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Selective Molecular Recognition Conjugated Nanoparticles for Biological Applications

Permanent Link: http://ufdc.ufl.edu/UFE0021266/00001

Material Information

Title: Selective Molecular Recognition Conjugated Nanoparticles for Biological Applications
Physical Description: 1 online resource (140 p.)
Language: english
Creator: Smith, Joshua Elliott
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bioanalytical, biology, cancer, cells, dna, fluorescence, magnetic, nanoparticles, peptide, protein
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The collection and enrichment of biomolecules from complex biological samples is critical for disease diagnostics, environmental analysis, and biotechnological applications. Current separation methods suffer from numerous limitations that include time consuming procedures, difficulties with handling complex samples, and lack of selective molecular isolation. The objective of my research is to create nanodevices for the selective separation and isolation of biological molecules and to evaluate their applications for biological studies. First, nanoharvesting agents (NHAs) were developed for the selective collection, separation, and detection of DNA and peptide targets. The NHAs are composed of silica coated magnetic nanoparticles bioconjugated with DNA probes on the nanoparticle surfaces. For peptide recognition, a DNA aptamer was used for the selective binding of the analytes. The NHA were demonstrated to have excellent specificity and sensitivity in both artificial buffer and complex biological samples for subsequent analysis by either fluorescence or mass spectroscopy. Additionally, the NHAs were functionalized with antibody molecules, and used in a high-throughput protein microarray to improve the detection limit. The NHAs were used as a tracer device, where the antibody on the particle surface was labeled with fluorescent dye molecules for signaling. Prior to analyses by the microarray device, the NHAs were extracted from large volume samples, concentrated to small volumes, and used directly for the detection of proteins. Furthermore, a novel method was developed for the rapid collection and detection of whole cancer cells using a two silica-based nanoparticle approach. Magnetic nanoparticles were used for target cell extraction, and fluorescent nanoparticles were employed for sensitive cell detection. The nanoparticles were conjugated with DNA aptamers for target recognition. This system was used for the multiple extraction and detection of cancer cells. These newly developed techniques will be useful in biotechnology, biomedical science, and disease diagnostics for the selective extraction and isolation of biological targets from complex samples. Future investigations will include the improvement and exploration of biological applications for nanoparticle based techniques.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Joshua Elliott Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021266:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021266/00001

Material Information

Title: Selective Molecular Recognition Conjugated Nanoparticles for Biological Applications
Physical Description: 1 online resource (140 p.)
Language: english
Creator: Smith, Joshua Elliott
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: bioanalytical, biology, cancer, cells, dna, fluorescence, magnetic, nanoparticles, peptide, protein
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The collection and enrichment of biomolecules from complex biological samples is critical for disease diagnostics, environmental analysis, and biotechnological applications. Current separation methods suffer from numerous limitations that include time consuming procedures, difficulties with handling complex samples, and lack of selective molecular isolation. The objective of my research is to create nanodevices for the selective separation and isolation of biological molecules and to evaluate their applications for biological studies. First, nanoharvesting agents (NHAs) were developed for the selective collection, separation, and detection of DNA and peptide targets. The NHAs are composed of silica coated magnetic nanoparticles bioconjugated with DNA probes on the nanoparticle surfaces. For peptide recognition, a DNA aptamer was used for the selective binding of the analytes. The NHA were demonstrated to have excellent specificity and sensitivity in both artificial buffer and complex biological samples for subsequent analysis by either fluorescence or mass spectroscopy. Additionally, the NHAs were functionalized with antibody molecules, and used in a high-throughput protein microarray to improve the detection limit. The NHAs were used as a tracer device, where the antibody on the particle surface was labeled with fluorescent dye molecules for signaling. Prior to analyses by the microarray device, the NHAs were extracted from large volume samples, concentrated to small volumes, and used directly for the detection of proteins. Furthermore, a novel method was developed for the rapid collection and detection of whole cancer cells using a two silica-based nanoparticle approach. Magnetic nanoparticles were used for target cell extraction, and fluorescent nanoparticles were employed for sensitive cell detection. The nanoparticles were conjugated with DNA aptamers for target recognition. This system was used for the multiple extraction and detection of cancer cells. These newly developed techniques will be useful in biotechnology, biomedical science, and disease diagnostics for the selective extraction and isolation of biological targets from complex samples. Future investigations will include the improvement and exploration of biological applications for nanoparticle based techniques.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Joshua Elliott Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021266:00001


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1 SELECTIVE MOLECULAR RECOGNITION CONJUGATED NANO PARTICLES FOR BIOLOGICAL APPLICATIONS By JOSHUA E. SMITH 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 2007 Joshua E. Smith

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3 To my family and friends

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4 ACKNOWLEDGMENTS Joshua is extremely grateful to everyone that was involved in this work. Their suggestions, efforts, and ideas were invaluable. He especia lly thanks his research advisor Dr. Weihong Tan for providing guidance, support, and continuous encouragement th roughout his graduate studies. Dr. Tan made performing this research possible, and Josh is apprecia tive for the opportunities that were available and provided to him as a member of the Tan research group. Josh thanks Dr. Charles Martin, Dr. Ben Sm ith, Dr. Tom Lyons, and Dr. Steve Pearton for agreeing to be members of his graduate committee. Josh thanks Dr. Frances Ligler for allowing him the opportunity to perform research with her group in Washington D. C. She and the rest of the research group had a profound and very positive impact on me. That expe rience really helped me to develop as a scientist and more importantly as a person. The work presented here would not have been possible without the help of many scientists. First, Josh acknowledges Dr. Tim Drake for introducing him to rese arch with nanomaterials and guiding him in the early stages of this research. Josh gives special thanks to Colin Medley for being such a reliable resource a nd for all his help with everything over the last few years. Josh recognizes Josh Herr for all his hard work and help while he was in the group. Joshua Smith gives thanks to Dr. Kim Sapsford for taking the tim e to work with him while he was at the Naval Research Laboratory and all the helpful discussi ons they had. Josh recognizes Dr. Jeremiah Tipton for his dedication a nd continuous assistance. Smiths time at graduate school was an enj oyable and a joyful experience thanks to his family, friends, and lab mates that helped keep everything in perspective. Without his parents, John and Jean, none of this would have been possi ble, so he thanks them for their continuous love, endless support, and constant encouragement. Josh thanks his brothe r and sisters and their families for being there for him when he needed them and for always just being themselves and

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5 making life more interesting. For the ones that he knew before graduate school, he thanks you for being there for him, particularly Rish, Laila, Se yb, Ganoe, and Scott. Smith gives thanks to his friends that he made while in graduate school, especially EJ, Br ent, Lisa, Alex, and Jeremiah. You really helped him to become who he is, and he will remember you always. Finally, Joshua thanks all the members of th e Tan group, past and pres ent for all the great times we spent together. It has been a privile ge being a member of the Tan group. To James, Marie, Alina, Karen, Tim, Shelly, and Colin you ma de Joshs time at the University of Florida enjoyable and pleasant. He also acknowledges Santra, Steve, Lisa, and Charles for all their help and support. There was a lot of fun both insi de and outside the lab. Keep up the newly established traditions.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............12 1 INTRODUCTION................................................................................................................. .14 Impact of Selective Recognition Nanopa rticles for Biological Applications.........................14 Standard Sample Treatment for Biological Analysis.............................................................15 Oligonucleotide Sample Preparation...............................................................................15 Protein Sample Preparation.............................................................................................17 Cellular Sample Preparation............................................................................................18 Recent Growth and Development of Nanoparticles...............................................................19 Fluorescent Semiconductor Nanocrystals.......................................................................19 Noble Metal Nanoparticles..............................................................................................21 Magnetic Nanoparticles...................................................................................................23 Silica Nanoparticle Fundamentals..........................................................................................25 Synthesis of Silica Nanoparticles....................................................................................25 Silica Nanoparticle Modifi cation and Conjugation.........................................................27 Characterization of Pr epared Nanoparticles....................................................................29 Selective Biorecognition Component.....................................................................................30 Research Scope................................................................................................................. ......33 2 PREPARATION OF SILICA COAT ED MAGNETIC NANOPARTICLES........................34 Introduction................................................................................................................... ..........34 Experimental................................................................................................................... ........35 Materials and Methods....................................................................................................35 Instruments.................................................................................................................... ..35 Nanoparticle Synthesis, Surface M odification, and Bioconjugation...............................36 Avidin physical adsorption.......................................................................................37 Biotin-avidin oligonuc leotide attachment................................................................37 Carboxyl modification..............................................................................................38 Direct oligonucleotide attachment...........................................................................38 Avidin Modification Determination................................................................................38 Investigation of DNA Modification................................................................................39 Optimization of DNA Functionalization.........................................................................39 Results and Discussion......................................................................................................... ..40 Iron Oxide Formation and Silica Coating.......................................................................40 Avidin Surface Modification...........................................................................................43 DNA Labeled Nanoparticles...........................................................................................44

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7 Nanoparticle Characterization and Optimization............................................................45 Carboxyl Modified Nanoparticles...................................................................................48 Conclusions.................................................................................................................... .........49 3 EXTRACTION OF OLIGONUCLEOTIDES USING SILICA COATED MAGNETIC NANOPARTICLES...............................................................................................................50 Introduction................................................................................................................... ..........50 Experimental................................................................................................................... ........52 Materials and Methods....................................................................................................52 Instruments.................................................................................................................... ..52 DNA Conjugated Magnetic Nanoparticles......................................................................52 Extraction of DNA Targets.............................................................................................53 Pure oligonucleotide extractions..............................................................................53 Large volume extractions.........................................................................................54 DNA mismatch targets.............................................................................................54 Multiple target extraction.........................................................................................55 Sample Preparation for Mass Spectrometry....................................................................55 Results and Discussion......................................................................................................... ..56 Fluorescence Detection of Extracted Samples................................................................56 Single oligonucleotide extraction.............................................................................56 Sample enrichment and mismat ch oligonucleotide extraction.................................58 Multiple oligonucleotide extraction.........................................................................60 Analysis by Mass Spectroscopy......................................................................................61 Conclusions.................................................................................................................... .........64 4 FUNCTIONALIZED NANOPARTIC LES FOR PEPTIDE EXTRACTION.......................65 Introduction................................................................................................................... ..........65 Experimental................................................................................................................... ........66 Materials and Methods....................................................................................................66 Nanoparticle Synthesis....................................................................................................67 Silica C18 functionalized nanoparticles....................................................................67 Magnetic aptamer nanoparticles...............................................................................67 Matrix and Analyte Preparation......................................................................................68 Instrumentation................................................................................................................69 Extraction Procedures......................................................................................................70 Results and Discussion......................................................................................................... ..71 Nonspecific Nanoparticle Applications...........................................................................71 Selective Nanoparticle Applications...............................................................................71 Conclusion..................................................................................................................... .........75 5 ANTIBODY-CONJUGATED MAGNETIC NANOPARTICLES FOR PROTEIN MICROARRAYS...................................................................................................................77 Introduction................................................................................................................... ..........77 Experimental................................................................................................................... ........81

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8 Materials and Methods....................................................................................................81 Magnetic Nanoparticle (MNP) Synthesis........................................................................81 Dye Labeling of Chick IgG.............................................................................................82 Optimized IgG Protocol..................................................................................................82 Slide Preparation, MNP Ex traction, and Immunoassay..................................................83 Immunoassay Array Imaging and Analysis....................................................................85 Results and Discussion......................................................................................................... ..85 Conclusion..................................................................................................................... .........97 6 APTAMER-CONJUGATED SILICA NA NOPARTICLES FOR CANCER CELL EXTRACTION AND DETECTION......................................................................................99 Introduction................................................................................................................... ..........99 Experimental................................................................................................................... ......101 Materials and Methods..................................................................................................101 Fluorescent Nanoparticle Synthesis..............................................................................102 Magnetic Nanoparticle Synthesis..................................................................................103 Magnetic Extraction......................................................................................................105 Cells.......................................................................................................................... .....105 DNA Aptamer Synthesis...............................................................................................106 Sample Assays...............................................................................................................106 Cell Imaging..................................................................................................................107 Flow Cytometry.............................................................................................................108 Microplate Reader.........................................................................................................108 Magnetic Extraction and Labeling................................................................................108 Results and Discussion.........................................................................................................110 Aptamer-Conjugated Nanopart icle Characterization....................................................110 Collection efficiency..............................................................................................110 Dye and nanoparticle fluorescent intensity comparison........................................111 Nanoparticle selectivity..........................................................................................112 Single cell type extractions....................................................................................113 Detection Limit..............................................................................................................116 Complex Sample Extractions........................................................................................117 Single type cell mixed sample extraction...............................................................117 Multiple cell type extraction method.....................................................................119 Multiple cell type mixed cell extractions-buffer samples......................................120 Multiple cell type mixed extractions-serum samples.............................................123 Whole blood sample assays....................................................................................125 Conclusions.................................................................................................................... .......126 LIST OF REFERENCES.............................................................................................................128 BIOGRAPHICAL SKETCH.......................................................................................................140

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9 LIST OF TABLES Table page 2-1 The zeta potential measurements obtained from various nanoparticle surfaces and coatings....................................................................................................................... .......41 2-2 The determined amount of DNA molecule s modified to the nanoparticle surface...........46 3-1 Representative normalized fluorescence intensities obtained from buffer and serum multiple target extraction...................................................................................................61 5-1 Carbodiimide hydrochloride protocols investigated for magnetic nanoparticles modification with Alex647-chick IgG...............................................................................90 5-2 The solution and surface measurements used to characterize the Alex647-chickmagnetic nanoparticle sample s pre and post extraction.....................................................94 6-1 The microplate reader data for the evaluation of single cell type extraction experiments.................................................................................................................... ..115 6-2 The microplate reader data obtained from buffer extracted cells by the multiple cell type extraction procedure.................................................................................................122 6-3 The microplate reader data obtained from serum extracted cells by the multiple cell type extraction procedure.................................................................................................124

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10 LIST OF FIGURES Figure page 1-1 The scheme representing the effect size has on magnetic materials in the absence of a magnetic field................................................................................................................. ....24 1-2 The scheme representing the Stber pro cess used to create silica nanoparticles..............26 1-3 The scheme representing the water-in-oil microemulsion system used to create silica nanoparticles.................................................................................................................. ....26 1-4 Organosilane functionalized and biomol ecule conjugated silica nanoparticles used for biological applications..................................................................................................27 1-5 Schematic representation of the selec tion process used to generate cell binding aptamers cell-SELEX.........................................................................................................32 2-1 The transmission electron microscope images of synthesized nanoparticles....................40 2-2 The X-ray Photoelectron Spectroscopy data collected from silica coated magnetite nanoparticles.................................................................................................................. ....42 2-3 The fluorescence data obtained from av idin modified silica magnetic nanoparticles treated with biotin-FITC....................................................................................................43 2-4 The microplate reader fluorescence intens ity data collected for FITC labeled DNA molecules...................................................................................................................... .....45 2-5 The fluorescent data obtained from batch-to-batch and sample-to-samples reproducibility studies........................................................................................................47 3-1 Fluorescence intensity data representi ng individually extracted DNA targets from buffer and serum samples..................................................................................................57 3-2 Enrichment study of extracting MnSOD fluorescent dye labeled DNA target from several different buffer volumes........................................................................................58 3-3 Fluorescence data obtained testing the ability of the nanoharvesting agent to collect and isolate mismatched DNA sequences...........................................................................59 3-4 Mass spectra demonstrating the extraction of the nonhuman mismatched DNA using the nanoharvesting agents..................................................................................................62 3-5 Mass spectra demonstrating the extrac tion of multiple DNA sequences using the nanoharvesting agents........................................................................................................63 4-1 The mass spectra of the Land Dvasopr essin before and after extraction are shown.....74

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11 5-1 The fluorescence image of the immunoassay treated with Chick-magnetic nanoparticles.................................................................................................................. ....86 5-2 Fluorescence images of the immunoa ssay treated with Alex647-chick-magnetic nanoparticles performed with various parameters.............................................................87 5-3 The fluorescence data showing the effect of the percent carboxyl terminated silane used to modify the surface of the magnetic nanoparticles.................................................89 5-4 The fluorescence data obtained to determine the effect of the carbodiimide hydrochloride exposure methods.......................................................................................91 5-5 The effect of the extraction time used to collect the Chick-ma gnetic nanoparticles on the final signal intensity obtai ned from the fluorescence image........................................92 5-6 Fluorescence image displaying the effect of the time from ex traction procedure to time the assay was preformed............................................................................................95 5-7 The fluorescence data obtained for the magnetic nanoparticles modified with Alex647-chick IgG and PEG molecules............................................................................96 6-1 The flow cytometry determination of ma gnetic nanoparticle colle ction and separation efficiencies for target and control cells............................................................................110 6-2 Fluorescence images and flow cytometry data of Rubpy dye and Rubpy doped nanoparticle treated cells..................................................................................................111 6-3 Fluorescence images and flow cytometry da ta of target and c ontrol cells extracted using aptamer conjugated-magnetic na noparticles and labe led with aptamer conjugatedRubpy nanoparticles.....................................................................................113 6-4 Fluorescence images of pure cell samples in buffer after magnetic extraction...............114 6-5 The limit of detection experiments for the stepwise addition of the magnetic and fluorescent nanoparticles using the mi croplate reader for detection...............................117 6-6 The confocal images for single cell mixed extraction samples.......................................118 6-7 The schematic diagram used for the extraction of multiple cells....................................120 6-8 The confocal images of buffer and se rum extracted mixed cell samples using the multiple extraction procedure..........................................................................................121 6-9 The confocal images of the cel ls extracted from whole blood........................................125

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12 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 SELECTIVE MOLECULAR RECOGNITION CONJUGATED NANO PARTICLES FOR BIOLOGICAL APPLICATIONS By Joshua E. Smith August 2007 Chair: Weihong Tan Major: Chemistry The collection and enrichment of biomolecules from complex biological samples is critical for disease diagnostics, environmental analysis and biotechnological applications. Current separation methods suffer from numerous limita tions that include time consuming procedures, difficulties with handling complex samples, and lack of selective molecular isolation. The objective of my research is to create nanodevice s for the selective separation and isolation of biological molecules and to evaluate thei r applications for biological studies. First, nanoharvesting agents (NHAs) were developed for the selective collection, separation, and detection of DNA and peptide targets. The NHAs ar e composed of silica coated magnetic nanoparticles bioconjugated with DNA probes on the nanoparticle surfaces. For peptide recognition, a DNA aptamer was used for the selective binding of the analytes. The NHA were demonstrated to have excellent specificity and sensitivity in both artificial buffer and complex biological samples for subsequent anal ysis by either fluorescence or mass spectroscopy. Additionally, the NHAs were f unctionalized with antibody mol ecules, and used in a highthroughput protein microarray to im prove the detection limit. The NHAs were used as a tracer device, where the antibody on the particle surface was labeled with fluores cent dye molecules for

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13 signaling. Prior to analyses by the microarray device, the NHAs were extracted from large volume samples, concentrated to small volumes, a nd used directly for the detection of proteins. Furthermore, a novel method was developed for the rapid collection a nd detection of whole cancer cells using a two silica-based nanoparticle approach. Magnetic nanoparticles were used for target cell extraction, and fluorescent na noparticles were employe d for sensitive cell detection. The nanoparticles were conjugated w ith DNA aptamers for target recognition. This system was used for the multiple extr action and detection of cancer cells. These newly developed techniques will be usef ul in biotechnology, biomedical science, and disease diagnostics for the selective extract ion and isolation of bi ological targets from complex samples. Future investigations will include the improvement and exploration of biological applications for nanoparticle based techniques.

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14 CHAPTER 1 INTRODUCTION Impact of Selective Recognition Nanopa rticles for Biological Applications The development of novel bioassays with sensitive and multiplexing capabilities that have simple detection and sample prep aration protocols are critical for high throughput evaluations of samples designed for clinical diagnosis, biomedi cal science, and other biotechnological fields. Such analyses rely immensely on the recognition of individual molecules based on physical and chemical interactions. Common bi ological analytes include oligonuc leotides, peptides, proteins, and even cells that often exist in low abundan ce in complex biological matrices. In addition, these analyte molecule types exist on a small si ze range, and it is this size scale that makes nanomaterials attractive for the development of new technologies involving selective recognition molecular probes for the analysis of biomaterials leading to crucial adva ncements in biological diagnostics.1,2 Selectivity and sensitivity of bioassays is dire ctly related to the critical interactions of molecular recognition elements with target mol ecules. The interactions involved with these devices use the selective rec ognition of receptor-ligand associ ations, antibody-antigen, and oligonucleotide hybridization. Addi tionally, designing bioanalysis techniques require the linking of the target binding action with an induced signaling event. Cons equently, signal transduction is what affects the overall sensitivity of these devi ces with optical and electrical methods being the most often used.3,4 However for complex biological sample s, before bioanalysis procedure can be performed a great deal of sample prepar ation is needed. Typical sample clean-up and purification techniques involve a variety of centrifugation, elec trophoresis, and chromatographic protocols. These techniques are often criticized because of the multiple steps and time

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15 consuming procedures that are often inadequa te for proper purifica tion. New materials or procedures capable of overcoming these issues ar e of great interest to performing bioanalysis. Typical pathogenic or clinical diagnosis requires the screening for multiple target molecules from a single sample as a means to de tect the presence of these marker molecules. This multiplexed analysis can provided crucial information to clinicians and scientists with the added understanding and ability to identify small differences in disease states.5 The difficulty with using the mentioned standard separation tec hniques is that with comp lex biological sample markers often occur in low abundance and an enrich ment step prior to analysis is necessary. In addition, the chance for sample loss as a result of complex sample preparation procedures is a major concern. These factors greatly limit the performance of current bioassays. The heart of this dissertation is the deve lopment of selective nanoparticles for the collection and subsequent detec tion of biological samples to overcome the challenges of the present sample preparation methods. The material s that follow highlight conventional biological sample treatment methods, recently developed na nomaterials with their respective applications, as well as the synthesis and char acterization of silica-based nanopa rticles. A brief discussion of the systematic evolution of ligands by expone ntial enrichment (SELEX) will follow, and the principles involved with selec ting oligonucleotide ligands for biological targets. Finally, a summary of the overall focus of this dissertation will be provided. Standard Sample Treatment for Biological Analysis Oligonucleotide Sample Preparation Oligonucleotide samples, which include DNA or RNA molecules, are obtained from whole cells. To acquire these samples fr om intact cells, a popular appro ach involves lysing the cell to release the genes from the interior of the cell. In general, the extraction and purification of oligonucleotides requires isol ation of the genes from cellu lar debris by a number of

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16 centrifugation based techni ques often from a kit. Oligonucleo tide molecules are used with a variety of methods for analysis, such as ge l electrophoresis, southern blot (for DNA) and northern blot (for RNA), polymerase chain r eaction (PCR), and real time PCR (RT-PCR) for sequence identification.6 Oligonucleotide gel separations, such as gel electrophoresis are most often carried out using either polyacrylamide or agarose polymeric material depending on sequence size. The gel material forms different sized pores that act as a molecular sieve isolatin g oligonucleotides as a function of their size (mass) and shape when an electric field is applied to the system. Although this technique does not have the ability to provide sequence identification of complex unknown samples containing thousands of gene fragme nts, it is a powerful tool for separating oligonucleotide samples.7 To obtain sequence recognition, an additional sample work up is necessary. One of the original and probably most famous techniques for obtaining sequence identification is called blotting. Once the oligonucleotides are isolated from cells using the above mentioned process, the isolated oligonucleotides ar e transferred to a membrane a nd treated with probe sequences. Labels on the probe sequence are often radioactive or fluorescent molecules, which allow for easy detection. Unfortunately when low amounts of viable cellular material are available, this method is not sensitive enough to provide the necessary sequence determination.8,9 In situations where low oli gonucleotide copy numbers are ava ilable, a technique termed polymerase chain reaction (PCR) is used to expo nentially increase the am ount of sequences that are available for detection. 10,11 PCR involves short predetermined synthetic sequences (primers), the four individual nucleotides, a nd an enzyme (polymerase) to cr eate the oligonucleotide copies. After amplification, a more sensitive sequence de termination is made by the blotting technique

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17 described previously. However to complete th is entire process, a lengthy and time consuming procedure is needed. A more recently developed procedure for th e identification and amplification of gene material is an extension of PCR, called real-time PCR (RT-P CR). RT-PCR involves the same amplification scheme as PCR. However, the ma jor difference between the two techniques is in the sequence determination. With PCR, sequence de termination can only be achieved at the endpoint, or plateau region, of the P CR reaction, but obtaining the quantit y of the original sample is not accurate. In the case of RT-PCR, the amp licon, amplified sequence, is monitored throughout the amplification process, and the quantity de termined during the PCR reaction. The process by which these copies are detected is via a repo rter probe molecule a dded to the PCR sample. Commonly, these probe molecules are based on fl uorescent signal transduction, where the signal increases as more target molecules are created.12,13 Protein Sample Preparation Protein samples are obtained fr om intact cells. These molecules are acquired by lysing the cell to release the proteins from the cellular matr ix. Proteins are isolated by centrifugation from cellular debris, and the protein fraction of interest is isolated using centrifugation based on their relative solubility. The protein samples from the cellular matrix are further separated sodium dodecyl sulfate polyacrylamide gel electrophores is (SDS-PAGE). SDS-PAGE has two main categories, one-dimensional and two-dimensi onal electrophoresis (2DE). One-dimensional PAGE is similar to gel electrophoresis descri bed above in the oli gonucleotide section. Twodimensional SDS-PAGE separates the samples us ing the isoelectric point (pI) and electric potential in orthogonal dire ctions, which separates by pH and size respectively.14,15 Separation is achieved with smaller polypeptides migrati ng faster and larger ones more slowly.7

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18 Electrophoresis is a powerful separation tool for proteins, but is not a reliable technique for identifying and quantifying complex samples containing multiple proteins. Traditionally, western blot is used methods for determining the identity and even quantifying proteins from cellula r lysates. Similarly to the o ligonucleotide blotting, the samples separated by electrophoresis are transferred from the gel to a membrane material by blotting. Once transfer is completed, the membrane is tr eated with labeled anti body probes that bind to proteins of interest. Labels used for anti body probes include radioa ctive or fluorescent molecules.16 This process uses a lengthy and complex procedure. Another method conventionally used for an alyzing protein extracts separated by gel electrophoresis is mass spectroscopy (MS). In genera l, the gel with the pr otein of interest is excised and treated with a protea se to digest the protein. The re sultant peptides are analyzed by MS. MS has the ability to provide the identifica tion and the quantity of the proteins in most cases.17,18 The sample preparation procedure has numerous steps and is time consuming. Cellular Sample Preparation There is a limited number of ways for harv esting cells from a live source. The first is through biopsy, where tissue cells are surgically removed. The second is by gradient density separation from biological fluids. For biopsy, there are three majo r classes. Incisional biopsies remove a small portion of tissues or organs for medical testing, excisiona l biopsies remove entire masses of cells for diagnosing medical conditions, and needle aspiration bi opsies collect tissue or fluid with a needle and are often used to avoid performing major surgical biopsies.19 For density gradient separation, a gradient of solution densities are layered in a centrifuge tube, and the cells are localized in the t ube based on their densities after cen trifugation. The two categories of density gradient separation are discontinuous and continuous. The difference between the two is that continuous density gradient separation has a continuous flow of the density solutions during

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19 centrifugation.20,21 Changing the centrifugation speed, cen trifugation time, sample volume and solution composition has a dramatic e ffect on the separation performance.21 After cell harvesting, one of the original analys is techniques for cells involves determination of the morphology, such as the shap e, structure, and pattern using an optical microscopy. However, further sample handling a nd preparation is required before the cells morphology can be determined. The cells are solidi fied, sliced, and placed on a microscope slide. Finally, the specimen is stained to reveal structural anomalie s that can be microscopically observed for scientific and diagnostic purposes.22 Selective cell staini ng methods use antibodies to specifically visualize and recognize proteins, car bohydrates, and lipids, called immunohistochemistry (IHC). These antibody molecules are labeled with fluorophores, radiolabels, or other molecules that produce a color response upon bindin g, and analyzed by the appropriate instrumentation. IHC is widely used for the diagnosis and treatment of diseases, such as leukemia. Leukemia is a cancer of the blood a nd detection entails a sophisticated version of IHC known as immunophenotyping. This process uses a panel of an tibodies that recognize cell membrane antigens, and a pattern develops th at specifically identifies the leukemia disease type.23,24 Recent Growth and Development of Nanoparticles Fluorescent Semiconductor Nanocrystals Quantum dots (QDs or qdots), fluorescent semi conductor nanostructures, have aided in the advancement of biological research. QDs are constr ucted in a core-shell structure, and with no further surface coating these materials range in size from 1 to 10nm in diameter. The shell coating provides protection and stability for the light emitting core component from environmental degradation. QDs have been found to be superior to traditional dyes by being brighter, more photostable, and ha ving longer fluorescence emission times.25 One of the more

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20 unique qualities of semiconductor nanocrystals is their size-dependent absorption and emission properties, where larger qdots emit light more in the red wavelengths compared to smaller ones. The result of all the extraordinary characteris tics of qdots proves the attractiveness of these structures for biolog ical applications. One such biological applicati on involves oligonucleotide de tection, a number of sensing platforms have been used in the development of these technologies.26 DNA modified QDs are used as fluorescent tracer agents for the analysis of target molecules on a slide surface or in a chip format.27 Simply, two short-complementary DNA se quences one attached to the QDs and the other to the slide surface se lectively recognize the target resulting in detection. More recently, quantum dot fluorescence resonance energy transfer (FRET) has been taken advantage of in oligonucleotide analysis.28,29 In this format two fluorescent species, an energy donor and acceptor, provide detection of the target DNA upon binding. The tw o fluorescent agents are each modified with complementary sequences. When target hybridization occurs, the donor and acceptor species are brought into close proximity to one another, during which the donor is excited at its excitation wavelength and transf ers its emitted light to the acceptor inducing fluorescence emission from the acceptor. Utilizing FRET, researchers have used QDs as both the donor and acceptor species and as the donor w ith a fluorophore acting as the acceptor species. Additional bioconjugated qdots have been de veloped for the detection and study of a number of other biomaterials and even for the study of cells and cellu lar processes. For the majority of these detection schemes, the dot s are labeled with an tibody and other protein molecules for target recognition.30,31 In cellular staining approaches, QDs have been used to process mammalian and pathogenic cell samples.31,32,34 In these studies, qdot s provide a level of observation not possible with c onventional dyes by providing long term, as much as several

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21 hours, observation times as a result of th e dots resistance to photobleaching. Also, biofunctionalized dots have been used to se lectively label both liber ated and cell surface proteins.35,36 Antibody conjugated QDs were employed to study receptor-mediated signals to identify cancer markers on cells, and for the detec tion of proteins harvested from lysed cells by western blot. QDs have been shown to be advanced vers ions of fluorescent labels for biological detection compared to dye molecules, but limita tions exist. Synthesizing these structures is difficult to reproducibly perfor m, and the surface chemistrie s are not well understood. QDs are notorious for their observable intermittent fluores cence or uncontrollable blinking effect, which can make obtaining reliable qua ntifiable data challenging. Perh aps the biggest hindrance to employing these dots in vivo is the uncerta inty of the toxicity these materials.26 Researchers are currently investigating ways to overcome these issues. Noble Metal Nanoparticles Metallic materials such as gold, silver, a nd platinum have been used to create nanoparticles. However, due to th e reactivity of silver and the expensive nature of platinum, gold (gold colloids) is the attractive choice for extensive investigations.37 Numerous studies have demonstrated the abundance of unique physical a nd chemical properties associated with these metallic nanoparticles. Focusing on the optical properties, gold nanoparticles have size dependent spectral character with the response bei ng red shifted as partic le size increases. In addition, this result is affected by particle shape, part icle-particle interact ion, and environmental conditions. These superior spectral properties are the result of light energy being transferred to surface electromagnetic waves and propagate al ong the metal interface. This phenomenon is called surface plasmon resonance. This process is associated with metal colloids, which allow them to be controllable scat ters and absorbers of light.38

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22 One bioassay developed using gold nanoparticles utilizes colorimetric detection of DNA. A set of thiol modified oligonucleotide mol ecules complementary to the DNA target were attached to 10-15 nm gold nanoparticles.39 In the absence of the target DNA, the colloid solution appears red in color with a narrow absorpti on maxima occurring around 520nm depending on the particle size. With addition of target DNA, the particles are for ced into close proximity to one another increasing the extinction coefficient producing a blue color change in the solution. Spectrally, the sample has a red shift that occurs as a result of binding obtained by absorbance measurements.40 This detection event has b een observed in the analysis of other targets, which include proteins and small organic compounds. In these instances, target recognition is achieved using DNA probes, called aptamers.41 In simple terms, an aptamer is an oligonucleotide sequence that forms a complex three-dimensional structure to selectively bind to a variety of targets, which will be discussed in more detail in a later section. Noble metal particles have also been used in detection sche me that involve more complex data acquisition methodologies than that for the colorimetric approach, namely surface enhanced Raman scattering (SERS). The normal Raman process is the result of a sm all fraction of light scattered at frequencies different from that of the incident light, most often at lower frequencies. However, Raman produces extremely weak sign als making it difficult to use in practical applications. On the other hand, SERS is one of the most sensitive techniques available for molecular analysis with enhancements as high as 1014 over the background. SERS takes advantage of the inelastic scattering process of the Raman effect, but in this instance the substance being detected is ad sorbed directly on or is with in a few Angstroms of a metal surface.42 Commonly used metal surfaces include gold a nd silver with silver having the strongest observable Raman scatter.43,44 The surface enhanced effect is th e result of incident light energy

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23 being transferred to electronic en ergy by the metal surface, often as surface plasmon resonance. This electronic energy is then transferred to the substance associated to the metal surface producing enhanced Raman scattering. A number of bioassays have been deve loped using SERS as the signaling motif.45-48 One of the more attractive features of SERS is that mo lecules with different chemical structures form characteristic spectra, much like a fingerprint based on that materials chemical makeup. For instance, unlabeled oligonucleot ides have been detected by SERS using gold nanoparticles. Unfortunately difficulties arise when sequence identif ication is required because of the structural similarities from one oligonucleotide to the next, which result in similar spectra.49,50 In addition, Raman-active molecules have been employed to selectively recognize analyte molecules to distinguish different targets fr om one another, such as pr oteins and oligonucleotides.45,51,52 In both circumstances, gold nanoparticles have been used as the SERS surface. Magnetic Nanoparticles Magnetic materials have been the subject of research interest fo r several decades. The interest in magnetic materials has been to gain an understanding of ma gnetic properties and to synthesize materials with specific magnetic prope rties. Until more recently magnetic materials have been studied as bulk substances.53 These materials are often divided into two categories of magnetism. Those that have no unpaired electro ns repulsed by an external magnetic field (diamagnetic), and those with unpaired electrons attracted to an external magnetic field (paramagnetic). For paramagnetic materials, no ma gnetization is retained by the substance in the absence of a magnetic field. Additional classes of magnetism include fe rromagnetic and ferrimagnetic substances. Ferromagnetic materials are attracted to a magnetic field, and retain magnetization after removing the magnetic field. Additionally, ferrom agnetic substances contain equal and opposite

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24 unpaired electrons on each of the atoms within th e metal complex structur e. Alternatively for ferrimagnetic compounds, the opposing magnetic mome nts associated with the atoms within the metal complex structure are unequa l resulting in a retained ma gnetism after removal of an induced magnetic field. Since ferrimagnetic ma terials have unequal opposing magnetic moments within the metal complex structure, these co mpounds are attracted to a magnetic field. As bulk substance, ferrimagnetic materials ar e divided into magnetic domains that have regions of aligned electron spins and a net magnetic moment.54 When demagnetized, the magnetic moments of the domains are randomly or iented, and the magnetic moments cancel each other resulting in no net magnetism. With the reduc tion of the size of the material or particle, there is a point when a single-domain is reached.55,56 Further reducing the size of the singledomain magnetic material creates a particle that can no longer sustain an induced magnetic moment for an extended amount of time (Figure 1-1). However, in the presence of a magnetic field the particles become magnetized. This phenomenon is known as superparamagnetism because there is no magnetic hysteresis. The material does not become magnetized by an external field and returns to its original stat e in the absence of the field, just like with paramagnetism.54 Figure 1-1. The scheme representing the effect size has on magnetic materials in the absence of a magnetic field. Bulk material domain Single domain Undefined domain randomly oriented reduce reduce Bulk material domain Single domain Undefined domain randomly oriented reduce reduce

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25 Countless superparamagnetic materials have be en synthesized using transition metals of iron containing compounds, such as magnetite (Fe3O4). These substances have become extremely attractive for biologi cal applications. This is due to the superparamagnetic characteristic, which is essential for avoiding part icle aggregation. Magnetite particles have been used in biology for numerous applications, wh ich include MRI contrasting agents, magnetic separations, and as dr ug delivery vehicles.57-59 For MRI contrast, iron oxi de particles darken the image acting as a negative contrasting agent. Ty pically, these particles have a reduced uptake by tumor tissue, and therefore appear bri ghter than the surr ounding health tissue.60 In drug delivery, drugs are chemically or physic ally bound to the magnetic part icles and a magnetic field is applied to direct the particles to the target tissue or organ.44 As in a magnetic separation, a targeting molecule is chemically bound to the ma gnetic materials to prov ide attachment to the biomolecules or cells of interest.58 These and other advancements in magnetic technology have provided powerful tools to a va riety of biology and biotechnol ogy related fields, but further research and improvements are still necessary to realize the full potential of these materials. Silica Nanoparticle Fundamentals Synthesis of Silica Nanoparticles Silica nanoparticles have been prepared by two common processes, microemulsion method and the Stber (sol-gel) approach. The Stbe r method involves an ethanol and ammonium hydroxide solution that hydrolyzes silane molecule s, such as tetraethylorthosilicate (TEOS), to form amorphous silica nanoparticle s (Figure 1-2). The Stber proce ss is relatively simple to perform and can be carried out in as little as a few hours. This me thod has been modified to form organic dye nanoparticles by c ovalently attaching the fluor ophore to the silica matrix.67-69 Additionally, magnetic nanoparticles Fe3O4 and Fe2O3 have been incorporated in the silica

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26 particles using this method.65 However, the nanoparticles formed via this method are often large and have non-uniform shapes.66 Figure 1-2. The scheme representing the Stber process used to create silica nanoparticles. An alternative synthesis of silica particles is to use the reverse-micelle approach, also known as water-in-oil microemulsion (w/o) met hod. This method consists of three primary components: water, oil, and a surfactant.67-69 These components form a single-phase microemulsion system which is both isotropi c and thermodynamically stable (Figure 1-3). Figure 1-3. The scheme representi ng the water-in-oil microemulsion system used to create silica nanoparticles. Nanodroplets of water form in the bulk oil phase, which act as a confined medium (nanoreactors) for discrete particle formation. Again, silica partic les form by the hydrolysis of silane molecules. The nanoparticles size can be cont rolled by altering the water -t o-surfactant molar ratio (Wo). Si OEt OEt OEt EtO hydrolysis EtOH, NH4OHSi OH OH OH HO condensationSi Si O doped silica nanoparticles Si OEt OEt OEt EtO hydrolysis EtOH, NH4OHSi OH OH OH HO condensationSi Si O doped silica nanoparticles Water pool (nanoreactor) surfactant molecules oil Reverse micelle Iron oxide doped silica nanoparticles TEOS NH4OH

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27 The microemulsion yields monodispersed and highly uniform nanopartic les taking anywhere from 24-48 hours to complete. Th e water-in-oil microemulsion s ynthesized silica nanoparticles have been doped with various inorgani c and modified organic dye molecules.70-73 In addition to the dye-doped particles, the same doping pr ocedure has been used to incorporate Fe3O4 and Fe2O3 nanoparticles in the silica matrix.74,75 Silica Nanoparticle Modification and Conjugation Figure 1-4. Organosilane functionalized and bi omolecule conjugated silica nanoparticles used for biological applications. For biological applications, the nanoparticle surface is often modifi ed with recognition elements, such as antibodies a nd oligonucleotides that selectively bind target molecules. This modification has been accomplished by both phys ical adsorption and covalent attachment. Physical adsorption uses electr ostatic, van der Waals, hydrophobi c and hydrophilic interactions to associate biomolecules to the particle surfac e. Where as, covalent attachment requires the modification of nanoparticles with organosilane molecules that contai n chemically reactive Antibody DNA Protein COO-NH2SH PO2C18

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28 functional groups such as thiols, amines, and carboxyls (Figure 1-4).76,77 These groups are applied to the surface either after synthesis by po stcoating with an organosilane or by postcoating during synthesis via cohydrolysis with TEOS.78 Another role of functionalizati on is to provide solubility a nd stability to nanoparticles in solution. When particles are modified with am ine groups as well as with biomolecules at physiological pH, the net surface charge of the co mplex is typically reduced. This causes the particle to be less stable in a queous solutions resulting in particle aggregation. To overcome this issue, additional chemical groups are added to the particle surface to restore the colloidal stability. For example, during postcoating ch arged-unreactive organosilane compounds or polymer-based stabilizing reagents have b een used to improve their dispersibility.79,80 Polymeric reagents, such as polyethylene glyc ol (PEG), have been used to block the adsorption of unwanted molecules to the particle surf ace and reduce adsorption of the pa rticles to detection surfaces. When using the unreactive charged silane groups, the desired charge need ed to disperse the particles is reestablish.81,82 To covalently label functionalized particle s with the preferred biological recognition molecules, chemical attachments are often used such as peptide and disulfide bonds along with numerous amine reactive linking reagents (Fig ure 1-4). For carboxylic acid functionalized nanoparticles, amine containing biomolecules ha ve been attached through peptide coupling by way of activating the carboxyl gr oup with carbodiimide reagents.83 Additional conjugations have been executed through the disulfide linkage, wher e nanoparticles coordinated with sulfide groups can react with sulfur containing biomolecule s to fix them to the particle surface.78 Succinimidyl esters and isothiocyanates are commonly used as cross linking reagents for attachment of a variety of molecules to amine labeled particle surfaces.84 A popular surface attachment method

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29 uses the natural interaction be tween biotin and avidin. In many instances, avidin protein molecules are physically adsorbed or covalently bound to a surf ace. The biorecognition molecule is modifying with biotin, and im mobilization of the biotinylated molecule is made possible via the biotin-avidin interaction. Th e decision as to which bioconjugation strategy to use depends on the application and expected func tion of prepared materials. Characterization of Prepared Nanoparticles Determining the physical and chemical propert ies of nanomaterials is essential. Since many of the characteristics are ba sed on physical size, one of the l eading categories of techniques used to determine particle size has been elec tron microscopy (EM). Nanoparticles have been imaged using transmission electron microscopy ( TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM).62-64,85-87 The limitation with EM techniques is that the images are obtained from a surface, which provides li ttle indication as to the dispersive nature of nanoparticles in solution. To so lve this problem, light scatteri ng can be used to measure both particle size and dispersi on of samples in solution.88 Another important characteristic of nanopartic les is the overall surface charge or zeta potential ( ), which is effected by the particle environment. The magnitude of the indicates the repulsive force that is present on the particle sample, and is used to determine the solution stability and dispersion of the particle sample. Some of the factors that effect particle stability are pH, salt content, and particle concentrat ion. Particles of high positive or negative values, 30mV or larger, repel each other and have a lowe r propensity for aggregation. In addition, measurements can be used to verify whether bioconjugation reactions or surface modifications have taken place. Throughout the synthesis a nd modification process the nanoparticle surface charge can be monitored after each step. Based on the materials coordinate d to the particles the

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30 will change with the addition of a charged species or by screening of the surface charge by these additional molecules, like charged organosilanes and oligonucleotides or polymers and proteins respectively.85 Other characterizations of na noparticles are dependent on the type of particles that are being analyzed. For dye-doped silica nanoparticles, the optical properties are of significant interest. Dyes incorporated in silica nanoparticle s have no significant ch anges to the absorption and emission properties with the ex ception of Rubpy nanoparticles, wh ere a slight red shift in the emission is observed. The photostability of the dy es trapped in the silica matrix was improved compared to free dye molecules. The dye -doped nanoparticles did not suffer from photobleaching after long periods of continuous-intense light exposure and have high signal intensities compared to free dye molecules.74,89 For magnetic nanoparticles (Fe3O4), the magnetic properties are of particular in terest. These properties are m easured using a superconducting quantum interference device (SQUID) magnetometer and is accomplished by varying the magnetic field applied to the sample and mon itoring the magnetic response of the magnetic material.75 Selective Biorecognition Component The essential component when developing t ools for bioanalysis deal with molecular interactions and using those interactions for r ecognition of disease states Diseases have been known to have specific markers associated with them. These biomarkers have aided researchers and scientists in diagnosing and treating di seases. The limitation for the development of diagnostic devices has been with the lack of known markers for th ese applications. The discovery of selective biomolecules as mark ers involves separating and identifying these molecules from complex biological samples. This requires a great deal of time and effort to

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31 avoid ascertaining a nonselective or even an inco rrect molecule. Two classes of molecules used for biorecognition are antibodies and aptamers. The most studied recognition molecules are an tibodies, which are large protein molecules produced by the body to identify and neutralize fo reign materials like diseases. The two classes of antibodies are polyclonal and monoclonal. Polyclonal antibodies ar e mixtures of these proteins that recognize different epitopes of the antigen. Polyclonal an tibodies are produced by many different cells by injecting the antigen into an animal a nd isolating the antibody from the animals blood. Monoclonal antibodies are homoge neous proteins that recognize the same epitope of an antigen and are isolated from a population of identical ce lls rather than from different cells. Since antibodies are produced as a biological response to antigens they are difficult to reproduce, difficult to chemically mo dify, sensitive to environmental conditions, and have limited shelf lives. A more recent development in molecular r ecognition has been with aptamers, which are single-stranded DNA, RNA, or peptides that se lectively bind to target molecules ranging from small organic compounds to proteins.90-92 Aptamer molecules have binding affinities and target recognition that is comparable to antibodies, and are able to disc riminate between protein targets that are highly homologous.93,94 This is due to the complex th ree-dimensional structures, which provide the basis for target recognition.95,96 Oligonucleotide aptamers are selected by a process called the Systematic Evolution of Ligands by Exponential enrichment (SELEX). Aptamers are selected from a library of around 1012-15 random sequences of synthetic oligonucleotides by repeatedly binding these molecules to the target of interest.90, 97-99 More recently, a cell-based aptamer process wa s developed to obtain molecular probes for profiling diseases, such as can cer. In cell-SELEX, whole cells are used to identify DNA

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32 aptamers that distinguish target cells from control or nontarge t cells (Figure 1-5). Through the cell-SELEX approach a panel of cell-selective ap tamers is screened. This is accomplished by using a subtraction strategy wher e aptamer candidates that intera ct with nontarget cells are removed. The aptamers that are screened are de termined without knowing the biomarkers that are present on the cells surfaces.100 The attractive features of DNA aptamers over antibodies are that they have low molecular weights allowing fo r fast tissue penetration, and are simple to synthesis and modify as a result of we ll established oligonuc leotide chemistries.101 Aptamers can also be used as tools for identifying new bi omarkers expressed in other disease states. Figure 1-5. Schematic representation of the se lection process used to generate cell binding aptamers (cell-SELEX). PCR amplification evolved pool sequencing target cells DNA library positive selection unbound DNA eluted DNA wash nontarget cells negative selection bound DNA retain unbound DNA cell SELEX PCR amplification evolved pool sequencing target cells DNA library positive selection unbound DNA eluted DNA wash nontarget cells negative selection bound DNA retain unbound DNA cell SELEX

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33 Research Scope The research in this dissertation describes the development of selective recognition conjugated nanoparticles for biot echnological, biomedical, and c linical applic ations. Silica coated magnetic nanoparticles were used to co llect and isolate oligonucleotides, peptides, proteins, and intact cells from complex biolog ical matrices. The collected oligonucleotide and peptide samples were analyzed by both optical and mass spect roscopic devices. Fluorescently labeled antibodies modified to silica nanoparticle s were used to improve the detection limit of protein microarray devices. A two particle, one fluorescent and one magnetic, approach was investigated for the multiplexed detection of cancer cells using a flow system, fluorescent imaging, and simple optical methods.

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34 CHAPTER 2 PREPARATION OF SILICA COATED MAGNETIC NANOPARTICLES Introduction Magnetic materials have been created from nume rous metallic elements, and until the past few decades these substances have been develope d and studied in bulk. Researchers have spent a large amount of time and effort employing metallic elements to construc t magnetic nanoparticles and molecules. One of the metals commonly used to produce these items has been iron, particularly as iron oxide. So me synthesis protocols used to make iron oxide nanoparticles include coprecipitate of Fe2+ and Fe3+ in alkaline or microemulsion systems, the oxidationreduction of suitable valence iron ions, and th e utilization of copolymer gels or other templates.54,57,102 The research that follows uses the c oprecipitation procedure. However, bare iron oxide objects are difficult to ta ke advantage of as is because th e ability to chemically modify the nanoparticle surfaces with reactive groups is not possible. To provide magnetic nanoparticles the ability to be chemically treated, one of the standard approaches has been to encapsulate these co mpounds with modifiable surface coatings. Iron oxide nanoparticles have been coated with a num ber of synthetic and bi ological polymers, some of which are commercially available, and with silica. The focus of this work was on coating these magnetic particles with a si lica surface. Iron oxide particles have been coated with silica using the microemulsion protocol for biol ogical functionalizations and applications.75 Silica nanoparticles have been created usin g a process called the Stber method.66 The magnetic particles in this work were coated using this procedure, and additional surface modifications were explored for covalently and physica lly attaching biomolecu les to the particle.

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35 Experimental Materials and Methods All materials were purchased from Sigma-Al drich (St. Louis, MO) unless other noted. Ammonium hydroxide (NH4OH), ethanol (EtOH), and hydrochloric acid (HCl) was purchased from Fisher Scientific (Fair Lawn, NJ, USA) The biotinylated DNA a nd fluorescein labeled sequences were obtained from GenoMechanix (Gainesville, FL). Other DNA molecules were synthesized in-house. A neodymium iron boron magnet purchased from Edmund Optics (12,200 gauss, Barrington, NJ). Carboxylet hylsilanetriol sodium salt ( carboxyl or COOH) was purchased from Gelest, Inc. (Morrisville, PA). 1Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) was purchased from Pierce Bi otechnology, Inc. (Rockford, IL). Avidin was obtained from Molecular Probes (Eugene, OR). A ll other chemicals were of analytical grade. Instruments A Hitachi H-7000 transmission electron microscope (TEM) was used to obtain the size and shape of the formed nanoparticles from dried sa mples. Light scattering and zeta potential ( ) measurements were made on a series 1270 Brookhaven Zeta Plus instrument to gain solution size and surface charge information, respectiv ely. A Perkin-Elmer PHI 5100 ESCA X-ray Photoelectron Spectroscopy (XPS) and a JEOL JSM 6335F SEM with Energy Dispersive X-ray Spectroscopy (EDS) instruments were used to demonstrate the chemi cal composition of the surface and interior of the synthesized silica nano particles. Additional sizing measurements were obtained on a Veeco Multimode with a Nanoscope IIIa controller atomic force microscope (AFM) of a dried sample. Fluorescence and absorbance measurements were obtained using a TECAN Safire microplate reader (R esearch Triangle Park, NC, USA).

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36 Nanoparticle Synthesis, Surface Modification, and Bioconjugation The iron oxide core magnetic nanoparticles were prepared by coprecip itation of iron oxide salts.54 The procedure was slightly modified from th e source materials. Solutions of ammonium hydroxide (2.5%) and iron chlorides were added together under nitrogen, and continuously stirred at 350 RPM using a mechanical stirre r for 10 minutes with a final volume of around 255 mL. The iron chloride solution was made from ferric chloride hexahydrate (0.5 M), ferrous chloride tetrahydrate (0.25 M) and HCl (0.33 M) at a final volume of approximately 100 mL. The ammonium hydroxide solution was diluted in a 500 mL beaker at a volume of about 155 mL. The bulk solution of the formed iron oxide nanoparticles was stor ed as is at room temperature until needed for further experiments. The iron nanoparticles were coated with silica using the Stber process, similar to the solgel approach.66 To begin, a 6 mL aliquot of the magne tic particle solution was magnetically extracted and washed three times with water and once with ethanol, and the washed samples were suspended in an ethanol solution cont aining ~1.2 % ammonium hydroxide. The final concentration of the washed particles was ar ound ~7.5 mg/mL, which contained 60 mg of iron oxide in 6 mL of solvent. To this sample, 210 L of tetraethoxyorthosil icate (TEOS) was added and then sonicated for 90 minutes to complete th e hydrolysis process to form the silica coating. A postcoating layer of pure silica was created by adding an additional 10 L aliquot of TEOS, and the sample was sonicated for an additional 90 minutes. The resulting silica nanoparticles were magnetically extracted and washed three times with a 6 mL aliquot of ethanol to remove excess reactants.

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37 Avidin physical adsorption For avidin protein coating by physical adso rption, a 0.1 mg/mL silica coated magnetic nanoparticle solution washed thre e times with 1 mL aliquots of 10 mM phosphate buffered saline (PBS) pH 7.4 and suspended in 12 mL of the buffe r. A 10 mL solution at a concentration of 5 mg/mL avidin was added to the nanoparticle solu tion and sonicated for 5 minutes. This solution was incubated at 4 C overnight. The particle s were magnetically extracted and washed three times with 5 mL aliquots of 100 mM phosphate buffered saline (PBS) pH 7.4. The sample was dispersed in a 30 mL solution of 1% glutaraldehyde diluted by 100 mM PBS and continuously mixed for 1 hour at 25 C to st abilize the avidin layer by cro ss-linking. After stabilization, the particles were magnetically extracted and washed three times with 5 mL aliquots of 1M Tris-HCl buffer pH 7.0, and the samples dispersed in 5 mL of the buffer then incubated for 3 hours at 4 C. Finally, the avidin coated partic les were washed three times with 5 mL aliquots of 20 mM TrisHCl, 5 mM MgCl2, pH 8.0 and suspended in 4 mL of th is buffer at a concentration of ~0.2 mg/mL. Biotin-avidin oligonucleotide attachment DNA modification was performed by adding 6 nmols of biotinylated DNA (5-biotin-TTT AAA TCT AAA TCG CTA TGG TCG C-3) to 500 L of the avidin na noparticles. This reaction was completed by incubating the sample at 4 C overnight. Three final washings of the particles were performed using 500 L aliquots of 20 mM Tris-HCl, 5 mM MgCl2 buffer and suspended in 500 L of this buffer. The final concentr ation of the DNA labeled particles was ~0.2 mg/mL and stored at 4 C until used.

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38 Carboxyl modification For covalently attachment after postcoating th e iron oxide particles with silica and washing with ethanol and 10 mM PBS, 80 L of the carboxyethylsilanetrio l sodium salt was added to 1 mL of 10 mg/mL silica-coated magnetic nanopa rticles (MNP) in 10 mM PBS, pH 7.4 and continuously mixed for four hours. Finally, the pa rticles were washed three times with 10 mM PBS and stored at room temperature until used. Direct oligonucleotide attachment To attach DNA, a 250 L solution of 4 mg/mL of the carboxyl-modified MNPs were washed three times with 250 L aliquots of a 0.5 mM MES, pH 5.0 buffer. To these particles, 50 L of a 20 mg/mL EDC solution to the washed pa rticles and incubated for fifteen minutes. A 10 L volume of amine modified DNA (5-ami ne-TTT AAA TCT AAA TCG CTA TGG TCG C3) at a concentration of 100 M was added to the activated particles. The solution was allowed to react for four hours with continuous mixi ng. The MNPs were magnetically extracted and washed three times with 500 L aliquots of the 10mM PBS buffer. The final concentration of the MNPs was 2 mg/mL in the 10mM PBS, and the samples were stored at 4 C until used. Avidin Modification Determination To confirm the presence and act ivity of the avidin layer on the nanoparticle surface, 50 L of 0.5 mg/mL biotin labeled with fluorescein isot hiocyanate (FITC) dissolved in deionized water was added to 100 L of 0.2 mg/mL avidin modified ma gnetic nanoparticles (MNP) into two separate particle samples incubated at different times, 24 and 4 hours respectively. Simultaneously, two samples of 100 L at 0.2 mg/mL silica magnetic nanoparticles were treated with 50 L of the biotin-FITC and one was incuba ted for 24 hours and the other for 4 hours. After incubation, the samples were magnetically extracted and washed three with 100 L of 20

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39 mM Tris-HCl, 5 mM MgCl2, pH 8.0. These samples were analy zed by the microplate reader to obtain the fluorescence data. The ze ta potential of the avidin modified MNP was measured to confirm the presence of the avidin coating. Investigation of DNA Modification To verify the presence of DNA and that th e DNA was functional, the zeta potential of biotin-DNA treated avidin na noparticles were measured, and the biotin-DNA treated nanoparticles (NPs) were treated with FITC la beled DNA, which were a target (5-FITC-GCG ACC ATA GCG ATT TAG A-3) and control (5-FITC-AAT CAA CTG GGA GAA TGT AAC TG-3) sequences. To 180 L of 0.2 mg/mL DNA modified magnetic nanoparticles called nanoharvesting agent (NHA), were treated with FITC-DNA to make a 100 nM final concentration of dye labeled DNA in a final volume of 200 L. These samples were typically incubated at room temperature for 1 hour, however shorter times have been used. These samples were magnetically extracted and washed three with 200 L aliquots of 20 mM Tris-HCl, 5 mM MgCl2, pH 8.0 buffer, and the extracted samples were heated to 70 C for 10 minutes to release the captured DNA from the NHA. The NHA materi als were magnetically removed from the sample while the sample was heated. The releas ed DNA sample was allowed to cool to room temperature and then analyzed by microplate reader. Optimization of DNA Functionalization For determining the optimum amount of biotinDNA attached to the na noparticle surface, this was accomplished by performing a doseresponse or saturation study of prepared DNA labeled magnetic nanoparticles. Two different factors were controlled and monitored to demonstrate the optimized DNA functionalizat ion to the nanopart icle surface. The concentrations of DNA per na noparticle were controlled by increasing the biotin-DNA amount

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40 from 1 M to 71 M (~330, 2000, 5100, 10300, and 23800 molecules). To determine the number of functional DNA molecules per particle, target DNA-FITC was added to seven aliquots of 180 L of the NHA at concentrations of 100, 250, 400, 550, 700, 850, and 1000 nM in a final volume of 200 L. These samples were incubated at room temperature for 1 hour, and the samples washed and heated as described previously. The DNA samples were analyzed by a microplate reader. Results and Discussion Iron Oxide Formation and Silica Coating Figure 2-1. The transmission electron microscope images of synthesized nanoparticles. A) Iron oxide nanoparticle images. B) Silica co ated iron oxide nanoparticle images. The uncoated iron oxide, magnetite (Fe3O4), and silica coated na noparticle size and shape were by transmission electron microscopy (TEM). The uncoated magnetite particle (Figure 2-1 A) size was found to be 10 2 nm in diameter and these particles had a variable shape. The representative image for silica coated magnetite pa rticles (Figure 2-1 B) determined the diameter to be 65 nm, and these particles were spherical in shape. However, the size of silica coated iron oxide particles was more variable. The smalle st silica coated partic les were found to be AB AB AB AB

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41 approximately 30 nm and some of the largest were 90 nm in diameter. This is caused by the Stber synthesis procedure because of the lack of control over nucleation of the silica nanoparticle formation. In addition to obtaining th e particle size, the TEM images showed that the iron oxide nanoparticles were encapsulated with the silica layer. This was seen by the differences in size and shape of the materi als observed in the TEM images. Additional techniques were used to further demonstrate th e particle size and that the magnetic nanoparticles were coated with silica. The silica coated magnetic nanoparticles (M NP) were analyzed using atomic force microscopy (AFM). The size of the particles obtained by this method showed diameters of approximately 30 5 nm with uniform spherical sh apes (image not shown). This technique again provided evidence that the magnetite nanomaterials were coated with silica. Both nanoparticle types were further characterized by light scattering and zeta poten tial measurements. The light scattering data indicated the hydrody namic particle sizes in soluti on of approximately 27 3 nm and 90 10 nm for the bare magnetic and silica co ated nanoparticles, respectively. These larger sizes of the analyzed samples were possibly the result of la rger or aggregated particles dominating the samples. Table 2-1. The zeta potential measurements obtai ned from various nanoparticle surfaces and coatings. Nanoparticle surface Zeta potential* (mV) Magnetite Silica Avidin-silica Biotin-DNA (biotin-avidin) Carboxyl-silica Amine-DNA (peptide bond) -30.09 -19.59 -4.37 -14.56 -43.27 -33.31 All measurement variations were within 1-3 mV in all samples. Zeta potential ( ) measurements (Table 2-1) taken of the bare iron oxide and silica coated particles indicated surface charges of -30.09 mV and -19.59 mV, respectively. This study

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42 indicates that uncoated particles have a bett er colloidal stability as a result of the and seen by the settling of the silica coated samples out-of-solution after several hours. Both the light scattering and zeta potential data provide evidence for successful coating of the iron oxide with a silica layer as seen by the differences in th e measured particle size and surface charge. X-ray Photoelectron Spectroscopy (XPS) a nd Energy Dispersive X-ray Spectroscopy (EDS) measurements were acquired to ensure th e elemental composition of the silica-magnetite nanoparticles. The atomic ratios obtained from the XPS plot (Figure2-2) indicated that the surface of these particles consist of 96.9% Si a nd 3.1% Fe, and atomic ratios attained from EDS measurements illustrate that the overall composit ion of the silica coated particle was 62.3% Si and 37.7% Fe. Based on these results, the synthesi zed nanoparticles had a hi gher Si to Fe ratio. The XPS and EDS analysis demonstrate a layere d composite structure with a successful silica coating of the iron oxide. Figure 2-2. The X-ray Photoelectron Spectroscopy data collected from silica coated magnetite nanoparticles. 0 10000 20000 30000 40000 50000 60000 0 200 400 600 800 1000 eV intensity Si Fe 0 10000 20000 30000 40000 50000 60000 0 200 400 600 800 1000 eV intensity 0 10000 20000 30000 40000 50000 60000 0 200 400 600 800 1000 eV intensity Si Fe

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43 Avidin Surface Modification After completing the avidin coating process, th e particles were analyz ed by zeta potential. The surface charge of avidin coated magnetic nanoparticles (MNP) was determined to be -4.37 mV (Table 2-1). With such a low zeta potential these particles were prone to aggregation. Comparing the silica coated nanopa rticles to the avidin modified samples; the zeta potential measurements indicated a decrease of more than 15 mV with immobilizat ion of avidin to the silica surface. The decrease in the overall surf ace charge is explained by the introduction of positively charged avidin protein molecules to the negatively charged silica surface. This change in surface charge provides evidence that the silica particles were covered w ith an avidin layer. Figure 2-3. The fluorescence data obtained from avidin modified silica magnetic nanoparticles (MNP) treated with biotin-FITC. The avidin coated particles were treated with biotin-FITC to further determine whether the avidin layer was added to the si lica surface, and the fluorescence data obtained (Figure 2-3). The information indicated that these nanoparticles have been modifi ed with avidin molecules as evidence of the increased fluorescence intensity obs erved in both of the biotin treated samples. The fluorescence analysis showed that the pa rticle bound avidin molecules remained active upon 0.0 0.2 0.4 0.6 0.8 1.0 1.2 MNPAvidin-MNP (4h) Avidin-MNP (24h) Relative Intensity Relative Intensity 0.0 0.2 0.4 0.6 0.8 1.0 1.2 MNPAvidin-MNP (4h) Avidin-MNP (24h) Relative Intensity Relative Intensity

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44 being immobilized to the partic le surface. Additionally, this e xperiment indicated that longer incubation times provide increased biotin-a vidin hybridization. The zeta potential and fluorescence analysis provide confirmation of the presence and activity of avidin on the nanoparticle surface. DNA Labeled Nanoparticles Creating DNA functional nanoparticles using bi otin-avidin linkage was assessed by first performing zeta potential measur ements. The surface charge of the DNA modified nanoparticles was -14.56 mV, which were 10 mV higher then the avidin only nanoparticles (Table 2-1). The increase of the surface charge can be explai ned by adding negatively charged DNA molecules, which improves the overall charge of the partic le. The DNA labeled nanoparticles had a lower propensity for aggregation than the avidin modi fied particles, but aggregated after a longer period of time. However, the sample was suspe nded by simply shaking or vortexing. The change in the particle charge provides evidence th at the sample was labeled with DNA through the biotin-avidin interaction. DNA functionalized magnetic na noparticles were treated with complementary DNA labeled with FITC (target). A random biotin-D NA sequence was modified to the particles and, these particles were treated with the FITC-DNA (control). This experiment was performed to confirm that the particles were modified with DNA and if the immobilized DNA remained functional (Figure 2-4). Based on the target sample, these nanopart icles have been labeled with DNA and the DNA collected the target DNA as s een by the fluorescence signal. Furthermore, this experiment showed that the DNA nanoparticles were selective for the target as determined from the lack of signal in the control sample and the intensity of signal in the target sample. At these experimental conditions, the collection effici ency was determined to be 95 3 %. The zeta

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45 potential and fluorescence data confirm the pres ence and binding activity of the immobilized DNA. Figure 2-4. The microplate reader fluorescence in tensity data collected for FITC labeled DNA molecules. Nanoparticle Characteriza tion and Optimization The density of the silica coated magnetic nanoparticles was determined to be approximately 2.87 g/mL by measuring the mass a nd acquiring the volume of those particles. From this data and the measured size of a singl e nanoparticle (~65 nm), the molecular weight of this material was calculated to be 2.44x107 g/mole. Using this molecu lar weight, the number of DNA molecules attached to the nanoparticle surface was determ ined by calculating the number of nanoparticles in 180 L at 0.2 mg/mL, and the number of biotin-DNA reacted with each avidin coated silica particle (Biotin-DNA) was calculated ba sed on the particle number. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 buffercontroltargetRelative Intensity

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46 These prepared samples were treated with FITC labeled DNA described in the experimental section. The DNA-FITC left in the sample after hybridizatio n and extraction of the DNA modified nanoparticles (Rem aining), the DNA-FITC extracted and removed from these particles by heating (Col lected) was monitored by microplat e reader fluorescence (Table 2-2). Saturation of the DNA conjugated nanoparticle wa s determined to be at ~1300 DNA molecules per particle by both methods, which were ~71 M and ~ 6 M by the remaining and collected procedures respectively. However, the point at which the samples become saturated differs depending on the method used to monitor the am ount of DNA collected. In this instance, the values obtained from the collected samples provi de a direct determination, and therefore more dependable versus the indirect sa mple determination (Remaining). Table 2-2. The determined amount of DNA mol ecules modified to the nanoparticle surface. Biotin-DNA (molecules) DNA-FITC remaining (molecules) DNA-FITC collected (molecules) 330 2000 5100 10300 23800 259 614 769 890 1289 296 1106 1098 1246 1347 The particle stability and repr oducibility of the prepared nano particles were next examined. For the particle stability, both DNA conjugated and avidin coated samples were prepared in bulk, where the avidin samples were treated with biotin-DNA when needed. These samples were stored at 4 C until used. Once a week 180 L of the DNA modified magnetic nanoparticles (NHA) were obtained, and these samples were tr eated the same as the previously described DNA-FITC samples. For both prepared particle t ypes, all samples resulted in similar signal responses for up to three months at which time no more sample remained to continue the investigation (data not shown). Therefore, the NHA and avid in modified nanoparticle have good stability.

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47 Particle batch-to-batch (Figur e 2-5 A) and sample-to-sample within a single batch (Figure 2-5 B) reproducibility was investigated. As determined from Figure 2-5 the NHA collection ability of DNA-FITC target samples from one samp le to the next within a batch and from one Figure 2-5. The fluorescent da ta obtained from batch-to-b atch and sample-to-samples reproducibility studies. A) Testing the batc h-to-batch reproducibility. B) Testing the sample-to-sample reproducibility w ithin a single nanoparticle batch. 0 0.2 0.4 0.6 0.8 1 1.2 0246810 sample numberRelative IntensityBA 0 0.2 0.4 0.6 0.8 1 1.20246810sample number Relative IntensityA 0 0.2 0.4 0.6 0.8 1 1.20246810sample number Relative Intensity

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48 batch to the next, the results were highly cons istent between samples. In both studies, the fluorescence signals collected from all samples produced nearly linear responses with some sample variability. The explanation for this irregu larity is that the avidin coating procedure for these samples was accomplished through physical ad sorption, and there is some difficulty in controlling the amount and the efficiency of adso rbing materials to the particle surface. Carboxyl Modified Nanoparticles As with the avidin treated a nd biotin-DNA conjugated partic les, the carboxyl modified nanoparticles with covalently attached capture DNA samples were characterized by zeta potential measurements (Table 2-1) and m onitoring collection of DNA-FITC by fluorescence (data not shown). Comparing the par ticle surfaces analyzed by zeta po tential, recall that the silica coated particles had an overall charge of -19.59 mV, carboxyl modified nanoparticles had a surface charge of -43.27 mV, and covalently conj ugated amine-DNA to the particle produced an overall charge of -33.31 mV. This translates to confirmation of the carboxyl surface modification, and these nanoparticles had an im proved dispersion compared to the silica only surface. The carboxyl surface has an increased char ge because at neutra l pH these functional groups are inherently negatively charged. Upon conjugation with amine-DNA, the surface charge has a slightly lower surface charge, whic h means that the particles are more prone to aggregation. However, the charge was large enough to avoid severe settling of the particles, but settling was observed after seve ral hours. The zeta potential measurements for the DNA conjugated particles provide evidence that the particles were modifie d. The decrease in the magnitude of the surface charge when labeled wi th amine-DNA is explained as the result of the reaction of the amine (NH2) and carboxyl (COO-) forming a peptide bond and removing or reducing the presence of the added negative charge. Furthermore, DNA molecules have an

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49 inherent negative charge, which s hould increase the overall charge of the particle. However, the charge added to the particles by DNA is not associated directly to the partic le surface, but instead is spread across the DNA molecule, which is no t compounded as a single charge point on the particle surface. When the covalently modified DNA magnetic nanoparticles (NHA) were treated with target and control DNA-FITC, similar results were obtained for this experi ment (data not shown) as with the biotin-DNA prepar ed particles in Figure 2-4. Th e covalent conjugated magnetic particles had similar characteriz ations of the partic les as the biotin-avidin conjugation. The biggest difference between the co njugation processes was the cova lently modified particles had lower DNA molecular densities by around an order of magnitude, and therefore, these particles saturate at lower target concentrations with the same amount of nanoparticles used in each sample. Conclusions Silica coated magnetite nanopartic les were successfully prepared using the Stber process, and the biotin-avidin DNA conjugation was dem onstrated. The DNA modi fied nanoparticles were characterized as these samples were c onstructed layer-by-layer using TEM, X-ray, zeta potential, and fluorescence analysis. The iron oxide particles were determined to have a full and complete silica coating with avidin and carboxyl f unctionalizations, which lends them to conjugations with target recogni tion molecules that are biotin and amine labeled, respectively. The conjugated nanoparticles proved to be exploitabl e for selectively bindi ng and isolating target molecules in this part icular instance was DNA.

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50 CHAPTER 3 EXTRACTION OF OLIGONUCLEOTIDES USING SILICA COATED MAGNETIC NANOPARTICLES Introduction The selective separation of biomolecules from bi ological fluids is of distinct interest for disease diagnosis, biomedical st udies, and biotechnology development. However, there is a lack of efficient biotechnology availa ble to gather a single target molecule, such as DNA, mRNA, peptides, and proteins, at low concentrations a nd in large volumes of complex sample solutions. To date, routine separation techniques used fo r DNA purifications have included HPLC and gel electrophoresis.103-105 These methods are used for the separation of high target capacity bulk samples.106 HPLC purifications separate DNA based on length and hydrophobic interactions and not by the oligonucleotide sequence.107 Gel separation has the same fundamental problem. When dealing with low-abundance target molecules in living cells, this separation issue becomes amplified. Thus, the selective extraction of olig onucleotide molecules from complex samples has been a difficult challenge for investigat ors that require innovative solutions. Nanomaterials have quickly become an excelle nt medium to act as a molecular carrying device for many biological applica tions. This is the result of their large surface-to-volume ratio and extremely small size.108 Nanoparticles have demonstrat ed unique advantages when combined with biomolecules as analyt e recognition elements for bioanalysis.72,110 In one instance, magnetic nanoparticles have been de veloped as a molecular carrying device for separating genes because of their cont rollable response to a magnetic field.111-113 Separation and detection of DNA molecules has been acco mplished by using avidin-coated magnetic nanomaterials,114 and this type of approach has been used for the collection and separation of single-base mismatched DNA using fl uorescence based detection scheme.75 However,

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51 fluorescence detection does not provide the ability to distinguish with any certainty the presence of the full complement or mismatch sequences. An alternative method for the detection of se parated DNA samples that has the ability to provide sequence identification is mass spectrom etry (MS). One area that MS detection of DNA has gained notoriety was in the characteriza tion of polymerase chain reaction (PCR) products. MS analysis compared to the traditional PCR de tection methods has the potential advantages of speed, sensitivity, and mass accuracy.115-119 MS has been shown to detect single-nucleotide polymorphisms (SNP), essentially single-base mismatch, and has emerged at the core of several SNP projects.120,121 Prepared PCR samples present a challenge, the efficient and rapid removal of the reaction components was necessary for MS anal ysis. The current limitations on MS analysis of PCR exist with the time required for sample clean-up.122 In this work, the development of a nanoharvesting agent (NHA) using silica coated magnetite nanoparticles functionalized with avidin and conjugated with biotinylated DNA to the particles surface. DNA hybridizati on provided sequence selectivity, and combined that with the exceptional separation capability of magnetic nanopart icles extracted target molecules with little sample clean-up required. Target DNA was sepa rated, collected, and analyzed from pure and mixed samples. To demonstrate the extrac tion properties of NHAs, single and multiple oligonucleotide targets in both buffer and complex samples, single target extractions in large volume from buffer and complex samples, and mismatch DNA experiments in buffer were investigated. The separated and collected ta rgets were monitored by fluorescence and mass spectrometry techniques.

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52 Experimental Materials and Methods The materials for these experiments were purch ased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicte d. Ammonium hydroxide (NH4OH), ethanol (EtOH) and hydrochloric acid (HCl) were obtained from Fi sher Scientific (Fair Lawn, NJ ). All the DNA molecules were synthesized in-house. The neodymium iron boron magnet used for extractions was purchased from Edmund Optics (12,200 gauss, Barrington, NJ). Clean resin used for desalting DNA samples was purchased from Sequenome (Newton, MA). The avidin protein was bought from Molecular Probes (Eugene, OR). AnchorChip ma trix assisted laser desorption-ionization (MALDI) plate was obtained from Bruker Daltonic s (Billerica, MA). A ll the chemicals used were of analytical grade. Instruments All fluorescence measurements were obtaine d using a TECAN Safire microplate reader (Research Triangle Park, NC, USA). The mass sp ectra were collected with a Bruker Microflex (Billerica, MA) MALDI time-of-flight (TOF) operated in negative ion reflectron mode. DNA Conjugated Magnetic Nanoparticles The DNA modified silica coated magnetic nano particle, referred to as nanoharvesting agent (NHA), synthesis was descri bed in detail in Chapter 2. Briefly, a solution of ammonium hydroxide (2.5%) and a solution containing ferric ch loride hexahydrate (0.5 M), ferrous chloride tetrahydrate (0.25 M), and HCl (0.33 M) were mi xed. The sample was stirred at 350 RPM using a mechanical stirrer for 10 minutes. Silica coa ting was made by washing the iron oxide sample and suspending them in ethanol contai ning ~1.2 % ammonium hydroxide with 210 L of tetraethoxyorthosilicate (TEOS) a nd sonicating for 90 minutes. Next, the sample was treated

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53 with an additional 10 L of TEOS with sonication for 90 minutes. The silica coated nanoparticles were washed with ethanol th en 10 mM phosphate buffer saline (PBS) pH 7.4. Avidin coating was completed by taking 1.2 mg of the silica coated magnetic nanoparticles with 5 mg of avidin and incubated at 4 C ove rnight. The particles we re washed with 100 mM PBS pH 7.4, and suspended in a 1% glutaraldehyde solution dilu ted in 100 mM PBS for 1 hour with continuous mixing at room temperature. Th e sample was washed and dispersed in 1M TrisHCl pH 7.0 for 3 hours at 4 C. These particle s were washed with 20 mM Tris-HCl, 5 mM MgCl2 pH 8.0 and suspended at a concentrati on of ~0.2 mg/mL. Biotin modified DNA, complementary to nonhuman (5-biotin-TTT AAA TCT AAA TCG CT A TGG TCG C-3), MnSOD (5-biotin-TTT AAA CAG TTA CA T TCT CCC AGT TGA TT-3), or -actin (5biotin-TTT AAA AGG AAG GAA GGC TGG AAG AG-3) truncated genes, was attached adding 6 nmols of DNA to 500 L of the avidin nanoparticles at 4 C overnight. Finally, the NHA were washed with 20 mM Tris-HCl, 5 mM MgCl2 buffer and suspended at a concentration of ~0.2 mg/mL. Extraction of DNA Targets Pure oligonucleotide extractions To 180 L of 0.2 mg/mL of the NHA, 20 L of each of the individual target DNA sequences, which were a nonhuman truncated gene (5-FITC-GCG ACC ATA GCG ATT TAG A-3) labeled with fluorescein isothiocyanate (FITC), the MnSOD truncated gene (5-cy3-AAT CAA CTG GGA GAA TGT AAC TG-3) la beled with cy3, and truncated -actin gene (5-Tx Red-CTC TTC CAG CCT TCC TTC CT-3) labeled w ith Texas Red (Tx Red) producing a final concentration of 50 nM. The prepared samples we re incubated at room temperature for 1 hour. The samples were magnetically extr acted and washed three with 200 L aliquots of 20 mM Tris-

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54 HCl, 5 mM MgCl2, pH 8.0 buffer. These samples were heated to 70 C for 10 minutes to release the captured DNA from the NHA. While the samp les were still hot, NHA were magnetically removed from the supernatant. The released DNA sample was allowed to cool to room temperature and then analyzed by the microplat e reader. Additionally, this experiment was performed in a more complex matrix, where 36 g of the NHA was suspended in a final volume of 200 L fetal bovine serum (FBS) c ontaining the target DNA. Af ter incubation, the samples were washed three times with 200 L of the 20 mM Tris-HCl, 5 mM MgCl2 buffer. These samples were then treated the same as the buffer incubated samples. Large volume extractions Large volume extraction experiments were investigated as well. The large volume buffer extractions were evaluated at final volumes of 10, 5, 1, and 0.2 mL. These samples were spiked with 20 L of target DNA at 500 nM, and contained 0.2 mg of the NHA, which is 1 mL at 0.2 mg/mL. The samples were incubated for 1 hour at room temperature. After target binding, the samples were magnetically extracted and washed sequentially with 1 mL 0.5 mL, and 0.2 mL of the 20 mM Tris-HCl, 5 mM MgCl2 buffer. As described above, the isolated DNA was removed from the NHA by heating to 70 C for 10 minutes, and the particles removed from the supernatant while the sample wa s still hot. The collected DNA sa mple was analyzed using the microplate reader. This experime nt was repeated using 10 mL of FBS as the matrix, and the target DNA extracted. DNA mismatch targets The NHA ability to bind and extract one (5-FITC-GCG ACC ATA TCG ATT TAG A-3) and two-base (5-FITC-GGG ACT ATA GCG ATT TAG A-3) mismatch DNA sequences versus the full complementary target was explor ed using FITC labels based on the truncated

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55 nonhuman gene. Individual sample s containing the 50 nM full complement, one-base mismatch, and two-base mismatch DNA in a final volume of 200 L with DNA labeled magnetic nanoparticles using the biotin-DNA sequences desc ribed above. These samples were extracted, collected, and isolated as described in previous sections. Each of the prepared samples was analyzed using the microplate reader. Multiple target extraction Using the sample extraction pr otocol presented previousl y, samples containing one, two, and three target DNA sequences using combinations of the nonhuman, MnSOD, and -actin. These samples were treated with 36 g, 180 L at 0.2 mg/mL, of each of the three prepared NHA types simultaneously. When extracted, the one two, and three target containing samples were heated to release the captured DNA targets from the nanopa rticle, and the isolated materials were analyzed by fluorescence using the microplate reader. Sample Preparation for Mass Spectrometry The DNA extraction experiments were perf ormed in the 20mM Tris-HCl, 5mM MgCl2 buffer as described previously and analyzed by MALDI-TOF. The samples analyzed were single sequence type extractions of the nonhuman target (5836.9 g/mol) and one-base mismatch (5811.9 g/mol) followed by mixtures of full complementary and one-base mismatch at various ratios (1:1, 2:1, and 5:1 respectively). The multip le target extraction experiments were executed as previously described. The average mass i ons for the truncated nonhuman gene were 5836.9 g/mol, MnSOD truncated gene was 7120.7 g/mol, and -actin 5905.9 g/mol. After extraction and washes with the 20mM Tris-HCl, 5mM MgCl2, the samples were dispersed in 20 L deionized water. These samples were heated to 70 C for 10 minutes in the deionized water. Desalting was performed prior to mass spectrometry analysis by using a weak anion exchange resin at half the

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56 total volume of the sample in a 0.5 mL centrifuge tubes. The samples were then mixed with 1:1 with 1 L of 10 mg/mL 3-hydroxypico linic acid (50/50 H2O/acetonitrile) and spotted (0.5 L) onto a dry pre-spotted (1 L) anchor-chip. Results and Discussion Fluorescence Detection of Extracted Samples Single oligonucleotide extraction Individual target extractions of three different oligonucleot ide sequences were performed in buffer and serum using DNA modified nanopart icles (NHA) labeled with capture sequences complementary to their respective target s, which include nonhuman, MnSOD, and -actin. These collected samples were analyzed by the microplat e reader and repeated 10 times. The control sequences for each of the nanopar ticle types was one of the other DNA sequences used in this study, for example MnSOD and -actin for the nonhuman capture NHA. Figure 3-1 displays representative 20 L aliquots of fluorescen tly labeled DNA targets extracted from buffer (solid bars) and serum (striped bars). The nonhuman DNA target was represented in blue, MnSOD DNA was represented in green, -actin was repres ented in red, and the control experiments in black. The control data shown here was th e nonhuman NHA treated with the dye labeled MnSOD oligonucleotide. The serum control experiment produced a slightly higher background signal than the buffer control. This increased background signal was most likely a result of the extraction being performed in a biological fluid, and after the wash steps some of the biomaterial remained in the sample. As seen in the figure, each of the NHAs select ively extracted their re spective targets, and a significant intensity difference was observed between the target and control samples for all NHAs. For all the control samples investigated, there was little to no signals observed. Due to

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57 Figure 3-1. Fluorescence intensity data representing individua lly extracted DNA targets from buffer (solid bars) and serum (striped bars) samples. the signals obtained, the target samples are easil y distinguishable from the control sample. The samples extracted from buffer had signal enhan cements of nearly 300-fold over the control samples, and for the serum extracted samples th e enhancements were at least 40-fold over the control samples. The difference in signal enhancem ents was that the set performed in the serum media resulted in higher background signals most likely due to resi dual serum materials. At these experimental conditions of 0.2 mL sample volumes with 36 g of NHA using 50 nM of target DNA, the collection efficiency from the buffer sa mples were determined to be more than 95% and the serum samples were estimated to be around 90% or higher (data not shown). For determining these collection efficiencies, the fluorescence signals for the DNA samples were 0.0 0.2 0.4 0.6 0.8 1.0 1.2 ControlNonhumanMnSODB-actinRelative Intensity Relative Intensity 0.0 0.2 0.4 0.6 0.8 1.0 1.2 ControlNonhumanMnSODB-actinRelative Intensity Relative Intensity

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58 obtained prior to and after extraction for both the buffer and serum samples, and the percent recovered was calculated fr om these obtained values. Sample enrichment and mismatch oligonucleotide extraction Figure 3-2. Enrichment study of extracting Mn SOD fluorescent dye labeled DNA target from several different buffer volumes. The NHAs were used to extract and concen trate 10 pmols of targ et DNA from various sample volumes, which were 10, 5, 1, and the standard 0.2 mL. The enriched samples were removed from the particles by heating into 0.2 mL sample volumes as described previously, and 20 L aliquots were analyzed by the microplate reader. Each of the target DNA samples was tested. Figure 3-2 shows the representative fl uorescence data obtained for the MnSOD enriched samples extracted from buffer, where the fluoresce nce intensities were obtained before extraction (striped bars) and after extraction (solid bars). The first set of samples is the 10 mL, the second set of samples is the 5 mL, the third set of samples is the 1 mL, and the last set of samples is the 0.2 mL sample volumes. The NHAs proportionally enriched the target molecules from the various sample volumes, as indicated in Figure 32. The samples were concentrated with a linear before extraction extracted10510.2 1.0 0.8 0.6 0.4 0.2 0.0 Volume (mL)Relative Intensity1.2 before extraction extracted10510.2 1.0 0.8 0.6 0.4 0.2 0.0 Volume (mL)Relative Intensity1.2

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59 response with respect to the signals observed pr ior to extraction, and th e fluorescence intensities of the enriched samples were measured to be the same for all NHA treated samples. The amount of the NHAs used in these studies was increased to 0.2 mg. This was done to ensure that sufficient collection of the target wa s obtained in the large vol ume samples. When the standard 36 g were used, the collection efficiency of the target DNA from the 10 mL sample decreased by more than that of the standard 0.2 mL volume. The amount of NHAs used was not optimized. The target DNA was enriched from 10 mL of serum, and similar results were obtained (data not shown). Figure 3-3. Fluorescence data obta ined testing the ability of th e nanoharvesting agent to collect and isolate mismatched DNA sequences. For the mismatch extraction study, 50 nM conc entration of the full complementary, onebase mismatched, and two-base mismatched DNA sequences labeled wi th a FITC fluorophore were added to three different NHA samples. The se t of sequences represented in Figure 3-3 were the nonhuman oligonucleotides. The fluorescence of each sample solution was determined after collection and removal of the extracted sequence from the particle into 200 L of buffer sample, and 20 L aliquots of the samples were analyzed by the microplate reader. The first intensity 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Target1-base mismatch2-base mismatchRelative Intensit y

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60 peak represents the full complementary nonhuman oligonucleotide (target), the second peak is the one-base mismatch oligonucleotide (1-base mism atch), and the third peak is the two-base mismatch oligonucleotide (2-base mismatch). As expected, the NHAs extracted each of the DNA sequences, but with different efficiencies. The ta rget was removed with the highest efficiency followed by the 1-base and 2-base mismatch sequences, respectively. Additionally, this presents a problem for fluorescence analysis when mismatch sequences are present in a sample with a target sequence. There is no way to determin e the identity of the sample extracted by fluorescence techniques. Multiple oligonucleotide extraction The multiple extraction and detection experiments were evaluated by creating mixtures of one, two, and three DNA targets at 50 nM final concentrations of each analyte used and adding 36 g of all three of the NHAs. These samples were prepared in either bu ffer or serum media. For the single target samples, the sample contained the MnS OD target. Additionally, double and triple target samples were evaluated, whic h were composed of the MnSOD and nonhuman sequences and all three targets respectively. All combinations of the one, two, and three target mixed samples were evaluated (data not s hown). Each sample was analyzed using 20 L of the isolated materials by the microplat e reader, and every sample was measured three times to check for the presence of each of the th ree dye-labeled oligonucleotides (Table 3-1). The first column represents the DNA samples that were analyzed, the second colu mn indicates the presence or absence of the nonhuman sequence, the third in dicates the MnSOD sequence, and the fourth represents the -actin sequence. The rows of the table display the different DNA samples that were investigated.

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61 The buffer extracted samples produced signal en hancements that were 200-fold and higher above the background signal, and the serum extracted samples had enhancemen ts of at least 35fold. In samples that contained the MnSOD but not the -actin targets, a small signal was measured. This was the result of the cy 3 fl uorophore on the MnSOD havi ng a slight excitation overlap with the excitation wave length used for the Texas Red dye. The determined standard deviations for these extractions were 8-12%. This data indicated that the NHAs were selective for the target DNA and reproducible in all evaluated samples. Table 3-1. Representative normalized fluoresce nce intensities obtained from buffer and serum multiple target extraction. Sample type Nonhuman MnSOD -actin Buffer one target two target three target Serum one target two target three target 0.0 1.0 1.0 0.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.1 0.1 1.0 0.1 0.1 1.0 Analysis by Mass Spectroscopy The mismatch samples were further investig ated by mass spectrometry. In this study, the sequences examined were the nonhuman target (m /z = 5836.9 g/mol) and its one-base mismatch (m/z = 5811.9 g/mol) sequence, where these samples were not fluorescently labeled. The oligonucleotide sequences were extracted from 200 L volumes of a 50 nM sample concentration. The extracted samples were released from the NHA into 20 L of deionized water. This was performed to reduce the amount of salt present in the isolated samples for analysis. The DNA sequences were extracted fr om samples of the nonhuman (target), the onebase mismatch, and a mixture of the two sequences For the mixed samples, three ratios of the target and mismatch sequences were investig ated, which were 1:1, 2:1, and 5:1 target-to-

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62 mismatch respectively. The targ et concentration was kept at 50 nM, and the mismatch concentration was varied accord ing to the ratios indicated. Figure 3-4. Mass spectra demonstrating the ex traction of the nonhuman mismatched DNA using the nanoharvesting agents. A) The full complement DNA target is the bottom spectrum and the 1-base mismatched DNA is the top spectrum. B) Mixed samples of the full complement and mismatched DNA ex tracted using the nanoharvesting agent. Figure 3-4 A contains the mass sp ectra obtained from the target sample (bottom) and onebase mismatch sample (top). These results indi cated that the NHA extracts both the target and mismatch sequences which confirms the fluores cence intensity data. However, Figure 3-4 B displays the mass spectrum obtaine d when both sequences were mixed into the sample at the 5:1 ratio. This spectrum provides the ability to diffe rentiate between the two sequences, which were not possible with the fluorescence measurements. The other ratios confirmed this conclusion as well. However, these ratios demonstrated that the mismatch sequence ionized better than the target DNA. Additional studies using this MALDI-TOF and the instrumental conditions indicated, this MS instrument can discriminate between sequences with mass differences around 9-10 mass units. The smallest possible mass differe nce obtainable betwee n two nucleotide bases is 9-10 mass units. The multiple DNA extraction study was furthe r investigated by MS. The sequences examined were the nonhuman (m/z = 5836.9 g/mol), MnSOD (m/z = 7120.7 g/mol), and -actin 5836.890 5872.499 0 500 1000 1500 2000 2500Intens. [a.u.] 5700 5750 5800 5850 5900 5950 6000 6050 m/z 5811.103 0 500 1000 1500 Intens. [a.u.] 5700 5750 5800 5850 5900 5950 6000 6050 m/z Relative Intensity 570057505800585059005950600060506100 A m/z 5838.616 5812.759 0 100 200 300 400 Intens. [a.u.] 5760 5780 5800 5820 5840 5860 5880 5900 5920m/z 5760 5920 5780580058205840586058805900Relative Intensitym/z B 5836.890 5872.499 0 500 1000 1500 2000 2500Intens. [a.u.] 5700 5750 5800 5850 5900 5950 6000 6050 m/z 5811.103 0 500 1000 1500 Intens. [a.u.] 5700 5750 5800 5850 5900 5950 6000 6050 m/z Relative Intensity 570057505800585059005950600060506100 A m/z 5838.616 5812.759 0 100 200 300 400 Intens. [a.u.] 5760 5780 5800 5820 5840 5860 5880 5900 5920m/z 5838.616 5812.759 0 100 200 300 400 Intens. [a.u.] 5760 5780 5800 5820 5840 5860 5880 5900 5920m/z 5760 5920 5780580058205840586058805900Relative Intensitym/z B

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63 (m/z = 5905.9 g/mol) sequence, where these samples were not fluorescently labeled. The oligonucleotide sequences were extracted from 200 L volumes of a 50 nM sample concentration. The extracted samples were released from the NHA into 20 L of deionized water. This was performed to reduce the amount of salt present in the isolated samples for analysis. Individual extractions were performed for each of th e DNA sequences and analyzed by MS. Mixed samples of the three DNA targets were extracted by treatment with one, two, or all three of the NHA as described above. The extracted samples were analyzed by MS. Figure 3-5. Mass spectra demonstrating the ex traction of multiple DNA sequences using the nanoharvesting agents. A) The nonhuman se quence is the bottom spectrum, MnSOD sequence is the middle spectrum, and -actin is the top spectrum obtained from extracted samples containing individual ta rgets. B) Multiple DNA extraction from a sample containing all three target se quences using the nanoharvesting agents. Figure 3-5 A contains the mass sp ectra obtained from the individual target samples treated with their corresponding NHA. The bottom spect rum is of the nonhuman oligonucleotide that was extracted, the middle spectrum displays the extracted MnSOD sequence, and the top spectrum is the extracted -actin sequence. These results in dicated that the NHA extract the target sequences, and confirm the fluorescence da ta. Figure 3-5 B displays the mass spectrum obtained from the multiple extraction samples wh en treated with all the NHAs simultaneously. MS provides the ability to differe ntiate between all the multiple extracted target sequences. This 5836.800 0 1000 2000 3000 Intens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 5905.653 7120.074 0 200 400 600 ntens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 5906.263 0 200 400 600 800 Intens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 40006000800010000120001400016000180002000022000 Relative IntensityA 5834.731 7116.949 0 500 1000 1500 2000 Intens. [a.u.] 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 m/z 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 BRelative Intensitym/z m/z B-actin Nonhuman MnSOD 5836.800 0 1000 2000 3000 Intens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 5905.653 7120.074 0 200 400 600 ntens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 5906.263 0 200 400 600 800 Intens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 40006000800010000120001400016000180002000022000 Relative IntensityA 5836.800 0 1000 2000 3000 Intens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 5905.653 7120.074 0 200 400 600 ntens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 5906.263 0 200 400 600 800 Intens. [a.u.] 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 m/z 40006000800010000120001400016000180002000022000 Relative IntensityA 5834.731 7116.949 0 500 1000 1500 2000 Intens. [a.u.] 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 m/z 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 5834.731 7116.949 0 500 1000 1500 2000 Intens. [a.u.] 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 m/z 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 BRelative Intensitym/z m/z B-actin Nonhuman MnSOD

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64 data confirms the experiments performed by fl uorescence. However, MS has the ability for increased multiplexed analysis of targets compared to fluorescence. Conclusions The selective and multiple extraction capab ility of the DNA conjugated silica coated magnetic nanoparticles (NHA) were demonstrat ed by analysis by both fluorescence and mass spectroscopy. These isolation experiments were demonstrated in buffer and serum for the fluorescence based analysis. Also compared to the fluorescence based detection method, MS has the capability for multiplexed analysis beyond th at of fluorescence. Fluorescence techniques detect no more than 5-6 DNA target molecu les simultaneously, where MALDI at these experimental conditions has the ability to dete ct dozens of DNA molecules. The limit to the number of DNA molecules to be detected by MALDI depends highly on the ionization capability of the targets. However, fluorescence analysis served well for characterizing the extraction characteristics of the NHA. The NHA was determ ined to extract mismatch DNA from samples containing these sequences. This data was coll ected by both fluorescence and mass spectrometry techniques. However, MS has the ability to di scriminate with certainty the presence of mismatched DNA in samples, which is nearly impossible for fluorescence-based detection. Efficient extraction of DNA targets from large volume samples for enriching the DNA analytes for analysis were demonstrated fluorescently in both buffer and serum sample matrices. The extra dispersion into deionized wa ter and desalting steps were added to the sample preparation to improve the ionization of the DNA molecule. The NHAs have been established as a powerful sample clean-up and preparation tool for oligonucleotide analytes.

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65 CHAPTER 4 FUNCTIONALIZED NANOPARTICLES FOR PEPTIDE EXTRACTION Introduction With the recent advancements in mass spectro metry, it has become a routine technique for the analysis of biological molecu les. However, for complex biologi cal samples the analysis time is limited more by sample separation and clean-u p processes than by mass analysis. In addition, as sample matrices become more complex and th e analytes of interest are in lower and lower abundance, methods for selective extraction and concentration must be developed. This work describes the use of silica nanoparticles for ta rget isolation and anal ysis by mass spectroscopy. To date, a number of separation procedures have been developed for mass spectrometry analysis. Solid phase extraction (SPE) tools are a popular tool for the removal of background materials from biological samples, such as Ziptips.122 SPE procedure involve multiple step procedures for analyte isolation. This method pr ovides sample clean-up and concentration, but SPE does not have the ability to sele ctively remove target molecules. Another approach related to SPE called su rface enhanced laser/de sorption ionization (SELDI), where a MALDI plate is functionalized to selectively retain the analyte molecules. After the sample is treated to the SELDI surface, the plate is washed prior to MS analysis. This technique provides increased throughput for bi ological applications in profiling studies. However, SELDI requires bringing the sample to the surface for extraction increases the risk of sample loss.123 Alternatively, microparticles have been used for the collection of analytes prior to MALDI analysis with a choice of functionalization ch emistries and particle recovery processes.124-125 For example, C18 modified magnetic microspheres were used in the profiling of proteomic targets.124 The C18 group adsorbs a fraction of the materials through weak van der Waals interactions and

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66 maintains the interactions during the washing procedures. For analysis, the retained molecules are removed from the particle by treatment with non-polar solvents, and the MALDI matrix is mixed with the isolated sample. Silica nanoparticles have been used in a num ber of analyte detection schemes. However, applications for mass spectrometry analysis have not been widely investigated. This work explores the selective extraction and concentr ation of target molecules for high sample throughput. Additionally, an atmospheric pressure liquid MALDI was used as a medium the direct analysis of the functi onalized nanoparticles to minimize sample loss and analysis time. Liquid matrices do not require the use of low pr essure sample chambers and avoid the problems of inhomogeneous matrices, as seen with solid -based MALDI. Atmospheric pressure sampling provides an alternative appro ach to allow for rapid and re producible sample analysis.126 The focus is on the aptamer conjugated magnetic nano particles for selective peptide extraction, but C18 functionalized silica was investigated as we ll. Aptamers have been composed of singlestranded nucleic acids that bind to target molecules. These aptamers form a complex threedimensional structure, enabling the aptamers to selectivity recognize a wi de range of molecules with high affinities. Experimental Materials and Methods Peptide standards (Sigma-Aldrich Corp., St. L ouis, MO, USA), angiotensin I, angiotensin II, bradykinin, bradykinin fragment 1-7, and L-vasopressin were pr epared as stock solutions of 500 pmoles/ L using either acetonitrile (ACN), for C18 nanoparticle extractions, or water, for aptamer nanoparticle extractions. D-vasopressi n (Genomechanix, Gainesville, FL, USA) was synthesized using conventional fluorenylmethoxy carbonyl chemistry, and dissolved in water for analysis. Biotinylated DNA was also purchased from Genomechanix (Gainesville, FL USA).

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67 Non-extraction analysis was conducted by spotting 0.5 L of matrix onto 0.5 L of analyte stock solutions. Nanoparticle Synthesis Silica C18 functionalized nanoparticles The silica C18 functionalized nanoparticles were prep ared using a previously reported synthesis procedure.76,127 Briefly; the nanoparticles were prepared using a water-in-oil microemulsion (W/O) with a water-to-surfactant molar ratio of 10:1. The synthesis produced uniform silica nanoparticles (60 5 nm in diamet er) as shown in Figure 4-1 A. Twenty hours into the synthesis, 40 L of octadecyltrimethoxysilane (Sigma -Aldrich Corp., St. Louis, MO, USA) and 10 L of ~30% ammonium hydroxide (Fisher Sc ientific, Fair Lawn, NJ, USA) were added to the microemulsion. The mixture was stirred for an additional 4 hours to yield a C18 outer coating of the silica core nanoparticles. Prior to peptide extracti on, the nanoparticles were washed with ethanol, acetone, a nd water three times each, and redi spersed in acetonitrile. The final concentration of the na noparticle suspension was approximated at ~6 mg/mL. Magnetic aptamer nanoparticles The iron oxide core magnetic nanoparticles were prepared using the Stber method.66 The magnetite core was formed by precipitating iron oxide through mixing ammonia hydroxide (2.5%) and iron chloride at 350 RPM using a mechanic al stirrer (10 minutes). The iron chloride solution contained ferric chlori de hexahydrate (0.5 M), ferrous chloride tetrahydrate (0.25 M), and HCl (0.33 M).128 After three washes with water and on ce with ethanol, an ethanol solution containing ~1.2 % ammonium hydroxide was added to the iron oxide nanopa rticles, yielding a final concentration of ~7.5 mg/mL.

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68 Tetraethoxyorthosilicate (200 L) was added to create the sili ca coating for the magnetite core particles. The mixture was sonicated for 90 minutes to complete the hydrolysis process, and the nanoparticles were washed three times w ith ethanol to remove excess reactants. Aptamers were immobilized onto the particle surface through avidin-biotin linkage (5' biotin-TCACGTGCAT GATAGACGGC GAAGCCGTCG AGTTGCTGTG TGCCGATGCA CGTA).129 For avidin coating, a 0.1 mg/mL silica coat ed magnetic nanoparticles solution and a 5 mg/mL avidin solution were sonicated in the presence of the partic les for 5 minutes and incubated at 4 C for 14 hours. The particles were magnetically separated and washed three times with 10 mM phosphate buffered saline (PBS) pH 7.4. The particles were redispersed at 1.2 mg/mL in 10 mM PBS and stab ilized by cross-linking the coat ed nanoparticles with 1% glutaraldehyde (1 hour at 25 C) After another separation, the particles were washed three times with 1M Tris-HCl buffer. For aptamer attachment the particles were disp ersed at 0.2 mg/mL in 20 mM Tris-HCl, 5 mM MgCl2, pH 8.0. Biotin labeled DNA was added to the solution at a concentration of 0.2 x10-6 M. The reaction was incubated at 4 C for 12 hours. Three final washings of the particles were perfor med using 20 mM Tris-HCl, 5 mM MgCl2 at pH 8.0. The nanoparticles were made to a final concentration of 0.2 mg/mL and stored at 4 C before use in the same buffer. Matrix and Analyte Preparation The liquid matrix was a UV absorbing form ulation developed for use with APMALDI.126 The matrix was prepared by mixing -cyano-4-hydroxycinnamic acid (CHCA) (Sigma-Aldrich Corp., St. Louis, MO, USA) with a liquid support containing a solvent liqui d, equal parts ethanol and water (Fisher Scientific, Fair Lawn, NJ, USA), and a viscous component, diethanolamine

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69 (DEA) (Sigma-Aldrich Corp., St. Louis, MO, USA) The matrix was soni cated and vortexed to ensure dissolution. Instrumentation The mass spectrometer used was an or thogonal acceleration time-of-flight mass spectrometer (TOFMS) (LECO Corporation, St. Jo seph, MI, USA). Ions are sampled through an atmospheric pressure interface using a heated cu rtain gas configuration with 5 L/min of 100 C nitrogen. Briefly, the source uses a 337 nm nitrogen laser (V SL-337-ND-S, Spectra-Physics, Mountain View, CA, USA), focused by a fused silica lens to ~ 250 m, to irradiate the sample on a stainless steel target 2 mm in diameter. Laser pulses were ~ 20 J as measured using a pyroelectic detector (J409-030, Molectron Detector, Inc., Santa Clara, CA, USA). The target (2 kV) was positioned on-axis ~1.5 mm from and ~1 mm below the MS orifice (400 V) using a motorized xyz translational stage (8302/IPic o Driver, New Focus, San Jose, CA, USA). During analysis, the laser was pulsed (20 Hz) asynchronously with the MS repeller pulse (5 kHz). The spectrometer data acquisition syst em is based upon a time-to-digital converter (TDC) multichannel plate (MCP) assembly. Th e data system is designed to producing 100 spectra per second; however, for t ypical analysis, spectra were expor ted and stored in an external computer at a rate of ~ 4 spectra per second. The spectra shown are an accumulation of summed spectra for 1-5 minutes. Fluorescence measurements for the extracted fl uorescein labeled angi otensin II were made using a TECAN Safire microplate reader (Research Triangle Pa rk, NC). Imaging of the C18 nanoparticles was conducted on an inverted Olympus microscope, 100X magnification, (Olympus, Melville, NY) using an intensified IPentamax III CCD (Roper Scientific, Trenton,

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70 NJ). Transmission electron micrographs (TEM) of nanoparticles were taken using a Hitachi H7000 transmission electron microscope. Extraction Procedures Two procedures were used for peptide extract ions: a centrifugation technique was applied for the silica C18 particles, while the magnetic particle s required only a magnetic separation. For the C18 functionalized particles, 90 L of nanoparticle solution (~4 x 10-8 M) was incubated with 10 L of stock analyte solution (~ 1:1000 particle-t o-analyte ratio) for 10 minutes. The mixture was centrifuged for 5 minutes at 14000 RPM. Afte r centrifugation, the supernatant was removed leaving ~1 L of silica particles. The particles we re washed to remove interferences and unabsorbed analyte. The wash solutions, 99 L of acetonitrile or wate r, were added and the mixture was vortexed ~5 seconds. The wash solution used was dependent upon the desired result, analyte removal or retention. The proced ure was repeated when additional washing steps were used. For nanoparticle extr action analysis, the si lica particles remaining in the centrifuge tube after supernatant remova l were applied directly to the MALDI target surface. The aptamer functionalized magnetic nanoparticle s allowed rapid separation using a simple magnetic extraction method. First, a buffer exchange was conducted using 50 L of aptamer conjugated nanoparticles (~1 x 10-8 M). The particles were magnetically extracted and washed with 5 mM phosphate buffer and then 3 mM MgCl2, pH 6.0. The buffer used was based upon the reported protocol for aptamer chir al separation of L and D vasopressin.130,131 The resulting nanoparticle suspension was used in an alyte extractions. For extraction, 10 L of 50 pmol/ L analyte solution was incubated with 10 g (50 L) of the aptamer conjugated nanoparticles for 10 minutes (~ 1:100 particle-to-anal yte ratio). A magnet directed the particles to the bottom of the vial for removal of the supernat ant. The remaining particles, ~1 L, were mixed with 5 L

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71 of water and 5 L of liquid matrix. Analysis was conducted by spotting 0.5 L of the aptamer particle/matrix mixture on the MALDI target. Results and Discussion Nonspecific Nanoparticle Applications A study of C18 modified silica nanoparticles were investigated for the nonselective extraction of peptide molecules investigated by Turney et al.65 The analyte bound silica-based nanoparticles were removed from solution by centr ifugation. In the initial investigation, the particles were characterized by fluorescent labeled peptide molecules to demonstrate that the peptides adhered to the nanoparticle surface. Thes e molecules not only adsorbed to the particle surface by hydrophobic interactions, but they were retained by the particles throughout the various wash steps. However, the FITC dye molecule labeled to the peptide may have contributed to the retention of the anal ytes to the hydrophobic nanoparticles. Further investigations involved using mass spectroscopi c detection and unlabeled targets. The C18 functionalized nanoparticles performed with the same principles as reversed-phase chromatography where the investigated peptides were retained by th e nanoparticles through nonpolar washes and then released with a polar so lvent. Additionally, varyi ng lengths of peptides were examined, where the smallest ones were re moved from the particles first followed by the largest ones. The C18 nanoparticles were used directly in the liquid atmospheric pressure MALDI to reduce the chance for sample loss. Where with vacuum based systems, require removal of the analytes prior to analysis. However, this appr oach provides a low resolution separation for the studied peptides. Selective Nanoparticle Applications The C18 functionalized nanoparticles utilize hydrophobic interacti ons for the collection and isolation of target molecules. However, the wash procedure uses centrifugation as the means to

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72 obtain the samples, which is time a consuming process. The DNA aptamer conjugated magnetic nanoparticles offer the possibility of a rapid-se lective extraction of analytes from complex samples. These recognition molecules have nano molar and picomolar dissociation constants for the best binding aptamers, which make them very attractive for numerous scientific and biotechnological devices. Aptamers have been us ed for investigating ce llular protein function and protein-ligand interactions.92,133,134 Common selective analyte extractions are achieved by using antibody-antigen interactio ns, where antibodies provide se lectivity through conformational binding sites with high Kd.133 However, limitations exist for antibody conjugated systems, such as diminished detection limits, extensive ch aracterization are neede d, and lengthy-complex procedures are required for obtaining these molecules.135 Aptamers have several advantages compared to antibodies. Since aptamers can be constructed of single stranded DNA or RNA molecules, they are simple and less costly to s ynthesize with increased shelf lives. Modifications of aptamers are easily performe d with site labeling because of the well understood-controllable oligonucleotide chemistry compared to engineer ing antibodies The aptame r used in this study was selected for D-vasopressin (H-Cys1-Tyr-Phe-Gln-Asn-Cys6-Pro-Arg-Gly-NH2), which has a binding efficiency of ~ 1000 times that of L-vasopressin.130,131 The silica coated magnetic nanoparticles serve as the solid support for conjugation of the aptamer to extract targets. The magnetic propert y of the nanoparticles redu ces the complexity of the separation protocol required for isolating the target molecules. Placing the particles directly into the liquid matrix allows a rapid analysis procedure that limits analyte loss allowing lower abundance samples to be detected. The D-vasopressin aptamer provides high se lectivity, but this aptamer has a large dissociation constant (KD ~ 1M), which allows for release of the analyte from the particles131.

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73 The aptamer was investigated previously when at tached to a surface, and it retained its binding functionality with little or no reduction in its binding ability.130 The selectivity of the nanoparticle bound aptamer was examined by conduc ting two control experiments. The first control used D-vasopressin aptamer conjugated ma gnetic particles, and they were treated with two control peptides, which were angioten sin II (ASP-ARG-VAL-T YR-ILE-HIS-PRO-PHE) and bradykinin fragment 1-7 (ARG-PRO-P RO-GLY-PHE-SER-PRO). These aptamer nanoparticles indicated no suffici ent binding of either peptide and no mass spectrometry signal was produced after extraction. The second sel ectivity control demonstrated whether the vasopressin analytes nonspecifically bound to a random aptamer conjugated nanoparticles, where individual aliquots of L-vasopr essin and D-vasopressin were incubated with an adenosine aptamer conjugated magnetic nanoparticles. This e xperiment demonstrated that the vasopressin molecules were not nonselectively extracted by apta mer conjugated nanopartic les as evidence of the lack of mass spectrometry signal. The KD values of the D-vasopressin aptamer for D-vasopressin compared to L-vasopressin were at a ratio of 1000:1130. This ratio provides an additional and interesting control for the Dvasopressin aptamer nanoparticles. The extract ion of L-vasopressin should produce a limited mass spectrometry signal, and extraction of D-vas opressin should be more efficient indicated by an increased mass spectroscopy signal. Figure 4-1 shows the mass spectra for Land Dvasopressin before and after treatment with th e D-vasopressin aptamer conjugated nanoparticles. Figure 4-1 A displays the L-vasopressin molecula r ion and sodium adducts before extraction the top spectrum. The bottom spectrum in Figure 4-1 A indicates the nanoparticle extraction for Lvasopressin. The limited signal intensity that was obtained confirmed lower binding of the aptamer modified nanoparticles for L-vasopressi n. Figure 4-1 B provides the molecular ion and

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74 Figure 4-1. The mass spectra of th e Land Dvasopressin before and after extraction are shown. A) L-vasopressin before extraction the t op spectrum and after extraction the bottom spectrum. B) D-vasopressin before extrac tion the top spectrum and after extraction the bottom spectrum. A B intensity intensity 106010701080 1090 1100 111011201130 1140 106010701080 1090 1100 111011201130 1140 m/z m/z A B intensity intensity 106010701080 1090 1100 111011201130 1140 106010701080 1090 1100 111011201130 1140 m/z m/z

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75 sodium adducts for D-vasopressin prior to extr action the top spectrum. The bottom spectrum in Figure 4-1 B shows the extraction for D-vasopr essin treated with the aptamer conjugated nanoparticles. The mass spectrometry intensities for the extracted sa mples are estimated to be an approximate ratio of ~ 1000:1 D-va sopressin to L-vasopressin, wh ich is consistent with the reported KD values for the aptamer. Additionally Figur e 4-1 B, the before extraction and after extraction spectra indicated that the aptamer binds and extracts only the disulfide bridge containing peptide. For this experiment, the orig inal sample solution c ontained both the oxidized and reduced forms of D-vasopressin, and after tr eatment and extraction of the sample with the nanoparticles the only oxidized peptide remained. This leads to the fact that the aptamer binds the tertiary structure of the target peptide and not the primary structure. Combining the selectivity of the aptamer c onjugated magnetic nanoparticles w ith AP liquid MALDI analysis provides rapid analysis usi ng by using magnetic extraction. Conclusion The use of nanoparticles has provided the de velopment of a rapid analyte extraction technique for MS analysis. These silica-based nanoparticles act as a powerful scavenging agent for peptide molecules. The use of these functionaliz ed particles with the liquid matrix allows for a rapid analysis procedure that limits analyt e loss by direct insert ion of the analyte bound nanoparticles. The C18 particles retained their chromat ographic properties as evident through non-polar and polar washing proced ures, and provided the extracti on capability for a variety of peptide molecules. The magnetic nanoparticles provided a simplif ied separation procedure through the use of magnetic extraction. These particle s were functionalized with aptamers for a more selective analyte-nanoparticle interac tion. The magnetic nanoparticles have the capability for wider

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76 applications for the analysis of low abundance analytes. The aptamer conjugated nanoparticles allowed for the selective extraction of D-vasopr essin, and the extraction of L-vasopressin was observed to the efficiency of the 1000:1 dissoci ation constant ratio fo r D-vasopressin to Lvasopressin.

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77 CHAPTER 5 ANTIBODY-CONJUGATED MAGNETIC NANOPARTICLES FOR PROTEIN MICROARRAYS Introduction Biosensors have been development for target screening in clinical, environmental, water and food samples. The essentia l components of these systems ar e the recognition elements for selective identification of targ et analytes, which are typically antibodies. Antibodies have demonstrated high binding affinities with extrao rdinary selectivity for target molecules in complex sample matrices and at low target con centrations. The Array Biosensor developed at the Naval Research Laboratory (NRL) have be en successfully used in increasingly more complex sample matrices for the detection of a variety of materials, su ch as protein toxins, organic molecules, biomarkers, viruses, and bacteria.136-138 The two-dimensional nature of the sensing surface facilitates simultaneous analysis of multiple samples for multiple analytes. The immunoassays developed to date ar e rapid (15-25 min) and simple to perform, with little-if any sample pretreatment prior to analysis. Much of the recent work in assay developm ent with the NRL Array Biosensor has been concerned with the rapid detec tion of food-borne contaminants.139-147 Limits-of-detection (LODs) obtained with the NRL A rray Biosensor typically fall short of those for the desired analyte application or LODs obtained by th e more complex-time consuming standard methodologies. However, the LODs for the NR L system are comparable to ELISAs and a number of other rapid biosenso r-based technologies. One way to improve the current LODs obtained by the NRL Array Biosensor would be to include a target pre-co ncentration step prior to the immunoassay. In order to keep the detec tion method practical, these steps must minimally increase the overall assay tim e or demands on the operator.

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78 One such preconcentration technique is immuno magnetic separation (IMS), this process is commonly used prior to detecti on. Magnetic particles have b ecome increasingly popular for automated separations.142,143 Commercially available magnetic particles are typically 1-2 ms in diameter and come with a variety of chemically active surfaces that can be used to functionalize the particle as desired. These magnetic partic les have been used in immunoassays as solid supports for target biomolecule capture and con centration from complex sample matrices, such as clinical, environmental, and food samples. This process was performed to improve the sensitivity of the resulting assay. Target bind ing to the antibody coated magnetic particles is usually performed in solution, and the sample is concentrated, and the target measured using a method independent of the magnetic particle s while generally still bound to the magnetic particles. Methods commonly used for the quantification of particle tr eated samples are performed independently of the magnetic particles. These analysis techniques include culture followed by flow cytometry analysis,150 PCR coupled with hybridization studies,151 or enzyme-linked immunosorbent assays (ELISAs) For fluorescence-based measurements, quantification of the resulting fluorescent immunoma gnetic-target complex is achieve d using simple solution-based fluorescence microplate reader 152-154 or flow cytometry.150,155-157 In the highlighted studies, the target remains bound to the magnetic particle during analysis, and additional steps are required to include fluorescently labeled primary or seconda ry antibodies for detection. The magnetic particles have also been measur ed using fluorescence microscopy,158,159 and spotted onto protein binding membranes with capillary blotting tech niques and the fluorescence from the resulting membrane were measured using a near IR sensitive photon counting system.160 Magnetic beads were coupled with ELISAs and detection of the target obtained by optical or electrochemical

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79 methods.161-163 In one study, immunomagnetic sepa ration coupled with PCR and ELISA detection gave LODs at 20 cfu/ ml for Mycobacterium in milk.161 In less common applications, researchers have us ed the properties of the magnetic particles themselves to determine the presence of the bound target. For example, giant magnetoresistive (GMR) sensors were used to quantify DNA hybridization,166 while an electromagnet has been used to read immunoassay sensor chips.164 Additionally, a superconducting quantum interference device (SQUID) was developed to monitor biotin-avidin binding by measuring the magnetic moments.165 Advances in microfluidics and integrated technologies have re sulted in the use of magnetic particles coupled with planar surfaces.161,164,166,167 Magnetically-assisted transport evanescent field fluoroimmunoassay (MATEFFs) demonstrated that magnetic beads functionalized with a fluorescence sandwich immunoassay complex could be transported into the region of an evanescent field for detection using an external magnetic field.168 A number of methods for interacting antibody-labeled magnetic particles with protein microa rrays have been investigated, where magnetic brushing, magnetic scanning, an d a push/pull method were used. In these methods, a magnet was used below the substrate to concentrate the beads to the surface and a magnet above the substrate to remove weak ly bound or non-specif ically bound magnetic particles.169 To date, there have been few studies inves tigating the direct interaction of magnetic particles with protein microarrays. This may be in part due to the rela tively large size of the commercial magnetic particles used in most st udies. The binding of large antigen-antibodymagnetic particle complexes to an antibody imm obilized on a sensor surface is subject to shearing in the flow conditions normally used in immunoassays One way to address this

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80 problem is to decrease the size of the magnetic particles used. Nano-sized magnetite particles enveloped in lipid membranes, have been used in a number of studies.170-172 Modifiable iron oxide-based magnetic nanoparticles have also be en synthesized with a well defined size and monodispersity.75,173-175 Magnetic nanoparticles are star ting to be used in ELISAs,163 but to date have not been coupled with protein microarrays. The work presented here represents a proof-of-concept approach for exploiting magnetic nanoparticles (M NPs) for sensitive multiplexed immunoassays. Unlike the previously mentioned MATEFFs, this technique does not use the magnetic nanoparticles (MNPs) as the delive ry method for localiza tion of the target to the evanescent field sensing surface, but instead uses target re cognition by surface-bound antibodies to localize the particles for signal generation in microarrays. Th ese MNPs were coated with fluorescent labeled antibodies for signal generation. The ultimate goal is to use these magnetic nanoparticles to preconcentrate target analytes to allow simplifi cation for subsequent dete ction with the aim of improving assay sensitivity without sacrificing ea se of operation. For initial optimization and proof-of-concept, a simple direct-binding assay system was studied. MNPs were functionalized with target chicken IgG fluorescently labeled with Alexafluor 647 (Alex647-chick-MNPs), and the resulting particles were passed over sensor surfaces patterned with rabbit-anti-chick IgG. Assay performance was optimized by evalua ting the MNPs surface composition, chick IgG conjugation conditions, the extraction time for c oncentration experiments and increased MNP concentration after extraction. Detection sign als were generated dir ectly by binding of the fluorescent MNPs to the slide surface.

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81 Experimental Materials and Methods Unless otherwise specified, chemicals were of reagent grade and used as received. All materials, such as tetraethoxyorthosilicate (TEO S) and 2-(N-morpholino)et hane sulphonic acid (MES), were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Poly(dimethyl)siloxane (PDMS), used for making the assay flow cells, was obtained from Nusil Silicone Technology (Carpinteria, CA). Borosili cate glass slides from Daigger & Co. Inc. (Vernon Hills, IL) were used as slides in al l the assays described. The 3-mercaptopropyl trimethoxy silane (MTS) and N-( -maleimidobutyryloxy) succinim ide ester (GMBS) were purchased from Fluka Chemical Co. (St. Louis, MO). Carboxyethyls ilanetriol sodium salt (Carboxy-silane) was purchased from Gelest, Inc. (Morrisville, PA). 1-Ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochlorid e (EDC), N-hydroxysuccinimide (NHS) and NeutrAvidin were purchased from Pierce Bi otechnology, Inc. (Rockford, IL). Ammonium hydroxide was obtained from Fisher, Inc. The bi otin-SP-conjugated rabb it anti-chicken IgY (Rbanti-chick IgG) and chicken IgY (chick IgG) were purchased from Jackson ImmunoResearch (West Grove, PA). Fluorescent labeling of the chick IgG was achieved using succinimide esterfunctionalized AlexaFluor647 (Alexa647), purchased from Molecular Probes (Eugene, OR). Magnetic Nanoparticle (MNP) Synthesis Iron oxide nanoparticles were synthesized by coprecipitating iron salts. Using a mechanical stirrer, ammonia hydroxide (2.5%) a nd iron chloride were mixed at 350 RPM for ten minutes, as described previously.176 Briefly, the iron salt soluti on contained ferric chloride hexahydrate (0.5 M), ferrous chlo ride tetrahydrate (0.25 M), and HCl (0.33 M). The iron oxide nanoparticles were washed with water three times and ethanol on ce. The MNPs were dispersed

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82 in an ethanol solution that c ontained ~1.2% ammonium hydroxide at a final concentration of ~7.5 mg/mL. The magnetite core particles were coated with silica by adding 200 L TEOS. The hydrolysis process was completed by sonicating for 90 minutes. Another aliquot of TEOS (10 L) was added and sonication was continued fo r an additional 90 minutes. This additional TEOS step was used to post-coat the nanoparticles. The sample was again washed with ethanol three times. An 80 L aliquot of the carboxy-silane was added to 1 mL of 10 mg/mL silicacoated MNPs in 10 mM PBS, pH 7.4 and con tinuously mixed for four hours. Finally, the particles were washed three times with 10 mM PBS and stored at room temperature until used. Dye Labeling of Chick IgG AlexaFluor labeling of the ch ick IgG prior to attachment to the MNPs was carried out according to the procedure of Anderson et al.178 Labeled antibodies were separated from unincorporated dye using size exclusion chromat ography (BioGel P10). Protein-to-dye ratios were determined using UV-Visible spectroscopy. Optimized IgG Protocol A 250 L solution of 4 mg/mL carboxyl-modified MN Ps were washed three times with 250 L aliquots of a 0.5 mM MES, pH 5.0 buffer. The protein, chick IgG, modification was carried out by adding 50 L of a 20 mg/mL EDC solution to th e washed particles and incubated for fifteen minutes. Next, 100 g of Alexa647-chick IgG with a 1-to-5 molar equivalent of a 5000 Da amine-PEG (polyethylene glycol) was adde d to the activated car boxyl particles. The solution was incubated for two hours with vorte xing every 15-to-30 minutes. The MNPs were magnetically extracted and washed three times with 500 L aliquots of 10 mM PBS buffer. After the third wash, the Alex647-chic k-MNP complex was resuspended in 500 L of 30 mM

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83 hydroxylamine with 1% BSA in 10 mM PBS pH 7.4 buffer and incubated for thirty minutes. Finally the Alex647-chick-MNPs were wash ed three times and resuspended in 500 L aliquots of 10 mM PBS with 0.05% Tween 20 and 0.1% BSA at pH 7.4. The final concentration of the MNPs was 2 mg/mL, and the samples were stored at 4 C until used. Slide Preparation, MNP Extraction, and Immunoassay Microscope slides, used as waveguides, were cleaned by immersion in a 10% (w/v) KOH in 2-propanol for 30 min at room temperature, followed by rinsing with deionized water and drying with a nitrogen stream. The slides we re immediately immersed in a toluene solution containing 2% MTS for 1 h under nitr ogen. The silanized slides were then rinsed with toluene, dried with nitrogen and immediatel y immersed in 1 mM GMBS in absolute ethanol for 30 min at room temperature. The slides were ri nsed with water and incubated in 25 g/mL NeutrAvidin in PBS overnight at 4 C before being washed in PBS and eith er used immediatel y for patterning or stored in PBS at 4 C until required. Patterning of the biotinylated Rb-anti-chick IgG (10 g/mL) in PBS + 0.05 % Tween (PBST) was carried out using a 6-channel patterning PDMS flow cell clamped onto the NeutrAvidin functionalized slide surface and injecti ng the biotinylated capture antibody into 4 or 5 of the channels.178 Biotinylated goat anti-mouse IgG (10 g/mL in PBST) was introduced into the remaining channels for use as a negative control (C). The slides were then incubated overnight at 4 C. After the flow cell channels were rinsed with 1 mL PBST, the slide was removed from the PDMS patterning template and placed in PBS blocking solution containing 1% casein. After ~1 h, the slides were rinsed with Milli-Q water and assembled in a 6or 12channel assay PDMS flow cell, with the flow channels orientated perpendicular to the stripes of immobilized biotinylated antibodies. Each channe l was hooked up to an ISMATEC multi-

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84 channel pump (Cole-Parmer Instruments Compa ny, Vernon Hills, IL) at one end (outlet) and syringe barrels (1 mL) were then attached at the opposite end (inlet), ready for the immunoassay. The Alex647-chick-MNPs were first sonicate d and vortexed briefly to resuspend the sample. The Alex647-chick-MNPs where then eith er directly diluted in 1mL of the PBS/0.1 % casein/0.05% deoxycholic acid (DOC) buffer for di rect immunoassays or extraction studies. A typical extraction procedur e involved diluting 200 L of the stock Alex647-chick-MNPs in 10 mL PBS/0.1% casein/0.05% DOC. The samples were prepared for the assay as follows: 1mL of the diluted sample was kept for the assay and re ferred to as As is. The As is Ex sample involved taking 1 mL of the diluted sample and ex tracting the MNPs using the Epindorf magnet. The liquid was removed and the Alex647-chickMNPs resuspended in 1 mL of PBS/0.1% Casein/0.05% DOC (x 0 concentration). For the extracted and concentrated sample, referred to as Ex, 6 x 1 mL of the diluted sample was ex tracted using the Epindorf magnet, the liquid was removed and the Alex647-chick-MNPs in each Epindorf resuspended in 0.2 mL of PBS/0.1% Casein/0.05% DOC before the contents of the 6 Epindorf tubes were combined (x5 concentration). This procedure was carried out the day before, and the samples stored at 4 C, or the day of the flow cell surface assay. The samples were sonicated for either 1 min or 5 min (5 min was found to be optimal), prior to use. The MNPs labeled with Alexa647-chick IgG, prep ared as described above, were applied to each channel (0.8 mL) at a flow rate of 0.1 mL/mi n. The channels were then washed with 1 mL at 0.25 mL/min. The PDMS flow cell was remo ved, and the slide was washed with Milli-Q water, dried with nitrogen and imaged on the Array Biosensor.

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85 Immunoassay Array Imaging and Analysis The slides were imaged using a Peltier-cooled CCD camera, as previously been described.179 Briefly, evanescent wave excitation of the surface-bound fluorescent species was achieved using a 635 nm, 12 mW diode laser (Las ermax, Rochester, NY). Light was launched into the end of the slide at an appropriate angle through a 1 cm fo cal length lens equipped with a line generator. The fluorescence emission was monitore d at right angles to the planar surface. A two-dimensional graded index of refraction (GRIN) lens array (Nippon Sheetglass, Summerset, NJ) was used to image the fluorescent pattern onto the Peltier-cooled CCD camera (Spectra Source, Teleris, Westlake Village, CA).84 Long-pass (Schott 0G-0665, Schott Glass, Duryea, PA) and band-pass filters (Corion S40-670-S, Franklin, MA) were mounted on the device scaffolding to eliminate excitation and s cattered light prior to CCD imaging. Data was acquired in the form of digital imag e files in Flexible Image Transport System (FITS) format. To analyze the images, a cu stom software application was written in LabWindows/CVI (National Instruments). The program creates a mask consisting of data squares (enclosing the areas wher e the capture antibody is patterned) and background rectangles which are located on either side of each data square. The averag e background value is subtracted from the average data square va lue, and net intensity value is calculated and imported into a Microsoft Excel file for data analysis. Results and Discussion The NRL Array Biosensor has demonstrated the rapid detection of a number of food-borne contaminants including bacterial cells, mycotoxins and bacterial protein toxins.139-147 In an effort to improve the limits-of-detection (LODs) while maintaining a rapid assay time, immunomagnetic concentration of the target s with simultaneous fluorescent labeling for biosensor analysis was investigated. For initia l optimization and proof-o f-concept, the direct-

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86 binding assay between Alex647-chick IgG functiona lized MNPs and Rb-anti-chick IgG modified surfaces was studied. Initial investigations with micron-sized magne tic particles demonstr ated successful binding under static conditions with bu ffer containing casein (1%) and deoxycholic acid (DOC; 0.05%). However, the direct binding assay performed poorly under flow conditions and resulted in low signal intensities in the regions of the slide patterned with Rb-a nti-chick IgG. This poor assay performance was probably due to the large diamet er of the magnetic beads and the shear force they experience at the surface under flow condition s. To address this problem using magnetic microparticles, MNPs synthesized in-house we re investigated. S ilica coated iron oxide nanoparticles, were functionalized with carboxysilane, and chick IgG was conjugated to the particles via EDC coupling chemistr y. These particles were found to be ~65 nm in diameter. To generate a signal in the evanescent field of the NRL Array Biosensor, the chick IgG attached to the MNPs was labeled with Alexafluor 647 dye The ratios were kept between 2-4 dyes molecules per chick IgG determined by UV-visibl e spectroscopy. This was done to ensure that free lysines were available for coupling to the MNP surface. Figure 5-1. The fluorescence image of th e immunoassay treated with Chick-MNP. The EDC activated MNPs were initially exposed to a total of 350 g Alexa647-chick IgG purified, and then 50, 20 or 5 L of the stock was diluted in 1 mL of the running buffer (PBS/0.1 50 L MNPs/mL 20 L MNPs/mL 5 L MNPs/mL Bt-Rb-anti-chick C

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87 % casein/0.1% DOC) for assay studies. A Neutra vidin slide was patterned with Rb-anti-chick IgG and blocked with a 1 % casein solution. The assay pattern was formed using a PDMS flow cell. The slide was then exposed to the purifie d-diluted Alex647-chick-MNPs at a flow rate of 0.1 mL/min. After washing with buffer, the flow cell was removed and th e slide imaged on the NRL Array Biosensor. The CCD image of these initial assays is show n in Figure 5-1. Strong signals are found in the Rb-anti-chick IgG functiona lized regions of the slid e, which decrease in intensity with decreased concen tration of the stock Alex647-chic k-MNPs. No signal is observed in the control lane (C), demonstrating the spec ificity of the interac tion. These experiments illustrate that these chick-MNPs work well under flow conditions. However, the non-uniformspeckled fluorescence signal observed in Figure 5-1 suggests that aggregation of the MNPs a still problem. Figure 5-2. Fluorescence images of the imm unoassay treated with Alex647-chick-MNPs run under various parameters. A) Ma gnetic nanoparticles exposed to 350 g Alex647chick IgG; assay buffer PBS/0.1 % Ca sein/0.1% Tween-20. B) Magnetic nanoparticles exposed to 350 g Alex647-chick IgG; a ssay buffer PBS/0.1 % Casein/0.1% DOC. C) Magnetic nanoparticles exposed to 100 g Alex647-chick IgG; assay buffer PBS/0.1 % Casein/0.05% DOC. To address aggregation, a number of parameters were investigated in cluding, changing the assay buffer conditions, the amount of Alexa647-chick IgG the MNPs were exposed to, and the density of carboxy-groups present on the surface of the MNPs (Figures 5-2 and 5-3). Chick350 g Alex647-chick IgG 100 g Alex647-chick IgG Bt-Rb-anti-chick C 350 g Alex647-chick IgG 100 g Alex647-chick IgG Bt-Rb-anti-chick C

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88 MNPs made using 350 g Alex647-chick IgG were diluted 50 L/1 mL in PBS/0.1% casein containing either 0.1% Tween (Figure 5-2 A) or 0.1% DOC (Figure 5-2 B), and passed over a rabbit-anti-chick IgG patterned s lide. Changing the surfactant does not eliminate the speckled fluorescence signal observed in the CCD images. However, when the amount of Alexa647-chick IgG exposed to the EDC activated MNPs was reduced from 350 g to 100 g (Figure 5-2 A versus 5-2 C), the resulting fluorescence signal is much more uniform in intensity w ithin individual data s quares, suggesting less aggregation of the chick-MNPs. The percen tage of carboxy-groups (COOH) present on the surface of the MNPs was also investigated. The silica coated MNPs surface was exposed to silane solutions containing 100, 75, 50 or 25 % COOH groups with the remainder of the solution consisting of silane containing the EDC-unreactive PO2 group. Either 350 g or 100 g of Alex647-chick IgG was exposed to the MNPs in the presence of EDC. The purified Alex647chick-MNPs were then diluted 150 L in 1 mL PBS/0.1% casei n/0.05% DOC, and 0.8 mL of this solution were passed over an antibody-functi onalized surface at a flow rate of 0.1 mL/min. The resultant bar graph (Figure 5-3) shows the re lative intensity of th e purified chick-MNPs, fabricated using 350 g (black) or 100 g (gray) of Alex647-chick Ig G captured by the surface as a function of their solu tion exposed COOH groups. As seen in the figure as the % COOH decreases so does the final intensity reached in the Rb-anti-chick IgG functionalized squares. Howe ver in the case of the MNPs exposed to 350 g of the Alex647-chick IgG, the decreased COOH concentration did not decrease the speckled nature of the fluorescence signal observed in the images, which suggests the aggregation was still present. As observed pr eviously, only reduction of the ex posed Alex647-chick IgG amount from 350 g to 100 g seemed to improve the uniform ity of the fluorescence signal.

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89 Figure 5-3. The fluorescence data showing the effect of the % COOH terminated silane used to modify the surface of the MNPs. Fluores cent signals are measured as the net intensity in the CCD image and normaliz ed relative to 100% COOH Alex647-chickMNPs. Error bars represent the standard deviation in net inte nsity values obtained from a minimum of 4 squares. The effect of the EDC exposure method used to attach 100 g of Alex647-chick IgG on the surface of 100% COOH MNPs was also inve stigated. In this study, the EDC was not removed from the MNP solution prior to the add ition of the Alex647-chick IgG (referred to as method a). Since the protein chic k-IgG contains both -COOH and -NH2 groups, it is likely that multilayers of Alex647-chick IgG are formed on the MNP surface. To inves tigate the effect of the EDC method on the amount of Alex647-chick IgG attached to the surface of the MNPs, five different procedures were investigat ed and summarized in Table 5-1. % COOH on MNPs 100%75%50%25% Relative Intensity 0 20 40 60 80 100 120 100%75%50%25% % COOH on MNPs 0 20 40 120 60 100 80Relative Intensity % COOH on MNPs 100%75%50%25% Relative Intensity 0 20 40 60 80 100 120 100%75%50%25% % COOH on MNPs 0 20 40 120 60 100 80Relative Intensity

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90 Table 5-1. EDC protocols inve stigated for MNP modification with Alex647-chick IgG. EDC exposure method EDC MNP activation procedure prio r to Alex647-chick IgG addition EDC a EDC b EDC c EDC d EDC e EDC/ resuspend in IgG solution. EDC/ collect magnet/ resuspend in IgG solution. EDC/ collect magnet/ PBS or MES wash/ collect magnet/ resuspend in IgG solution. EDC + NHS/ collect magnet/ PBS wash/ collect magnet/ resuspend in IgG solution. EDC [x5]/ collect magnet/ MES wash/ collect magnet/ resuspend in IgG solution. The purified Alex647-chick-MN Ps were then diluted 100 L in 1 mL PBS/0.1% casein/0.05% DOC and 0.8 mL of th is solution passed over a Rb-a nti-chick IgG functionalized surface at a flow rate of 0.1 mL/min. The result ing bar graph (Figure 5-4) shows the relative intensity of the Alex647-chick-MN Ps prepared using the variou s EDC methods a-e captured by the rabbit-anti-chick IgG functi onalized surface. The net intensities obtained from the images were normalized to the EDC a procedure, which produced the brightest fl uorescence intensity on the slide surface. Slides 1 (black) and 2 (light gray) were washed with PBS for EDC-c, whereas Slide 3 (dark Gray) used an MES wash. As e xpected, the EDC-a procedure resulted in the brightest signals obtained from the Rb-antichick IgG functionalized regions. The EDC-b procedure included removal of the EDC prior to addition of the Alex647-chick IgG, and resulted in a significant decrease in the overall intensity of the MNPs. A further reduction in signal was observed in EDC-c, where the MNPs were washed with PBS to IgG exposure (compare the black bars EDC a-c). The EDC reactive intermediate was found to be more stable using a wash step at lower pH (MES 5.0-5.5 versus PBS pH 7.4) by comp aring the EDC-c (black and dark gray bars) by the higher relative intensity observed from the CCD intensity.

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91 Figure 5-4. The fluorescence data obtained to determine the eff ect of the EDC exposure methods a-e. Neither the addition of NHS EDC-d (light gray bar), which is repor ted to stabilize the reactive EDC intermediate or an excess of EDC as in method EDC-e (dark gray bar) seemed to increase the intensity of the signal obtained relative to procedure EDC-c. Solution UV-Visible absorption experiments also confirmed that more IgG was removed from the reaction solution when EDC-a versus EDC-c was used (data no t shown). While EDCa probably results in multilayers of Alexa647-chick IgG on the surface of the MNP, it also produces the brightest fluorescence signals. Therefore EDCa, along with the addition of 100 g Alexa647-chick IgG to the EDC activated MNPs was c hosen as the optimized protocol. The next step was to investigate the extr action and concentration procedures of the Alex647-Chick-MNPs to determine their performance in the direct immunoassays. Alex647Chick-MNPs were diluted 150 L in 10 mL PBS/0.1% casei n/0.05% DOC. The Alex647EDC Method EDC aEDC bEDC cEDC dEDC e Relative Intensity 0 20 40 60 80 100 120 0 20 40 120 60 100 80Relative Intensity EDC method EDC Method EDC aEDC bEDC cEDC dEDC e Relative Intensity 0 20 40 60 80 100 120 0 20 40 120 60 100 80Relative Intensity EDC method

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92 Chick-MNPs were extracted from 1 mL of sa mple for 3, 7, 15, or 30 min using an Epindorf magnet, which can hold up to 6 x 1.5 mL Epindorf tubes. The liquid was then removed and the Alex647-chick-MNPs resuspended in 0.5 mL of PBS/0.1% casein/0.05% DOC, which is a potential concentration factor of 2. The ex tracted Alex647-Chick-MNPs and a portion of the pre-extraction sample (both 0.5 mL) were passed over an antibody patterned surface at a flow rate of 0.1 mL/min. The resulting bar graph (Fig ure 5-5) shows the rela tive signal intensity of the Alex647-chick-MNPs obtained from the CCD im age as a function of the extraction time. These intensities were normalized to the pre-ex traction sample. Relative signal intensity versus extraction time suggests that 15 min extractions were optimal for the extraction procedure. Figure 5-5. The effect of the extraction time used to collect the Chick-MNPs on the final signal intensity obtained from the CCD image. The resulting bar graph shows the relative intensity, normalized to the pre-extracted sample, of the Chick-MNPs captured by the Rb-anti-chicken IgG functionalized surface as a function of their extraction time. However, Figure 5-5 demonstrates that although the Alex647-Chick-MNPs were concentrated by factor of two, this did not transl ate to an increased intensity from the CCD array biosensor image for the concentrated samples. In fact, the data suggests that the intensities are Extraction Time (min) PRE3MIN7MIN15MIN30MIN Relative Intensity 0 20 40 60 80 100 120 0 20 40 120 60 100 80Relative Intensity pre 371530 Extraction Time (min) Extraction Time (min) PRE3MIN7MIN15MIN30MIN Relative Intensity 0 20 40 60 80 100 120 0 20 40 120 60 100 80Relative Intensity pre 371530 Extraction Time (min)

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93 slightly lower than the original sample (PRE). Sonication of the concentrated Alex647-chickMNPs for 5 min versus the original 1 min gave a signal intensity improved up to a factor of ~2, which suggests that the drop in si gnal that is observed when th e samples are extracted may be due to aggregation. Aggregation of the Alex647chick-MNPs leads to la rger particles, which may not be as effectively captured by the surf ace under the shear force of the flow conditions. A number of extraction experiments were performed using the optimized Alex647-chickMNPs with 5 min sonication pr ior to analysis by the immunoa ssay. Alex647-chick IgG (100 g) was attached to the surface of 100% COOH modified MNPs using the EDC a method (no wash). The purified Alex647-chick-MNPs (200 L) were then diluted into 10 mL PBS/0.1% casein/0.05% DOC. The samples were prepared fo r the assay as follows. The As is samples were simply 1 mL of the diluted sample kept for the assays. For the Ex samples, 6 x 1 mL of the diluted sample was extracted using the Epin dorf magnet for 15 min. The liquid was removed and the Alex647-Chick-MNPs in each Epindorf t ube were resuspended in 0.2 mL of PBS/0.1% casein/0.05% DOC. The contents of the 6 Epindorf tubes were combined to give a 1 mL sample with a potential concentration factor of 5. After sonication for 5 min, 50 l was diluted in 950 l of milli Q water for both the As is and Ex samples for UV-Visible and fluorescence spectroscopy measurements. Table 5-2, summarizes the relative ratio of the Ex versus the As is samples for solution and surface characterization for an average of five separately pr epared batches of these Alex647chick-MNPs. The UV-Visible measurements at 4 00 nm demonstrates an increase in the amount of MNPs in solution following extraction, the solution fluorescence at 650 nm illustrates a respective increase in concentration of Alexa647-chick-MNPs in solution, and the intensity taken from the CCD images from the rabbit-anti-ch icken IgG functionalized regions following the

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94 immunoassay demonstrated the effectiveness of capture of the extracted Alex647-Chick-MNPs. As shown in Table 5-2, both the solution absorb ance and the fluorescence data show an increase in intensity following extraction. This demons trates that the MNPs were collected and concentrated using the magnet. Table 5-2. The solution and surface measuremen ts used to characterize the Alex647-chick-MNP samples pre and post extraction. Chick-MNP sample # of batches Solution absorbance Solution fluorescence emission Surface fluorescence MNP MNP overnight 5A:1P PEG A 1A:1P PEG A 1A:5P PEG A 1A:10P PEG A 5A:1P PEG B 1A:1P PEG B 1A:5P PEG B 5 2 1 3 2 1 1 1 1 3.0 + 0.3 2.9 + 0.6 3.0 4.0 + 0.4 4.5 + 0.5 4.3 2.8 3.6 3.7 2.3 + 0.3 2.1 + 0.6 1.6 2.4 + 0.4 2.6 + 0.4 1.9 1.4 2.4 2.0 1.2 + 0.4 1.6 + 0.5 0.9 1.3 + 0.4 1.5 + 0.1 0.6 0.6 1.0 0.9 However, this did not translate to an incr ease in CCD fluorescence signal generated from the immunoassay captured Alex647-Chick-MNPs. Th is is likely a result of MNP aggregation that leads to larger particles wh ich may not be as effectively capt ured by the surface. Note that this observation was also noted for samples prep ared using the EDC-c method. Therefore, the potential for multilayers of chicken IgG on the surface of the MNPs causing these observations can be eliminated as the possible reason. To determine if this lower than expected in crease in CCD fluorescence signal was a result of the extraction or the concentration of th e MNPs, an extra control was included in the immunoassay called the As is Ex sample. Here 1 mL of the diluted sample was extracted using the Epindorf magnet, the liquid was removed, and the Alex647-chick-MNPs resuspended

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95 in 1 mL of PBS/0.1% Casein/0.05% DOC. This means the sample was extracted, but not concentrated. While the absorbance measurements suggested that the conc entration of the chickMNPs in the solutions As is and As is Ex we re the same, both the so lution fluorescence and surface intensity taken from the CCD image s uggest a drop in the fluorescence for the chickMNPs following extraction. This is seen by the lower half of Figure 5-6 of the CCD image. This suggests that the extraction process itself is a ffecting the fluorescence from the MNPs in some manner. Aggregation of the Alex647-chickMNPs may produce particle s that are too large to remain bound to the surface due to the shear fo rce that occurs at the surface under the flow conditions of the assay, which may explain th e decrease in signal from the surface. Figure 5-6. Fluorescence image di splaying the effect of the time from extraction procedure to time the assay was preformed. Additionally after extraction, the samples were allowed to sit ove rnight in the fridge before the analysis on the immunoassay. A significant incr ease of nearly double th e fluorescent signal obtained from the CCD image of the surface was observed. The MNP overnight samples are As is As is Ex Ex As is As is Ex Ex Bt-Rb-anti-chick C Extraction carried out day before the assay Extraction carried out day of the assay As is As is Ex Ex As is As is Ex Ex Bt-Rb-anti-chick C Extraction performed the day before the assay Extraction performed the day of the assayAs is As is Ex Ex As is As is Ex Ex Bt-Rb-anti-chick C Extraction carried out day before the assay Extraction carried out day of the assay As is As is Ex Ex As is As is Ex Ex Bt-Rb-anti-chick C Extraction performed the day before the assay Extraction performed the day of the assay

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96 displayed in Figure 5-6 and Table 5-2. This would suggest that if MNP aggregation was the cause of these lower signal intensities that it is at least partially reversible. Figure 5-7. The fluorescence data obtained fo r the magnetic nanoparticles (MNPs) modified with Alex647-chick IgG and PEG molecules. A) The resulting fluorescence image for the different Alex647-chick IgG/PEG (MW 500 0) modified MNPs. B) A bar graph of the net intensities take n from the fluorescence image of the different Alex647chick IgG-MNPs, where the 5000 MW PEG samples are black and the 10,000 MW PEG B samples are gray. A Chick-IgG (A):PEG (P) Ratio 5A:1P1A:1P1A:5P1A:1P WASH Net Intensity 0 5000 10000 15000 20000 25000 30000 B Bt-Rb-anti-chick C 100 ng/mL Alex647-chick IgG 1A:5P PEG A 1A:5P PEG A PBSTB 1A:1P PEG A 5A:1P PEG A 1A:1P PEG A EDC c Chick-IgG (A):PEG (P) Ratio 30000 25000 20000 15000 10000 5000 0Net IntensityA B C Bt-Rb-anti-chick 100ng/mL Alex647-chick IgG 1A:5P PEG A 1A:5P PEG A PBSTB 1A:1P PEG A 5A:1P PEG A 1A:1P PEG A EDC-c A Chick-IgG (A):PEG (P) Ratio 5A:1P1A:1P1A:5P1A:1P WASH Net Intensity 0 5000 10000 15000 20000 25000 30000 B Bt-Rb-anti-chick C 100 ng/mL Alex647-chick IgG 1A:5P PEG A 1A:5P PEG A PBSTB 1A:1P PEG A 5A:1P PEG A 1A:1P PEG A EDC c Chick-IgG (A):PEG (P) Ratio 30000 25000 20000 15000 10000 5000 0Net IntensityA B C Bt-Rb-anti-chick 100ng/mL Alex647-chick IgG 1A:5P PEG A 1A:5P PEG A PBSTB 1A:1P PEG A 5A:1P PEG A 1A:1P PEG A EDC-cA Chick-IgG (A):PEG (P) Ratio 5A:1P1A:1P1A:5P1A:1P WASH Net Intensity 0 5000 10000 15000 20000 25000 30000 B Bt-Rb-anti-chick C 100 ng/mL Alex647-chick IgG 1A:5P PEG A 1A:5P PEG A PBSTB 1A:1P PEG A 5A:1P PEG A 1A:1P PEG A EDC c Chick-IgG (A):PEG (P) Ratio 30000 25000 20000 15000 10000 5000 0Net IntensityA BA Chick-IgG (A):PEG (P) Ratio 5A:1P1A:1P1A:5P1A:1P WASH Net Intensity 0 5000 10000 15000 20000 25000 30000 B Bt-Rb-anti-chick C 100 ng/mL Alex647-chick IgG 1A:5P PEG A 1A:5P PEG A PBSTB 1A:1P PEG A 5A:1P PEG A 1A:1P PEG A EDC c Chick-IgG (A):PEG (P) Ratio 30000 25000 20000 15000 10000 5000 0Net IntensityA B C Bt-Rb-anti-chick 100ng/mL Alex647-chick IgG 1A:5P PEG A 1A:5P PEG A PBSTB 1A:1P PEG A 5A:1P PEG A 1A:1P PEG A EDC-c

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97 The main issue with extracting and leaving samp les overnight is that it is not conducive to the rapid analysis time expected for many applica tions. Furthermore, investigations involved the addition of PEG to the surface of the Alex647-ch ick-MNPs to determine if this would help prevent aggregation. Carboxy MNPs activated using either the EDC-a (no wash) or EDC-c (MES wash) protocols were simultaneous ly exposed to Alex647-chick IgG (100 g), and either amine-PEG A (5,000 MW) or amine-PEG B (10,000 MW ) at different mole ratios. Purified Alex647-chick-MNPs-PEG were then diluted 50 L in 1 mL PBS/0.1% casein/0.05% DOC. The samples (0.8 mL) were passed over an antibody patte rned surface at a flow rate of 0.1 mL/min. The resulting image for the different Alex647-ch ick IgG-PEG A or PEG B (PEG B image not displayed) modified MNPs are sh own in Figure 5-7 A. The bar graph (Figure 5-7 B) plots the net intensities taken from the CCD image for th e different Alex647-chick-MNPs modified with either PEG A (black) or PEG B (Gray) molecules. As illustrated in Figure 5-7 B, the smaller PEG A modified Alex647-ChickMNPs produces slightly stronge r fluorescent signals from the CCD image than the corresponding ratio of PE G B modified Alex647-Ch ick-MNPs. For both PEG molecules, the fluorescence intensity increases slightly with increasing PEG ratios. Data from the extraction experiments are su mmarized in Table 5-2 for both the solution and surface characterization. The extraction e xperiments with Alex647-chick-MNPs modified with the smaller of the amine-PEG molecules (PEG A = 5,000 MW) showed promising data. The Alex647-chick-MNPs modified with PEG A at a ratio of 1 chick IgG:5 PEG A molecules provided a similar enhancement in CCD fluorescent signal in less time than the regular Alex647Chick-MNPs extracted and left overnight. Conclusion The use of MNPs for target preconcentrati on and signal generation in biosensor assays performed under flow conditions was demonstrat ed in a direct binding assay format. The

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98 optimal conditions for synthesizing the MNPs were determined by exploring the surface composition, antibody immobilization procedures, and various blocking buffers. In addition to investigating the MNP synthesis, the best extraction time and method for introducing the concentrated MNPs to the biosensor were as certained. However, the magnetic concentration process had an adverse effect on the modified MNPs, and further study into nanoparticle surface treatment was performed by adding PEG pol ymers along with the antibody during the immobilization step. Using MNPs in conjunction with sensi ng, under flow has not been previously demonstrated. MNPs were essential to this work mainly due to their small size, which reduced the shearing effect of the fluid flow on the surface bound particles compared to standard magnetic particles. By including the MNPs in th e assay instead of removing the bound target or requiring the addition of a seconda ry fluorescent species minimized the number of steps in the assay and reduces the chance of losing the target an alyte. Furthermore, little time is added to the overall assay protocol and the target is concentr ated prior to performing the analysis. The next step will be to demonstrate sa ndwich assays where the analyte is pulled out of solution by MNPs coated with fluorescent antibodies and con centrated prior to biosensor analysis.

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99 CHAPTER 6 APTAMER-CONJUGATED SILICA NA NOPARTICLES FOR CANCER CELL EXTRACTION AND DETECTION Introduction The need for accurate-sensitive diagnosis and understanding of human diseases at the molecular level has been limite d by the lack of probes availa ble to recognize the distinct molecular features of diseases. Diseases like can cer originate due to muta tions and alterations at the genetic level. These changes cause the infected cells to beha ve differently at the molecular level, which can facilitate the effective treatment programs implemented by clinicians. Determining the molecular characteristics of can cers, particularly knowing the characteristic biomarkers associated with a specific cancer can be of great benefit. These differences contain significant potential for aiding th e understanding of diseases ba sed on the biological processes and mechanisms, which are vital for disease diag nosis, prevention, and treatment. The detection of leukemia uses standard analysis methods fo r bone marrow and periph eral blood cytochemicals by karyotyping,180 immunophenotyping,181 microarrays,182 and amplification mutated genes by PCR.183 Immunophenotypic analyses uses antibody recogn ition elements commonly labeled with fluorescent dye molecules to differentiate diseas e cells from healthy cells by profiling with a panel of antibodies. The major limitations of th is method are the lack of sensitivity and it is subject to false positive results. In addition, antibody in tegrity is continually in question due to the lifetime of the sources needed to obtain a relia ble antibody. PCR based methods are highly sensitive analys is techniques for cel lular recognition by amplifying low copy numbers of critical gene mutations.183-185 This technique requires complexlengthy sample preparation procedures for analysis to be performed. In addition, PCR is limited by inconsistent sensitivities. These limitations result in false-negative results especially in

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100 samples with low-level signals even after amplification.183 Therefore, a need to develop new technologies for rapid-simple cell detection is of great interest. Aptamer-conjugated nanoparticles (ACNPs) we re used for the rapid extraction and detection of acute leukemia cells using high-a ffinity DNA aptamers for recognition. Aptamers form complex three-dimensional structures creating hydrophobic, hydrophilic, and electrostatic interactions for distinct target binding recogni tion. These recognition molecules were selected using intact cultured tumor cells through the process of cell-SELEX.186 The aptamer oligonucleotides used here were selective fo r CCRF-CEM acute leukemia (CEM), Burkitts lymphoma (Ramos), and non-Hodgkins B cell ly mphoma (Toledo). The sequences were attached to magnetic nanoparticles (MNPs) and fluorescent nanoparticles (F NPs) to act as the solid platform to collect and detect the respec tive intact cancer cells from simple buffer and biologically complex samples. Combining the selectivity of aptamers w ith the power of MNP based separation has produced a selective and sensitive method for co llecting, enriching, and su bsequently detecting targets. In this work, the aptamers were attach ed to three spectrally different FNPs to provide enhanced signaling. Fluorophore doped silica nanopa rticles (NPs) have been used to increase signals and ease for bioconjugation co mpared to single dye molecules.73,187,188 Using the functional groups on the particle surface has pr oven useful for oligonucleotide detection,73,189 protein, and antigen detection.189-192 Each NP contains thousands of dye molecules, and provide increased signal for every cell binding event through the aptamer recognition. Aptamer-conjugated MNP based cell sorting was employed in this work for the selective isolation of target cells. Ho wever, magnetic based cell coll ection has been used for the enrichment of a number of different cell types from a variety of species.193-198 These methods

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101 used micrometer-sized magnetic particles, where the magnetic particles studied here were 65 nm silica coated MNPs. The small size of these particles provide surface area to volume ratios, which provide enhanced extraction capabi lities compared to the microspheres.74 Magnetic extraction reduces the need for complex presam ple clean-up by removing FNP aggregates and any material not associated w ith the magnetic nanoparticles. Fluorescence based imaging, flow cytometry, a nd a microplate reader spectrometer were used to detect the extracted cell samples by th e ACNP technique. Using FNPs and the three aptamers, allowed for the demonstration of th is method for both inor ganic and organic dyedoped silica NPs, and the multiple extraction of th ree different related cell lines from the same sample. This method demonstrates the ability to reproducibly extract targ et cells from complex mixtures and biological fluids establishing a foundation for the relevance of this method for clinical applications. Experimental Materials and Methods All materials were purchased from Sigma-Aldr ich (St. Louis, MO) unless otherwise noted. Fetal bovine serum (FBS) was obtained from I nvitrogen (Carlsbad, CA). Whole blood samples were obtained from Research Blood Components, LLC (Brighton, MA). Fluo-4 was purchased from Molecular Probes (Eugene, OR). Carboxylet hylsilanetriol sodium salt was purchased from Gelest, Inc. (Morrisvil le, PA). 1-Ethyl-3-[3-dimethylam inopropyl] carbodiimide Hydrochloride (EDC) was purchased from Pierce Biotechnology, Inc.( Rockford, IL) and ammonium hydroxide was obtained from Fisher Scientific (Fair Law n, NJ). Cy5-NHS was purchased from Amersham Biosciences and Tetramethyl rhodamine succinim idyl ester (TMR SE) (mixed isomers) was purchased from Molecular Probes. Deoxyribonucleotides, 5`-a mino-modifiers, and biotin phosphoramidite were purchased from Glen Research (Sterling, Va).

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102 Fluorescent Nanoparticle Synthesis Rubpy dye-doped nanoparticles (NPs) were sy nthesized by the reverse microemulsion method.176 Briefly the NPs were synthesized by adding 1.77 mL Triton X-100, 7.5 mL cyclohexane, 1.6 mL n-hexanol to a 20 mL glass vial with conti nuous magnetic stirring. Next 400 L of H2O and 80 L of 0.1M tris(2,2 bipyridyl) di chlororuthenium (II) hexahydrate (Rubpy) dye (MW=748.63) were added. Followed by the addition of 100 L tetraethyl orthosilicate (TEOS) the material s stirred for thirty minutes of stirring. To initiate silica polymerization, 60 L NH4OH was added. After 18 hours, a pos t-coating of carboxyl modified silica was performed by adding 50 L TEOS and 50 L carboxylethylsilanetriol sodium salt. This polymerization was allowed to proceed fo r 18 hours. The particles were centrifuged, sonicated, and vortexed four times with 10 mL aliquots of fresh 95% ethanol, followed by a wash with a 10 mL aliquot of H2O. Each wash step was perfor med from the addition of fresh ethanol or H2O with sonication and vortexing to the next centrifugation step t ypically within 3-5 minutes. The DNA modification was carried ou t by adding 1.2 mg EDC, 0.5 nmoles DNA, and 2 mg of particles to 1.5 mL of 10 mM MES buf fer (pH= 5.5). The solution was vortexed for three and a half hours. Partic les were washed by centrifugi ng at 14000 rpm and dispersing in 200 L of 0.1 M Phosphate Buffered Saline (PBS ) (pH=7.2) three times. Rubpy NPs were stored at 4 C and dispersed in cell media buffer at a final concentration of ~10 mg/mL. Tetramethylrhodamine (TMR SE) and Cy5 doped NPs were synthesized according to the following: TMR SE and Cy5-NHS were each di ssolved in DMSO at a concentration of 5 mg/mL and 3-Aminopropyltriethoxysilane (APT S) was added at a molar ratio of 1.2:1 APTS:dye. The APTS was allowed to conjugate to the amine reactive dye for 24 hours in the dark with shaking prior to synthe sis of the particles. Glass re action vessels and Teflon coated

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103 magnetic stir rods were washed with 1M NaOH solution for 30 minutes, rinsed with DI water and ethanol, and allowed to dry. This wash step was performed to clean the glass vessel and stir rods and smooth the inside surface of the glass vessel which prevents unwanted seeding and NP formation. After conjugation, 4.19 mL of ethanol was mixed with 239 L of ammonium hydroxide solution in the reaction vessel. 36 L of TMR-APTS conjugate or 54 L of Cy5APTS conjugate were added to th e reaction vessels, yielding 3.44 x 10-7 moles of dye per reaction (A ratio of 2300 moles of silica per mole of dye). 177 L of TEOS was added rapidly to the reaction mixture and the vessels were sealed The reaction was allowed to proceed for 48 hours in the dark before the particles were recovered by centrifugati on at 14000 rpm. The particles were washed three times with phosphate buffer to remove any dye molecules that are weakly bound. The synthesis method was found to reproducibly produce a number average particle size of 50 nm 5 nm with a mono-modal distribution wh en measured with a Honeywell UPA 150 dynamic light s cattering instrument. To modify with carboxy groups with these NPs, a 2mg solution of NPs were diluted in 200 L of 10 mM PBS, pH 7.4, and 40 L of the carboxyl silane were continuously mixed for four hours. The NPs were washed three times and resuspended in 200 L aliquots of 10 mM PBS by centrifuging at 14,000 RPM for fifteen minutes and stored at room temperature until use. DNA modification was completed as described above. Magnetic Nanoparticle Synthesis Iron oxide core MNPs65 were synthesized by coprecipita ting iron salts. A mechanical stirrer was used to mix ammonia hydroxide (2.5%) with an iron chloride solution at 350 RPM for ten minutes. The iron chloride solution containe d ferric chloride hexahy drate (0.5 M), ferrous chloride tetrahydrate (0.25 M), and HCl (0.33 M). The iron oxide NPs were washed three times

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104 with 5 mL aliquots of H2O and once with a 5 mL aliquot of ethanol. Each wash was performed by decanting the supernatant, adding fresh wash solution, and redispersing in the fresh solution typically within 3-5 minutes. Next the iron ox ide NPs were dispersed in an ethanol solution containing ~1.2% ammonium hydroxide at a final concentration of ~7.5 mg/mL. The magnetite core particles were coated w ith silica by adding tetraethoxyorthosilicate (200 L), and the mixture sonicated for 90 minutes to complete the hydrolysis process. An additional aliquot of TEOS (10 L) was added and again sonicated for 90 minutes to add a postcoating to the NPs. The sample was washed thre e times with ethanol to remove excess reactants. A solution of 0.1 mg/mL silica coated magnetic nanoparticles (MNPs) solution in 10 mM PBS, pH 7.4, and a 5 mg/mL avidin solution in 10 mM PBS, pH 7.4 were vortexed for 5-10 minutes to initiate an avidin coating. The re sulting sample was incubated at 4 C for 12-14 hours. Next the particles were washed thr ee times and dispersed at 1.2 mg/mL with 100 mM PBS. The avidin coating was stabilized by cros s-linking the coated NPs with 1% glutaraldehyde (1 hour at 25 C). Again the particles were magnetically separated, washed three times, and dispersed in 1M Tris-HCl buffer. The samples wa s incubated in the 1M Tris-HCl buffer (3 hours at 4 C), followed by three additional washes with 20 mM Tris-HCl, 5 mM MgCl2, pH 8.0 at a concentration of ~0.2 mg/mL. Finally, the DNA was attached to the particle s by adding biotinylated DNA (3 pmol) to a solution of 500 L at 0.2 mg/mL in 20 mM Tris-HCl, 5 mM MgCl2, pH 8.0 avidin coated MNPs. The attachment was performed at 4 C for 12 hours, and three final washes were performed using 20 mM Tris-HCl, 5 mM MgCl2 at pH 8.0. The MNPs were st ored at 4 C and used at a final concentration of ~0.5 mg/mL.

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105 Magnetic Extraction Two different magnetic extraction procedures were used, and each were performed by adding the specified amounts of MNPs to each sample as described in the experimental sections for the respective extraction procedures. For the initial studies, aptamer conjugated magnetic nanoparticles were then incubated with the target cells for 5 minutes unless specified otherwise. After the incubation period a magnetic field was applied to the side of the sample container. After a minute the non-magnetic materials were removed with a pasteur pipette and then fresh buffer was added and the magnetic field was rem oved. The materials were mixed in the buffer and previous steps were repeated for a total of three times to remove anything nonspecifically bound to the magnetic nanoparticles. For later ex traction studies, the aptamer-conjugated MNPs were incubated with the cell samples for 15 min, a magnetic field was introduced to the sample container, and after 2-5 minutes the nonmagne tic materials were decanted using a pasteur pipette. To complete the wash process, the magnetic field was removed and the samples were redispersed in 200 L fresh media buffer and this pr ocess was repeated three times. Cells CCRF-CEM cells (CCL-119 T-cell, human acu te lymphoblastic leukemia), Ramos cells (CRL-1596, B-cell, human Burkitts lymphoma ), and Toledo cells (CRL-2631, non-Hodgkin's B cell lymphoma) were obtained from ATCC (Ameri can Type Culture Association). The cells were cultured in RPMI medium supplement ed with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin-Streptomycin. The cell density was determined using a hemocytometer, and this was performed prior to any experiments. After which, approximately one million cells dispersed in RPMI cell media buffer were centrifuged at 920 rpm for five minutes and redispersed in cell media three times, and were then redispersed in 1 mL cell media buffer. During all experiments, the cells were kept in an ice bath at 4 C.

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106 DNA Aptamer Synthesis The following aptamers have been selected for the CCRF-CEM, Ramos, and Toledo cells respectively, 5-TTT AAA ATA CCA GCT TAT TCA ATT AGT CAC ACT TAG AGT TCT AGC TGC TGC GCC GCC GGG AAA ATA CT G TAC GGA TAG ATA GTA AGT GCA ATC T-3, 5-AAC ACC GGG AGG ATA G TT CGG TGG CTG TTC AGG GTC TCC TCC CGG TG-3, and 5ATA CCA GCT TAT TCA ATT ATC GTG GGT CAC AGC AGC GGT TGT GAG GAA GAA AGG CGG ATA ACA GAT AAT AAG ATA GTA AGT GCA ATC T3. Both the amine and biotinylated versions of the aptamer sequencers were synthesized inhouse. An ABI3400 DNA/RNA synt hesizer (Applied Biosystems, Foster City, CA) was used for the synthesis of all DNA sequences. A ProSta r HPLC (Varian, Walnut Creek, CA) with a C18 column (Econosil, 5u, 250.6 mm) from Alltech (Deerfield, IL) was used to purify all fabricated DNA. A Cary Bio-300 UV spectromete r (Varian, Walnut Creek, CA) was used to measure absorbances to quantify the manufact ured sequences. All oligonucleotides were synthesized by solid-state phosphoramidite chemistry at a 1 mol scale. The completed sequences were then deprotecte d in concentrated ammonia hydroxi de at 65 C overnight and further purified twice with reve rse phase high-pressure liquid chromatography (HPLC) on a C-18 column. Sample Assays To determine the extraction and detection ca pabilities in an artificial complex sample, equal amounts of CEM and Ramos cells were mixe d and tested using the assay. Approximately 105 cells of each type were mixed, followed by magnetic and fluorescent nanoparticle incubation for five minutes. Magnetic extraction proce dures were performed three times to remove

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107 unbound cells. A 2 L aliquot of the redispersed extracted sample was then imaged by confocal microscopy. To show applicability in r eal biological samples, whol e blood was spiked with 105 CEM cells. Fluorescent and magnetic nanoparticles were then incubated for five minutes with spiked and unspiked blood samples, followed by three ma gnetic extractions. Confocal imaging was then used to characterize cell extractions. Collection efficiency was measured from pure cell samples and spiked blood samples. For efficiency studies, cell samples subjected to na noparticle incubation a nd magnetic extractions were compared to samples not subjected to any separations by magnetic extraction. For pure cell analyses, 5 30 g of magnetic nanoparticles were individually incubated in 5 g increments with approximately 105 cells initially, and subjected to ma gnetic extractions after five minute incubation. The efficiency of cell extraction fr om the spiked blood sample was determined by incubating magnetic nanoparticles (30 g) with 500 L whole blood spiked with 105 CEM cells. Cells were counted by flow cytometry for pure samples, and by imaging for blood samples. Various magnetic nanoparticle concentrations were used to determine maximum collection efficiency and optimal separation efficiency. Cell Imaging Fluorescence imaging was conducted with a conf ocal microscope setup consisting of an Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and three lasers, a tunable Argon Ion laser (458nm, 488nm, 514nm), a green HeNe laser (543nm), and a red HeNe laser (633nm) with three separate photomu ltiplier tubes (PMT) for detection. The cellular images were taken w ith a 10x objective. The Rubpy NPs were excited with 488nm line of the Argon ion laser and em ission was detected using a 610nm long pass

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108 filter. The TMR NPs were excited with the 543n m laser line and were detected with a 560600nm band pass filter. The Cy5 NPs were exci ted with the 633nm laser line and the emission was detected with a 660nm long pass filter. Flow Cytometry Fluorescence measurements were also made using a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). To support imaging data, Rubpy fluorescence of pure sample s initially containing 105 cells were measured by counting 30000 events. Cell experiments were performed exactly as stated for imaging experiments, except all solutions were diluted to a final volume of 200 L. Cell sorting allowed for accurate quantitative analysis of cell samples, as well as a plat form for collection efficiency determination. Microplate Reader All plate reader experiments were conducted with a Tecan Sa fire microplate reader with 384 well Corning small volume plates. The exci tation and emission wavelength used were the same as those in the fluorescent imaging. For each experiment 20 L of the extracted cell solution was placed in the well and the fluoresce nce of the sample was measured immediately. Magnetic Extraction and Labeling To establish the extraction a nd detection capabilities of th e method, equal amounts of cells in media were tested using the two NP approac h. When using the CEM cells as the target the Ramos and Toledo cell types were used as the co ntrol (nontarget) cells, when the Ramos cells were used as the target the CEM and Toledo cell t ypes were used as the co ntrol cells, and finally when using the Toledo cells as the target th e CEM and Ramos cell types were used for the control experiments. The extraction of the multiple individual cell types (CEM, Ramos, and Toledo) using their respective aptamer conjugated NPs was performed by the following procedure. Approximately

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109 105 of each cell type were obtained in individu al test tubes. To the cell samples, 5 L of the MNP solution was added, and the mixture was inc ubated for 15 min. After incubation, the cells were washed by magnetic extraction with 200 L of fresh cell media three times, and resuspended in 200 L of the media buffer. The wash was performed by removal of the supernatant, addition of fresh buffer, and the sa mple resuspended in the fresh buffer typically with in 3-5 minutes. To complete the stepwise process, 2 L of FNPs were added and incubated for 5 min. The concentration of MNPs to FNPs in the samples was 2:1. Again the sample was washed three times with 200 L of cell media as described prev iously, and then dispersed in 20 L of media for imaging and microplate reader an alysis. The FNPs used to label the Ramos cells were doped with cy5, and the fluorescent dye doped in the NPs for Toledo and CEM cells were Rubpy and TMR, respectively. All pure cell samples contained 1.0 x 105-5.0 x 105 cells before NP incubation. The multiple cell type extr action procedure will be described in a later section. For determining the detection limit, the extr action was performed by first determining the number of cells in 1 L of stock cell solution. The total number of cells was counted. This process was repeated five times, the determined va lues were averaged, and extrapolated to obtain the number of cells per microliter. The cell samples were diluted to 200 L with cell media accordingly. Next, cell samples were subjected to MNP incubations for 15 minutes followed by magnetic extraction and washing as described pr eviously, and FNP incubations for 5 minutes with magnetic extraction and washing as describe d above. All cell samples were treated with MNPs and FNPs at a ratio of 2:1 using the stepwi se format, and extracted samples were analyzed by the microplate reader.

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110 Results and Discussion Aptamer-Conjugated Nanoparticle Characterization Collection efficiency Figure 6-1. The flow cytometry determina tion of magnetic nanopart icle collection and separation efficiencies for target (CEM) and control (Ramos) cells. The collection efficiency was obtained by incubating increasing amounts of magnetic nanoparticles (MNPs) with the CEM and Ramos cel ls. The samples were extracted and counted using the flow cytometry by the counting of signal events. A 200 L aliquot of the target and control cell samples were counted before extracti on, and these values were used as the reference cell amounts in each 200 L volume. The collection efficiency was calculated by dividing the number of cells collected by the reference ce ll number. Figure 6-1 di splays the collection efficiency of target (CEM) cells and control (Ram os) cells. The collection efficiencies for target cells increased from 30-80% and plateau at ar ound 80%. The collection efficiencies for the control cells were no higher than 5% for th e maximum amount of MNPs added. This data indicates that the CEM cells are preferably extracted with litt le nonselective extraction of the

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111 control cells. The 10 L magnetic nanoparticle sample had the highest separation efficiency with minimal nonselectivity for that sample. Theref ore, this amount of MNPs was used for the remainder of these experime nts unless otherwise noted. Dye and nanoparticle fluorescent intensity comparison The increased fluorescent signals of the aptamer-conjugated Rubpy doped nanoparticles were compared to individual Rubpy dye mol ecules linked to the DNA aptamer. Equal concentrations of magnetic a nd Rubpy nanoparticles (0.5 nM) we re incubated with the CEM cells, then washed by magnetic extraction with 500 L media buffer three times, and redispersed in 20 L buffer for imaging and 200 L buffer for flow cytometric analysis. Figure 6-2 A and Figure 6-2. Fluorescence images and flow cytometry data of Rubpy dye and Rubpy doped nanoparticle treated cells. A) Magnetic nanopa rticle extracted cells treated with 40 M Rubpy dye-aptamer conjugates. B) Magne tic nanoparticle extr acted cells treated with 0.5 nM Rubpy nanoparticle-aptamer conj ugates. C) Flow cytometry comparison of dye labeled cells to nanoparticle labeled cells extracted using magnetic nanoparticles.

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112 Figure 6-2 B show the extracted cell samples extr actions labeled with fl uorescent nanoparticles and with Rubpy dye, respectively. An observable signal difference was s een in the confocal images. The flow cytometry data (Figure 62 C) confirmed the Rubpy nanoparticles provided increased fluorescence. Figure 62 C indicated over a 100-fold si gnal difference produced by the Rubpy nanoparticle labeled cells. This figure also shows the nanoparticle labeled cells in an apparent bimodal distribution. These experime nts illustrated the advantage of using the fluorescent nanoparticles compared to the individual fluorophores. Nanoparticle selectivity CEM and Ramos cell solutions were treated with the two particle procedure and the samples analyzed by fluorescence imaging and flow cytometric. Fluorescent and magnetic nanoparticles were added to the cell solutions at a 20:1 ratio, respectively. After five minutes, the cells were magnetically extracted and washed as described, and the samples suspended in 20 L buffer for imaging and 200 L buffer for flow cytometric analyses. The cell samples contained 1.0 x 105 5.0 x 105 cells before nanoparticle trea tment. These experiments were repeated 10 times. Figure 6-3 contains 2 L aliquots of target cells (A), and control cells (B) after five minute incubation. This experiment demonstrates a notable differe nce in the amount of ce lls collected and the fluorescent signal obtained for these samples. The magnetic collection approach removed few control cells while a large numbe r of target cells were extract ed. In addition, the extracted control cells were labe led with only a few FNPs producing li ttle or no fluorescent signal. The target CEM cells that were extracted had inte nse fluorescent signals that made them easily distinguishable from the control cells. The flow cytometry data confirmed the results obtained

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113 by confocal imaging Figure 6-3 C. There were little cells co unted in the control samples compared to the target samples. Additionall y, a large signal was observed for the CEM cells. Figure 6-3. Fluorescence images and flow cytometr y data of target (CEM) and control (Ramos) cells extracted using aptamer conjugatedmagnetic nanoparticles and labeled with aptamer conjugatedRubpy nanoparticles. A) Fluorescence image of CEM cells extracted using magnetic nanoparticles a nd labeled with Rubpy nanoparticles. B) Fluorescence image of Ramos cells tr eated with the aptamer conjugatednanoparticles. C) Flow cytometry compar ison of the CEM and Ramos cells treated with the aptamer-conjugated nanoparticles. Single cell type extractions To expand the concept of the two particle -based magnetic collection and detection technique to include three di fferent leukemia cell lines by us ing three different aptamer molecules for three different cell lines. CEM, Ramos, and Toledo cell samples were extracted using ACNPs followed by fluorescent imaging and an alysis by the microplate reader. Each pure cell sample extraction was repeated 10 times. As was mention in the methods section, the control cells used for the CEM experiments we re the Ramos and Toledo cell types, for Ramos were the CEM and Toledo, and for the Toledo we re the CEM and Ramos. Figure 6-4 shows representative conf ocal images of 2 L aliquots of the CEM target cells (left) and Ramos nontarget cells (right) using CEM ACNPs (red) (A), Toledo target cells (left) and CEM nontarget cells (right) using Toledo ACNP s (green) (B), and Ramos target cells (left) and CEM nontarget

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114 cells (right) using Ramos ACNPs (blue) (C) after NP incubations and magnetic washes. The figure indicates Ramos, CEM, and CEM as the resp ective controls for those experiments. The Figure 6-4. Fluorescence images of pure cell sa mples in buffer after magnetic extraction. A) Fluorescence image of CEM nanoparticle treate d samples with target cells on the left and Ramos nontarget cells on the righ t. B) Fluorescence image of Toledo nanoparticle treated samples with target cells on the left and CEM nontarget cells on the right. C) Fluorescence image of Ramos nanoparticle treated samples with target cells on the left and CEM nontarget cells on the right. A B C A B C

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115 other control cell type experiment s were performed and resulted in the same responses as the one presented (data not shown). In addition, some fluorescence spots were obs erved in the images. However when the samples were analyzed with the microplate reader and compared to sample blanks treated with the MNPs and FNPs, the leve ls of fluorescence signal were the same (images not shown). Table 6-1 provides the fluorescence data obtained from the microplate reader. The first column represents the cell sample that was analyze d, the second column represents the signal produced by the CEM NPs, the third column represents the signal produced by the Toledo NPs, and the fourth column represents the signa l produced by the Ramos NPs. The rows in the table display the cell samples that were investigated using the ACNPs. Table 6-1. The microplate reader data for the evaluation of singl e cell type extraction experiments. Sample cells CEM NP signal Toledo NP signal Ramos NP signal Ramos 945 965 7,574 CEM 48,967 1,056 314 Toledo 1,075 36,728 438 Based on the fluorescence images a significant di fference is evident in both the amount of cells extracted and fluorescent signal present between the target and control cells in all samples. However, some control cells that were inadverten tly collected and even labeled with some FNPs, but no significant signal was indi cated by the microplate reader da ta for those samples producing signals in the same realm as sample blanks (image s not shown). On the c ontrary, the ta rget cells subjected to this procedure had very intense fluor escent signals that made them easily discernible from the control cells. A closer look at the characterization of expanding the ACNP technique to multiple cell types the microplate reader data (Table 6-1) demonstrated that when using 100,000

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116 cells in each of the pure cell samples at a collect ion efficiency of 85%, all target cell samples produced signals in upwards of 24-fold enhancements above the background and as high as 50fold. The target samples indicated in Table 6-1 for this experiment were the CEM cells (row 2) for the CEM NPs (column 1), the Toledo cells (row 3) for the Toledo NPs (column 2), and the Ramos cells (column 3) for the Ramos NPs (c olumn 3). The control samples for these experiments were represented in the remainder of the table for each of the NPs and cell types. The signals for the control samples at the c onditions mentioned above and the collection efficiency for the MNP amounts used in these expe riments for the control cells determined to be no greater than 5%, resulted in fluorescence signals at the same level as a buffer blank sample treated with the ACNPs. This data indicates that the MNPs we re both selective for the target cells by discriminating against the control cells and repr oducible in all sample types investigated. Detection Limit The limit of detection (LOD) was determined using pure cell samples, and the extractions were performed as described previously. The lim it of detection threshold was taken to be three standard deviations above the blank, and because of this any residual fluorescence in the blank was accounted for. The LOD was performed using CEM target cells. Each of the samples was then analyzed with NPs using the previously me ntioned protocols with th e fluorescence intensity being determined on the microplate reader follo wing completion of the ACNP technique. The detection limit was computed by plotting the fl uorescence intensity versus the cell number present in the sample. Consequently, the plo tted data produced a linear response as seen in Figure 6-5 A, and Figure 6-5 B disp lays the data zoomed in on the lower sample concentrations. From this plot the detection limit was determin ed to be approximately 250 cells with a dynamic range covering more than two orde rs of magnitude. This indicates that the ACNP system has the ability to sensitively det ect low amounts of intact targets cells from a given sample, and that a

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117 wide range of cell concentrations can be anal yzed by this method with little to no sample preparation depending on the amount of ACNPs used. Figure 6-5. The limit of detection experiments for the stepwise addition of the magnetic and fluorescent nanoparticles using the micropl ate reader for detection. A) The full calibration curve obtained using the two nanoparticle approach. B) An inset displaying a zoomed image of the lower concentration range. Complex Sample Extractions Single type cell mixed sample extraction Complex samples were tested to determine ex traction and detection capabilities of the two particle approach in complex matrices. Figure 6-6 shows the results from a mixed cell sample extraction, where equal numbers of CEM and Ramo s cells were mixed together. The two cell types were differentiated by trea ting the Ramos cells with Fluo-4, which is a fluorescent calcium A B 45000 40000 35000 30000 25000 20000 15000 10000 5000 0Fluorescence Intensity 50000 40000 30000 20000 010000 Number of Cells 6000 5000 4000 3000 2000 1000 0 0200040006000 A B 45000 40000 35000 30000 25000 20000 15000 10000 5000 0Fluorescence Intensity 50000 40000 30000 20000 010000 Number of Cells 6000 5000 4000 3000 2000 1000 0 0200040006000

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118 ion indicator. Fluo-4 labeled co ntrol cells were mixed with th e unlabeled CEM cells, and the data is shown in Figure 6-6 A. The mixed cell sample was treated with the magnetic and fluorescent nanoparticles simultaneou sly, and the sample incubated at 4 C for five minutes. A magnetic field was applied to remove the unbound cells from the sample, where 2 L aliquots of the extracted sample was analyzed by monito r the Fluo-4 and Rubpy fluorescence by confocal imaging, as seen in Figure 6-6 B and C respect ively. The image indica ted that the magnetic nanoparticles were able to collect the CEM ce lls, and the fluorescence signal from the Rubpy nanoparticles made them easily distinguishable from the control samples. This experiment was Figure 6-6. The confocal images for single cel l mixed extraction samples. A) The confocal image of a 1:1 ratio of targ et cells (CEM) mixed with Fluo-4 stained control cells (Ramos). B) Confocal image analyzing for the Fluo-4 signal after extraction. C) Rubpy signal obtained after extraction of the CE M target cells. D) Confocal images of a 1:1 ratio of Fluo-4 stained target cells mixed with control cells. E) Confocal image analyzing the Fluo-4 signal after ex traction. F) Rubpy signal obtained after extraction of the CEM cells.

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119 repeated by labeling the CEM cells with the Fluo-4 dye (Figure 6-6 D). The cells were separated by the magnetic nanoparticles as se en in Figure 6-6 E, and all th e extracted cells exhibited the Fluo-4 signal. In Figure 6-6 F shows the simu ltaneous labeling of the extracted cells by the Rubpy nanoparticles. The Fluo-4 fluorescence confirms that only the CEM cells were collected and imaged. The lack of Fluo-4 signal in Figu re 6-6 B, along with the presence of the Fluo-4 signal in Figure 6-6 E prove that the target cells were extracted using this method from 1:1 cell mixtures. These samples were repeat ed 5 times producing similar results. Multiple cell type extraction method Determining the multiple extraction and detec tion capability of the ACNP was performed by creating artificial complex sa mples of CEM, Ramos, and Toledo cells. Figure 6-7 displays the schematic diagram of the multiple cell extrac tion procedure that was employed. The samples with one, two, and three cell types were analyzed using the ACNPs. The samples were prepared by obtaining approximately 105 cells of each type for the respective sample type. The stepwise extraction protocol was performed by adding th e specified amounts of MNPs for Ramos cells, followed by CEM aptamer-conjugated MNPs, and fi nally with Toledo specific MNPs. Each set of MNPs were incubated with the cell sample s separately for 15 minutes. After the Ramos MNPs were incubated with the cell samples, magnetic extraction was performed, and the supernatant kept to be treated with the CEM specific MNPs. The remainder of the magnetic extractions was carried out as described in th e magnetic extraction section. The sample was redispersed in 200 L cell media, followed by addition of the Ramos aptamer-conjugated FNPs with 5 minute incubation, and magnetic extrac tion procedure performed. Similarly, the respective CEM and Toledo aptamer-conjugated FNPs were subsequently introduced to their samples. After the final wash, the cell sample was dispersed in 20 L media buffer. The

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120 samples were analyzed by confocal imaging with 2 L aliquots and plate reader spectrometer with 20 L aliquots. Figure 6-7. The schematic diagram used for the extraction of multiple cells. Multiple cell type mixed ce ll extractions-buffer samples The power of the multiple extraction procedur e needed to be evaluated using complex sample mixtures. Figure 6-8 A, B, and C demo nstrates the results from artificial complex samples by mixing equal amounts of the appropriat e cell types for the th ree different multiple extraction samples diluted in cell media buffer, where CEM, Ramos, and Toledo cells were mixed and the ACNP process applied as describe d above. A total of 100,000 cells in all samples was used, cell and buffer volumes were adjusted a ccordingly. To exhibit that the MNPs indeed have the ability to selectively differentiate the cells from one another in a multiple cell mixture format, single, double, and triple cell mixed samples were evaluated. Figure 6-8 A illustrates the selective nature of the technique by performing the ACNP steps with a single cell sample, Ramos cells. The single cell sample was first treat ed with CEM ACNPs followed by Toledo ACNPs, and finally Ramos ACNPs. The samples were in cubated at 4 C with the MNPs and FNPs as expressed in the previous section. Based on th e fluorescence images, this method was able to

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121 selectively collect the Ramos cells (blue) only when the Ramos ACNP were introduced to the cell sample Figure 6-8 A. The Toledo and Ramo s cells were used in single cell sample Figure 6-8. The confocal images of buffer and serum extracted mixed cell samples using the multiple extraction procedure. A) Confocal image of sample containing only Ramos cells in buffer. B) Confocal image of sample containing CEM and Toledo cells in buffer. C) Confocal image of sample c ontaining CEM, Toledo, and Ramos cells in buffer. D) Images of samples containing only Ramos cells in serum. E) Images of samples containing CEM and Ramos cells in se rum. F) Images of samples containing CEM, Toledo, and Ramos cells in serum. extractions as well (image not shown). This method was further tested by performing the ACNP steps with a mixture of two di fferent cell types, CEM and Tole do. Figure 6-8 B displays the selective nature of this technique for the cel ls indicated. The fluorescence images again demonstrate selective isolation of the CEM (red) and Toledo (gr een) cells. Other CEM, Toledo, and Ramos two cell type mixed samples were perfo rmed as well (image not shown). The final test was to perform this technique with a mixture of all three cell types in the same sample using the CEM, Toledo, and Ramos cells at the same time. Figure 6-8 C reveals the selective nature of the method for each of the cells indicated. Fl uorescence images again depict the selective CEM NPToledo NP Ramos NP CEM NPToledo NP Ramos NP A B C D E F CEM NPToledo NP Ramos NP CEM NPToledo NP Ramos NP A B C D E F

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122 isolation of the CEM, Toledo, and Ramos cells, Fi gure 6-8 C left (red), middle (green), and right (blue) images respectively. Table 6-2. The microplate reader data obtained from buffer extracted cells by the multiple cell type extraction procedure. Sample cells CEM NP signa l Toledo NP signa l Ramos NP sign al Ramos 1,281 1,040 7,862 CEM 44,972 920 375 Toledo 1,025 34,972 320 CEM, Ramos 46,874 2,367 7,385 CEM, Toledo 43,890 37,896 414 CEM, Toledo, Ramos 42,145 32,945 7,524 Microplate reader data was in complete ag reement with the confocal image data and presented in Table 6-2. The firs t column represents the cell samples that were analyzed, the second column represents the si gnal produced by the CEM NPs, th e third column represents the signal produced by the Toledo NPs, and the fourth column represents the signal produced by the Ramos NPs. The rows in the table display the cell samples that were investigated using the ACNPs. With 100,000 total cells present in all samples, samples containing target cells produced signals in upwards of 24-fold enhancements above the background and as high as 47fold with the exception of the Toledo nontarget sample, sample 4. The signals for the control samples at the conditions mentioned above resulted in fluorescence signals at the same level as a buffer blank sample treated with the ACNPs w ith the exception of the Toledo nontarget sample in sample 4. Standard deviations determined fo r all these samples were determined to be 8-12%.

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123 This data indicates that the MNPs were both se lective for the target cells by discriminating against the control cells and repr oducible in all sample types inve stigated. However, the Toledo nontarget sample in sample 4 produced a bac kground signal above the background observed for other Toledo nontarget samples. The Toledo apta mer is less selective then the other aptamers that were used, which would explain the higher background produced in this particular sample. Multiple cell type mixed extractions-serum samples To show applicability of the stepwise proce ss in real biological samples, fetal bovine serum (FBS) was used. FBS was spiked with each of the respective cell types for the corresponding one cell, tw o cell, and three cell ex traction experiments (500 L). The process was performed as described above. Confocal imaging and fluorescence microplate reader were used to characterize cell extractions. Figure 6-8 D, E, and F illustrate the results of the FBS spiked complex samples by mixing equal amounts of the indicated cells at a total cell concentration of approximately 100,000 cells. Figur e 6-8 D, E, and 4F present the selective nature of the technique for the single, double, an d triple mixed cell type samples. The samples were treated with CEM ACNPs followed by Toledo ACNPs, and finally Ramos ACNPs. For the single cell sample experiment, the sample wa s first treated with CEM ACNPs followed by Toledo ACNPs, and finally Ramos ACNPs. The sa mples were incubated at 4 C with the MNPs and FNPs as expressed previously. Figure 6-8 D fluorescence image shows the sample contains Ramos cells extracted and labeled only afte r being treated with Ramos ACNP (blue). Extractions with the CEM and Toledo cell types were completed as well (images not shown). Figure 6-8 E left image (red) and right image (blue) show the CEM cells and Ramos cells extracted when treated with CEM and Ramos ACNP for the two cell type extraction experiment. Other two cell type extractions with the CEM, Toledo, and Ramos cell types were performed as

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124 well (images not shown). Figure6-8 F left (red), middle (green), and right (blue) images show the extraction of all three cells treated with all the ACNP. The fluorescence imaging data was confirme d by collecting fluores cence data using the microplate reader, Table 6-3. The table layout wa s the same as in the previous table: first column was cell samples, second column was th e CEM NP signals, third column was Toledo NP signals, and fourth column was the Ramos NP signa l. The rows in the table display the cells mixed to make the samples that were analyzed. The standard deviations determined to be 8-12% for all samples measured in the FBS. With 100,000 total cells present in each sample dispersed in FBS, the signal enhancements determined above the background ranged from 10 to about 24. In all cases, the signals for all ta rget samples were lower than t hose for the cell media buffer, and the background signals were all higher. The Toledo samples produced the lowest enhancement of all the extracted samples, which produced the highest background signal of the three ACNPs pairs. The fluorescence images and microplate r eader data demonstrated that the MNPs were both selective for the target ce lls by discriminating against the control cells and reproducible even in spiked FBS samples. The performan ce of the Toledo aptame r with nontarget samples was again evident in FBS compared to the ot her aptamers, which would be further evidence pointing to the lower selectivity of th is aptamer compared to the others. Table 6-3. The microplate reader data obtained from serum extracted cells by the multiple cell type extraction procedure. Sample cells CEM NP signa l Toledo NP signa l Ramos NP signa l Ramos 1,845 3,241 6,776 CEM, Ramos 43,835 3,554 6,980 CEM, Toledo 40,767 31,240 452 CEM, Toledo, Ramos 42,973 33,112 7,078

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125 Whole blood sample assays Figure 6-9. The confocal images of the cells extr acted from whole blood. A) Confocal image of cells extracted from target cell spiked w hole blood. B) Image of aptamer conjugatednanoparticle treated unspiked whole blood samp les. C) and D) Magnified images of extracted cells from whole blood spiked samples. For further determination of complex biologi cal solution extraction, spiked whole blood samples were investigated. The aptamer sequen ce used was stable in serum for up to 2 hours based on performed control experiments. The targ et cells were spiked in to whole blood samples (500 L). This sample was compared to unspiked samples. Figure 6-9 indicated the

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126 nonselective extraction of red blood cells from a ll samples. However, there was no fluorescence signal observed by the presence of the FNPS on the nontarget cells. For the spiked whole blood samples, 40% of the spiked target cells were r ecovered. This was estimated after accounting for washing and dilution, which is comparable to extraction efficiencies reported by immunomagnetic separation.194, 196 These experiments were repeated five times and similar results were obtained for each sample. Conclusions ACNPs were demonstrated to selectively extract and detect multiple intact target cells from complex samples with limited sample prepara tion. The inherent advantages of the ACNP include the aptamer-conjugated FNPs produce hi gh signal intensity and signal stability, and aptamer-conjugated MNPs allow for the selectiv e extract of analytes. Magnetic extraction allows for the easy of sample clean-up by removing excess FNPs and other unbound fluorescent materials resulting in a lower background. Empl oying magnetic extractions to such a system provides a separation and scavenging capability that is unlike any other method available in that the MNPs can be introduced to the sample of interest, locate the ta rget, and remove the bound substance from the remainder of the sample. This isolation step can also be used to enrich or concentrate the sample after removal of the unw anted sample components with provided ease of washing due to the ability to manipulate the bound samples with a magnetic field. The MNPs have also demonstrated high coll ection efficiencies for biologi cal species. The detection limit studies resulted in a wide ranged linear response with a good detection limit. The ACNP detection process has been performed relatively quickly in as little as 30 minutes in complex samples of FBS and whole blood containing target cells, as opposed to the hours needed for immunophenotyping or PCR-based met hods. Therefore, the ACNP procedure has been used as a rapid detection method for cance r detection. Nanoparticle bound aptamers retain their binding

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127 properties with the intact cel ls. Using the two nanoparticle s add an additional level of selectivity, where only cells th at are magnetically extracted and are bound with FNPs are recognized as target cells.

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140 BIOGRAPHICAL SKETCH Joshua Elliot Smith was born in Clari on, PA on August 21, 1976 to John and Dorothy Smith. He was raised in Rimersburg, PA and graduated in 1995 from Union High School. He entered Clarion University of Pennsylvania (C UP) to study chemistry and mathematics. After Josh received his Bachelors of Science and B achelors of Education in Chemistry and Mathematics from CUP in May 2002, he began hi s doctorate study under the supervision of Dr. Weihong Tan at the University of Florida in August that year. He earned his Doctorate of Philosophy in Analytical Chemistry in August 2007.