height is related to the analyte concentration. A well designed FIA

system has an extremely rapid response in the range of 5-20 s with injected volumes between 1

CLL to 200 CLL.101 This makes FIA a high throughput technique with minimum reagent and sample

requirement. FIA systems are designed for the purpose of analyzing the maximum number of

samples at short time and minimum reagent and sample volumes.

FIA is governed by three basic principles as described by Ruzicka and Hansen.102 These

are reproducible timing, sample injection and controlled dispersion. The purpose of transporting

sample solution using an FIA is to treat the sample material in such a way that it will yield not

only a sensitive output but also reproducible. In an FIA system, physical mixing of the reagent

and sample is carried out reproducibly. The use of an injection valve prevents variation in the

sample volume. In such systems, band broadening or sample zone dispersion is controlled to suit

the detection and chemistry associated with it.

The process of dispersion, as the sample plug is transported through FIA system, can be

quantitated using the dispersion coefficient. Dispersion is defined as the amount by which the