Citation
Phage Display Techniques for Selection of Inorganic Binding Peptides with Electroactive Properties for Biosensor Applications

Material Information

Title:
Phage Display Techniques for Selection of Inorganic Binding Peptides with Electroactive Properties for Biosensor Applications
Creator:
Yeh, Ya-Wen
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (167 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biomedical Engineering
Committee Chair:
GOWER,LAURIE B
Committee Co-Chair:
ORMEROD,BRANDI K
Committee Members:
TSENG,YIIDER
GULIG,PAUL A
Graduation Date:
12/13/2013

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Bacteriophage M13 ( jstor )
Bacteriophages ( jstor )
Biosensing techniques ( jstor )
Chemicals ( jstor )
Electric fields ( jstor )
Elution ( jstor )
Fluorescence ( jstor )
pH ( jstor )
Sensors ( jstor )
Biomedical Engineering -- Dissertations, Academic -- UF
biosensor
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Biomedical Engineering thesis, Ph.D.

Notes

Abstract:
In biosensor devices, functionalization of the surface with covalent linkers is usually employed. However, it is difficult to avoid the loss of activity following the bioreceptor-analyte binding event, which limits the lifetime of the device. The goal of this project was to use phage display to biopan for inorganic binding peptides that are reversible upon application of an electric field. This technique could provide dynamic functionalization of surfaces, with applications such as reconfigurable and self-cleaning devices.  In this study, biopanning was performed to select for phage with displayed peptides that bind strongly to indium zinc oxide (IZO), a transparent semiconducting oxide which is an attractive electrode for biosensor applications. IZO binding phage collected by two different chemical elution methods, low pH or high salt elution buffer, were identified. It was found that phage clones eluted with high salt buffer were more selective in their binding to IZO than the clones eluted by low pH buffer. An electro- releasing device was then developed to select from those clones the ones that could be released upon application of an electric field.  In an alternative method, because a strong binding peptide may not be a reversible peptide, a new biopanning protocol was developed with an electro-elution process instead of the regular chemical elution. Using a small electronic device in the biopanning media, phage that desorb upon application of a field could be collected directly. Clones selected by electro-elution displayed a different composition of amino acids as compared to those from the chemical elution approach.  Based on the studies of phage clones selected from chemical elution and electro- elution biopanning processes, the amino acid sequences of the displayed peptides were then synthesized to further test the binding and release properties of the peptides in isolated form.  The binding affinity of the synthetic IZO binding peptides was confirmed. It was found that both the charge character and the secondary structure of a peptide seemed to play a role in affecting the electroactive properties. In conclusion, my work demonstrates that phage display with a novel electro-elution step can select peptides that are more sensitive to an electric field. ( en )
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.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: GOWER,LAURIE B.
Local:
Co-adviser: ORMEROD,BRANDI K.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-12-31
Statement of Responsibility:
by Ya-Wen Yeh.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
12/31/2015
Classification:
LD1780 2013 ( lcc )

Downloads

This item has the following downloads:


Full Text

PAGE 1

1 PHAGE DISPLAY TECHNIQUES FOR SELECTION OF INORGANIC BINDING PEPTIDES WITH ELECTROACTIVE PROPERTIES FOR BIOSENSOR APPLICATION S By YA WEN YEH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PA RTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

PAGE 2

2 2013 Ya Wen Yeh

PAGE 3

3 To my parents

PAGE 4

4 ACKNOWLEDGMENTS First and foremost, many thanks should be given to my advisor Dr. Laurie Gower for her mentorship and support over the past five years. I appreciate her patience as I explored this research and her encouragement whenever I met with some difficulties. Without all her support and direction, this work wou I would also like to thank my committee members, Dr. Paul Gulig, Dr. Yiider Tseng, and Dr. Brandi Ormerod for all their advice and help. I especially appreciate Dr. Gulig for his great help on my experiment, scientific thinking, and his valuable advice and time. I also want to thank his students for their help in his lab, especially Julio Martin. I would like to thank Dr. David Norton, his student Seonhoo Kim and Kyeongwon Kim for their great help on designing electronic devices and int roducing me to the fabrication of semiconductors. I also thank Bill Lewis, David Hays, and Alvin Ogden for all the help in the cleanroom and NRF facility. I would like to thank Dr. Fan Ren for pointing out the most critical point for this research and all the help he rendered. I would like to thank Dr. Josephine Allen for advice and help on my experiment. I want to thank Dr. Mark Davidson for his great help on electronic devices. I would like to thank Dr. Stephen Hagen for letting me use circular dichroism at his lab. I would like to thank Marjorie Chow in ICBR for the help on Biacore system. Many thanks should be given to all the members in the Gower group, especially Chih Wei Liao for teaching me how to run phage display. I want to thank all of my labmate s for not only providing scientific help, but also being my friends and always being supportive, especially Douglas Rodriguez Taili Thula, and MyongHwa Lee I also

PAGE 5

5 appreciate undergrads who worked with me for all their help, especially Teresa Rowe and Asa f Mor. I would like to thank all the friends I met in Gainesville. All of them are so warm and nice. My life in Gainesville would be different without any of you. Sharing everything with my girlfriends was the best time I had in Gainesville. Special thanks should be given to Yu Ciao Kuo for taking care of me. I would also like to thank all of my friends back in Taiwan for always being so supportive. Last but not least, a great many thanks should be given to my family. I would like to thank my fianc Yi Chu ng Wang for all his care, support, and company. I would like to thank the most important people to me, my parents Ming Kuei Yeh and Pi Hsia Lin, for everything they did for me. They always reserve the best for me and dedicate all they have to groom me into whom I am today. I am so lucky to have grown up in a warm and loving family. Without all the effort they put into me, I would not be the same person today.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 15 ABSTRACT ................................ ................................ ................................ ................... 17 C H A P T E R 1 INTRODUCTION ................................ ................................ ................................ .... 19 1.1 Overview ................................ ................................ ................................ ........... 19 1.2 Display Techniques for Selecting Pept ides Recognizing Specific Targets ........ 19 1.3 Screening Targets by Phage Display Technique ................................ .............. 21 1.4 In Vivo versus in Vitro Display Techniques ................................ ....................... 22 1.5 Evaluation of Binding Characteristics ................................ ................................ 23 1.6 Application of Inorganic Binding Peptides ................................ ......................... 25 1.7 App lication of Inorganic Binding Peptides Fused to Proteins ............................ 27 1.8 Nucleic acid Aptamers ................................ ................................ ...................... 30 2 SCREENING FOR INDIUM ZINC OXIDE BINDING PEPTIDES VIA PHAGE DISPLAY TECHNIQUE WITH STANDARD CHEMICAL ELUTION ........................ 44 2.1 Overview ................................ ................................ ................................ ........... 44 2.2 Background and Significance ................................ ................................ ........... 44 2.2.1 M13 Phage and M13 Phage Display ................................ ....................... 44 2.2.2 Indium Zinc Oxide ................................ ................................ ................... 48 2.3 Materials and Methods ................................ ................................ ...................... 49 2.3.1 Materials ................................ ................................ ................................ .. 49 2.3.1.1Sapphire substrate ................................ ................................ .......... 49 2.3.1.2 IZO target for sputtering ................................ ................................ 50 2.3.1.3 P hage d ispl ay p eptide l ibrary kit ................................ .................... 50 2.3.1.4 Buffer solutions ................................ ................................ .............. 50 2.3.1.5 Elution solutions ................................ ................................ ............. 51 2.3.1.6 Culture medium ................................ ................................ .............. 51 2.3.1.7 Stock solution ................................ ................................ ................. 52 2.3.2 Methods ................................ ................................ ................................ ... 52 2.3.2.1 Fabrication of IZO thin films for biopanning targets ........................ 52 2.3.2.2 Phage display protocol ................................ ................................ ... 53 2.3.2.3 Blue/White scre en protocol and DNA sequencing ......................... 56 2.3.2.4 Enzyme linked immunosorbent assay (ELISA) .............................. 60

PAGE 7

7 2.3.2.5 Immunofluorescence (IF) analysis ................................ ................. 61 2.3.2.6 Calculation of surface coverage of phage clones ........................... 62 2.4 Results and Discussions ................................ ................................ ................... 62 3 SCREENING FOR INDIUM ZINC OXIDE BINDING PEPTIDES WITH ELECTROACTIVE PROPERTIES USING A NOVEL EL ECTRO ELUTION APPROACH TO PHAGE DISPLAY ................................ ................................ ........ 87 3.1 Overview ................................ ................................ ................................ ........... 87 3.2 Background and Significance ................................ ................................ ........... 88 3.2.1 Biosensors ................................ ................................ ............................... 88 3.2.2 Monolayers used for Sensing Dev ices ................................ .................... 89 3.2.3 Electrodesorption ................................ ................................ .................... 91 3.3 Materials and Methods ................................ ................................ ...................... 92 3.3.1 Materials ................................ ................................ ................................ .. 92 3.3.1.1 P hage display peptide library and E.coli. ................................ ....... 92 3.3.1.2 Power supply ................................ ................................ ................. 92 3.3.1.3 Polydimethylsiloxane (PDMS) ................................ ........................ 92 3.3.2 Methods ................................ ................................ ................................ ... 93 3.3.2.1 Fabrication of IZO device ................................ ............................... 93 3.3.2.2 Phage display protocol with electro elution ................................ .... 94 3.3.2.3 Zeta potential measurement on IZO thin film ................................ 95 3 .3.2.4 Electro releasing test by immunofluorescence (IF) analysis .......... 96 3.4 Results and Discussion ................................ ................................ ..................... 96 4 CHARACTERIZATION OF SYNTHETIC INDIUM ZINC OXIDE BINDING PEPTIDES ................................ ................................ ................................ ............ 120 4.1 Overview ................................ ................................ ................................ ......... 120 4.2 Background and Significance ................................ ................................ ......... 121 4.3 Material and Methods ................................ ................................ ..................... 123 4.3.1 Materials ................................ ................................ ................................ 123 4.3.1.1 Inorganic binding peptide ................................ ............................. 123 4.3.1.2 Surface pla smon resonance measurement ................................ .. 123 4.3.1.3 Circular dichroism (CD) spectrometer ................................ .......... 123 4.3.1.4 Plate reader for fluorescence analysis ................................ ......... 123 4.3.1.5 Dicing saw ................................ ................................ .................... 124 4.3.1.6 Fluorescence microscopy of bound peptides ............................... 124 4.3.2 Methods ................................ ................................ ................................ 124 4.3.2.1 Surface plasmon resonance (SPR) measurement ....................... 124 4.3.2.2 Circular dichroism (CD) measurement ................................ ......... 125 4.3.2.3 Fluorescence intensity measurement ................................ ........... 125 4.3.2.4 Flu orescence imaging of peptide binding to substrates ............... 125 4.3.2.5 Electro releasing test by fluorescence microscopy ...................... 126 4.4 Results and Discussion ................................ ................................ ................... 126 5 CONCLUSIONS ................................ ................................ ................................ ... 150

PAGE 8

8 Future Work ................................ ................................ ................................ .......... 153 LIST OF REFERENCES ................................ ................................ ............................. 156 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 167

PAGE 9

9 LIST OF TABLES Table page 2 1 P h age titers for the I ZO target system, eluted with low pH buffer ....................... 69 2 2 P hage titers for the I ZO target system, eluted with high salt solution ................. 69 2 3 The amino acid sequence of 12 mer peptide regions displayed by IZO binding phage eluted with low pH buffer, and their binding affinity ..................... 70 3 1 Number of charged amino acid residues of peptide sequences selected by low pH elution biopanning ................................ ................................ ................ 103 3 2 Number of charged amino acid residues of peptide sequences selected by high salt elution biopanning ................................ ................................ .............. 103 3 3 Number of charged amino acid residues of peptide sequences selected by electro elution biopanning. ................................ ................................ ............... 104 4 1 Amino acid sequence of synthe tic IZO binding peptides. ................................ 134 4 2 Sequence of synthetic IZO binding peptides with their double repeat analogue. ................................ ................................ ................................ .......... 134

PAGE 10

10 LIST OF FIGURES Figure page 1 1 Principles of phage display and cell surface display techniques. ........................ 33 1 2 Principles of Ribosome display and mRNA display techni ques.. ........................ 34 1 3 Images demonstrate the specificity of material recognition of inorganic binding phage. ................................ ................................ ................................ .... 35 1 4 AFM images of 3rGBP peptide self assembled on Au ................................ ........ 36 1 5 Time lapse AFM images of GrBP5 assembly. ................................ .................... 37 1 6 Schematic showing the layer by layer assembly process of silica. ..................... 38 1 7 PEG peptide modified gold surf ace showed a bio inert surface while RGD peptide immobilized quartz/titanium resulted in a bio active surface. ................. 39 1 8 With gold binding peptide additives, gold crystals formed with platy morph ologies similar to gold formed by reducing AuCl 3. ................................ ..... 40 1 9 TEM images of gold nanoparticles synthesized with A3 dodecapeptide. ........... 40 1 10 One peptide can form gold nanoparticles with different morphologies by changing pH condition and HAuCl 4 concentration of reaction solution. .............. 41 1 11 Schematic of a pulling method to form a phage film of condensed and aligned phage. ................................ ................................ ................................ .... 42 1 12 Schematic of the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. ................................ ................................ ............................... 42 1 13 Schematic of an electrochemical sensor using a molecularly labeled aptamer. ................................ ................................ ................................ ........................... 43 1 14 Schematic of a label free electrochemical aptasensor. ................................ ...... 43 2 1 Schematic of an M13 phage showing the 5 different structural protein regions (not to scale). ................................ ................................ ................................ ...... 71 2 2 Schematic of the pVIII protein of M13 phage. ................................ ..................... 71 2 3 Phage display protocol used in the present study for the traditional chemical elution. ................................ ................................ ................................ ................ 72

PAGE 11

11 2 4 Different potential structures th at can be used for a peptide insert into pIII proteins, where randomly generated peptides are expressed within either a linear or cyclic conformation ................................ ................................ ............... 73 2 5 Life cycle of the non lytic M13 pha ge ................................ ................................ 74 2 6 Serial dilution of phage samples, as done in a 96 well plate. ............................. 75 2 7 Blue plaques displayed on an agar plate during th e blue and white experiment for identifying bacterial plaques obtained from selected and amplified phage clones. ................................ ................................ ...................... 75 2 8 Schematic of enzyme linked immunosorbent assay (ELISA). ............................ 76 2 9 Schematic of immunofluorescence (IF) analysis. ................................ ............... 76 2 10 XRD data of the IZO coated glass. ................................ ................................ ..... 77 2 11 AFM image of a representative IZO thin film. ................................ ..................... 77 2 12 12 mer amino acid sequences of peptides displayed on pIII proteins of IZO binding phages selected by phage display using low pH elution buffer. ............. 78 2 13 Images of the immunofluorescence analysis for t he negative control experiment ................................ ................................ ................................ ......... 79 2 14 IF i mage of M13KE. It is a negative control for non binding phage. ................... 79 2 15 IF analysis for t he phage clon e TKNMLSLPVGPG ................................ ........... 80 2 16 IF analysis of t he phage clone MNRPSPPLPLWV ................................ ............. 81 2 17 Surface coverage percentage of IZO binding clones panned with low pH buffer elution. ................................ ................................ ................................ ...... 82 2 18 Images of ELISA plates containing phage clones eluted from IZO with low pH elution buffer, with the corresponding substrates indicated for each row, and the 12 mer peptide indicated for each column. ................................ ................... 8 3 2 19 Absorbance of the enzymatic TMB substrate solution from ELISA plates of IZO binding clones obtained by low pH elution buffer, as shown in Figure 2 12. ................................ ................................ ................................ ...................... 84 2 20 Phot ograph of t he etched IZO that was caused by using the low pH elution buffer. ................................ ................................ ................................ ................. 84 2 21 12 mer amino acid sequences of peptides displayed on pIII proteins of IZO binding phages selected by phage display using high salt elution. ..................... 85

PAGE 12

12 2 22 Absorbance of the enzymatic TMB substrate solutions from ELISA plates of IZO binding clones obtained by high salt elution. ................................ ............... 85 2 23 IF images for t he phage clone TLMYAQPHQSKT from high salt elution. ........... 86 2 24 IF images for t he phage clone SHAPDSTWFALF from high salt elution ............ 86 3 1 Schematic of self cleaning, renewable biosensor. ................................ ............ 105 3 2 Graph of the value of biosensor market in US$ millions ................................ ... 105 3 3 Schematic of biosensor components. ................................ ............................... 106 3 4 Schematic of a dynamic patterning surface. ................................ ..................... 106 3 5 Schematic of SAMs.. ................................ ................................ ........................ 107 3 6 Schematic of different methods for preparation of organic monolayer films ..... 107 3 7 Schematic of the surface functionalization and releasing of PPS PEG from an ITO surface. ................................ ................................ ................................ 108 3 8 A plot of the thickness of the PPS PEG adlayer at different applied potentials measured by spectroscopic ellipsmetry ................................ ............................ 108 3 9 Schematic of electrodesorption of proteins.. ................................ ..................... 109 3 10 Schematic of our newly developed phage d isplay technique with an electro elution biopanning step that replaces the commonly used chemical elution, which is enabled using an electroreleasing device as the target substrate, in order to screen for peptides with electroactive properties. ............................... 110 3 11 Schematic of our design of an IZO coated electro elution device. .................... 111 3 12 The amino acid sequences of 12 mer IZO binding peptides selected by two rounds of electro elution biopanning. ................................ ................................ 112 3 13 Quantification of the amount of phage clones with the indicated peptide inserts, bound to three inorganic surfaces, as determined by absorbance of the enzymatic substrate solution from ELISA plates of clones selected from the first electro elution biopanning round. ................................ ......................... 113 3 14 Quantification of the amount of phage clones w ith the indicated peptide inserts, bound to three inorganic surfaces, as determined by absorbance of the enzymatic substrate solution form ELISA plates of clones selected from the second electro elution biopanning round. ................................ ................... 114 3 15 IF analysis of electro elution clone SRRLILQMLNRI incubated on IZO, SiO 2 and Si surfaces.. ................................ ................................ ............................... 114

PAGE 13

13 3 16 Composition percentage of each amino acid of IZO bind ing peptides from phage selected by electro elution biopanning. ................................ ................. 115 3 17 Composition percentage of each amino acid of IZO binding peptides from phage selected by chemical elution biopanning. ................................ .............. 115 3 18 Measurement of the zeta potential pH profile of an IZO thin film deposited on wafer. ................................ ................................ ................................ ................ 116 3 19 Fluorescence images of ele ctro eluted phage clone with displayed peptide MLPIIRNLIHTT before and after applying an electric field of 1000 mV for 30 seconds.. ................................ ................................ ................................ .......... 116 3 20 Schematic of an electro releasing device de signed to have a non conductive spacer to separate the areas for applying an electric field on only one side.. ... 117 3 21 Electro releasing test using electro eluted phage clone with displayed p eptide MLPIIRNLIHTT. ................................ ................................ ................... 118 3 22 Electro releasing test using chemical elution phage clone with displayed peptide: TKNMLSLPVGPG ................................ ................................ .............. 119 4 1 Schematic of an IZO coated SPR sensor chip. ................................ ................. 135 4 2 Atomic force microscopy images on the IZO coated SPR sensor chip surface. ................................ ................................ ................................ ......................... 136 4 3 SPR sensograms of a 15% sucrose solution flowed across all four flow cells (FC1 FC4). ................................ ................................ ................................ ....... 137 4 4 SPR sensogram of IZOLpH4 at different concentrations. ................................ 138 4 5 SPR sensograms of peptides selected by chemical elution biopanning. .......... 139 4 6 Binding response after peptide desorption for chemical elution peptides. ........ 140 4 7 SPR sensograms of peptides selected by electro elution biopanning. ............. 141 4 8 Binding response after peptide desorption for el ectro elution peptides. ........... 142 4 9 SPR sensograms of single repeat and double repeat peptides. ....................... 143 4 10 Binding response after pept ide desorption for single and double repeat electro eluted peptides IZOEE1, 2r IZOEE1, IZOEE4, and 2r IZOEE4. ........... 144 4 11 Representative CD spectra of IZOLpH3, which showed a random coil conforma tion, as was the case for all the peptides derived by chemical elution. ................................ ................................ ................................ .............. 144

PAGE 14

14 4 12 CD spectra of peptides selected by electro elution biopanning. ....................... 145 4 13 CD spectra of single repeat and double repeat peptides derived from electro elution biopanning. ................................ ................................ ........................... 146 4 14 Fluorescence intensity of IZOEE1 and IZOEE4 bound to the IZO s urface measured at different time points. ................................ ................................ .... 147 4 15 Fluorescence images of two regions of IZOEE4 bound to an IZO coated wafer. ................................ ................................ ................................ ................ 147 4 16 Fluorescence image of IZOHSE2 incubated with an IZO coated wafer, showing very little binding, except at the edge of the Si wafer due to structure ................................ ................................ ................................ ......................... 148 4 17 Electro releasing test of IZ OEE4 by comparing fluorescence images before and after application of the electric field.. ................................ ......................... 148 4 18 Electro releasing test of IZOLpH4 by comparing fluorescence images before and after applicat ion of the electric field. ................................ .......................... 149

PAGE 15

15 LIST OF ABBREVIATIONS A A A MINO A CIDS B/W S CREEN B LUE W HITE S CREEN B LAST B ASIC L OCAL A LIGNMENT S EARCH T OOL B SA B OVINE S ERUM A LBUMIN C D C IRCULAR D ICHROISM D I D EIONIZED E LISA E NZYME L IKED I MMU NOSORBENT A SSAY F ITC F LUORESCEIN I SOTHIOCYANATE H RP H ORSERADISH P EROXIDAE I CBR T HE I NTERDISCIPLINARY C ENTER F OR B IOTECHNOLOGY R ESEARCH I F I MMUNOFLUORSCENCE I GG I MMUNOGLOBULINS I PTG I D 1 THIOGALACTOPYRANOSID E I TO I NDIUM T IN O XIDE I ZO I NDIUM Z INC O XIDE M NPS M AGNETIC I RON O XIDE N ANOPARTICLES M S M INIMAL SALTS N RF N ANO R ESEARCH F ACILITY N CBI N ATIONAL C ENTER F OR B IOTECHNOLOGY I NFORMATION N P N ANOPARTICLE N W N ANOWIRE P DMS P OLYDIMETHYLSILOXANE P ZT P IEZOELECTRIC T RANSDUCER

PAGE 16

16 Q CM Q UARTZ C RYSTAL M ICROBALANCE Q CM D Q UARTZ C RYSTAL M ICROBALANCE D ISSIPATION S AM S ELF A SSEMBLY M ONOLAYER S PARC S ECRETED P ROTEIN A CIDIC A ND R ICH I N C YSTEINE S PR S URFACE P LASMON R ESONANCE S SDNA S INGLE S TRANDED D EOXYRIBONUCLEIC A CID S WNTS S INGLE W ALLED C ARBON N ANOTUBES T FTS T HIN F ILM T RAN SISTORS T MB 3, 5, T ETRAMETHYLBENZIDINE X GAL 5 BROMO 4 CHLORO 3 INDOLYL D GALACTOPYRANOSIDE

PAGE 17

17 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PHAGE DISPLAY TECHNIQUES FOR SELECTION OF INORGANIC BINDING PEPTIDES WITH ELECTROACTIVE PROPERTIES FOR BIOSENSOR APPLICATION S By Ya Wen Yeh December 2013 Chair: Laurie Gower Major: Biomedical Engineering In biosensor devi ces, functionalization of the surface with covalent linkers is usually employed. However, it is difficult to avoid the loss of activity following the bioreceptor analyte binding event, which limits the lifetime of the device. The goal of this project was t o use phage display to biopan for inorganic binding peptides that are reversible upon application of an electric field. This technique c ould provide dynamic functionalization of surfaces with applications such as reconfigurable and self cleaning devices. In this study, biopanning was performed to select for phage with displayed peptides that bind strongly to indium zinc oxide (IZO), a transparent semiconducting oxide which is an attractive electrode for biosensor application s. IZO binding phage collected by two different chemical elution methods, low pH or high salt elution buffer, were identified. It was found that phage clones eluted with high salt buffer w ere more selective in their binding to IZO than the clones eluted by low pH buffer. An electro rele asing device was then developed to select from those clones the ones that c ould be released upon application of an electric field.

PAGE 18

18 In an alternative method, because a strong binding peptide may not be a reversible peptide a new biopanning protocol was de veloped with an electro elution process instead of the regular chemical elution Using a small electronic device in the biopanning media, phage that de sorb upon application of a field could be collected directly Clones selected by electro elution displaye d a different composition of amino acids as compared to those from the chemical elution approach. Based on the studies of phage clones selected from chemical elution and ele c tro elution biopanning process es the amino acid sequences of the displayed pepti des were then synthesized to further test the binding and release propert ies of the peptides in isolated form The b inding affinity of the synthetic IZO binding peptides was confirmed. I t was found that both the charge character and the secondary structur e of a peptide seemed to play a role in affecting the electroactive properties. In conclusion my work demonstrate s that phage display with a novel electro elution step can select peptides that are more sensitive to an electric field.

PAGE 19

19 CHAPTER 1 INTRODUCT ION 1. 1 Overview Molecular recognition is one of the central features of biological systems. It provides the molecular basis of biological processes and delivers the specificity and diversity in biochemical reactions. Well studied systems include the work on binding events with DNA and protein or interactions between antibody and antigen, but more recently researche r s have been focused on examining molecular recognition between organic and inorganic surfaces. To enhance cell attachment on biomaterial surfa ces, growth factors, peptides, and proteins have been ionically or covalently attached to target surfaces. While proteins are difficult to purify and degrade easily, small peptides exhibit higher stability with heat treatment and pH variations. Also, pept ides have long ter m stability and are cost effective, which make them a better choice for interacting with materials for many applications [1] In the last two decades, with the utilization of display techniques, s ome breakthrough s i n understanding the interaction s between protein s and inorganic surface s have been achieved Display techniques which are based on a combinatorial approach, provide an easier way to obtain a specific protein or peptide sequence which has specific binding affinity to inorganic materials. This dissertation further explores the binding of peptide s to inorganic material s by applying phage display to a new electronic material, Indium Zinc Oxide (IZO). I n addition, i t also develops a new and no vel strategy for selecting inorganic binding peptide s with electroactive properties 1 2 Display T echniques for S electing P eptides R ecognizing S pecific T arget s Bacteriophages are virus particles that infect bacteria. The term is commonly used in its sho rtened form, phage. Since the first introduction of phage display by G.

PAGE 20

20 Smith in 1985 [2] display technologies have been confirmed to be a useful tool for biological and biotechnological applications. Study of protein ligand interactions, characterization of antibody binding and receptor binding sites, and isolation and evolution of proteins or enzymes improve binding feature for their ligands [3] Common display technologies are phage display, microbial cell surface display, ribosome display, and mRNA display, with phage display being the most utilized so far. Phage display and cell surface display are two approac hes based on the link between phenotype and genotype, but are of different organisms. Phage display uses bacteriophages as vectors while cell surface display uses bacterium or yeast [4] By genetic engineering of bacteriopha ge genomes or bacterium plasmids, a randomized gene region can be created to obtain a library [5] And this random peptide sequence will be expressed on the surface of a phage coat protein or a bacteria l flagella protein to achieve the display (Figure 1 1). In the case of pha ge display, b y incubating the phage library with a specific target, washing away unbound or we a k binders, and collecting the bound ones, several strong binding phage clones can be collected and amplified using their bacterium host. Then the peptide sequen ce s with high binding affinity can be identified by DNA sequencing of the selected phage clones If one wishes to have high selectivity between different materials, the phage can be selected from a pool of strong binder clones, those that only bind to the specific target of interest (such as by using immunofluorescent screening on the different surfaces). Ribosome display and mRNA display are both in vitro selection techniques and scription of the DNA library to produce mRNA. Ribosome display has a random peptide fold, and it

PAGE 21

21 displays on the ribosome by fusing the DNA library to a C terminal spacer region lacking making a complex of protein ribosome mRNA (ARM). Once the complex interacts and binds with an immobilized target, mRNA can be dissociated; and by reverse transcription, the sequence of the peptide can be determined [6] The main difference between ribosome display and mRNA display is that for mRNA display, a DNA spacer linker called puromycin is involved. It forms a covalent link between the RNA and polypeptide. Also the mRNA polypeptide complex will be released from the ribosome for further screening steps (Figure 1 2) [7] Ribosome display and mRNA display are believed to be the next generation of display technologies for antibody screening due to the fact that they have a larger library size (up to 10 15 ), less expression bias, and a cell free system [6] 1 3 Screening T argets by P hage D isplay T echnique Display technique s have been wid e ly used to identify peptide binders for both organic and inorganic targets. Using phage display for selection of human antibodies is one of the most successful applications. It is used to isolat e monoclonal antibodies from large collections of antibody fragments. Adalimumab, named Humira is a human IgG1 specific for human tumor necrosis factor (TNF) [8] and has been approved by the Food and Drug Ad m i n istration ( FDA ) for arthritis treat ment while there are many more antibodies in clinical trials now [9] Phage display is also used to identify sequences for discovery of enzyme substrates [10, 11] and inhibitors [12, 13] to target cell s to identify protein protein interaction s [14, 15] to design vaccine s [16] and to map epitope s of an antigen [17] Some tissues have also been screened for binding peptides [18] such as muscle s [19] kidney tubules [20] bone marrow [21] and cartilage [22]

PAGE 22

22 In addition to the biopolymer applications mentioned above, m any research groups have used display techniques to identify peptide sequences that have strong binding affinity for various materials. Organic targets include, polyvinyl chloride, polystyrene [23] and poly(methyl methacrylate) [24] Inorganic targets include carbon based material ( carbon nanotube s [25] ), noble metals (Au [26 28] Ag [29, 30] and Pt [31] ), metal oxides (SiO 2 [32] ZnO [33] Cu 2 O [33] TiO 2 [34 37] Fe 2 O 3 [38] ), minerals ( hydroxyapatite [39] calcite [40] sapphire [41] ) and semiconductors (GaAs [42] ZnS [43] CdS [43] ) [44] The sequence of amino acids with specific binding is defined by as a geneticall y engineered peptide for inorganics (GEPIs) because a directed evolution is used in the biopanning approach The target substrate can be rough like powders, or well defined like a single crystal or a nanostructure [45] Phage can be very selective i n their recognition of different materials which would seemingly have similar surface properties For example, F igure 1 3 shows fluorescently labeled GaAs clones on a patterned surface. The clones recognized GaAs surface specifically but not the SiO 2 surface The clones were element specific that bind to GaAs but not AlGaAs [42] In addition to solid surface s phage display can also be used to identify volatile compounds. For example, 2 4 6 trinitrotoluene (TNT) and 2,4 dinitrotoluene (DNT) have been screened by phage display for selective detection of explosives [46] 1. 4 I n V ivo v er s us i n V itro D isplay T echniques As men tioned in section 1. 3, in vivo display techniques like phage display and cell surface display rely on the gene modification of vectors to create a library. One of the main disadvantages is the process in which phage s or cell s uptake naked DNA, resulting in a library which contains only up to 10 13 varieties [3] Another drawback is that phage display and cell surface display are more sensitive to bias. The fact that

PAGE 23

23 phages need to infect host bacteri a to amplify leads the possibility of poor infectivity or los s of some clones even though they may have been strong binders to the target of interest Also, some phages tend to yield a larger progeny than other phages, which causes some clones to be identified repeatedly. Howev er, even though in vivo display as high a level of expertise as ribosome display and mRNA display. Therefore, c ommercialized phage display and cell surface display library kits make them the most co mmon ly used display techniques [3] For screening of inorganic target material s even though both phage display and cell surface display can be applied for most of the cases, the structure of the target material should be considered to select a suitable technique. For example, when biopanning on materials in powder form, a centrifuge step is required to separate the eluted phages and powders. Phage display would be a better choice since the flagella might be shear ed off from the cell by centrifugal forces [3] 1. 5 Evaluation of B inding C haracteristic s Although peptides with specific binding have been identified for many different inorganic materials, how peptides recognize inorganic su rfaces remains unclear. The mechanism of the specificity in binding may be either by chemical or structural recognition or both, such as H bonding, polarity, charge effects, or conformation of peptide versus structure and morphology of the inorganic [45] To understand the nature of the binding event c haracterization of the chemistry and structure of the binders and the inorganic surface properties have become important issue s When powders of various particle sizes and shapes are used for biopanning, the sequence is expected to be more diverse due to t he variation of local surface structures On the other hand,

PAGE 24

24 materials of carefully controlled size, crystallography or morphology may select peptides with higher sequence homology [3] There are many useful techniques t o qualitatively and quantitatively rank the affinity and specificity for peptide attach ment to surfaces. To qualitatively rank the binding, immunofluorescence (IF) analysis and enzyme linked immunosorbent assay (ELISA) are the most common methods to assess the affinity levels of individual phage clones. Scanning probe microscopy (SPM), like atomic force microscopy (AFM) [47] and scanning tunneling microscopy (STM), can also be utilized to investigate the peptide assembly. Surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) are used to quantitatively characterize the peptide adsorption kinetics [48] To develop an understanding of the organic inorganic molecular recognition liquid and solid state nuclear magnetic resonance (NMR) spectroscopy can help to understand the molecular structure of the peptide in solution and as it interacts with the surface Along w ith such experimental result s simulation is a useful tool for characterization the interface between peptide and inorganic surface. The conformation and orientation of peptides can be simulated for be tter understanding of the affinity and selectivity of identified peptides sequences based on the energy landscape [49] A bioinformatics approach has also been developed to computationally design new peptides with high er binding efficiency to a target material based on the sequence similarities between a collected peptide pool Sarikaya group has used a similarity matrix which compares strong binders to weak binders, to randomly generate new quartz binding sequences [32] The beaut y of the bioinformatics approach is that it can

PAGE 25

25 possibly design a peptide with multifunction ality and it can cont ribute to simulation studies. 1 6 Application of Inorganic B inding P eptides Inorganic binding peptides open a new chapter for controlling immobilization of organic compounds on inorganic surface s It provides the possibility of replacing self assembled monolayers (SAMs) to link nanocomponents onto solid substrates. So et al. tested the self assembly property of a gold binding peptide (GBP) in a triplet repeated form, 3rGBP1 (MHGKTQATSGTIQS) 3 on a Au (111) substrate. Based on high resolution AFM images, it shows that 3rGBP1 forms a ~1.5 nm thick monolayer on the gold surface from which the dimensions indicate that 3rGBP1 has its backbone laid down on the surface, enabling access of the functional groups in its side chains to interact with the surface. Th e six fold symmetry observed f r om the 3rGBP monolayer also indicates that the peptide recognizes the gold surface lattice ( F igure 1 4 ) [50] The s ame group further tested a graphite binding peptide GrBP5, and the result s show a transition between an amorphous phase of binding to an ordered phase within an hour, and then a complete ordered monolayer is formed with in three hours (Figure 1 5 ) [51] With the self assembly property and high specificity to target material s inorganic binding peptides can be used for functional ization of surface s a nd even monitoring of defects at surfaces Chemical composition defects at the m range can be detected by a fluorescent ly tagged inorganic binding peptide using simple fl u orescence microscopy [52] Multilayer nanostructure s can be achieved with layer by layer assembly. Sano et al. utilized a Ti binding peptide TBP 1 (RKLPNAPGMHTW), which is a bifunctional peptide that not only binds to Ti, Ag and Si, but also acts as a mediator for mineralization, to

PAGE 26

26 create a layer by layer structure on substrate s (Figure 1 6 ) [37] Khatayevich et al. covalently bound poly(ethylene glycol) (PEG), a polymer that prevent s cell adhesion, on gold and platinum substrate s covered with gold and platinum binding peptide s, respectively. O n the other hand, to achieve a cell binding surface, they attached an arginine glycine aspartic acid (RGD) peptide a cell adhesion motif, to quartz and titanium binding peptide s, and ha d these bifunctional peptide s self assemble o nto quartz and titanium surface s, respectively. In a cell study, they found the PEG peptide modified gold/platinum surface showed a bio inert surface while RGD peptide immobilized quartz/titanium resulted a bio active surface (Figure 1 7 ) [53] Another application for inorganic binding peptide s is i n synthesis of materials. Taking gold as an example, Brown et al. first published the work of a dodecapeptide affecting gold crystal growth. By using gold binding peptides as additives gold crystal s formed in a thin, large, hexagonal shape, with expression of (111) faces, similar to gold formed by reducing AuCl 3 (Figure 1 8 ) [54] Slocik et al. later used a peptide motif A3 (AYSSGAPPMPPF) which ha d been found to bind to silver and gold particles [55] to synthesi ze gold nanoparticles in a more simple way. When A3 dodecapeptide reacts with chloroauric acid (HAuCl 4 ), gold nanoparticles formed in fiv e minutes and c ould be suspended in solution for at least several days while gold nanoparticles synthesized by using a nonspecific peptide tend ed to aggregate. Also, gold nanoparticles synthesized by A3 we re round in shape while the others we re irregular (Figure 1 9 ) [56] Kim et al. further proved that inorganic binding peptide s can affect the shape and s ize of synthesized gold nanoparticles. By changing pH condition s and HAuCl 4 concentration of the reaction solution, the same peptide form ed gold nanoparticles with different

PAGE 27

27 morphologies (Figure 1 10 ). Alternatively, by changing only one amino acid of a pe ptide sequence, under the same synthetic condition s the nanostructure of gold nanoparticles was found to be different [57] 1. 7 Application of I norganic B inding P eptide s F used to P roteins I nstead of utilizing small peptide s some researc hers have focused on using the whole bacteriophage body as a template to display inorganic binding peptide s The main idea of these researche r s is that by genetic ally engineer ing the sequence of binding peptides o nto a virus particle surface, the protein c oat can then be functionalized and act as a linker for different applications. Modif ying the phage body has been shown to control the morphology of cells. M13 phage, a filamentous phage, with RGD peptide s displayed on its major coat protein pVIII has been used to interact with different cell lines. The Lee group first developed a shearing method to make aligned phage films with all the long rod shape d phage bod ies lying i n the same direction. Hippocampal neural progenitor cells (NPCs) were seeded on the al igned films. The result s show ed that the aligned film can support cell proliferation and control cell behavior as the neurite extensions we re parallel to the aligned phage bodies [58] They later developed a pulling method from a phage containing solution to create a film with different phage self templated structures (Figure 1 1 1 ) Phages assembled with each other once they reached the air liquid solid phase. By controlling the pulling speed, concentration of phage solution, an d properties of substrate surface, phages form ed different order s of alignment and different optical properties, and further affect ed the cell growth [59] These researche r s have show n promising approaches for developing biomedical materials with controlled structure s and optical propert ies

PAGE 28

28 L ayer by lay er phage assembly provides a useful method for fabricating novel material s for different applications. Lee et al. fused a gold binding peptide into the pVIII protein of M13 phage. By reduction of Au 3+ ions, this M13 phage was covered with gold nanoparticle s (NPs) to form Au nanowires (NWs). Phage provides a template for synthesizing Au NWs in a uniform way and the size can be well controlled. Au Pt core shell NWs can be further synthesized and make a promising material for ethanol fuel cells [60] Instead of gene tically engineer ing only one protein of bacteriophage, some groups have developed phages with multiple binding sites. The most common one is a two binding site system where the pIII coat protein s of M13 phage display one target binding peptide, and the major coat protein s pVIII display another target binder in order to achieve the goal of connecting or c arrying two different materials. Yoo et al. engineered a tetra glutamate peptide into the pVIII coat protein which made this E4 phage have extra COOH groups so that it was negative ly charged and c ould bind with positive ly charged nanoparticles [61] Lee et al. engineered peptides that bind to single walled carbon nanotubes (S WNTs) into the gene of the pIII coat protein. Silver NPs were formed on the E4 phage coat protein s, and amorphous iron phosphate (a FePO 4 ) was then synthesized on to it. This modified phage has higher electronic conductivity and makes SWNTs well dispersed [62] The FePO 4 /SWNT hybrid electrode has been utilized to light up a green light emitting diode (LED) and is definitely a success story for applying biological system s for development of novel fuel cell materials Ghosh et al. fused pIII protein with a SPARC (Secreted Protein, Acidic and Rich in Cysteine) binding peptide onto E4 phage to make it multifunctional. SPARC is a

PAGE 29

29 prototypic matricellular protein that is overexpressed in many cancers [63] When in the presence of magnetic iron oxide nano particles (MNPs), this phage can carry around 26 particles, and can be used as an image probe for magnetic resonance (MR) scan ning They confirmed that by injecting this SPARC targeted MNPs phage in to mice, certain cancer s can be targeted. They also found that with phage bodies as templates, the targeting and imaging ability is actually better than a complex of SPARC binding peptides and MNPs due to the size of the phage relative to peptides [63] Two peptide sequences for two different targe t materials can also be linked and fused to one gene of the phage for multifunctional application s Nam et al. linked tetra glutamate and a Au binding peptide (LKAHLPPSRLPS) together into the pVIII coat protein of M13 phage to make the phage bind on oxide material but also ha ve specific binding to Au NPs. The Au E4 phage can form Co 3 O 4 NWs with Au NPs, and it wa s found that with different Co 3 O 4 concentration s the resulting NWs have different morphologies. Co 3 O 4 NWs i nteract with each other and form a self assembled monolayer that can be used as an anode for Li ion batter ies [64] Other than the l iterature I mentioned above, there are lots of different applications being explored such as photocatalytic structures sensors surface structure d iscrimination and separation of different materials In fact, prior work in our group has shown that franc olite mineral can be selectively floated from phosphate ore containing dolomite impurities, which is a system that is notoriously difficult for traditional surfactants to discriminate between due to the similar surface charge characteristics of the two min erals [65] Recently, more gene tically engineered sites of M13 phage ha ve been studied [66] The further exploitation of rational ly designed virus particles should provide more possibilit ies for

PAGE 30

30 applications incorporating biolog ical system s into functional biomaterial s Many of these examples demonstrate that peptides can be used as linkers between particles and This nice feature of GEPIs forms the basis of the work here, which is targ eted toward biopanning for peptides that can be used as linkers, but with the novel aspect that the linkers are reversible upon application of an electric field. 1.8 Nucleic acid A ptamers Other than peptide s another rapidly developing research area is ap tamer based molecular recognition. Ellington et al. and Tuerk et al. published the first paper demonstrating that RNA sequences can bind to target molecules in 1990 [67, 68] DNA sequences have also been found by El lington et al. in 1992 [69] These oligonucleotides a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). This SELEX process is similar to the biopanning process of display techniques. A random nucleic acid library contains 10 13 to 10 15 different sequences of ssDNA or RNA. The l ibrary is incubate d with a target, followed by separat ion of the bound and unbound nucleotides The bound sequence will then be eluted, collected, and amplified by polymerase chain reaction (PCR). Amplified sequences are then incubate d with the target ag ain to perform the next screening cycle. Generally, 6 20 cycles will be performed to get aptamers that have strong binding affinity to the target (Figure 1 12 ). Nucleic acid aptamer s ha ve been identified to bind on many different targets, including small organic molecules [70, 71] proteins [72, 73] peptides [74] microorganisms [75] and cells [76] In c omparing to protein recognition, apta mers have many advantages. Aptamer s ha ve high affinity and high specificity to a target; it is

PAGE 31

31 chemically stable and easy to synthesi ze and modify. There have been many developments using aptamer based biosensors. Due to the fact that the conformation of a n aptamer can change after binding to a molecul e it provides aptamer s with many different sensing applications [77] For example, Figure 1 13 shows an electrochemical detection method using a molecular ly labeled aptamer. A redox active methylene blue (MB) is attached to the aptamer and thi s complex is immobilized on an electrode. After the thrombin and aptamer binding event, the aptamer changes its conformation and interrupt s the electron transfer of MB to achieve a signal off state [78] Label free aptasensors have also been developed for many applica tions (Figure 1 14 ). Compared to peptide s one of the short coming s of using aptamer s in sensing applications is that it requires a further functionalization process for immobilization onto an inorganic surface. A thiol group is usually used to have the ap tamer bind onto metallic surfaces [79] or a pH modified surface is needed. For instance, a titanium surface and aptamer are both negatively charged at neutral pH. But a titanium surface is positive ly charged in a pH 4 solution A phosphate labeled aptamer can then be adsorptive ly immobilized o nto a titanium surface by electrostatic interactions [80] Silane SAMs can also be used for immobilization o f aptamers on silicon surface s [ 79] In conclusion, peptide s are as flexible and stable as aptamer s but greater flexib ility and multifunctional ity can be achieved by including both a target binding site for surface im mobilization and a functional domain site at the other end, all in o ne synthesized peptide chain. We also believe that peptide s, with 20 possible amino acids of variable polarity, which can to be used in various combination s, and the inherent conformation

PAGE 32

32 and structures provided by proteins, makes peptides an exciting buil ding block for many different materials applications.

PAGE 33

33 Figure 1 1. Principles of phage display and cell surface display techniques [3] Reprinted from Sarikaya M, Tamerler C, Schwartz DT, Baneyx FO. Materials assembly and formation using engineered polypeptides. Annual Review of Materials Research. 2004;34:373 408. With permission from Annual Review.

PAGE 34

34 Figure 1 2. Principles of Ribosome display and mRNA display techniques Blue wave indicate s RNA; blue straight li ne indicate s DNA; blue folding structure indicates peptide ; brown circle indicates ribosome [7] Reprinted from Current Opinion in Chemical Biology, 6, William J Dower,Larry C Mattheakis, In vitro selection as a powerful tool for the applied evolution of prote ins and peptides, 390 8, Copyright (2002), with permission from Elsevier.

PAGE 35

35 Figure 1 3. Images demonstrate the specificity of material recognition of inorganic binding phage. A) A substrate was patterned with 1 m GaAs li nes and 4 m SiO 2 spaces. Fluorescently labeled GaAs clone bound to GaAs but not SiO 2 B) A diagram illustrating the selective binding of phage to the lanes of GaAs, recognition biological system s, can also be achieved with inorganic materials C) The GaAs clones were tagged with 20 nm gold particles. The nanoparticles were imaged by SEM and showed the recognition on GaAs layers but not AlGaAs layers. [42] Reprinted (adapted) with permission from Whaley SR, English DS, Hu EL, Barbara PF, Belcher AM. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature. 2000;405:665 8. Copyright 2000 Nature Publishing Group.

PAGE 36

36 Figure 1 4 AFM images of 3rGBP peptide self ass embled on Au. AFM scan o ver an area of 4 m 2 showing the long range ordering of the peptide in both height (A B ) and amplitude (C) imaging mode. B) and C) are the higher magnification images of the region marked with a box in A). O ne can see t he peptide monolayer exhibits a six fold symmetry that correlates with the underlying Au [ 111] surface lattice. [50] Reprinted (adapted) with permission from So CR, Kulp JL, Oren EE, Zareie H, Tamerler C, Evans JS, et a l. Molecular recognition and supramolecular self assembly of a genetically engineered gold binding peptide on Au [81] ACS Nano. 2009;3:1525 31 Copyright (2009) American Chemical Society.

PAGE 37

37 Figure 1 5 Time lapse AFM image s of GrBP5 assembly. A) AFM images for 10, 60, and 180 minutes binding time. The structural assembly begins with discrete peptide clusters (left); growth of both amorphous (AP) and ordered phases (OP) (middle); and complete OP monolayer (right). B) Schematic of peptide self as sembly process. [51] Reprinted with permission from So CR, Hayamizu Y, Yazici H, Gres swell C, Khatayevich D, Tamerler C, et al. Controlling Self Assembly of Engineered Peptides on Graphite by Rational Mutation. 2012. Copyright (2012) American Chemical Society

PAGE 38

38 Figure 1 6 Schematic show ing the layer by layer assembly process of silic a The first monolayer is formed by minTBP 1 to Ti surface (i). A silica layer is deposited by mineralization mediated by minTBP 1 (ii). The second monolayer is deposited by the interaction between minTBP 1 and silica (iii). The next silica layer is deposi ted on the second minT1 LF layer (iv) And the process is repeated to form a third monolayer. Different types of minT1 LF containing different metal compounds (Fe@minT1 LF, CdSe@minT1 LF, and Co@minT1 LF) were used to construct multilayer nanostructures (i iii, v) [37] Repri nted with permission from Sano K, Sasaki H, Shiba K. Utilization of the pleiotropy of a peptidic aptamer to fabricate heterogeneous nanodot containing multilayer nanostructures. J Am Chem Soc. 2006;128:1717 22. Copyright (2006) American Chemical Society.

PAGE 39

39 Figure 1 7 PEG peptide modified gold surface showed a bio inert surface while RGD peptide immobilized quartz/titanium resulted in a bio active surface. A) Number of adhered cells in serum free and 1% FBS on gold surface. B) SEM images of bare and 3G BP PEG modified gold su r faces. C) Fluorescen ce microscope images of cells on different ly modified surfaces. D) Number of adhered cells on different ly modified surfaces [53] Reprinted from Khatayevich D, Gungormus M, Yazici H, So C, Cetinel S, Ma H, et al. Biofunctionalization of materials for implants using engineered peptides. Acta Biomater. 2010;6:4634 41. With permission from Elsevier.

PAGE 40

40 Figure 1 8 With gold binding peptide additi ve s, gold crystal s formed with platy morphologies similar to gold formed by reducing AuCl 3. A and B) The formation of large, thin, hexagonal crystals were stimulated by RP1 and RP2 peptides C) Gold crystal formed by reducing AuCl 3. [82] Reprinted from Brown S, Sarikaya M, Johnson E. A genetic analysis of crystal growth. J Mol Biol. 2000;299:725 35. Copyright (2000), with permission from Elsevier. Figure 1 9 TEM image s of gold nanoparticl es synthesized with A3 dodecapeptide. A) With a scale bar of 100 nm. B and C) W ith a scale bar of 20 nm. [56] Reprinted from Slocik JM, Stone MO, Naik RR. Synthesis of Gold Nanoparticles Using Multifunctional Peptides. Small.1:1048 52. Copyright (2005), with permission from Wiley.

PAGE 41

41 Figure 1 10 One peptide can form gold nanoparticles with different mor phologies by changing pH condition and HAuCl 4 concentration of reaction solution. A) at pH 1.0. B) at pH 3.0. C) at pH 5.0. D and E) at pH 5.4. F) at pH 7.0. [57] Reprinted from Kim J, Rheem Y, Yoo B, Chong Y, Bozhilov KN, Kim D, et al. Peptide m ediated shape and size tunable synthesis of gold nanostructures. Acta Biomater. 2010;6:2681 9. With permission from Elsevier.

PAGE 42

42 Figure 1 1 1 Schematic of a pulling method to form a phage film of condensed and aligned phage The polarized optica l microscopy image shows iridescent color s from the liquid crystal phase. [59] Reprinted from Chung W J, Oh J W, Kwak K, Lee BY, Meyer J, Wang E, et al. Biomimetic self templating supramolecular structures. Nature. 2011;478:364 8. Copyright (2011), with permission from Nature Publishing Group Figure 1 1 2 Schematic of the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. [83] Cass AEG, Zhang Y. Nucleic acid aptamers : ideal reagents for point of care diagnostics? Faraday Discuss. 2011: 49 61.

PAGE 43

43 Figure 1 13 Schematic of an electrochemical sensor using a molecular ly labeled aptamer. A redox active methylene blue (MB) is used to label the aptamer (green) and this complex is immobilized on an electrode. After a thrombin (which was screene d for by the aptamer approach) binds to the aptamer, the aptamer changes its conformation which interrupt s the electron transfer of MB to achieve a signal off state [77] Reprinted from Shiping Song LW, Jiang Li, Chunhai Fan, Jianlong Zhao. Aptamer based biosensors. TrAC T rends in Analytical Chemistry. 2008;27:108 17. With permission from Elsevier Figure 1 14. Schematic of a label free electrochemical aptasensor. In a SWCNT FET sensor, a binding target (pink) with the aptamer (green) changes the conductance through the device to enable the detection of the target. [77] Reprinted from Shiping Song LW, Jiang Li, Chunhai Fan, Jianlong Zhao. Aptamer based biosensors. TrAC Trends in Analytical Chemistry. 2008;27:108 17. With permission from Elsevier

PAGE 44

44 CHAPTER 2 SCREENING FOR INDIUM ZINC OXIDE BINDING PEPTIDE S VIA PHAGE DISPLAY TECHNIQUE WITH STANDARD CHEMICAL ELUTION 2. 1 Overview Development of new approaches toward screening for inorganic binding peptide s, and understanding how the chemical sequences influence their binding behavior, wou ld accelerate the applications in material sciences and biotechnology. In this study, i ndium z inc o xide (IZO) was chosen as a target material for panning with a M13 phage library to screen for IZO binding peptides. IZO is rising in popularity as a material among transparent conducti ng oxide s (TCO s ) IZO has high electron mobility and high transparency IZO has the useful properties of chemical stability and thermal stability These properties make IZO attractive for applications like thin film transistors ( TFTs) and transparent electrodes in various optoelectronic devices and chemical sensors. IZO is also a promising candidate for the transducer element in a biosensor. On the other hand, while relative materials such as zinc oxide [84] and indium tin oxide (Sarikaya group, u npublished) have been screened for binding peptides, IZO has never been explored as a target for inorganic binding peptide s In this chapter, the focus is on selection of IZO binding peptides and testing the binding affinity of the selected clones. 2. 2 Ba ckground and Significan ce 2.2. 1 M13 Phage and M13 Phage Display Phage display involves the expression of peptide s equences within the proteins on the surface of the phage. The most common phages used in phage display are filamentous phage (M13 and fd phage used. The g enome of a filamentous phage is inserted with DNA sequences of interest and the encoded peptide sequence can then be ed ed on the surface

PAGE 45

45 of phage coat proteins [85] One of the advantages of using phage display technique is that it is very convenient to purchase commercially available libraries so researchers construct their own libraries to display peptide s for selection. Pop ular M13 filamentous phage libraries include Ph.D. TM 7, Ph.D. TM C7C, and Ph.D. TM 12 from New England Biolabs and T7 phage library such as T7Select from EMD Millipore. In this dissertation we used the Ph.D. TM 12 library, from New England Biolab s for the reasons described below M13 phage is a well understood and characterized strain which has a total of 11 proteins, pI XI with five different structural protein regions ( pIII p VI p VII pVIII and p IX ) t hat display random exogenous peptides (F igure 2 1). A phage body is composed of around 2700 copies of the p VIII major coat protein which comp rise s over 99% of a phage body. p VIII protein is a n helical chain with three segments and tilts at a 20 degree angle around the phage body. p VIII protein mainly makes up the majority of the phage body which is approximately 880 nm in length and 6.5 nm in diameter (Figure 2 2) [86] The p III and pVI protein s are located at one end o f M13 phage and resemble The p VII and pIX coat protein s are believed to be the first two coat proteins when phage secretion initiates [4, 87] Although a phage body has thousand s of copie s of pVIII there are only five copies each of the other four minor coat proteins. Generally, a phage display screening ( bio panning) round includes the following steps (Figure 2 3 ) : 1 ) a phage library is either purchased or is constructed by inserting the desired gene segment into the phage genome for displaying random peptides on the surface of the phages [6]; 2 ) phages are exposed to a target which is the material

PAGE 46

46 that binding is sought for; 3 ) the non binding phages are removed by washing with buffer s olution containing detergent ; and 4 ) the bound phages are eluted (typically with a low pH or high salt solution), and infected into the host bacteria to amplify the number of phage [7]. These biopanning rounds are repeated three to six times to evolve phag es with strong binding affinities. The DNA of the selected phages is sequenced to identify the peptide sequences presented on the phage coat protein that ha ve the desirable high binding affinity to the target. For constructing a n M13 library, all of the fi ve coat proteins of M13 phages have been successfully utilized to display peptides in either C terminal or N terminal regions [ 88] The most commonly used system is the p III protein because it is commercially available, and there are fewer proteins (only 5 copies) at the tip of the filamentous phage A randomly generated peptide can be expressed in either a linear or cyclic struc ture in the p III protein providing researchers more options i n selecting a suitable library (Figure 2 4 ) [5]. The s econd most common approach use s the pVIII protein. The N termin us of pVIII proteins is located on the outer surface of the whole phage body. In c ompar ison, t he pVIII protein has only 50 amino acids, while the p III protein has 406 amino acids, but only five copies. Also because there are only five copies of p III protein, it is actually more suitable for screening for a high affinity binder as compar ed to 2700 copies of p VIII which due to the sheer number of combined interactions, may yield individual peptides that have a lower binding strength In this study, we chose the 12 mer linear M13 pIII system based on the reasoning that it is commerc ialized, and we believed that a 12 mer could provide us with more combination s of amino acid

PAGE 47

47 sequences and conformations that may assist in the challenging task of finding a reversible peptide binder The e lution step is critical for phage display select io n In biopanning for inorganic binding peptide s the most common way to perform elution is by incubating the phage bound target with an extreme pH buffer for example a pH 2 .0 glycine solution, which was used in this study High ionic strength, ultrasonic a tion and dithiothreitol can be utilized as well These approaches are based on weaken ing the interaction between the target surface and phage to achieve a nonspecific elution [2] Adding host bacteria or using a polymerase chain reaction (PCR) driven method to sequence binding phage directly can also be used [89] To perform the ampl ification step, it involves the life cycle of M13 phage. D etail s of the amplification process are described in Figure 2 5 [87, 90] M13 phage is a nonlytic phage apart the host bac teria when it is releas ed This nonlytic feature makes the amplification step of M13 phage display protocol much simpler as compared to a lytic phage, because there is no other bacteria l membrane or DNA that need s to be worried about when separating phage from host bacteria. The first phage will be amplified in around ten minutes after infection, and one bacteri um can secrete an average of 1000 M13 phages every hour. Blue and white screening is a cell culture technique used to isolate and collect the amplif ied M13 phage. The E. Coli host strain for the Ph.D. TM library is ER2738 a lacZ gene in its DNA. When ssDNA of M13 phage which contains lacZ fuses into ER2738, the enzyme beta galactosidase can be secreted in the presence of an induce r i sopropyl D 1 thiogalactopyranoside (IPTG) Beta

PAGE 48

48 galactosidase can decompose X gal (5 bromo 4 chloro 3 indolyl D galactopyranoside) to produce a blue product (5 bromo 4 chloroindole) [91] X gal and IPTG are premixed into an agar plate. Blue plaques on the agar plate indicate M13 phage from library kit, and white plaques might be some other bacteria l contaminants from the environment. 2.2. 2 Indium Zinc Oxide Transparent conducting oxide (TCO) th in films are important for many optoelectronic applications. Features that require consideration for TCOs are their s tructural, optical, compositional, and electrical properties Recently, many researchers have focused on IZO because it is a promising new material due to its high carrier mobility (10 50 cm 2 /Vs) high transparency, durab i l ity thermal stability, smooth surface, eas e of etch ing eas e of preparation at room temperature, and it has high resistance to H plasma [92 94] Based on these properties, IZO is attractive for applications like thin film transistors (TFTs) and transparent electrodes in various optoelectronic devices and chemical sensors, and was therefore chosen as the target substrate for this stud y. For TFTs, the film s need to have a band gap Eg greater than 3 eV to be transparent under visible light, and also the film has to be conductive and amorphous when deposit ed at room temperature. Many conducting oxides are polycrystalline as deposited such as ZnO. Amorphous In 2 O 3 becomes polycrystalline w hen increasing the oxygen ratio. Indium tin oxide (ITO) has been demonstrated to be a material t hat can be readily prepared as an amorphous and transparent film However, IZO is easier to etch and cheap er in price compared to ITO [95] With all the advantages, IZO has been gain ing attention among the various conducting oxides for TFT applications and was therefore chosen for our studies

PAGE 49

49 There are many different ways to prepare a TCO thin film. It can be prepared by R F magnetron sputtering, molecular beam epitaxy (MBE), atomic layer epitaxy (ALE), pulsed laser deposition (PLD), electron bea m evaporation (EBE) and low pressure metal organic chemical vapour deposition (MOCVD) [93] Different preparation methods at different operating condition s lead to differ ing degrees of crystallin ity of the thin film s For example, PLD deposited IZO thin film is amorphous when deposit ed at room temperature, but is polycrystalline when deposit ed at a temperature higher than 200 C [96] For this study, an amorphous structure withou t grain boundar ies or crystal defect s was used because we felt it would provide a more homogeneous target surface. It has been reported that IZO films with amorphous surface s can be obtained with high reproducibility at different deposition conditions by R F magnetron sputtering [97] Therefore, R F magnetron sputtering wa s chosen to deposit amorphous IZO thin film s on sapphire substrates and devices, to serve as our biopanning target O n the other hand, the optical and electrical properties of IZO thin fil ms are highly reli ant up on film thickness. A thicker IZO film has higher conductivity b ut poorer transparency, so the thickness needs to be well controlled for testing. 2. 3 Materials and Methods 2.3. 1 Materials All aqueous solutions were prepared using N ANO pure Diamond TM RO deionize (DI) water with a resistivity a (Barnstead). 2.3.1.1 Sapphire s ubstrate Double side d polished amorphous sapphire substrate s from Saint Gobin w ere used as the solid substrates upon which an IZO thin film was deposited as the phage target

PAGE 50

50 2.3.1.2 IZO t arget for s puttering A f our inch In 2 O 3 /ZnO sputtering target purchased from Kurt J. Lesker was used provided by the Nanoscale Research Facility (NRF) at University of Florida The ra t io of In 2 O 3 to ZnO is 90:10 by weight. 2.3.1.3 P hage d isplay p eptide l ibrary kit A Ph.D. TM 12 phage display kit was pu rchased from New England Biolabs. It is based on an M13 phage vector modified for pentavalent display of linear twelve mer peptides as N terminal fusions to the minor coat protein pIII The library contains 100 of 2 x 10 13 pfu/ml (1.29 x 10 9 12 mer peptide sequences supplied in TBS with 50% glycerol). There is a short linker sequence Gly Gly Gly Ser between the displayed peptide and pIII The E. Coli Er2738 host strain ( proAB) thi mcrB)5 (rk mk McrBC ), was also supplied in this kit as a 50% glycerol culture 2.3.1.4 Buffer solutions PC b uffer Three gram s of KH 2 PO 4 (Fisher), 1.90 g of Na 2 CO 3 (Fisher), and 3.50 g NaCl (Fisher) were dissolved in 400 ml distilled water to get 55 mM KH 2 PO 4 45 mM Na 2 CO 3 and 200 mM NaCl. A n a ppropriate amount of detergent was added from the detergent s olution into the PC buffer according to the desired detergent concentration (e g. 0.02%, 0.1%, 0.5%). PC buffers were sterilized by using a 0.2 use sterile syringe tip filter. Detergent s olution Two ml of Tween 20 (Enzyme grade, Fisher) and 2 ml of Tween 80 (for molecular biol o gy, Sigma Aldrich) were added into 6 ml DI water to get a 20 w/v % Tween 20 : 20 w/v % Tween 80 solution

PAGE 51

51 PEG NaC l Twenty gram s of Poly(ethylene glycol) (PEG) 8000 (Sigma) and 14.61g NaCl (Fisher) were dissolved in distilled water up to 100 ml and sterilized for 15 minutes under 1.5 atm at 121C. 2.3.1.5 Elution s olutions Low pH e lution b uffer s olution Based on the el ution condition used by the Sarikaya group [31] t hree g ram s of glycine (Sigma Aldrich) and 4 00 mg BSA ( Sigma Aldrich) were dissolved in distilled water up to 200 ml to get 0.2 M glycine and 1 mg/ml BSA, and the pH was adjusted to 2. 0 The solution was filter ed by using a single use sterile syringe tip filter. High s alt e lution b uffer s olution Based on the elution condition used by the Sarikaya group, a 5 M MgCl 2 solution was prepared and filter ed using a use sterile syringe tip filter. 2.3.1.6 Culture m edium Lauria Bertani ( LB ) Lennox m edium Twenty gram s LB Lennox (Fisher Brand) (trypton: yeas t extraction: NaCl = 2:1:1) was dissolved in 1 liter of DI water and adjusted in pH to 7.0 7.5. The LB medium was sterilized for 15 minutes at 1.5 atm (121 o C) in an autoclave ( Tuttnauer 2540M ). LB agar medium Forty gram s LB agar (Fisher brand) w as dissolv ed in 1 liter DI water until the solution become transparent by heating. The pH was adjusted to 7.0 7.5 and the solution was sterilized for 15 minutes at 1.5 atm (121 o C) in an autoclave. Top agar medium Twenty gram s LB (Fisher brand) and 15 g agar were dissolved in 1 liter DI water by heating until the solution became transparent. The t op agar medium was sterilized for 15 minutes at 1.5 atm (121 o C) in an autoclave.

PAGE 52

52 10x Minimal salts (MS) Three gram s Na 2 HPO 4 7H 2 O (Fisher brand), 1.5g KH 2 PO 4 (Fisher bran d), 0.25g NaCl (Fisher brand), and 0.5g NH 4 Cl (Fisher brand) were dissolved in 50 ml DI water. M9 Solid medium plate Ten ml MS and 1.5 g agar (Fisher brand) were dissolved in 89 ml DI water by heating until the solution became transparent and sterilized for 15 minutes at 1.5 atm (121 o C) in an autoclave. Sterilized 1M MgSO 4 6 H 2 O (Sigma Aldrich), 1M CaCl 2 (Fisher brand), and 40(w/v) % glucose (Fisher brand) were filtered by using 0.2 use sterile syringe tip filter. Then 0.2 ml sterilized 1M MgSO 4 6 H 2 O, 0.01 ml sterilized 1M CaCl 2 and 0.5 ml sterilized 40 % (w/v) glucose were added into the agar/MS mixture. Finally, liquid M9 medium was poured onto a 90 mm petri dish until solidification. 2.3.1. 7 Stock s olution Tetracycline. Twenty mg/ml tetracycline hydrochloride (Sigma Aldrich) was dissolved in 70% ethanol and stored at 20 o C. Xgal/ IPTG stock Five gram IPTG (ultrapure grade, dioxane free, Molecul A) and 4 g Xgal (Molecul A) were dissolved in 100 ml DMF (Sigma Aldrich) and stored at 20 o C. Glycerol s tock s olution Eighty ml glycerol (Sigma Aldrich) and 20ml DI water wer e mixed to make 80% (w/v) and sterilized for 15 minutes at 1.5 atm (121 o C) in an autoclave. 2.3. 2 Methods 2.3.2.1 Fabrication of IZO thin film s for b iopanning targets Wafer cleaning process Sapphire wafers were cut into 5 mm x 5 mm square s Sapphire substrates w ere washed with trichloroethylene, acetone, and methanol in an

PAGE 53

53 ultrasonic bath for 5 minutes respectively to remov e contamination s from the sapphire surface, and then were dried by a nitrogen gas stream. D eposition of IZO t hin f ilm s IZO thin films were deposited on sapphire wafers in argon plasma using RF magnetron sputtering at the Nanoscale Research Facilities (NRF) at UF Before deposition, the sputter chamber was pumped down to less than 5x10 6 Torr. In addition, a commercial 3 inch diameter IZO pell et was used as a target for producing IZO vapor by applying 1 2 5 W power under 5 mTorr working pressure The sapphire substrate was turned over and the coating procedure repeated in order to coat both sides with IZO, to avoid exposure of sapphire to the pha ges when it is immersed in the biopanning media. There is, however, a small amount of sapphire surface remaining at the edges of the thin plate. 2.3. 2.2 Phage display protocol The standard p hage display protocol and the other methods applied during the phage disp lay procedure are discussed in detail as follows. All chemicals and labware were autoclaved, and the reactions performed in an Airclean 600 PCR workstation laminar flow hood. DI water was obtained from ultrapure water system N anopure D iamond D11931 Bindi ng s tep 12 in 990 l PC buffer was exposed to an IZO coated sapphire substrate in a 1.5 ml microfuge tube. IZO was coated on both the top and b o ttom of the sapphire plate although thin edges of sapphire remain non coated The microfuge tube containing the IZO phage solution was rotated by an rotisserie type agitator (Lab Q uake, Barnste a d Thermolyne) for 60 min in order to obtain sufficient time for phage IZO interaction.

PAGE 54

54 Washing s tep After a 60 min rotation, severa l washing cycles were performed in order to remove the non specific binding phage from IZO surface. These washing cycles were repeated for thirteen times for each biopanning round. T he first supernatant was put into another microfuge tube for confirming th e phage library and the IZO sheet was washed with 1 ml PC buffer containing 0.1 % detergent. The washing steps were repeated 6 times with fresh buffer changes between each From the seventh wash, detergent was incubated on an agitator for 30 min to remove unbound phage again This step was repeated 7 more times, including one time for overnight washing. Elution s tep After the washing steps, strongly bound phages were recovered from the IZO surface through elution. The strong interaction between phage and IZO surface was disrupted using a low pH buffer solution. The elution procedure i s described as follow s : After the last washing step, 1 ml of low pH elution buffer solution was added i nto the IZO sheet with bound phages in a microfuge tube. The m icrofuge tube containing the IZO sheet with bound phages and low pH elution buffer was put the on an agitator for 8 minutes to elute the bound phages from IZO surface. The supernatant including eluted bound phages was transferred into a fresh microfuge tube Then, 100 l supernatant was transferred into E. coli ER2738 culture (LB Lennox medium: E. coli ER2738 overnight culture = 100:1) in preparation for the incubation step The remaining 900 Another 1 ml of low pH elution buffer was put into the microfuge tube and incubated for another 8 minutes, followed by the same neutralized step. These elution steps were repeated for four times.

PAGE 55

55 Amplification and p urification s teps Eluted phage samples were infec ted into the host strain E. coli ER2738 and amplified. Before the beginning of the amplification period, the E. coli host strain ER2738 was cultured to reach the OD 600 ~ 0.5 (the best phage host strain propagation period) and then eluted phage solutions w ere transferred to the bacteria culture. The incubation period was approximately 4.5 hours at 37C and phage E. coli host strain solution was shaken at 250 rounds per minute ( rpm ) on a platform shaker (Max Q 2000, Barnstead Lab Line) The E. coli host str ain ER2738 used in amplification is a robust F+ strain with a rapid growth rate and is particularly well suited for M13 propagation. ER2738 is a recA+ strain and the F factor of ER2738 contains a mini transposon, which confers tetracycline resistance. Afte r amplification eluted phages were purified from host cell s according to the following procedure : First, E.coli phage culture was transferred to 50 ml sterilized centrifuge tubes after 4.5 hours of the growth period S amples were centrifuged at 8000 rpm for 10 min and the s upernatant was transferred to 50 ml sterilized centrifuge tubes. PEG/NaCl was added (1:6) into supernatant to precipitate phage overnight at 4 The next day, s amples were centrifuged at 10 000 rpm for 20 min Then, the s upernatant was discarded and a phage pellet was resuspended with 5 ml PC buffer by pipetting to remove any remaining E.coli ER2738 Next, s amples were centrifuged at 10 000 rpm for 10 min. After that, the s upernatant was transferred to 50 ml sterilized centrifuge tubes. PEG/NaCl was added (1:6) into the solution to precipitate phage and the solution was le ft for 2 hours at 4 Samples were centrifuged at 10 000 rpm for 10 min. The s upernatant was discarded and phage pellet was resuspended by pipetting with 1 ml PC buffer (no detergent) to remove E.coli ER2738 Samples were centrifuged at 10 000 rpm for 10 min and

PAGE 56

56 supernatant was transferred to sterilized microfuge tubes. PEG/NaC l solution was added (1:6) into the microfuge tube to precipitate phage, sample was vortex ed 5 sec, and left in this solution in the air for 10 min. Samples were centrifuged at 13 200 rpm for 3 min to compact the phage s The s upernatant was discarded and p hage pellet was resuspended with 0.2 ml PC buffer (no detergent) by pipetting gently. Samples were centrifuged at 13 200 rpm for 3 min. The s upernatant was transferred to sterilized microfuge tubes and stored at 20 2.3.2.3 Blue/White s creen p rotocol and DNA s eq uencing This experiment was performed to estimate the phage titers at the end of each biopanning round. The b lue white screening experiment has three fundamental steps, namely preparation of Xgal/IPTG plates, serial dilution of eluted phage samples, and e stimation of phage titers for each round. Preparation of LB Xgal/IPTG p lates warm liquid LB agar in a 150 ml glass medium flask which was then poured in to 60 mm plastic sterile petri dishes. Plates were wrapped with P arafilm and Aluminum foil and stored at 4 in the dark. Serial dilution of phage samples 198 l PC buffer (no detergent) and 2 l phage w ere added into the A1 well of a 96 well plate. 180 l PC buffer (no detergent) was put into wells from A2 to A12. Ten fold dilutions were made from A1 to A12 by taking 20 l samples from each of the preceding wells (Figure 2 6) Calculation of Phage Titers After serial dilutions, LB Xgal/IPTG plates that were prepared previously and stored at 4 C were acclimated to room temperature. Three ml melt top agar was aliq u o t ted into 15 ml cell culture tube s, and these tubes

PAGE 57

57 were put in a 55 water bath to prevent the solidification until other processes were carried out. E. coli ER2738 from overnight culture were inoculated into 5 ml LB medium in 50 ml centrifuge tube s The culture was incubated at 37 C and 250 rpm until mid log phase (OD 600 ~ 0.5). After the E. coli diluted phage sample were put into a 1.5 ml test tube. Phage E. coli ER2738 mixture was added into 3 ml melt top agar and poured onto LB Xgal/IPTG petri dishes. After the petri dishes solidified, they were kept up side down and incubated at 37 overnight. After incubation, blue plaques were obtained (Figure 2 7 ). The plate with 30~100 plaques from each eluted p h age solution (E1 E4) were cho sen to calculate the amount of phage using the equation as follow s : (pfu: plaque forming unit) (2 1) For example, consider a 10 l diluted phage solution from A9 well was added into 3 ml top agar. After vo r texing, the mixed solution was pour ed onto a LB Xgal/IPTG plate After incubation overnight, 100 phage plaques were counted on this LB Xgal/IPTG plate The calculation of the amount of phage was as follows : A9 well represents 10 10 dilution. (100 phage plaques/0.01 ml diluted phage solution) x 10 10 = 1.00 x 10 14 pfu/ml Saving p hage c lones for s equencing Preparation procedure of saving clones for sequencing i s described in the following subsections. First, of 0.02 % PC buffer was put i nto each well of a 96 well plate. Each phage plaque from big plates was picked and put into different well s of a 96 well plate. Twenty four plaques were picked per each elution step. At the end of the third round 288 plaques were picked. Next, a

PAGE 58

58 96 well plate containing phage clones was placed into the inc ubator at 60 for 45 min, and then the 96 well plate was left at 4 overnight. After that, glycerol solution was put into each well in two sets of 96 well plates ( o verall glycerol concentration was kept as 50% in stocks). Subsequently, of e ach phage clone was added from storage plate to glycerol containing plates. Plates were covered and stored at 80 After saving clones, phages DNA were isolated for DNA sequencing. DNA isolation was performed using the QIAprep Spin M13 kit (QIAGEN) proc edure. The procedure is described in detail as follows A 10 l sample was taken from the glycerol stock of a single phage clone, and was added into the 3ml E. coli ER2738 culture (LB medium: E. coli ER2738 overnight culture = 100:1), which was incubated u ntil mid log phase (OD 600 ~ 0.5). After this step, the culture was incubated 4.5 hours for phage E. coli ER2738 infection. Next, the c ulture was centrifuged at 5000 rpm for 15 minutes at room temperature. The s upernatant containing M13 bacteriophage was tr ansferred to a fresh reaction tube. During transferring the supernatant, care was taken to not disturb the bacterial pellet. This step can be repeated twice. Buffer MP was added in 1/100 volume (i.e. 10 l per 1 ml of phage supernatant) to the supernatant in reaction tube. It was mixed by vortexing and incubated at room temperature for at least 2 min. During this step, bacteriophage particles were precipitated from the culture medium. A QIAprep spin column was placed in a 2 ml microfuge tube and 0.7 ml of s ample was applied to the QIAprep spin column. The r eaction tube was centrifuged for 15 sec onds at 8000 rpm and flow through was discarded from the collection tube. During this step, intact bacteriophage was retained on the QIAprep silica gel membrane. This centrifuge step was repeated until all supernatant passed through the QIAprep spin column. Then

PAGE 59

59 0.7 ml MLB buffer was added for M13 lysis and binding of DNA to the QIAprep spin column which was then centrifuged for 15 sec. at 8000 rpm. This step creates appropriate conditions for binding of the M13 DNA to the QIAprep silica gel membrane. When bacteriophage lysis begins, another 0.7 ml MLB buffer was added to the QIAprep spin column which was then incubated for 1 minute at room temperature to lyse bacteri ophage completely. QIAprep spin column was then centrifuged for 15 second at 8000 rpm. M13 single stranded DNA is released from bacteriophage particles and adsorbed to the QIAprep silica gel membrane. 0.7 ml b uffer PE was added and centrifuged for 15 seco nd at 8000 rpm to remove residual salt. Buffer PE was discarded from the collection tube and the spin column was centrifuged again for 15 sec. at 8000 rpm to remove residual buffer PE. It is important to dry the QIAprep membrane with a quick micro centrif ugation step. This prevents residual ethanol from being carried over into subsequent reactions. The QIAprep spin column was placed in a clean 1.5 ml microfuge tube. 100 l EB buffer (10 mM Tris.Cl, pH 8.5) was added to the center of the column membrane to elute the DNA. Incubation of elution buffer in the QIAprep spin column significantly increases the recovery of single stranded M13 DNA molecules, which adsorb tightly to the silica membrane. The DNA can also be eluted with water. When using water for eluti on, the pH of water should be in the range 7.0 8.5 because the efficiency is dependent on pH where the maximum elution efficiency is achieved within this pH range. DNA s equencing The Interdisciplinary Center for Biotechnology Research ( ICBR ) in the UF pro vided service in PCR and DNA sequencing for single strain DNA of M13 phage clones. 96 gIII sequencing primer ( 5 HOCCC TCA TAG TTA GCG TAA

PAGE 60

60 CG ry Kit was used in PCR. DNA samples were sequenced by using ( Roche Applied Science). Determination of e xpressed 12 mer p eptides The DNA codes of every M13 phage in the library pool are the same except the 12 mer pep tide gIII fu sion for each phage. In order to identify the 12 mer peptide gIII fusion, two segments of DNA sequences in the immediate vicinity of the gIII fusion domain were used and aligned with any DNA sequence of phage clones by using Nucleotide alignment in Basic L ocal Alignment Search Tool (BLAST) from National Center for Biotechnology Information (NCBI): http://blast.ncbi.nlm.nih.gov/ After obtaining the DNA sequence of the gIII fusion, DNA codes of gIII fusion can b e translated into the desired 12 mer amino acid sequences. 2.3.2.4 Enzyme linked i mmunosorbent a ssay (ELISA) A phage single clone (10 9 pfu/ml) in 1 ml PC buffer was incubated with 5 mm x 5 mm IZO, sapphire (0001), silicon (100), or silicon oxide (amorphous) sheet for 1 hour on an agitator. After incubation, supernatant was discarded and sheets washed with 1 ml PC buffer with a suitable concentration of detergent three times. Monoclonal anti M13:HRP (GE Health) which is the secondary antibody conjugated to horserad ish peroxide (HRP) for M13 phage detection (anti M13:HRP : PC buffer = 1:2500) was incubated with inorganic sheets for 20 minutes. Subsequently, inorganic sheets were again washed with 1 ml PC with a suitable concentration of detergent three times to remo ve unbound anti M13:HRP. Inorganic sheets were then transferred to a fresh microfuge tube. The biochemical s ubstrate for the development reaction for HRP conjugated secondary enzymes was prepared by dissolving one capsule of phosphate

PAGE 61

61 citrate buffer with s odium perborate (Sigma Aldrich) in 100 ml distilled water (0.05 M tetramethylbenzidine (TMB) substrate (Sigma Aldrich) was added to 10 ml of the buffer to give a f inal concentration of 1 mg/ml. 1 ml TMB substrate solution was added into the microfuge tube containing inorganic sheet and was developed for 20 minutes (Figure 2 8 ). The s upernatant from each tube was transferred into a 96 well plate. The plate was read i n an ELx 800UV plate reader (BioTek) at 630 nm. 2.3.2.5 Immunofluorescence (IF) a nalysis At the beginning of the fluorescence microscopy experiment, a negative control experiment was carried out to decide on the right procedure during the fluorescence experiment. In the negative control experiment, IZO sheet was incubated with Anti M13 pIII monoclonal antibody (GE Lifescience) ( Anti M13 monoclonal antibody: PC buffer = 1: 2500) which is specific to the M13 pVIII protein for 20 minutes on an agitator. After the p rimary antibody binding step, IZO thin film was wash ed by PC buffer with a suitable concentration of detergent (0.1 %, 0.3 %, and 0.5 % for phage clones from first, second, and third biopanning round respectively). Subsequently, anti mouse IgG FITC as sec ondary (Sigma Aldrich) antibody (anti mouse IgG FITC : PC buffer = 1 : 500) was incubate d with IZO in 1 ml PC buffer for 20 minutes on an agitator. After 20 minute s incubation, the IZO thin film was washed by PC buffer with a suitable concentration of dete rgent three times. IZO was visualized at 20X magnification and 2 sec exposure time with a fluorescence microscope (Nikon Eclipse E600). For the detection of phage binding, IZO was incubated with a single phage clone (10 9 pfu/ml) in 1 ml PC buffer o n an agitator for 1 hour, follow ed by washing steps to remove unbound phage. Anti M13 pIII monoclonal antibody and anti mouse IgG FITC

PAGE 62

62 were applied as primary and secondary antibody as in the same procedure mentioned for the negative control. These comple xes were visualized at 20X magnification and 2 sec exposure time by fluorescence microscop y Figure 2 9 shows a schematic of the IF analysis steps. 2.3.2.6 Calculation of surface coverage of phage c lones Image processing software, Image J, was used to measure th e surface coverage of phage clones on the various substrate s A fluorescence image of each phage clone adsorbed on to the IZO sheet was converted to a 32 bit black and white image. The r atio of black to white area which indicates the exist ence of phage to the whole picture area was measured using Image J This ratio represents surface coverage of phage clones on the substrate sheets. 2 4 Results and Discussions In this study, indium zinc oxide (IZO) was chosen as a target material to select phage clones wi th 12 mer expressed peptides showing binding affinity to IZO. The t arget material IZO was first analyzed. When sputter ing at room temperature, the IZO coating wa s confirmed to be amorphous since it displayed no Bragg peak s in an x ray diffraction scan (Fig ure 2 10) An AFM was used to measure the roughness of the deposited IZO thin film over a 5 m x 5 m area which appeared to be fairly uniform (Figure 2 1 1 ) The IZO thin film was determined to have a r oot mean square (RMS) roughness value of 0.552 nm. In a 200 nm thick film, we obtained an electron mobility of 1 0 cm 2 V 1 s 1 by Hall measurement. Amorphous IZO is anticipated to provide a more homogeneous surface than a crystalline one because it should not have grain boundaries and crystal defects which might affect the binding of phage This has been

PAGE 63

63 proposed as a reason that inorganic materials d o not always converge to a consensus sequence as readily as organic molecules [49] After each biopanning round of the phage display protocol, a concentration of eluted phages was calculated by the blue and white screen protocol. Table 2 1 shows the result of phage titer s when using a low pH el ution buffer. Based on the results, the level of phage concentration was over 10 11 pfu/ml. This indicates that amplification efficiency of phages within E.Coli host bacterial ER2738 strain was promising. And based on the concentration of eluted phages, the volume of each elution solution can be determined to make an equal contribution of phage amount to phage pool. After phage titer measurements some phage clones were picked for DNA sequencing to deduce the IZO binding peptide sequences. We performed three phage display biopanning round s Biopanning round s should not be repeat ed more than four times because too many rounds of selection tend to collect phage that elute or amplif y better rather than the strong binders [98] Figure 2 1 2 shows the expressed 12 mer peptide sequences translated from the DNA of phage clones selected from IZO thin film s F r o m the amino acid sequences shown in Figure 2 1 2 most of the peptide sequences contained a block of hydrophobic amino acids, and one or two charged amino acids. In addition, approximately 4 to 5 polar amino acids were separately distributed within the 12 mer expressed peptides. Also, in comparing the clones collected from second and third biopanning round s we found one consensus sequence [49] the amorphous nature of the IZO may have provided a ver y uniform surface.

PAGE 64

64 It is important to note that a collected phage clone does not necessary mean that it is a strong binder to a target material. A collected se quence could represent an expressed peptide from the most enriched phage clones after many amplification steps during different rounds if it somehow biases the amplification process Thus, immunofluorescence (IF) analysis and enzyme linked immunosorbent a ssay (ELISA) were used to evaluate the properties of phage clones with respect to both binding affinity and specificity to the target material. The specificity of various clones to the IZO surface as well as three inorganic substrates were compared, sapph ire (0001) face, silicon (Si) (100) face, and amorphous silicon dioxide (SiO 2 ). Sapphire was chosen because it was present on the side edges of the IZO coated substrates; Si and SiO 2 are commonly used materials in electronics devices. In order to visualize the phage binding on inorganic substrates, immuno fluorescence analysis (IF) was used with a fluorescence tag (FITC) conjugated to anti mouse IgG, to indicate the location of M13 phage on the inorganic substrate surface. However, Anti mouse IgG FITC may h ave a binding affinity to some inorganic substrates directly. Thus, it was necessary to test anti mouse IgG FITC to see if it has the ability to bind onto IZO, sapphire, Si, and SiO 2 in the absence of M13 phage and anti M13 primary antibody. Figure 2 1 3 sh ows that anti mouse IgG FITC did not tend to bind onto those inorganic substrates because no green spots were observed under fluorescence light without loading M13 phage and anti M13 primary antibody. Binding affinity of phage M13KE was also examined by IF analysis. M13KE is the Ph.D. library cloning vector, and was used as a control in this study. Figure 2 1 4 shows a

PAGE 65

65 fluorescence image of M13KE phage on IZO thin film, which confirmed that the M13KE phage has no specific binding affinity to IZO. In IF anal ysis, phage clone TKNMLSLPVGPG had high binding affinity to IZO, but showed minor binding affinity to the other materials (Figure 2 1 5 ). For the phage clone: MNRPSPPLPLWV, it showed preferential binding affinity to sapphire, rather than IZO (Figure 2 1 6 ). Program Image J was utilized to calculate the surface coverage of each clone on IZO thin films from the corresponding fluorescence images. We qualitatively defined their coverage between 80 100% as a strong binder, 60 80% as a moderate binder, and lower th an 60% as a weak binder (Table 3 and Figure 2 1 7 ). In ELISA test ing a secondary antibody conjugated with horseradish peroxide (HRP), anti M13:HRP, was used to quantitatively measure M13 phages absorbed onto the surface of the inorganic substrates. When H RP catalyze s the enzymatic reaction of tetramethylbenzidine (TMB) substrate, the oxidized product of TMB ha s a deep blue color. A deep blue color indicates a higher density of phages on those substrates. The degree of blue color can be quantif ied by absorbance measurement at 630 nm. Figure 2 1 8 (a) and (b) show image s of ELISA plates of seven representative phage clones eluted by the low pH buffer solution. Figure 2 1 9 show s the absorbance value at 630 nm of the enzymatic substrate solution extr acted from the wells of the represent at i ve plate s (Figure 2 1 8 ). Based on UV absorbance measurements (Figure 2 1 9 ) and IF analysis (Figure 2 1 5 ) one can see that t he phage clone with displayed amino acid sequence TKNMLSLPVGPG was strongly bound strongly t o IZO, but also displayed a moderately high binding affinity to sapphire. With respect to another phage clone with displayed amino acid sequence MNRPSPPLPLWV, it tended to bind much

PAGE 66

66 more strongly onto sapphire, even though IZO was the chosen target materia l (Figure 2 1 6 ) The reason might be that IZO was sputtered onto the top and bottom surfaces of a sapphire plate which left some edges of the sapphire plate expose d to the phage library during the biopanning process. This might cause a few of the clones t o be selected from this region of the substrate, which apparently had a strong binding affinity to sapphire. As judging from Figure 2 1 9 it seems that sapphire ( Al 2 O 3 ) may ha ve a surface chemistry that is simply fa vorable for peptide adsorption. In addition, other phage clones wit h different types of expressed 12 mer peptides were also explored for their binding affinity to IZO in ELISA the results of which are shown in Figure 2 1 9 In one interesting comparison Figure 2 12 shows the polarity of the amino acids by color coding, wh ere it can be seen that peptide ASQITHFPRPPW (6 th row of Second Round) contained more polar amino acid residues (with two positive charges and 3 polar) than peptide TKNMLSLPVGPG (5 th row of Third Round with one positive charge and 3 polar AAs ) yet they h ave similar properties regarding binding affinity to the IZO material (Figure 2 1 9 ) It was discovered after repeating the elution steps for four times with low pH elution buffer, that the IZO thin film s w ere becoming etched (Figure 2 20 ). Some phages wi th binding affinity to IZO were released because of the etching This might not be a benefit for the development of an electro active peptide linker due to the excessively strong binding of these clones which are only removed along with the dissolution of substrate Thus, alternative approaches, such as either a high pH or high salt elution buffer w ere considered for future use in the elution step to avoid this potential problem

PAGE 67

67 A high salt elution buffer, 5M MgCl 2 solution was chosen for elution steps for a different approach to avoid the extreme pH buffer We first made sure that the high salt elution buffer would not etch the IZO thin film s Using high salt elution, similar amount s of phage w ere eluted as compared to the phage amount eluted by a low pH buffer solution (Table 2 2). This indicated that the high salt elution buffer could also provide enough diversity of eluted phage clone s for subsequent biopanning. Some expressed 12 mer peptide sequences eluted from high salt elution buffer are shown in Figure 2 2 1 The h igh salt elution evolved four consensus peptide sequences between the second and third biopanning round s : AGFPWSTHSSWL, SHAPDSTWFALF, TNSSSQFVVAIP, and ALDDLRARFLPP. It was found that most of the peptide sequences obtained by high salt elution were comprised of a block of hydrophobic amino acids (2 5 residues) which also appeared in peptide sequences obtained by the low pH elution. In addition, a block of hydrophilic amino acids (3 5 residues) were usually followed by a block of hydrop hobic amino acids. Interestingly, the a mino acid s histidine serine, threonine and proline frequently appeared in most IZO binding peptide sequences from both low pH and high salt elution method s Some metalloproteins have been shown to coordinate to zinc cations through basic amino acid residue s such as histidine [84, 99] Thus, it may be that the residue histidine in our IZO binding peptides may function by interact ing with the zinc components with in IZO through so me similar type of coordination binding. An evaluation of representative phage binding affinit ies panned with the high salt elution, as measured through ELISA is shown in Figure 2 2 2 Most of the representative 12 mer expressed peptide sequences display significantly higher

PAGE 68

68 preferential binding to IZO compared to other material s except for the consensus peptide sequences AGFPWSTHSSWL and ALDDLRARFLPP. AGFPWSTHSSWL shows a similar binding affinity to IZO and s apphire, while ALDDLRARFLPP tends to bind to IZO, sapphire, and Si wafer. Clone s SHAPDSTWFALF and TLMYAQPHQSKT show a distinct binding affinity to IZO as compared to other materials. IF analysis was also performed to evaluate the binding affinity of each clone. Figure 2 23 shows the binding of clone TLMYAQPHQSKT, which showed the highest ELISA signal, also displays a relatively bright fluorescence image with IF analysis on IZO substrate. Howe ver, the SHAPDSTWFALF appeared to have a little more fluorescence intensity, and was therefore examined for sp ecificity. Figure 2 2 4 shows that the phage clone SHAPDSTWFALF displayed a high preferential binding affinity to IZO, which matches the result obtained by the ELISA test. To sum marize peptide sequences with strong and spe cific binding affinity to IZO ha v e been identified by the phage display technique. With two different elution approaches, we found that the binding affinity of phage clones eluted from high salt elution was more selective to IZO than the clones collected b y low pH elution. This indicates that the high salt elution approach possibly avoided the selection of phage clones with strong binding affinity to many differ ent inorganic substrates.

PAGE 69

69 Table 2 1 P hage titers for the I ZO target system eluted with low pH buffer Elution F irst round S ec ond round T hird round C um p (pfu/ml) C am p (pfu/ml) V t ot al C um p (pfu/ml) C am p (pfu/ml) V to t al C um p (pfu/ml) C am p (pfu/ml) E1 (1 st elution) 2.7x10 5 3.3x10 14 37.5 1.0x10 5 2.0x10 17 1 8.0x10 4 3.4x10 14 E2 (2 nd elution) 1.9x10 4 3.8x10 16 1 1.0x1 0 4 7.0x10 13 150 1.5x10 4 7.1x10 14 E3 (3 nd elution) 2.0x10 4 1.2x10 15 12.5 5.0x10 4 1.0x10 13 175 4.5x10 2 1.8x10 11 E4 (4 nd elution) 1.0x10 4 2.2x10 15 75 1.1x10 4 1.0x10 14 150 2.0x10 2 8.2x10 11 Total 126 476 C um p : The concentration of u n amplified phages C am p : The concentration of amplified phages V to t al : The total taken volum e of eluted phages Table 2 2. P hage titers for the I ZO target system eluted with high salt solution Elution F irst round S econd round T hird round C um p (pfu/ml) C am p (pfu/ml) V to t al C um p (pfu/ml) C am p (pfu/ml) V to t al C um p (pfu/ml) C am p (pfu/ml) E1 (1 st elution) 1.2x10 5 1.8x10 18 1 2.1x10 5 1.3x10 13 75 8.2x10 4 1.0x10 13 E2 (2 nd elution) 2. 3 x1 0 4 3.9x10 18 1 6.1x10 4 6.7x10 12 150 3.3x10 4 8.4x10 12 E3 (3 nd elution) 4.0x 10 4 1.2x10 17 10 5.6x10 4 1.6x10 16 1 1.2x10 4 8.1x10 12 E4 (4 nd elution) 1.0x10 4 1.4x10 15 100 1.3x10 4 1.2x10 15 1 1.1x10 4 5.2x10 12 Total 112 227 C um p : The concentration of un amplified phages C am p : The concentration of amplified phages V to t al : The tot al taken volum e of eluted phages

PAGE 70

70 Table 2 3. The amino acid sequence of 12 mer peptide regions displayed by IZO binding phage eluted with low pH buffer and their binding affinity Sequence Binding % Affinity T F K YS H E L E S R G 99 S F NG RH GTT D H P T 96 S T E A HR Q S M T L T W 94 S STT L NNTT W R L T 94 S G L GSN M T AP K L E 92 S A SQ I T H FP R PPW 91 S N M T M S FP TY PIA 90 S S V S L ST MLPIP Q 89 S GN H STTN M H PPL 86 S QTG H W N A E W H T R 85 S T K N ML S LPV G P G 83.7 S TN PL SS W T FP TY 68 M IL TSS K TYT I S A 66.4 M T PLLF S M T AA R G 62 M V D G L T P HR G L K L 36 W A ffinity : S = strong binding, M = moderate binding, W = we a k binding

PAGE 71

71 Figure 2 1. Schematic of an M13 phage showing the 5 different structural protein regions (not to scale) [88] The p III proteins were used for peptide display in the present work. Reprinted from Sidhu SS. Engineering M13 for phage display. Biomol Eng. 2001;18:57 63 w ith permission from Elsevier. Figure 2 2. Schematic of the p VIII protein of M13 phage. A) M13 phage is about 880 nm in length and ~6.6 nm in diameter B) Side view and C) cross section view of p VIII coat protein. D) p VIII tilts about 20 degree s from the long axis of the phage body [86] Reprinted from Lee BY, Zhang J, Zueger C, Chung W J, Yoo SY, Wang E, et al. Virus based piezoelectric energy generation. Nature Nanotechnology. 2012;7:351 6. With permission from Nature Publishing Group.

PAGE 72

72 Figure 2 3 Phage display protocol used in the present study for the traditional chemica l elution

PAGE 73

73 Figure 2 4 Different potential structures that can be used for a peptide insert into p III protein s where r andomly generated peptide s are expressed with in either a linear or cyclic conformation

PAGE 74

74 Figure 2 5. Life cycle of the non lytic M13 phage [90] M13 phage infect the host bacteria Escherichia coli ( E. Coli ) through the F pilus. The p III protein is responsible for the attachment to the F pilus. The pilus retracts after phage have bound onto it, which allows the pIII to attach to TolA, an E. Coli membrane protein. Once pIII attach to TolA, single stranded DNA (ssDNA) of the phage enters into the host cell, and the phage body disassembles. ssDNA then converts to double stranded DNA (dsDNA), and starts to synthesiz e proteins defined by the M13 genome. Coat proteins of M13 phage s will be synthesized and await assembl y in the inner membrane of bacteria. Once the synthesized pV reach a certain amount, ssDNA stops converting to dsDNA, which makes the phage packaging sta rt. When assembly starts, pV on ssDNA is stripped off. pVII and pIX are located at either end of the newly forming phage, while pVIII is assembled and elongates the phage body. When reaching the end of ssDNA, pIII and pVI are added on. The pIII is involve d with the releasing of phage from the bacterial host membrane Reprinted with permission from Kehoe JW, Kay BK. Filamentous phage display in the new millennium. Chem Rev. 2005;105:4056 72. Copyright 2005 American Chemical Society.

PAGE 75

75 Figure 2 6. Serial dilution of phage samples as don e in a 96 well plate Figure 2 7. B lue plaques displayed on an agar plate during the b lue and white experiment for identifying bacterial plaques obtained from sel ected and amplified phage clones

PAGE 76

76 Figure 2 8 Schematic of enzyme linked immunosorbent assay (ELISA) Commercially available antibodies with attached enzyme (HRP) were used to recognize and bind to M13 phage. The reaction product of the enzyme with a particular substrate (TMB) produces a blue color from which the absorbance can be measured at a wavelength of 630 nm. Figure 2 9 Schematic of immunofluorescence (IF) analysis. An antibody raised against the phage was used to selectively bind to M13 phage. The antibody contains a docking domain for attachment of a secondary antibody that contains a fluorescent tag (FITC). Fluorescence analysis can be done qualitatively on a fluorescence microscope to compare binding to select substrates or quanti fied on an overall surface using image analysis software

PAGE 77

77 Figure 2 10. XRD data o f the IZO coated glass. A) Glass was used rather than sapphire, to avoid XRD peaks from the underlying crystalline substrate. The lack of peaks in dicate s that IZO was amorphous, as anticipated. B) XRD pattern of c rystalline IZO [100] Reprinted from Jiansheng Jie GW, Xinhai Han, Qingxuan Yu, Yuan Liao, Gongpu Li, J.G. Hou. Indium doped zinc oxide nanobelts. Chemical Physics Letters. 2004;387:466 70., with permission from Elsevier. Fig ure 2 1 1 AFM image of a representative IZO thin film. The r oot mean square (RMS) roughness of the IZO thin film was determined to be 0.552 nm over a 5 m x 5 m area.

PAGE 78

78 Figure 2 1 2 12 mer amino acid sequence s of peptides displayed on pIII proteins of IZO binding phages selected by phage display using low pH elution buffer.

PAGE 79

79 Figure 2 1 3 Images of the immunofluorescence analysis for t he negative control experiment O nly monoclonal anti M13 antibody and monoclonal anti mouse IgG FITC were incubated with IZO, sapphire, Si, and SIO 2 respectively. No fluorescence was observed from these inorganic substrates. Scale bar s : 100 Figure 2 1 4 IF image of M13KE It is a negative control for non binding phage. Scale bar: 2 00

PAGE 80

80 Figure 2 1 5 IF analysis for t he phage clon e TKNMLSLPVGPG It showed binding affinity to the both of IZO and sapphire However, this phage clone preferentially b ound to IZO because the intensity of fluorescence from IZO is markedly stron ger than sapphire. Scale bar: 20 0

PAGE 81

81 Figure 2 1 6 IF analysis of t he phage clone MNRPSPPLPLWV It showed preferential binding affinity to sapphire, rather than IZO. This suggests that this phage clone might have be en selected from the exposed sapphire edge s without IZO coating. Scale bar: 100

PAGE 82

82 Figure 2 1 7 Surface coverage pe rcentage of IZO binding clones panned with low pH buffer elution. Coverage was determined by Image J. A fluorescence image was converted to a 32 bit black and white image, and the ratio of black area to the whole picture was measured.

PAGE 83

83 Figure 2 1 8 I mage s of ELISA plate s contai ni ng phage clones eluted from IZO with low pH elu t ion buffer with the corresponding substrates indicated for each row and the 12 mer peptide indicated for each column A ) I mage of ELISA plate containing 4 phage clones from the third biopanning round after 20 minute development time B ) I mage of ELISA plate containing 3 phage clones from the second biopanning with low pH buffer elution after 20 minute development time

PAGE 84

84 Figure 2 1 9 Absorbance of the enzymatic TMB substrate solution f ro m E LISA plates of IZO binding clones obtained by low pH elution buffer, as show n in F igure 2 1 2 By two way ANOVA with different materials and clones as factor P<0.05. Figure 2 20 Photograph of t he etch ed IZ O that was caused by using the low pH elution buffer The color of IZO at an initial thickness of 200 nm is normally light brown.

PAGE 85

85 Figure 2 21 12 mer amino acid sequen ce s of peptides displayed on pIII proteins of IZO binding phages selected by phage display using high salt elution. Figure 2 2 2 Absorbance of the enzymatic TMB su bstrate solution s f ro m ELISA plates of IZO binding clones o btained by high salt elution By two way ANOVA with different materials and clones as factor, P<0.05.

PAGE 86

86 Figure 2 2 3 IF image s for t he phage clone TLMYAQPHQSKT from high salt elution Scale bar: 100 m. Figure 2 2 4 IF image s for t he phage clone SHAPDSTWFALF from high salt elution. Scale bar: 250 m

PAGE 87

87 CHAPTER 3 SCREENING FOR INDIUM ZINC O XIDE BINDING PEPTIDE S WITH ELECTROACTIV E PROPERT IES USING A NOVEL ELECTRO ELUTION APPROACH TO PHAGE DISPLAY 3 1 Overview For biosensor devices, functionalizing the surface with covalent linkers is usually employed. However, covalent bonds are permanent, which makes the functionalized surface permanent. This may not always be desirable. For example, in biosensors, it is difficult to avoid the loss of activity following the bioreceptor analyte binding event, which limits the lifetime of the device. The goal of this chapter is to use phage display to biopan for inorganic binding peptides that can serve as non covalen t linkers that are reversible upon appl ication of an electric field. This can provide dynamic functionalization of microelectronic surfaces, with applications such as self cleaning devices dynamic patterning and micro transport For example, in biosensors when the bioreceptor becomes clogged, the peptide linker may be released by triggering an electric field to generate a non binding state for release and removal of the clogged bioreceptor A fresh surface of bioreceptors c ould then be applied via a flow through setup (Figure 3 1). Peptides that bind strongly to IZO have been obtained as described in the previous chapter. In this chapter, because a strong binding peptide might not necessar il y be a reversible peptide, a novel phage display biopanning proto col was developed where an electro elution process replaces the regular chemical elution step, in order to directly obtain a n electroactive IZO binding peptide.

PAGE 88

88 3. 2 B ackground and S ignifican ce 3.2. 1 Biosensor s Biosensors are analytical devices that can s pecifically, rapidly, and continuously convert a biological/chemical response into a readable signal. Since the first biosensor reported in 1962 by Leyland C. Clark, biosensors are becoming increasingly important in many applications such as medicine, food science, environmental monitoring and basic research. Figure 3 2 shows the market of biosensor s in millions of US dollars in the past twenty years and the predict ed value in the future [101] In general, a biosensor device consists of a sensing component chemically or physically immobilized onto the transducer element which converts the biological, chemical, or biochemical signal to an optical or electrical signal (Figure 3 3). Due to the size and expense of optical components, there is a distinct advantage to preparing electronic biosensors, as long as a means for sign al transduction can obtained. Biosensors can be classified by the type of receptor or the type of transducer. Bioreceptors can be enzymes, antibody or antigen, cells, nucleic acids, and biomimetic molecules Metabolism sensors are devices where the signal is measured by the difference of product concentration after a chemical reaction produced by the analyte and receptor binding event. Catalytic sensors detect the signal of converting an auxiliary substrate without changing the analyte chemically [102] Affinity sensors are focused on detecti on of the binding event, and immunosensors are antibody ba sed sensors. Immobilization methods for bioreceptors o nto the transducer surface are covalent binding, crosslinking, or simply adsorption through secondary interactions Many different ways can be used to achieve transduction. It can be an electrochemical detection, optical detection or a mass detection method [102, 103] To date, one of the

PAGE 89

89 challenges of current biosensors is the lack of continuous detection. It is difficul t to avoid the loss of activity following a bioreceptor analyte binding event, which limits the lifetime of the device. On the other hand, some of the biosensor devices are disposable, one time detection devices, if they can be made cheaply Also, with the increasing demand for sensors, and the variation between batch to batch which can generate false positive or false negative responses, high quality reusable biosensor s are be ing explor ed Hence, in this study, we bring up a new idea to achieve the goal of a self cleaning device by releasing and restoring the immobilized bioreceptors And another possible application of electroactive peptides is the possibility of a dynamic ally pattern ed surface By spatial control on a microe lectronic device, electroacti ve peptide s c ould act as a cargo to carry linked receptor s or other components to a target location. A temporal and spatial control c ould be achieved by electrically activat ing specific area s, or pixels, for binding and releasing the peptide linkers with t heir attached cargo (Figure 3 4) 3 .2. 2 Monolayer s used for S ensing D evice s Self assembled monolayer s (SAM s ) ha ve been widely used to functionalize a transducer surface of biosensors with various attached organic molecules or receptors This is based on th e idea of making organic thin films. SAM s are easily prepared by immersing a substrate into a solution of organic molecule s which can self assemble to form a very dense film by the chemisorption between the headgroup of organic molecule and the substrate [104] For example, the most commonly used SAMs are on gold and silica substrates, where t hiol has been found to preferentially attach to noble metal s and silane has been found to attach to oxide s [105, 106] Thi ol linked SAMs have high stability on Au surface s due to the S Au bond and the multitude of secondary van der Waals interaction s between the aligned hydrocarbon tails, which cause them to

PAGE 90

90 form an ordered and densely packed monolayer structure. In an oxide free surface, thiol can also bind to many different materials, such as Ag, Cu, Pt, GaAs, and InP [107] Silane SAMs are more complex than thiol SAMs. Silane SAMs attach to silica and glass by covalent bonding to hydroxyl groups at the surface [108] By modif ying the free solution facing end group of these organic molecules with a probe pro tein or a functional group for some desired chemistry functional ized surface s can be achieved (Figure 3 5 ). Langmuir Blodgett films are an other method to link a biomolecule to material surface. First, a Langmuir film is created at the air water interfac e which is based on the use of amphiphilic molecules where the hydrophobic endgroups oriented towards the air, while the hydrophilic headgroups face down in to the solution, or water phase Langmuir Blodgett films are created by transfer ring Langmuir films to a solid substrate that is slowly dipped through the interface containing the Langmuir film (Figure 3 6 ) [109] However, compared to SAMs, SAMs have better stability and have more choices of substrate materials which make SAMs a preferred method for many different applications [104] SAMs have been utilized in ma ny sensing devices including biosensors, such as dopamine sensors and glucose sensors, pH sensors, and chemical sensors, such as detection of electrochemical reactions [110 112] SAMs have also been combined with m olecules to enhance many surface characterization techniques like SPR, QCM, and AFM [112] Even though SAMs provide a stable way to create modified and functionalized surface s there are some significant shortcomings. First of all, SAMs cannot be specifically b ou nd to certain material s For example, thiol s c annot tell Au and Pt apart.

PAGE 91

91 Secondly, SAMs are rigid, narrow and one dimensional. Also, attachment o f endgroup s can only be linked chemically, and cannot be modified genetically. SAMs are formed in a nonbiological environment, and most of the SAMs are toxi c which also limit s the ir use for in vivo applications [3, 44] I norganic binding peptide s can avoid some of these disadvantages and provide a more specific linker for possible biosensor applications where one ca n connect a biological probe to new target material s due to the high material specificity and affinity of the peptides One could even have multiple components immobilized onto patterned surfaces of differing inorganics, as nicely demonstrated by the mole cular construction [45] 3.2. 3 Electrodesorption Some reports mention the use of electric fields to release a bound organic, where biofouling surfaces. Tang et al. [113] f ound that electrical stimulation could remove a triblock copolymer of co(propylene sulfide block ethylene glycol) (PPS PEG) from ITO surfaces (Figure 3 7 ). These copolymers have been studied as protein resistant coatings because the PPS component ha s origi nally been found to chemisorb onto gold surfaces while the PEG component prevents protein adsorption This group applied an ascending anodic electrical stimulus to the surface of the modified samples, and found that the copolymer was steadily removed, wit h the complete loss of a polymeric monolayer at a potential of 2000 mV (Figure 3 8 ). However, the mechanism of the electro desorption was unk n own. Yeh. et al. ha ve also demonstrated an electric field desorption of the protein bovine serum albumin (BSA) fro m a lead zirconate titanate substrate, which is a piezoelectric transducer (PZT), coated with either fired silver or titanium (Figure 3 9 ).

PAGE 92

92 Through modeling, they believed that the applied electric potential was the major contributor in reducing the adhesi ve force between protein and surface, where the desorbed protein was then removed by an acoustic streaming shear stress. They also described a mechanism where charge polarity of the PZT would be the same as that of the adsorbed proteins, leading to repulsi ve forces between surface and protein [114] These reports demonstrate that the electrodesorption principle is feasible and compared to either synthetic copolymer or protein, we anticipate that peptides with a smaller size should be desorbed more quickly and completely and might even be desorbed at a lower potential. In this chapter, we developed a novel phage display ap proach using electro elution biopanning to select clones that are more sensitive to an electric field, and further examined the electroactive propert ies by using an electroreleasing test to test individual phage clone s 3. 3 Materials and Methods 3.3. 1 Ma terials 3.3.1.1 P hage d isplay p eptide l ibrary and E.coli. Phage display related materials are the same as mentioned in Chapter 2. 3.3.1.2 Power s upply Keithley Series 2400 SourceMeter was used for current voltage measurement. 3.3.1.3 Polydimethylsiloxane ( PDMS ) Sylgard 184 sili cone elastomer kit was purchased from Dow Corning.

PAGE 93

93 3.3. 2 Methods 3.3.2.1 Fabrication of IZO device To perform the electro elution biopanning, we designed an IZO coated device. This device was composed of a two electrode system separated by a block of PDMS to seal in the solution (Figure 3 11). Devices coated with IZO for electro elution biopanning A SiO 2 / Si wafer was cut into 25 mm x 25 mm squares Wafers were cleaned in an ultrasonic bath for 5 minutes each in trichloroethylene, acetone, and methanol, and were blown dry by nitrogen gas to remove organic or inorganic contamination. Ti and Au thin films were deposited by KJL CMS 18 Multi source sputtering system. Ti was deposited with 10 nm and Au was deposited with 80 nm thickness. Ti layer was for Au adhesion. Final metal layers were Ti / Au / Ti. After metal deposition, photoresist ( PR ) S1813 was used to block an area to later connect gold wire to apply the potential IZO was deposited last, on top of the conducting metal layers. Fabrication of gold electrode A SiO 2 / Si wafer was cut into 25 mm x 25 mm squares Wafers were cleaned in an ultrasonic bath for 5 minutes each in trichloroethylene, acetone, and methanol, and were blown dry by nitrogen gas to remove organic or inorganic contamination. Ti and Au thin films were deposited by KJL CMS 18 Multi source sputtering system. Ti was deposited with 10 nm and Au was deposited with 80 nm thickness. Gold surface was then blocked, and an area of Ti was deposited for connecting a gold wire later D evice s coated with IZO for electro releasing experiments In the fabrication of the IZO devices, glass slides 25 mm x 25 mm in size were used as substrates Substrates were cleaned in an ultrasonic bath for 5 minutes each in trichloroethylene,

PAGE 94

94 acetone, and methanol, and were blown dry by nitrogen gas to remove organic or inorganic contamination. Substrates were heated in an oven at 105 o C for 10 minutes to remove moisture on the substrate surface. S1813 positive photoresist (PR) (Shipley) was coated onto the substrate by a spi nner ( Headway ) at 5000 rpm for 50 seconds Subsequently, PR coated substrates were baked at 100 o C for 90 seconds on a hot plate. Then, PR covered substrates were exposed to light with wavelength in 365 nm for 15 seconds by Karl Suss MA 6 Contact aligner sy stem with hard contact mode under exposure intensity in 8 m W/cm 2 To develop the photoresist after exposure, AZ 300 developer was used. We developed for 50 seconds and washed for 2 minutes in deionized (DI) water. After the developing process, we used the sputtering system to make metal electrodes. Ti and Au thin films were deposited by KJL CMS 18 Multi source sputtering system. Ti was deposited with 10 nm and Au was deposited with 80 nm thickness. Final metal layers were Ti / Au / Ti. After metal depositi on, a circ ular pattern was made with the PR (S1813) to connect gold wires later, which would then be removed after the IZO film deposition PDMS block Silicone elastomer base and silicone elastomer curing agent Sylgard 184 were mixed in a 10 to 1 radio. Mix was poured into a mold, and cured at 60 degree Celsius for overnight. 3.3.2.2 Phage display protocol with e lectro elution Phage display protocol and the other methods applied during the phage display procedure are discussed in detail as follows. All chemicals and labware were autoclaved, and the reactions performed in an Airclean 600 PCR workstation laminar flow hood.

PAGE 95

95 Binding s tep In this step, a 1 12 in 990 l PC buffer was exposed to an IZO coated device by placing the solution within a PDMS rubber ring which could be sealed to the surface of the device in order to hold the phage solution during incubation. The IZO coated d evice was shaken by a platform shaker ( New Brunswick Scientific ) for 60 min in order to obtain sufficient time for phage IZO interaction. Washing s tep Washing steps are the same as mentioned in Chapter 2. Elution s tep After the washing steps, strongly bo und phages were recovered from the IZO surface through electro elution. A power supply was used for eluting bound phage. During appl ication of an electric field, a micropipette was used to create a gentle flow across the surface of the IZO device and to c ollect the solution containing the eluted phage The solution containing electro eluted phages was transferred into a fresh microfuge tube Then, 100 l supernatant was transferred into E. coli ER2738 culture (LB Lennox medium: E. coli ER2738 overnight cul ture = 100:1) and it was incubated for 4.5 hours at 37C and 250 rpm. These electro elution steps were repeated for four times. Amplification and p urification s teps These steps are the same as the description in Chapter 2. 3.3.2.3 Zeta potential m easurement on IZO thin film Paar Physica Electro Kinetic Analyzer in the Particle Engineering Research Center at UF was used to measure the zeta potential of a representative IZO thin film. The IZO thin film was fabricated by sputtering IZO onto the top of a 20 mm x 40 mm silicon wafer. A 1.0 mM KCl solution was used as the electrolyte solution (as required

PAGE 96

96 for these measurements) and the pH was adjusted from pH 2.0 11.0 with 0.1N HCl or 0.1N NaOH. 3.3.2.4 Electro releasi ng test by immunofluorescence (IF) a nalysis An IZO coate d electro releasing device was incubated with a single phage clone (10 9 pfu/ml) in 1 ml PC buffer on a shaker for 1 hour, follow ed by washing steps to rem ove unbound phage. Anti M13 pIII monoclonal antibody and anti mouse IgG FITC were applied as primary a nd secondary antibod ies, respectively, according to the same procedure described in Chapter 2. These complexes were visualized at 20X magnification and 2 sec exposure time with a fluorescence microscope (Nikon) After taking fluorescence image s an electri c field was applied to the device with flow through created by the micropipette. Washing steps was performed and the device was visualized again by the fluorescence microscope. 3. 4 Result s and Discussion In this chapter, we utilized a novel phage display protocol to select clones that directly release from simple electronic device when an electric field is applied during the elution step of biopanning ; we therefore have called this an electro elution biopanning method. In the traditional approach, in order to collect phages that bind onto the target in a biopanning round, a number of treatments can be applied to elute the bound phage as previously described, such as l owering or increasing pH or eluting with a high salt solution Because a strong binding p eptide might not necessarily be a reversible peptide, an electro elution process was used to replace the regular chemical elution step, in order to directly obtain an electroactive IZO binding peptide. For the electro elution approach we developed, an elec tric field was applied across an IZO coated device to achieve the elution step. In other words, after washing away unbound phage in the first

PAGE 97

97 phage display step, the device was then activated to apply an electric field to release the tightly bound phage. The ones that release were collected and amplified for further study (Figure 3 10 ). To perform the electro elution biopanning, we designed an IZO coated device. This device was composed of a two electrode system separated by a block of PDMS to seal phage in the solution One electrode was coated with IZO to adsorb the phage, and the other electrode was coated with a Au layer only to provide a difference of electric potential (Figure 3 1 1 ). These two parallel electrode plates can provide a direct electric field across the solution, and therefore across the adsorbed phage, from the positive to negative potential electrodes The e lectric field was calculated to be around 10 3 Vm 1 at a distance of 1 mm between two parallel electrodes. After the electro elutio n step, phage clones were picked for DNA sequencing to deduce the IZO binding peptide sequences that provided this electroactiv e property. We performed the phage display biopanning for two round s After the electro elution step of each round, a chemical el ution with low pH buffer was performed to confirm the phage library and amplification by E.Coli. host bacterial ER2738 strain was still working properly (i.e., the phage were not damaged by the electric field) Figure 3 1 2 shows the expressed 12 mer peptid e sequences translated from the DNA of phage clones collected by first and second round electro elution biopanning. Encoded colors indicate the different chemical properties of the individual amino acid residue s To examine binding affinities of each of t he selected clones, an ELISA test was performed. In the ELISA test, a secondary antibody conjugated with horseradish peroxide (HRP), anti M13:HRP, was used to quantitatively measure M13 phages

PAGE 98

98 absorbed onto the surface of inorganic substrates. When HRP cat alyze s the enzymatic tetramethylbenzidine (TMB) substrate, the oxidized product of TMB has a deep blue color. A deep blue color indicates a higher density of phages on those substrates. The degree of blue color can be quantified by absorbance measurement at 630 nm. Figure s 3 1 3 and 3 1 4 show the absorbance value at 630 nm of the enzymatic substrate solution from represent ative clones selected from first and second biopanning rounds M13KE is the Ph.D. library cloning vector, and was used as a control in this study. The specificity of various clones to the IZO surface as compared to two inorganic substrates, Si (100), and SiO 2 (amorphous) was compared A higher absorbance value means the peptide had a higher binding affinity as it wa s incubated on the indicated target. Even though IZO binding peptides obtained by electro elution 2 and Si, all the clones show a high binding affinity to IZO as compared to M13KE. In order to visua lize the phage binding on inorganic substrates, immunofluorescence analysis (IF) was used by utilizing a fluorescen t tag (FITC) conjugated with anti mouse IgG to indicate the location of M13 phage on the inorganic substrate surface as viewed under fluores cence microscop y. Figure 3 1 5 shows that clone SPRLILQMLNRI from the first biopanning round has a preferential binding affinity to IZO and Si, but not SiO 2 Interestingly, it was observed that there is a high prevalence of repeated arginines in clones s eleced by electro elution biopanning as has also been seen in the literature, where many ZnO binding peptides show arginine arginine ( RR ) in sequence [115] Also serine and leucine were observed frequently with electro elution approach a nd they were also found in ZnO binding peptides in the literature using chemical

PAGE 99

99 elution [116] Proline and threonine frequently appeared which w as the same as the peptides selected by chemical elution. Another interesting result was that based on calculation of the AAs composition of all the peptides, we found that the sequences of peptides collected by electro elution biopanning displayed quite a different composition of a mino acid residues: histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, and threonine (Figure 3 1 6 ), as com pare d to the chemical elution clones (Figure 3 1 7 ). For chemical elution clones, 19 out of 20 amino acids were expressed except cysteine. It might be because cysteine in the random peptide sequence interferes with phage infectivity of the bacterial host, result ing in the low frequency observed in the Ph.D 12 library [98] Comparing phage clones collected by electro elution biopanning and traditional chemical elution biopanning discuss ed in Chapter 2, we found the clones were different. We found that there were fewer hydrophobic residues, where the chemically eluted phage tended to have sequences of green colored AAs ( see Figures 2 12 and 2 21). Table 3 1 3 2 and 3 3 show the number of charged amino acid residue s found in both approaches and all of the electro elution clones have positive charged residues and lack of negative charged residues while this is not found on all chemical elution clones. Our hypothesis i s that charged clones might play an important r o le for peptide releas e by an electric field. However, this result also indicate s that electrostatic interaction s might be one of the rea sons for the initial peptide to binding on IZO but that the degree and type of charge groups differs significantly when the phage are electro eluted To explore the electrostatic interaction s between IZO and peptide binders, the charge character of the p eptides and the inorganic surface need s to be confirmed. The

PAGE 100

100 collected IZO binding peptide sequences were calculated using comput ation programs on http://expasy.org (Table 3 1 3 2, and 3 3 .) For the charged character of IZO, zeta potential was used to determine the surface charge in different pH condition s We used Paar Physica Electro Kinetic Analyzer in the Particle Engineering Research Center at UF to measure the zeta potential profile of our deposited IZO thin film as shown in Figure 3 1 8 The point of zero charge of IZO is around pH 4.0, and at around pH 7.7 (the biopanning pH) the zeta potential is around 20.0 mV. Combining the result s from the zeta potential and the property of the collected clones, we believe t hat electrostatic interactions play an important role for the IZO binding peptides where the positive charge of the numerous basic residues may be attracted to the negatively charged IZO And based on literature, sapphire has a similar zeta potential prof ile, which might be the reason some IZO binders tend to bind to the sapphire surface as well [117] To test the releasing property of clones selected by electro elution we first utilized IF analysis with the devices we designed for performing an electro elution step (Figure 3 1 1 ). A scratch was m ade in the middle of IZO coated electrode to make sure we were taking image s at the same spot because phage tend to be trapped by defect s in the structure. Figure 3 1 9 shows an electro releasing test for consensus electro elution clone: MLPIIRNLIHTT. The first image was taken following the primary and secondary anti body binding step before appli cation of a voltage on the device (Figure 3 1 9 (a)). After applying 1V for 30 seconds, an image was taken again (Figure 3 1 9 (b)). The bright line in both Figure 3 1 9 (a) and (b) indicates the scratch which trapped many phage clones just due to the structure. As judging from visual inspection of the images before and after the voltage was applied, it appears that the electro releasing of clones

PAGE 101

101 was fairly effective. A nd based on the remaining green line of the scratch, it was confirmed that the fluorescent dye was not photobleached during the time of the procedure (which was approximately 15 minutes ) After the preliminary tests, we designed a device with two regions on the surface, with one having an current passing through it, next to a region that is similarly prepared, but not connected to the electrodes, so it would not produce an electric field so that when we image d with fluorescence, we c ould image both region s at the same time, to demonstrate selective release in the r e gion with the field. This was to avoid issues associated with fluorescence, such as variability in contrast, which can vary with the setup conditions, and photobleaching. Figure 3 20 is a schem atic of the device. This device was built on a glass substrate instead of Si wafer to avoid any current at the bottom ; a non conductive region, around 150 m in width, was used to separate the two deposited sides. By utilizing IF ana lysis on the device, F i gure 3 2 1 (b) and (c) show s a n electro releas e experiment o f phage clone display ing peptide sequence: MLPIIRNLIHTT Note, the separation region between the two sides appears green was from the autofluoresence of petri dish from the bottom of the device, vi sualized through glass substrate of the device. This green region provided a more clear view of two separated regions. O n the left region which had an electric field applied, there was a large reduction in fluorescence suggesting good release However, t here also seemed to be some release on the right side, which we believe may have been due to carryover of electric field since the separation distance was not that large. It seem ed to stop about 100 microns from the separation gap. This result demonstrated the releasing of phage clones selected by the electro elution biopanning approach. The s ame electro releasing

PAGE 102

102 test was performed on phage clones selected by the chemical elution approach. Figure 3 22 (a) and (b) shows an electro releasing experiment of ph age clone displaying peptide sequence: TKNMLSLPVGPG before and after applying an electric field on the left side of the device However, no releasing was observed. To sum up, in this chapter, we demonstrated the feasibility of a novel phage display protoco l with an electro elution process. And by utilizing this protocol, IZO binding peptides with electroactive propert ies w ere obtained.

PAGE 103

103 Table 3 1. Number of charged amino acid residues of peptide sequences selected by low pH elution biopanning *: indicates consensus sequence from second and third biopanning round Table 3 2. Num ber of charged amino acid residues of peptide sequences selected by high salt elution biopanning Chemical elution (high salt buffer) Number of charged amino acid 12 mer amino acid sequence positive negative S H AP D ST WFALF 1 1 TNSSSQ FVVAIP 0 0 AL DD L R A R FLPP 2 2 H P K P S L D AA R LV 3 1 T LM Y A Q P H GS K T 2 0 A G FPW ST H SS WL 1 0 *: indicates consensus sequence from second and third biopanning round Chemical el ution (low pH buffer) Number of charged amino acid 12 mer amino acid sequence positive negative T E A HR QS M T L T W 2 1 GN H STTN M H PPL 2 0 F NG RH GTT D H P T 3 1 TN PL SS W T FP TY 0 0 G L GSN M T AP K L E 1 1 A SQ I T H FP R PPW 2 0 S V S L ST MLPIP Q 0 0 T PLLF S M T AA R G 1 0 H QSG K G W T I H Y E 3 1 STT L NNTT W R L Y 1 0 V D G L T P HR G L K L 3 1 T F K YS H E L E S R G 3 2 T K N ML S LPV G P G 1 0 IL TSS K TYT I S A 1 0 N M T M S FP TY PIA 0 0 QTG H W N A E W H T R 3 1 M N R P S PPLPLWV 1 0

PAGE 104

104 Table 3 3 Number of charged amino acid residues of peptide sequences selected by e l ectro elution biopanning Elec tro elution Number of charged amino acid 12 mer amino acid sequence positive negative MLPII R N LI H TT* 2 0 K NT RR IP S H P T I 4 0 R PL HHRRRHH S P 8 0 L R M K PL R TT RR Q 5 0 RR I H T L Q I RRR S 6 0 R I SQ P N L R I K T P 3 0 S RR LIL Q ML N R I 3 0 R M N RK T L Q RR P 5 0 HK P R PP T I S IIL 3 0 R NT KK M R L S KR Q 6 0 N R S R IPLI HR II 4 0 R I R N H IL QT I R S 4 0 I T L RH P RRR LI R 6 0 : indicates consensus sequence from first and second biopanning round

PAGE 105

105 Figure 3 1. Schematic of self cleaning, renewable biosensor. T he red linker peptide is derived from the phage display approach by screening for inorganic binding peptides identified as being electroactive which can be reversibly bound to the target surface Figure 3 2. Graph of the value of biosensor market i n US$ millions [101] Turner APF. Biosensors: sense and sensibility Chemical Society Review. 2013;42:3175 648.

PAGE 106

106 Figure 3 3. Schematic of biosensor components Figure 3 4. Schematic of a dynamic patterning surface. The concept is that a peptide linker (with its attached cargo) can be moved to different locations on a pixelated device, where the electroactive properties can be selectively activated by electronic processing. It may be desirable to have an antifouling compound, such as polyethylene glycol (PEG) in the remaining areas to avoid non specific adsorption, where it may have the opposite activation properties to bind in locations where the cargo is released.

PAGE 107

107 Figure 3 5. Schematic of SAM s Headgroups bind to the substrate while endgroups can be different chemistries to ach ieve a chemical ly functional ized monolayer surface Figure 3 6. Schematic of different methods for preparation of organic monolayer films [109] Reprinted from Schreiber F. Structure and growth of self assembling monolayers. 2000;65:151 257. With permission from Elsevier.

PAGE 108

108 Figure 3 7. Schematic of the surface f unctionalization and releasing of PPS PEG from an ITO surface. (a) A 100 nm thick ITO coat ing deposited on a silicon wafer. (b) An adlayer of PPS PE G block copolymer chemisorbed on to the ITO surface. (c) After applying a direct current voltage, PPS PEG is removed from the surface. [113] Reprinted from Tang C, Feller L, Rossbach P, Keller B, Voros J, Tosatti S, et al. Adsorption and electrically stimulated desorption of the triblock copolymer poly(propylene sulfide bl ethylene glycol) ( PPS PEG) from indium tin oxide (ITO) surfaces. Surface Science. 2006;600:1510 7. With permission from Elsevier. Figure 3 8. A plot of the thickness of the PPS PEG ad layer at different applied potential s measured by spectroscopic ellipsmetry [113] Reprinted from Tang C, Feller L, Rossbach P, Keller B, Voros J, Tosatti S, et al. Adsorption and electrically stimulated desorption of the triblock copolymer poly(propylene sulfide bl ethylene glycol) (PPS PEG) from indium tin oxide (I TO) surfaces. Surface Science. 2006;600:1510 7. With permission from Elsevier.

PAGE 109

109 Figure 3 9. Schematic of electrode s orption of proteins. Ad s orbed proteins can be desorbed by an electric field and then removed by acoustic streaming generated by vibration. [114] Reprinted from Yeh PYJ, Kizhakkedathu JN, Madden JD, Chiao M. Electric field and Vibration assisted nanomolecule desorption and anti biofouling for biosensor applications. Colloids and Surfaces B Biointerfaces. 2007;59:67 73. With permission from Elsevier.

PAGE 110

110 Figure 3 10. Schematic of our newly developed phage display technique with an electro elution biopanning step that replaces the commonly used chemical elution, which is enabled using an electroreleasing device as the target substrate, in order to screen for peptides with electroactive properties

PAGE 111

111 F igure 3 11. Schematic of our design of an IZO coated electro elution device. The peptide inserted pIII proteins (not drawn to scale). The dimension of the liquid cell spacing b etween two electrodes is 1 mm.

PAGE 112

112 Figure 3 1 2 The amino acid sequences of 12 mer IZO binding peptides selected by two rounds of electro elution biopanning.

PAGE 113

113 Figure 3 13 Quantification of the amount of phage clones with the in dicated peptide inserts, bound to three inorganic surfaces, as determined by a bsorbance of the enzymatic substrate solution f r om E LISA plates of clones selected from the first electro elution biopanning round. M13KE which is phage that does not contain in serted peptide, was used as a negative control, to make sure it does not inherently have good binding characteristics. By two way ANOVA with different materials and clones as factor, P<0.05.

PAGE 114

114 Figure 3 1 4 Quantification of the amount of phage clones with the indicated peptide inserts, bound to three inorganic surfaces, as determined by a bsorbance of the enzymatic substrate solution form ELISA plates of clones selected from the second electro elution biopanning round M13KE which is phage that does not co ntain inserted peptide, was used as a negative control, to make sure it does not inherently have good binding characteristics. By two way ANOVA with different materials and clones as factor, P<0.05. Figure 3 15 IF analysis of electro elution clone SRRLILQMLNRI incubated on IZO, SiO 2 and Si surfaces Scale bar: 200 m.

PAGE 115

115 Figure 3 1 6 Composition percentage of each amino acid of IZO binding peptides from phage selected by electro elution biopanning. A strong preference fo r R, arginine, is seen, as well as several showing pronounced lysines, L, and h istidine, H. Figure 3 1 7 Composition percentage of each amino acid of IZO binding peptides from phage selected by chemical elution biopanning.

PAGE 116

11 6 Figure 3 1 8 Measurement of the zeta potential pH profile of an IZO thin film deposited on wafer. At pH 7, which is the pH of the biopanning buffer, the zeta potential of IZO is around 20 mV. Figure 3 1 9 Fluorescence images of electro eluted phage clone with displayed peptide MLPIIRNLIHTT before (a) and after (b) applying an electric field of 1000 mV for 30 seconds. Scare bar: 100 m.

PAGE 117

117 Figure 3 20. Schematic of a n electro releasing device designed to have a no n conductive spacer to separat e the areas for applying an electric field on only one side The separation gap is about 150 m.

PAGE 118

118 Figure 3 2 1 Electro releasing test using electro eluted phage clone with displayed peptide MLPIIRNLIHTT Fluorescen ce images taken before ( A ) and after ( B and C ) applying an electric field of 1000 mV for 30 seconds on th e left side of the device (C) Image was taken at a lower magnification to show a larger region, where phage are still adsorbed further away from the separation gap, but apparently some release occurred near the gap since there seems to be a zone just to t he right that did release the phage. Scale bar: 100 m for images in A B, and C.

PAGE 119

119 Figure 3 22. Electro releasing test using chemical elution phage clone with displayed peptide : TKNMLSLPVGPG Fluorescence images taken befo re (A) and after (B) applying an electric field of 1000 mV for 30 seconds on the left side of the device Scale bar: 100 m

PAGE 120

120 CHAPTER 4 CHARACTERIZATION OF SYNTHE TIC INDIUM ZINC OXIDE BINDING PEPTID ES 4. 1 Overview I n previous chapters, IZO binding phage clones have been screened by two phage display approaches The 12 mer amino acid sequences displayed by the pIII tail proteins on M13 phage have been identified by DNA sequencing and the binding affinity of each sequence displayed by the clones has been t ested by IF and ELISA. In comparison between chemical elution versus electrical elution biopanning a difference between the types of amino acids within the sequences obtained by the two approaches has been revealed. However, since a whole phage particle i s much larger than the 12 mer displayed peptide, the results of binding analysis of M13 phages does not provide direct evidence of a binding affinity of the peptide alone Also, the specificity of a peptide for an inorganic surface is likely to be rooted i n its molecular structure which may be impacted by the surrounding pIII protein sequence and conformation Therefore, t he binding and electro releasing properties of the isolated peptide s also need to be examined because it is the peptides alone that are intended to serve as linkers to bioreceptors or other various bionanotechnology applications In this case, o ne might anticipate that a short peptide might be easier to releas e from a surface than the much larger phage body which could have multiple att achment sites, or could lay flat on the surface and simply bind through non specific reduction in surface energy In this chapter, we will confirm the binding affinity of the peptide sequences obtained from the previous chapter s by examining synthetic pept ides and also test the electroacti ve properties of the peptides that appear most promising

PAGE 121

121 4. 2 Background and S ignifican ce Since the first f in d ing of RGD (arginine glycine aspartic acid) peptide in 1984 by Pierschbacher et al [118] cell recognition peptide s ha ve drawn t he attention of the bioengineering community Scientists from different fields su ch as biology, chemistry, biochemistry, pharmacy, material science, and surface engineering have studied the interactions between protein s or peptide s with cell or material surface s They first focused on protein interactions. Laminin, fibronectin, and col lagen are widely used cell adhesive proteins [1] However, because of the natur al properties of proteins, they have many disadvantages. Firs t, proteins need to be isolated and purified from some organism, which is challenging and has the risk of immune response for any in vivo applications. Second, proteins are easy to denature or degrade. Therefore, l ong time usage of proteins will often requ ire replenish ment with fresh ones. Also, only certain domains of proteins have the adhesion function which leaves many possible varieties of adhesion on other domains Proteins can change orientations or conformations with in different environmental condit ions [18] Considering these disadvantages, peptide mimic s have become a promising way to functionalize a surface. Peptides can sometimes mimic the ac tive domain of a protein to provide the same functionality yet while being smaller and less sensitive to the environment Peptides can be made with well developed synthe tic techniques, without requiring development of recombinant protocols necessary for f ull proteins The RGD peptide motif has been widely used for enhancing cell adhesion. Different combinations of RGD containing peptides were tested for different cell lines. For example, it has been shown that RGD containing peptides can promote cell atta chment and differentiation on cartilage [119, 120] bone [121, 122] neural [123]

PAGE 122

122 and endothelial tissues [124] In addition to RGD containing peptide s there are many other peptide sequences that have been selected from natural protein s and prove n functional. For example, YIGSR and IKVAV derived from laminin have been prove n to increase human foreskin fibroblast adhesion and neurite extension respectively [125, 126] FHRRIKA and KRSR derived from heparin binding domain s, have been prove n to increase osteoblast adhesion and mineralization [127, 128] REDV derived from fibronection ha s been prove n to increase endothelial cell adhesion [129] Even though peptides selected from protein domains can be used to functional surface s phage display provides a de novo route which can provide more combination s of sequences for selecting and design ing functional peptide s At present, the effect of the primary and secondary peptide structure on peptide material interactions is not well understood, but some researchers have indicated that the molecular conformations and architectures have a major effe ct on the binding behavior [130] For many types of peptides, circular dichroism (CD) has been used to study the global effect of different peptid e conformations on nanoparticle binding and adsorption characteristics [131] CD is an excellent tool for determining the secondary structure of peptides and proteins. Briefly, CD is the differ ential absorption of left and right handed circularly polarized light. The effect will occur when a light wave interacts with a symmetric molecules. In the far UV region (190 250 nm), a CD spectrum can be used to analy ze the secondary structure of a protein or peptide. In the near UV region (260 320 nm), the CD signal provides the tertiary structure of the protein [132, 133] In this study, we were only focused on the far UV regain, which can reveal secondary

PAGE 123

123 structure s, such as alpha helix, beta sheet, or random coil, of our peptides in order to elucidate if there are any structur al effec t s influencing the binding affinity 4. 3 Material and Methods 4. 3 .1 Materials 4.3.1.1 Inorganic binding p eptide Peptides were synthesi zed by ChinaPeptides Co. The N terminal region of each peptide was tagged with 5 fluorescein isothiocyanate (5 FITC) for fluoresc ence studies 4.3.1.2 Surface plasmon resonance measurement Biacore system. A Biacore 3000 instrument available in the Proteomics Division of the Interdisciplinary Center for Biotechnology Research (ICBR) at UF was used. Sensor chip. A Biacore SIA Kit Au was purchased from GE Healthcare Life Sciences. B uffer s PC buffer ( 55 mM KH 2 PO 4 45 mM Na 2 CO 3 and 200 mM NaCl ) was utilized as the running buffer. PC buffer with 0.5% Tween20 was prepared as the regeneration buffer. 4.3.1.3 Circular dichroism (CD) spectrometer A c ircular dichroism spectrometer AVIV model 202 was used for measurement s of peptide conformation lab in the Department of Physics at UF 4.3.1.4 Plate reader for fluorescence analysis A BioTek Synergy H 1 plate reader was used to read the fluorescent intensity of the adsorbed peptides, kindly made available in the lab of Dr. Josephine Allen at the UF

PAGE 124

124 4.3.1.5 Dicing saw ADT 7100 series was used to cut substrate into 3mm x 3mm squares at the NRF at UF 4.3.1.6 Fluorescen ce microscopy of bound peptides Olympus microscope BX60 was used to visualize bound peptides. 4.3 .2 Methods 4.3.2.1 Surface plasmon resonance (SPR) measurement Sensor chip preparation. Gold sensor s need to be stored at 4 o C. Before IZO deposition, the sensor packe t was warmed to room temperature for 30 minutes to allow the sensor surfaces to be released from the gel, and to prevent condensation on the sensor surfaces. The packet was opened in a cleanroom right before the deposition step to minimize dust on the surf ace. IZO thin films were deposited on the gold side of the sensor surface in argon plasma using RF magnetron sputtering at the NRF at UF. A sensor chip was placed on a sensor chip support to have the adhesive strip put on top. The IZO coated gold sensor w as then adhered to the sensor chip. The glass side of the sensor surface was cleaned by a lint free cloth if necessary. The sensor chip with mounted sensor was then inserted into a protective sheath. SPR measurement. A PC buffer was made fresh, filtered a nd degassed. The p eptide being examined was dissolved with the degassed PC buffer at a certain concentration. When docking the sensor chip, the degassed PC buffer was connected to the Biacore system as a running buffer. Whenever docking a new sensor, the s ystem was primed with running buffer, so that it would then be cleared and ready to run a new SPR measurement.

PAGE 125

125 4.3.2.2 Circular dichroism (CD) measurement For CD measurements, a q uartz cuvette with a 1 mm pathlength was used. The p eptide was dissolved in 10 mM ph osphate buffer at a concentration of 0.2 mg/ml. The CD system was purged with N 2 for 30 minutes before turning on. Spectra were recoded over the wavelength range of 190 to 250 nm. Four scans were averaged using one nm bandwidth at a scan rate 0.5 nm/s. Te mperature was set at 25 o C. Each peptide and buffer background were measured three times. Upon finishing the measurement s N 2 was purged into the system for 5 more minutes after tuning the instrument off. 4.3.2.3 Fluorescen ce intensity measurement The p eptide s be ing examined were each dissolved in PC buffer at 0.1 mg/ml concentration. Substrates were cut into 3 mm x 3 mm squares with a dicing saw at the NRF at UF. To remove contamination from the surface, s ubstrates were washed with trichloroethylene, acetone, and methanol in an ultrasonic bath for 5 minutes each, respectively and then were dried by a nitrogen gas stream. The s ubstrate was incubated with the peptide solution for an hour with an agitator (Labqueake, Barnestend Thermolyne). After incubation, the su bstrate was rinsed three times with PC buffer containing 0.1% detergent to remove unbound ones and then was dried by a nitrogen gas. The d ried substrate was placed in a black 96 well plate. An excitation and emission wave length of 488/ 528 nm respectivel y, were used to obtain the fluorescen ce intensity of each represent at i ve well. 4.3.2.4 Fluorescence imaging of peptide binding to substrate s Substrate s coated with IZO w ere washed with trichloroethylene, acetone, and methanol in an ultrasonic bath for 5 minutes ea ch, respectively for removing contamination from surface, and then were dried by a nitrogen gas stream. The cleaned

PAGE 126

126 s ubstrate s w ere incubated with each peptide solution at a concentration of 0.1mg/ml for an hour. After incubation, the substrate s w ere rins ed three times with PC buffer containing 0.1% detergent to remove non binding peptide. Substrate s w ere then visualized at 20x magnification and 2 sec exposure time by a fluorescen ce microscope. 4.3.2.5 Electro releasing test by fl uorescence microscop y The IZO co ated electro releasing device was incubated with the peptide solution ( 0.1mg/ml) in PC buffer on a shaker for 1 hour, follow ed by washing steps to remove unbound peptide These complexes were visualized at 20X magnification and 2 sec exposure time by a flu orescence microscope. After taking fluorescence image s an electric field was applied to the electrodes of the device (see Figure 3 11 ) with flow through created by a micropipette. Washing steps w ere performed as before, and the device was visualized again by fluorescence microscop y 4. 4 Results and Discussion In this chapter, we first focused on the binding properties of IZO binding peptides selected by chemical and electro elution phage display biopanning described in the previous chapters. Table 4 1 show s synthetic peptide sequences and whether it was selected by a low pH elution buffer, a high salt elution buffer, or by the electro elution biopanning approach. These peptide sequences were chosen for their strong binding properties based on IF analysis an d ELISA tests of phage clones that showed strong affinity for the IZO surface. For describing the results more easily, we named the peptides by elution method and numbers, such as IZO LpH# stands for low pH elution, IZOHSE# stands for high salt, and IZOEE# stands for electro elution. To analyze the adsorption of the synthesized peptides on an IZO surface, surface plasmon resonance (SPR) was utilized. SPR detects adsorption via changes in

PAGE 127

127 the refractive index of the interface. The reflected light intensity f rom the interface between metal and the glass substrate at a specific incident angle is measured as a result of the optical excitation of surface plasmon waves. As molecules bind to the surface, a shift in the wavelength occurs, which increases as the mole cular binding accumulates on the metal surface of the SPR chip, allowing the monitoring of molecular adsorption to, or desorption from, the surface. However, not every material is capable of generating surface plasmon effects, gold surface is mostly used. Other materials, even the noble metals Pt or Pd, have poor plasmonic properties. Seker et at. demonstrated that with a 2 nm ultrathin Pt coating, a SPR sensor chip can still provide the SPR signal from the underlying gold layer [134] This provides the design principle for the coating of an additional ultrathin layer of target material onto the gold chip. In this study, we deposited an ultrathin IZO layer on the gold SPR sensor chip and detected the binding of the synthetic peptides using a Biacore SPR system. A 10 nm thick IZO coating was deposited on a commercially purchased 50 nm thick gold sensor chip to perform the binding test. Figure 4 1 shows a schematic of the designed sensor. Atomic force microscopy (AFM) was used to characterize the IZO coated sensor chip because it is important to make sure a uniform coverage was achieved in this extremely thin layer. AFM height image and 3D image with corresponding su rface profile are shown in Figure 4 2. The root mean square (RMS) roughness value of the film was determined to be 0.687 nm, and the whole surface appeared to be uniformly covered by IZO. In the Biacore SPR system, there are four flow cells (Fc1 Fc4) o n each sensor. To first verify that IZO coated sensor chips can obtain an SPR signal, a sucrose solution

PAGE 128

128 with known refractive index was first injected into all the flow cells. An approximately 20,000 increase in resonance units (RU) was observed in the SP R sensograms of all four cells after the sucrose injection, as shown in Figure 4 3. One resonance unit (RU) corresponds to 10 6 refractive index units (RIU), and is commonly assumed to correspond to 1 pg of bimolecular per mm 2 binding [135] A 20,000 RU increase matches the predicted difference of refractive index between sucrose solutio n and water, which was running through the system as a baseline. The same response of RU between all four flow cells also proved that the different surface coatings with IZO all provided the same signal response s We first injected a peptide, IZO LpH 4, at f our different concentrations (from 0.1 to 0.4 mM) for 600 seconds at a 2 l per second injection rate to study the effect of different concentrations (Figure 4 4). The results showed that with increasing peptide concentration, the response rate of RU incre ased, which means that more peptide became bound to the IZO surface, and at a faster rate. It also indicated that there was a two phase binding event, a quick binding at the first 250 seconds, and a reduced binding rate afterward, for this specific peptide And even at the different concentrations, the same peptide had a similar binding pattern, but at higher levels. groups based on the elution method of the phage display biopan ning (chemical versus electro elution). The injection amount and time was set at 600 seconds at a 2 l per second injection rate, and each peptide was prepared at a 0.1 mM concentration which was found in the first study to yield a sufficient signal that lies in the desirable range Figure 4 5 shows the binding response of peptides selected by low pH elution buffer

PAGE 129

129 (IZO LpH1 to IZO LpH 4) and high salt elution buffer (IZO HSE1 to IZO HSE4 ). Based on the RU response, IZO LpH1 IZO LpH 2, IZO LpH 3, and IZO HSE1 had a rapid binding response in the first couple seconds, but then IZO LpH 2 and IZO LpH 3 started to level off to a plateu with a much reduced rate of binding. In contrast, IZO LpH 4 showed a relatively steady binding rate over the whole injection process, with a s light decline (as described for this same peptide in Figure 4 4 ). IZO HSE3 seemed to start very slowly, but strangely showed an increasing rate of binding after around 180 seconds of injection. For IZO HSE2 even though it had a small response in the beginni ng, with continuous injection, the binding decreased, and it seemed the peptide was washed away as the RU dropped close to 0, which seems to indicate that the IZO HSE2 binding to IZO was very weak. After each peptide injection, a buffer solution without pe ptide was injected continuously to examine the desorption. Injection rate started from 2 l per second to 20 away, which makes the RU response decrease. After the desorption process, if the RU response reaches a plateau, this indicates the retention of that amount of peptide on the IZO sensor surface. This data provides the final binding amount of each peptide. Response RU in Figure 4 6 indicates the final amount of peptide bo und onto the IZO surface after the desorption process. However, with careful examination of the amino between the binding affinity and the final binding amount to the pr operties of the peptide sequence.

PAGE 130

130 For peptides selected by the electro elution approach, Figure 4 7 shows the SPR binding profile of each peptide. In this group, IZO EE5 bound to the IZO surface at a relatively steady rate, which was quite high, so it reac hed the highest binding RU response among all the peptides. It was also observed that IZO EE3 and IZO EE4 were very similar in their binding behavior, with a rapid initial increase, followed by leveling off to a high plateau level. IZO EE1 and IZO EE2 shared a similar pattern as well, but with a lower plateau, at about half the RU level. In this set of electro eluted peptides, there does seem to be a consistent trend. A possible reason that IZO EE5 exhibited such a rapid and high rate of binding might be that it has 8 charged amino acids in its sequence, which is the highest among all the peptides, while IZO EE3 and IZO EE4 have 5 and 6 positively charged amino acids, respectively, and IZO EE1 and IZO EE2 have only 2 and 3 positively charged amino acids, respectively This suggests that after the initial binding in the first 10s of seconds (where the trend with charge does not hold), peptides with more charged amino acids ultimately have a higher adsorption affinity to to the peptide group selected from chemical elution biopanning. Also, regarding the final peptide binding amount after desorption, the trend did not fully hold, although two of the peptides with the highest (IZO EE5 ) and second highest charge (IZO EE3 ), did remain at the highest level. The IZO EE4 peptide was lower (Figure 4 8), even though it had nearly identical binding behavior as IZO EE3 during adsorption (Figure 4 7). To study the effect of sequence length on surface binding, doubly repeated peptides of IZO EE1 and IZO EE4 were synthesized, named 2r IZO EE1 and 2r IZO EE4 respectively (Table 4 2). Peptides were injected at the same concentration of 0.1 mM.

PAGE 131

131 SPR sensograms showed a significant increase in the RU response for the double repeat peptides (Figure 4 9 ). For IZO EE1 the binding affinity pattern changed with the repeat, with a continual increase with no plateau for the double peptide, 2r IZO EE1 The IZO EE4 and 2r IZO EE4 system showed a similar binding pattern, with a plateau, but at a higher level for the double peptide, 2r IZO EE4 Both 2r IZO EE1 and 2r IZO EE4 had around a 5 fold increase in the binding amount after desorption (Figure 4 10), which indicates that by repeating an inorganic binding peptide, the binding property can be enhanced. In literat ure, So et al. found that a triply repeat gold binding peptide can have a two fold increase in binding by a SPR study [136] Based on the results of different SPR adsorption patterns for different peptides, we were interested in seeing if there was a structural factor of the synthetic peptide th at might influence the binding on IZO. Circular dichroism (CD) in the far UV region (190 250 nm) was utilized to analyze if there is any secondary structure in the synthetic peptides. For peptides collected by chemical elution (IZO LpH and IZO HSE ), all of the CD spectra exhibited a negative band near 200 nm and low ellipticity above 210 nm, which represents a random coil structure. As an example, figure 4 11 shows a CD spectrum of IZO LpH 3. These peptides were intrinsically disordered in aqueous conditions, which is common to other inorganic binding peptides [31, 137, 138] This is perhaps not surprising given the relatively short length of the peptides, which may not have a sufficiently long hydrophobic domain to cau se folding, as occurs in proteins. Among peptides collected by electro elution, the CD spectrum of IZO EE1 shows a different band position, which indicates that there was some partial helical or beta sheet structure (Figure 4 12). When further comparing th e CD spectra of one repeat (IZO EE1

PAGE 132

132 and IZO EE4 ) versus double repeat peptides (2r IZO EE1 and 2r IZO EE4 ), 2r IZO EE4 exhibited a negative band around 200 nm, which was the same as IZO EE4 However, 2r IZO EE1 had broad negative bands close to 208 nm and 222 nm, and a positive band helical structure (Figure 4 13) [139] Combining the information from the CD spectra a nd SPR sensograms, the change in structure might be the reason for the change of binding kinetics between IZO EE1 and 2r IZO EE1 peptide was synthesized with a FITC tag at the N terminus. A FITC molecular probe by IZO surfaces. To obtain a sufficient time for the peptide to be detected under fluorescence on the IZO surface, we first did a fluorescence intensity measurement tested at different time points of incubation. Figure 4 14 shows the fluorescence intensity reading of peptides as they adsorb to an IZO thin film from five minutes to an hour. Even though the intensity reached a plateau in five minutes, based on literature reports, it could take up to an hour for peptides to form a uniform layer on a surface of material, thus we decided to incubate the peptides with the substrates for an hour to be on the safe side. The peptides were seen to fully coat the IZO surface, as seen by the green fluorescence. Figure 4 15 shows fluorescence images of an IZO coated w afer after incubating with IZOEE4 for one hour. Figure 4 16 shows images of an IZO coated wafer after incubating with IZO HSE2 for one the result from the correspond ing SPR sensogram (Figure 4 6). However, the peptide seems to bind on the edge of the Si wafer due to the structure.

PAGE 133

133 We also did some electro releasing tests on synthetic peptides b y examining with the fluorescence microscope. In Figure 4 17(a), two devices coated with IZO were incubated with IZO EE4 an electro elution derived peptide, and images were taken side by side. After applying an electric potential on the left side of the de vice, the fluorescence image was taken again to identify if there was any release of the bound peptide. In Figure 4 17(b), the left side of the image revealed some release of IZO EE4 after applying an electric field. But by utilizing the same approach, two devices coated with IZO LpH 4 a chemical elution derived peptide, showed less change after applying an electric potential to the left side of the device (Figure 4 18), although there appear to be some regions where the peptide coating was thinner that becam e dark upon release. This result suggests that IZO binding peptides selected by the electro elution biopanning approach might be more readily released from the surface. We believe that with a device designed to be smaller, which can be fit into a well plat e, it might provide a more precise method to study the release by fluorescent intensity readings.

PAGE 134

134 Table 4 1. Amino acid sequence of synthetic IZO binding peptides. Amino acid sequence of synthetic peptide Biopanning method Name STT L NNTT W R L Y low pH elution IZO LpH 1 A SQ I T H FP R PPW low pH elution IZOLpH 2 T K N ML S LPV G P G low pH elution IZOLpH 3 T F K YS H E L E S R G low pH elution IZOLpH 4 S H AP D ST WFALF high salt elution IZOHSE1 TNSSSQ FVVAIP high salt elution IZOHSE2 T LM Y A Q P H QS K T high salt elution IZOHSE3 MLPII R N LI H TT electro elution IZOEE1 R I R N H IL QT I R S electro elution IZOEE2 L R M K PL R TT RR Q electro elution IZOEE3 I T L RH P RRR LI R electro elution IZOEE4 R PL HHRRRHH S P electro elution IZOEE5 Table 4 2. Sequence of synthetic IZO binding peptides with their double r epeat analogue. Amino acid sequence of synthetic peptide Name MLPII R N LI H TT IZO EE1 MLPII R N LI H TT MLPII R N LI H TT 2r IZOEE1 I T L RH P RRR LI R IZOEE4 I T L RH P RRR LI R I T L RH P RRR LI R 2r IZOEE4

PAGE 135

135 Figure 4 1 Schematic of an IZO coated SPR sens or chip. When the chip is inserted into the instrument, there are four flow cells attached on top of the sensor.

PAGE 136

136 Figure 4 2 Atomic force microscopy images on the IZO coated SPR sensor chip surface. AFM height image (A) and topology image (B) on 1.0 m x 1.0 m square, and corresponding line scan surface profile (C).

PAGE 137

137 Figure 4 3 SPR sensograms of a 15% sucrose solution flowed across all four flow cells (FC1 FC4). RU stands for response units, which corresponds to 1 0 6 refractive index units (RIU) A 20,000 RU increase matches the predicted difference of refractive index between sucrose solution and water, which was running through the system as a baseline. The same response of RU between all four flow cells also proved that the different surface a rea coatings with IZO all provided the same signal responses.

PAGE 138

138 Figure 4 4 SPR sensogram of IZO LpH4 at different concentrations. The results showed that with increasing peptide concentration, the response rate of RU increased, which means that more peptide became bound to the IZO surface, and at a faster rate. And even at the different concentrations, the same peptide had a similar binding pattern, but at higher levels.

PAGE 139

139 Figure 4 5 SPR sensograms of peptides selected by chemical elution biopan ning. Based on the RU response, IZOLpH1, IZOLpH2, IZOLpH3, and IZOHSE1 had a rapid binding response in the first couple seconds, but then IZOLpH2 and IZOLpH3 started to level off to a plate a u. In contrast, IZOLpH4 showed a relatively steady binding rate ov er the whole injection process. IZOHSE3 seemed to start very slowly, but strangely showed an increasing rate of binding after around 180 seconds of injection. IZOHSE2 seem ed to bind to IZO very weak ly

PAGE 140

140 Figure 4 6 Binding response after peptide desorption for chemical elution peptides. This data provides the final binding amount of each peptide after running buffer is run through the system to remove weak binders, until a steady state value is obtained

PAGE 141

141 Figure 4 7 SPR sensograms of peptides selected by electro elution biopanning. IZOEE5 bound to the IZO surface at a relatively steady rate, which was quite high, so it reached the highest binding RU response among all the peptides. It was also observed that IZOEE3 and IZOEE4 were very similar in their binding behavior, with a rapid initial increase, followed by leveling off to a high plateau level. IZOEE1 and IZOEE2 shared a similar pattern as well, but with a lower plateau.

PAGE 142

142 Figure 4 8 Binding response after peptide desorption for electro elution peptides. This data provides the final binding amount of each peptide after running buffer is run through the system to remove weak binders, until a steady state value is obtained

PAGE 143

143 F igure 4 9 SPR sensograms of single repeat and double repeat peptides. The IZOEE4 and 2r IZOEE4 system showed a similar binding pattern, with a plateau, but at a higher level for the double peptide, 2r IZOEE4. In contrast, t or IZOEE1, the binding affinit y pattern changed with the repeat, with a continual increase with no plateau for the double peptide, 2r IZOEE1.

PAGE 144

144 Figure 4 1 0 Binding response after peptide desorption for single and double repeat electro eluted peptide s IZO EE1 2r IZO EE1 IZO EE4 and 2r IZO EE4 Both 2r IZOEE1 and 2r IZOEE4 had around a 5 fold increase in the amount of binding after desorption, which indicates that by repeating an inorganic binding peptide, the binding property can be enhanced. Figure 4 1 1 Representative CD spectra of IZO LpH 3, which showed a random coil conformation, as was the case for all the peptides derived by chemical elution.

PAGE 145

145 Figure 4 1 2 CD spectra of peptides selected by electro elution biopan ning Most of the peptides showed a random coil conformation except IZOEE1 whcih shows a different band position that indicates that there was some partial helical or beta sheet structure

PAGE 146

146 Figure 4 1 3 CD spectra of single repeat and double repeat peptides derived from electro elution biopanning. The 2r IZOEE4 peptide exhibited a negative band at around 200 nm, which was the same as IZOEE4. However, 2r IZOEE1 had broad negative bands close to 208 nm and 222 nm, and a positive band at 193 nm, which helical structure when the peptide was doubled in length, which was not seen in the singlet peptide

PAGE 147

147 Figure 4 1 4 Fluorescence intensity of IZOEE1 and IZOEE4 bound to the IZO surface measured at dif ferent time points. Figure 4 1 5 Fluorescence images of two regions of IZO EE4 bound to an IZO coated wafer. (A) with a scale bar: 200 m. (B) with a scale bar: 100 m.

PAGE 148

148 Figure 4 1 6 Fluoresc ence image of IZO HSE2 incubated with an IZO coated wafer, showing very little binding, except at the edge of the Si wafer due to structure. Scale bars: 1 00 m. (A) in white light. (B) in fluorescent light. Figure 4 17 El ectro releasing test by comparing fluorescence images before and after application of the electric field. Fluorescence image taken after incubating the device with IZO EE4 which coated both sides of the device (A), and after applying an electric field to t he left side of the device (B). Scale bar: 200 m.

PAGE 149

149 Figure 4 18 Electro releasing test by comparing fluorescence images before and after application of the electric field. Fluorescence image taken after incubating the dev ice with IZO LpH4 which coated both sides of the device (A), and after applying an electric field to the left side of the device (B). Scale bar: 200 m.

PAGE 150

150 CHAPTER 5 CONCLUSIONS I n this dissertation, phage display technique s w ere utilized for selection of i norganic binding peptides for biosensor applications. A traditional phage display protocol was used to select peptides that have binding affinity to indium zinc oxide (IZO). To select a peptide that is more sensitive to an electric field, a novel phage dis play protocol with an electro elution process was developed. IZO binding peptides derived from phage clones with good binding affinity were synthesized to examine the binding and electroactive properties of the isolated peptides I n the first part of this study, an amorphous IZO was chosen as the target material to select inorganic binding peptides. Among many different display techniques, M13 phage library Ph.D. 12 was chosen because it is su it able for bio panning with inorganic material s I t is commerciall y available and has longer displayed peptide sequences (12 mers) as compared to other phage display librar ies on the market. Amorphous IZO was anticipated to provide a more homogeneous surface than a crystalline material because it should not have grain bo undaries and crystal defects which might affect the binding of phage Indeed, the amorphous surface might have contributed to the consensus peptide sequences we obtained from both low pH and high salt elution biopanning, because inorganic materials do not always converge to a consensus sequence During the phage display process, our first approach was to use the traditional chemical elution, where either a low pH buffer or high salt elution buffer were used for eluting phages from IZO surface. B ased on the sequence of selected peptides, it was found that IZO binding peptides obtained by the chemical elution approach tend to have

PAGE 151

151 blocks of hydrophobic amino acids, and blocks of hydrophilic amino acids. Histidine, serine, threonine, and leucine frequently app eared in these IZO binding peptide s I t was found that binding affinity of phage clones eluted from high salt elution buffer was more selective to IZO than the clones collected by the low pH elution buffer In the second part of this study, a novel phage display biopanning protocol was developed where an electro elution process replaces the regular chemical elution step A device comp ris ed of two parallel electrodes was fabricated to test the phage display protocol with an electro elution step. By two roun ds of biopanning one consensus sequence was obtained. It was found that IZO binding peptides collected by applying an electric field share some of the same frequently appear ing amino acids, like serine and leucine, as compared to the peptides selected by traditional chemical elution phage display. Peptides selected by electro elution had connected sequences of arginines, and all of the clones ha d several positive ly charged amino acid sequences and no negatively charged amino acids This led to the hypothe sis that positively charged peptides might play an important role for enabling peptides to be released by an electric field. Also we found that all the sequences were composed of only a specific set of 11 amino acids. Even though IZO binding peptides ob tained by electro show a high selectivity between IZO, SiO 2 and Si all the clones had a moderate to strong binding affinity to IZO. By characterization of zeta potential of IZO, we believe that electrostatic interactions play ed a n important role for the peptide binding ability on IZO surface s An electro releasing device with two separate areas was designed to test

PAGE 152

152 the releasing of phages. By using immunofluoresc e nce (IF) analysis, a release of phages display ing the peptide sequen ce: MLPIIRNLIHTT was visualized After identification of good IZO binding phage the corresponding peptide sequences were synthesized for further characterization. SPR sensor chip s coated with a n ultrathin layer of IZO was used to examine the binding aff inity behavior With time dependent SPR sensograms, it was found that even though a peptide displayed by a phage might have a strong binding affinity to IZO the peptide by itself might not exhibit the same good binding affinity. Nevertheless, many of the phage derived peptides did show good binding affinity to IZO. For one peptide system, it was found that the s ame peptide at different concentration s led to a similar ly shaped profile in the SPR sensogram. However, in comparing different peptide s, there was considerable diversity in the shapes of their binding kinetics curves B y doubling the peptide sequence, it was found that the binding affinity can be markedly enhanced. Combining SPR sensograms and circular dichroism (CD) spectr a it was found that the s econdary structure of a peptide might be playing a role in affect ing the binding propert ies P eptide sequence: ITLRHPRRRLIR (IZO EE4 ), obtained by electro elution, was visualized with fluorescence microscopy using an electro releasing device, and was found to show some release after applying an electric field. There may have been a small amount of release for the chemical elution derived peptide TFKYSHELESRG (IZO LpH 4), but it was less obvious. In summary IZO binding peptides were identified by two different approaches C hemical elution phage display provides a large number of IZO binders and many (5) consensus sequences, which can contribute to future bioinformatics study as discussed

PAGE 153

153 below This work demonstrated the feasibility of a novel electro elution approach for selecting binding peptides more sensitive to an electric field. We also demonstrated that by coat ing an ultrathin layer of IZO onto an SPR sensor chip the SPR signal can still be utilized for affinity test ing of inorganic binding peptide s Ev en though this work was focused on selection of peptides with binding and electroactive properties, the releasing mechanism of peptides is still not resolved We believe that by designing a device that can apply an electrical stimulus combin ed with sensiti ve surface characterization techniques the electroactive propert ies of peptides can be further studied. Future Work To continue this study, a bioinformatics approach would be valuable. Binding affinity of each peptide displayed by the phage to an IZO surf ace has been examined. Based on surface coverage, a strong binder, a moderate binder or a weak binder can be identified. T his result can be used as input data for defining of a set of scoring matrices which include similarities within strong binding sequen ces and the difference s between strong and weak binders. A lthough most of the IZO binding peptides had a preferential binding affinity to our target, there were still some minor binding affinities to other inorganic materials. By using a bioinformatics app roach with scoring matrices to computationally design peptide sequences one can potentially eliminat e bind ing to other inorganic materials, such that the affinity and specificity of the IZO binding peptide could be improved. With our IZO modified SPR sensor chip, time dependent binding affinity can be obtained This sensor chip can be used not only on testing binding behavior of peptides but also to examine binding of the whole phages that display the peptides However, the concentration of injected phages and the injection rate need to be well defined. This

PAGE 154

154 is because even though the phage has a higher molecular weight to generate a larger SPR response, the long filamentous shape of M13 phage can make the phage rotate slowly. The p III coat protein o f M13 phage which displays the binding peptide might require a slower injection of running buffer to allow sufficient time for the phage to rotate and bind on to the surface. For synthetic peptide s the surface coverage can be obtained with an AFM study. Wi th the data of surface coverage, SPR data can be fit to a Langmuir adsorption model to calculate the adsorption and desorption rate using a least square curve fitting algorithm. To characterize the binding peptide as is done for SAMs, and to examine the e lectro releasing property of inorganic binding peptide s quantitatively some surface characterization techniques should be applied. The thickness of the binding peptide can be measured by spectroscopic ellipsometry. The initial thickness of peptide adsorpt ion can be measured, and the releasing behavior of the peptide can be studied with reduction of thickness over time. A study of the release characteristics with variations in applied electric field would be interesting. X ray photoelectron spectroscopy (XP S) can also be used to detect the different chemical states of elements on the surface. These sensitive surface studies can provide more insight into the electroactive properties and the mechanism behind achieving them. After careful studie s of IZO electr o releasing peptides obtained by one or a combination of many experiments mentioned above, peptide sequences with strong binding potential can be modified to identify the key amino acids analogous to site directed mutagenesis Single amino acids or connec ted amino acids can be replaced to Repetition of sequences

PAGE 155

155 can also be utilized as the secondary structure might change in accordance with the length of a peptide chain as demonstrated in o ur study of the double repeats Last but not least, a functionalized binding peptide would definitely be the next step toward biosensor applications if these electroactive peptides are to serve as linkers. Connecting peptides with other functional groups or amino acids would be a reasonable start, such as the arginine glycine aspartic acid (RGD) peptide. With RGD peptide, cells could possibly attach more preferentially to targeted surfaces, and then be triggered for release. How attached biomolecules affe ct the electroactive properties also need to be studied. By all these studies, a peptide with reversible binding and releasing characteristics can be achieved and applied for biosensor applications. The mechanism behind the binding and electroactive proper ties can also be further clarified.

PAGE 156

156 LIST OF REFERENCES [1] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials. 2003;24:4385 415. [2] Smith GP. FILAMENTOUS FUSION PHAGE novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228:1315 7. [3] Sarikaya M, Tamerler C, Schwartz DT, Baneyx FO. Materials assembly and formation using engineered polypeptides. Annual Review of Materials Re search. 2004;34:373 408. [4] Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997;15:553 7. [5] Devlin JJ, Panganiban LC, Devlin PE. Random peptide libraries: a source of specific protein bindi ng molecules. Science. 1990;249:404 6. [6] He M, Khan F. Ribosome display: next generation display technologies for production of antibodies in vitro. Expert Rev Proteomics. 2005;2:421 30. [7] Dower W. In vitro selection as a powerful tool for the applied evolution of proteins and peptides. 2002;6:390 8. [9] Hoogenboom HR. Selecting and screening recombinant antibody libraries. Nature Biotechnology. 2005;23:1105 16. [10] Sedlacek R, Chen E. Screening for protease substrate by polyvalent phage display. Comb Chem High Throughput Screen. 2005;8:197 203. [11] Deperthes D. Phage display substrate: a blind method for determining protease specificity. Biol Chem. 2002;383:1107 12. [12] Kay BK, Hamilton PT. Id entification of enzyme inhibitors from phage displayed combinatorial peptide libraries. Comb Chem High Throughput Screen. 2001;4:535 43. [13] Hyde DeRuyscher R, Paige LA, Christensen DJ, Hyde DeRuyscher N, Lim A, Fredericks ZL, et al. Detection of small mo lecule enzyme inhibitors with peptides isolated from phage displayed combinatorial peptide libraries. Chem Biol. 2000;7:17 25. [14] Sidhu SS, Koide S. Phage display for engineering and analyzing protein interaction interfaces. Curr Opin Struct Biol. 2007;1 7:481 7.

PAGE 157

157 [15] Hertveldt K, Belien T, Volckaert G. General M13 phage display: M13 phage display in identification and characterization of protein protein interactions. Methods Mol Biol. 2009;502:321 39. [16] De Berardinis P, Haigwood NL. New recombinant vac cines based on the use of prokaryotic antigen display systems. Expert Rev Vaccines. 2004;3:673 9. [17] Rowley MJ, O'Connor K, Wijeyewickrema L. Phage display for epitope determination: a paradigm for identifying receptor ligand interactions. Biotechnol Ann u Rev. 2004;10:151 88. [18] Segvich S, Kohn DH. Phage Display as a Strategy for Designing Organic/Inorganic Biomaterials. Biological Interactions on Materials Surfaces: Understanding and Controlling Protein, Cell, and Tissue Responses. 2009:115 32. [19] Sa moylova TI, Smith BF. Elucidation of muscle binding peptides by phage display screening. Muscle Nerve. 1999;22:460 6. [20] Odermatt A, Audige A, Frick C, Vogt B, Frey BM, Frey FJ, et al. Identification of receptor ligands by screening phage display peptide libraries ex vivo on microdissected kidney tubules. J Am Soc Nephrol. 2001;12:308 16. [21] Nowakowski GS, Dooner MS, Valinski HM, Mihaliak AM, Quesenberry PJ, Becker PS. A specific heptapeptide from a phage display peptide library homes to bone marrow and binds to primitive hematopoietic stem cells. Stem Cells. 2004;22:1030 8. [22] Rothenfluh DA, Bermudez H, O'Neil CP, Hubbell JA. Biofunctional polymer nanoparticles for intra articular targeting and retention in cartilage. Nat Mater. 2008;7:248 54. [23] Ad ey NB, Mataragnon AH, Rider JE, Carter JM, Kay BK. Characterization of phage that bind plastic from phage displayed random peptide libraries. Gene. 1995;156:27 31. [24] Serizawa T, Sawada T, Matsuno H, Matsubara T, Sato T. A Peptide Motif Recognizing a Pol ymer Stereoregularity. 2005. [25] Wang S, Humphreys ES, Chung SY, Delduco DF, Lustig SR, Wang H, et al. Peptides with selective affinity for carbon nanotubes. Nat Mater. 2003;2:196 200. [26] Lu ZJ, Murray KS, Vancleave V, Lavallie ER, Stahl ML, McCoy JM. E xpression of thioredoxin random peptide libraries on the escherichia coli cell surface as functional fusions to flagellin a system designed for exploring protein protein interactions. Bio Technology. 1995;13:366 72.

PAGE 158

158 [27] Hnilova M, Oren EE, Seker UOS, Wi lson BR, Collino S, Evans JS, et al. Effect of Molecular Conformations on the Adsorption Behavior of Gold Binding Peptides. Langmuir. 2008;24:12440 5. [28] Huang Y, Chiang CY, Lee SK, Gao Y, Hu EL, De Yoreo J, et al. Programmable assembly of nanoarchitectu res using genetically engineered viruses. Nano Letters. 2005;5:1429 34. [29] Naik RR, Stringer SJ, Agarwal G, Jones SE, Stone MO. Biomimetic synthesis and patterning of silver nanoparticles. Nature Materials. 2002;1:169 72. [30] Nam KT, Lee YJ, Krauland EM Kottmann ST, Belcher AM. Peptide mediated reduction of silver ions on engineered biological scaffolds. Acs Nano. 2008;2:1480 6. [31] Seker UOS, Wilson B, Dincer S, Kim IW, Oren EE, Evans JS, et al. Adsorption behavior of linear and cyclic genetically eng ineered platinum binding peptides. Langmuir. 2007;23:7895 900. [32] Oren EE, Tamerler C, Sahin D, Hnilova M, Seker UOS, Sarikaya M, et al. A novel knowledge based approach to design inorganic binding peptides. Bioinformatics. 2007;23:2816 22. [33] Thai CK, Dai HX, Sastry MSR, Sarikaya M, Schwartz DT, Baneyx F. Identification and characterization of Cu2O and ZnO binding polypeptides by Escherichia coli cell surface display: Toward an understanding of metal oxide binding. Biotechnology and Bioengineering. 20 04;87:129 37. [34] Dickerson MB, Jones SE, Cai Y, Ahmad G, Naik RR, Kroger N, et al. Identification and design of peptides for the rapid, high yield formation of nanoparticulate TiO2 from aqueous solutions at room temperature. Chemistry of Materials. 2008; 20:1578 84. [35] Sano KI, Shiba K. A hexapeptide motif that electrostatically binds to the surface of titanium. Journal of the American Chemical Society. 2003;125:14234 5. [36] Sano KI, Sasaki H, Shiba K. Specificity and biomineralization activities of Ti binding peptide 1 (TBP 1). Langmuir. 2005;21:3090 5. [37] Sano K, Sasaki H, Shiba K. Utilization of the pleiotropy of a peptidic aptamer to fabricate heterogeneous nanodot containing multilayer nanostructures. J Am Chem Soc. 2006;128:1717 22. [38] Brown S. Engineered iron oxide adhesion mutants of the Escherichia coli phage lambda receptor. Proc Natl Acad Sci U S A. 1992;89:8651 5.

PAGE 159

159 [39] Gungormus M, Fong H, Kim IW, Evans JS, Tamerler C, Sarikaya M. Regulation of in vitro calcium phosphate mineralization by combinatorially selected hydroxyapatite binding peptides. Biomacromolecules. 2008;9:966 73. [40] Gaskin DJH, Starck K, Vulfson EN. Identification of inorganic crystal specific sequences using phage display combinatorial library of short peptides: A feasibi lity study. Biotechnology Letters. 2000;22:1211 6. [41] Krauland EM, Peelle BR, Wittrup KD, Belcher AM. Peptide tags for enhanced cellular and protein adhesion to single crystal line sapphire. Biotechnology and Bioengineering. 2007;97:1009 20. [42] Whaley SR, English DS, Hu EL, Barbara PF, Belcher AM. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature. 2000;405:665 8. [43] Lee SW, Mao CB, Flynn CE, Belcher AM. Ordering of quantum dots using genetically eng ineered viruses. Science. 2002;296:892 5. [44] Tamerler C, Khatayevich D, Gungormus M, Kacar T, Oren EE, Hnilova M, et al. Molecular Biomimetics: GEPI Based Biological Routes to Technology. Biopolymers. 2010;94:78 94. [45] Sarikaya M, Tamerler C, Jen AKY, Schulten K, Baneyx F. Molecular biomimetics: nanotechnology through biology. Nature Materials. 2003;2:577 85. [46] Jaworski JW, Raorane D, Huh JH, Majumdar A, Lee SW. Evolutionary screening of biomimetic coatings for selective detection of explosives. Lang muir. 2008;24:4938 43. [47] Chen HB, Su XD, Neoh KG, Choe WS. QCM D analysis of binding mechanism of phage particles displaying a constrained heptapeptide with specific affinity to SiO2 and TiO2. Analytical Chemistry. 2006;78:4872 9. [48] Pande J, Szewczyk MM, Grover AK. Phage display: Concept, innovations, applications and future. Biotechnology Advances. 2010;28:849 58. [49] John Spencer Evans RS, Tiffany R. Walsh, Ersin Emre Oren and Candan Tamerler. Molecular Design of Inorganic Binding Polypeptides. MRS Bulletin. 2008;33:514 8. [50] So CR, Kulp JL, Oren EE, Zareie H, Tamerler C, Evans JS, et al. Molecular recognition and supramolecular self assembly of a genetically engineered gold binding peptide on Au{111}. ACS Nano. 2009;3:1525 31. [51] So CR, Hayamiz u Y, Yazici H, Gresswell C, Khatayevich D, Tamerler C, et al. Controlling Self Assembly of Engineered Peptides on Graphite by Rational Mutation. 2012.

PAGE 160

160 [52] Vreuls C, Zocchi G, Genin A, Archambeau C, Martial J, Van de Weerdt C. Inorganic binding peptides as tools for surface quality control. J Inorg Biochem. 2010;104:1013 21. [53] Khatayevich D, Gungormus M, Yazici H, So C, Cetinel S, Ma H, et al. Biofunctionalization of materials for implants using engineered peptides. Acta Biomater. 2010;6:4634 41. [54] Br own S, Sarikaya M, Johnson E. A genetic analysis of crystal growth. Journal of Molecular Biology. 2000;299:725 35. [55] Naik RR, Stringer SJ, Agarwal G, Jones SE, Stone MO. Biomimetic synthesis and patterning of silver nanoparticles. 2013. [56] Slocik JM, Stone MO, Naik RR. Synthesis of Gold Nanoparticles Using Multifunctional Peptides. Small.1:1048 52. [57] Kim J, Rheem Y, Yoo B, Chong Y, Bozhilov KN, Kim D, et al. Peptide mediated shape and size tunable synthesis of gold nanostructures. Acta Biomater. 20 10;6:2681 9. [58] Merzlyak A, Indrakanti S, Lee S W. Genetically Engineered Nanofiber Like Viruses For Tissue Regenerating Materials. 2009. [59] Chung W J, Oh J W, Kwak K, Lee BY, Meyer J, Wang E, et al. Biomimetic self templating supramolecular structures. Nature. 2011;478:364 8. [60] Lee Y, Kim J, Yun DS, Nam YS, Shao Horn Y, Belcher AM. Virus templated Au and Au Pt core shell nanowires and their electrocatalytic activities for fuel cell applications. 2012. [61] Yoo PJ, Nam KT, Qi J, Lee S K, Park J, Belcher AM, et al. Spontaneous assembly of viruses on multilayered polymer surfaces. Nature Materials. 2006;5:234 40. [62] Lee YJ, Yi H, Kim W J Kang K, Yun DS, Strano MS, et al. Fabricating Genetically Engineered High Power Lithium Ion Batteries Using Multiple Virus Genes. 2009. [63] Ghosh D, Lee Y, Thomas S, Kohli AG, Yun DS, Belcher AM, et al. M13 templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer. Nature Nanotechnology. 2012;7:677 82. [64] Nam KT, Kim D W, Yoo PJ, Chiang C Y, Meethong N, Hammond PT, et al. Virus Enabled Synthesis and Assembly of Nanowires for Lithium Ion Battery Electrodes. 2006. [65] Liao C W, Ye h Y W, El Shall H, Gower L. M13 Phages Selected by the Phage Display Technique as Microbial Bio Amphiphiles for the Specific Separation of Mineral Particles. Applied Material and InterfaceSubmitted.

PAGE 161

161 [66] Ghosh D, Kohli AG, Moser F, Endy D, Belcher AM. Refa ctored M13 Bacteriophage as a Platform for Tumor Cell Imaging and Drug Delivery. 2012. [67] Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346:818 22. [68] Tuerk C, Gold L. Systematic evolution of lig ands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249:505 10. [69] Ellington AD, Szostak JW. Selection in vitro of single stranded DNA molecules that fold intospecific ligand binding structures. Nature. 1992;355: 850 2. [70] Shi Y, Dai H, Sun Y, Hu J, Ni P, Li Z. Fluorescent sensing of cocaine based on a structure switching aptamer, gold nanoparticles and graphene oxide. Analyst. 2013. [71] Rankin CJ, Fuller EN, Hamor KH, Gabarra SA, Shields TP. A simple fluorescen t biosensor for theophylline based on its RNA aptamer. Nucleosides Nucleotides Nucleic Acids. 2006;25:1407 24. [72] Ramos E, Pineiro D, Soto M, Abanades DR, Martin ME, Salinas M, et al. A DNA aptamer population specifically detects Leishmania infantum H2A antigen. Lab Invest. 2007;87:409 16. [73] Lee S, Kim YS, Jo M, Jin M, Lee DK, Kim S. Chip based detection of hepatitis C virus using RNA aptamers that specifically bind to HCV core antigen. Biochem Biophys Res Commun. 2007;358:47 52. [74] Niu W, Jiang N, H u Y. Detection of proteins based on amino acid sequences by multiple aptamers against tripeptides. Anal Biochem. 2007;362:126 35. [75] Ikanovic M, Rudzinski WE, Bruno JG, Allman A, Carrillo MP, Dwarakanath S, et al. Fluorescence assay based on aptamer quan tum dot binding to Bacillus thuringiensis spores. J Fluoresc. 2007;17:193 9. [76] Shangguan D, Li Y, Tang Z, Cao ZC, Chen HW, Mallikaratchy P, et al. Aptamers evolved from live cells as effective molecular probes for cancer study. National Academy of Scien ces. 2006;103:11838 43. [77] Shiping Song LW, Jiang Li, Chunhai Fan, Jianlong Zhao. Aptamer based biosensors. TrAC Trends in Analytical Chemistry. 2008;27:108 17. [78] Xiao Y, Lubin AA, Heeger AJ, Plaxco KW. Label free electronic detection of thrombin in b lood serum by using an aptamer based sensor. Angew Chem Int Ed Engl. 2005;44:5456 9.

PAGE 162

162 [79] Balamurugan S, Obubuafo A, Soper SA, Spivak DA. Surface immobilization methods for aptamer diagnostic applications. Anal Bioanal Chem. 2008;390:1009 21. [80] Ke Tai G uo DS, Bernd Schwenzer, Gerhard Ziemer, Hans P. Wen. The effect of electrochemical functionalization of Ti alloy surfaces by aptamer based capture molecules on cell adhesion. Biomaterials. 2007;28:468 74. [81] Estephan E, Larroque C, Bec N, Martineau P, Cu isinier FJG, Cloitre T, et al. Selection and Mass Spectrometry Characterization of Peptides Targeting Semiconductor Surfaces. Biotechnology and Bioengineering. 2009;104:1121 31. [82] Brown S, Sarikaya M, Johnson E. A genetic analysis of crystal growth. J M ol Biol. 2000;299:725 35. [83] Cass AEG, Zhang Y. Nucleic acid aptamers : ideal reagents for point of care diagnostics? Faraday Discuss. 2011:49 61. [84] Rothenstein D, Claasen B, Omiecienski B, Lammel P, Bill J. Isolation of ZnO binding 12 mer peptides a nd determination of their binding epitopes by NMR spectroscopy. J Am Chem Soc. 2012;134:12547 56. [85] Azzazy HME, Highsmith WE. Phage display technology: clinical applications and recent innovations. Clinical Biochemistry. 2002;35:425 45. [86] Lee BY, Zha ng J, Zueger C, Chung W J, Yoo SY, Wang E, et al. Virus based piezoelectric energy generation. Nature Nanotechnology. 2012;7:351 6. [87] Weiss GA, Roth TA, Baldi PF, Sidhu SS. Comprehensive mutagenesis of the C terminal domain of the M13 gene 3 minor coat protein: the requirements for assembly into the bacteriophage particle. J Mol Biol. 2003;332:777 82. [88] Sidhu SS. Engineering M13 for phage display. Biomol Eng. 2001;18:57 63. [89] R. R. Naik SEJ, C. J. Murray, J. C. McAuliffe, R. A. Vaia, M. O. Stone. P eptide templates for nanoparticle synthesis derived from polymerase chain reaction driven phage display. Advanced Funtional Materials. 2004:25 30. [90] Kehoe JW, Kay BK. Filamentous phage display in the new millennium. Chem Rev. 2005;105:4056 72. [91] Weav er R. Molecular Biology: McGraw Hill Science/Engineering/Math; 2011. [92] Craciun D, Socol G, Stefan N, Miroiu M, Craciun V. Structural investigations of InZnO films grown by pulsed laser deposition technique. Thin Solid Films. 2010;518:4564 7.

PAGE 163

163 [93] Ramamo orthy K, Kumar K, Chandramohan R, Sankaranarayanan K. Review on material properties of IZO thin films useful as epi n TCOs in opto electronic (SIS solar cells, polymeric LEDs) devices. Materials Science and Engineering B Solid State Materials for Advanced Technology. 2006;126:1 15. [94] D.Y. Ku IHK, I. Lee, K.S. Lee, T.S. Lee, J. h. Jeong, B. Cheong, Y. J. Baik, W.M. Kim. Structural and electrical properties of sputtered indium zinc oxide thin films. 2006;515:1364 9. [95] Terzini E, Nobile G, Loreti S, Mina rini C, Polichetti T, Thilakan P. Influences of Sputtering Power and Substrate Temperature on the Properties of RF Magnetron Sputtered Indium Tin Oxide Thin Films. Japanese Journal of Applied Physics 38 (1999). 1999. [96] N. Naghavi AR, C. Marcel, C. Gury J.B. Leriche, J.M. Tarascon. Characterization of indium zinc oxide thin films prepared by pulsed laser deposition using a Zn3In2O6 target. 2000;360:233 40. [97] T Sasabayashia NI, E Nishimuraa, M Kona, P.K Songa, K Utsumib, A Kaijoc, Y Shigesato. Compara tive study on structure and internal stress in tin doped indium oxide and indium zinc oxide films deposited by r.f. magnetron sputtering. 2003;445:219 23. [98] PhD. Phage Display Libraries Instruction Manual. New England Biolab Inc.; 2012. [99] Vodnik M, Z ager U, Strukelj B, Lunder M. Phage display: selecting straws instead of a needle from a haystack. Molecules. 2011;16:790 817. [100] Jiansheng Jie GW, Xinhai Han, Qingxuan Yu, Yuan Liao, Gongpu Li, J.G. Hou. Indium doped zinc oxide nanobelts. Chemical Phys ics Letters. 2004;387:466 70. [101] Turner APF. Biosensors: sense and sensibility. Chemical Society Review. 2013;42:3175 648. [102] Corcuera JIRD, Cavalieri RP. Biosensors. Encyclopedia of Agricultural, Food, and Biological Engineering: Marcel Dekker, Inc. ; 2003. [103] Vo Dinh T, Cullum B. Biosensors and biochips: advances in biological and medical diagnostics. Fresenius J Anal Chem. 2000;366:540 51. [104] Nirmalya K Chaki KV. Self assembled monolayers as a tunable platform for biosensor applications. 2002; 17:1 12. [105] Ulman A, Kang JF, Shnidman Y, Liao S, Jordan R, Choi GY, et al. Self assembled monolayers of rigid thiols. J Biotechnol. 2000;74:175 88.

PAGE 164

164 [106] Wasserman SR, Tao YT, Whitesides GM. Structure and reactivity of alkylsiloxane monolayers formed b y reaction of alkyltrichlorosilanes on silicon substrates. Langmuir. 1989. [107] Vericat C, Vela ME, Benitez G, Carro P, Salvarezza RC. Self assembled monolayers of thiols and dithiols on gold: new challenges for a well known system. Chem Soc Rev. 2010. [1 08] Haensch C, Hoeppener S, Schubert US. Chemical modification of self assembled silane based monolayers by surface reactions. Chemical Society Review. 2010. [109] Schreiber F. Structure and growth of self assembling monolayers. 2000;65:151 257. [110] Male m F, Mandler D. Self assembled monolayers in electroanalytical chemistry: application of .omega. mercapto carboxylic acid monolayers for the electrochemical detection of dopamine in the presence of a high concentration of ascorbic acid. Analytical chemistr y. 2002. [111] Stephen E. Creager KGO. Self assembled monolayers and enzyme electrodes: Progress, problems and prospects. Electrochemistry. 1995;307:277 89. [112] S. Flink FCJMvV, D. N. Reinhoudt. Sensor Functionalities in Self Assembled Monolayers. Advanc ed Materials. 2000;12:1315 28. [113] Tang C, Feller L, Rossbach P, Keller B, Voros J, Tosatti S, et al. Adsorption and electrically stimulated desorption of the triblock copolymer poly(propylene sulfide bl ethylene glycol) (PPS PEG) from indium tin oxide ( ITO) surfaces. Surface Science. 2006;600:1510 7. [114] Yeh PYJ, Kizhakkedathu JN, Madden JD, Chiao M. Electric field and Vibration assisted nanomolecule desorption and anti biofouling for biosensor applications. Colloids and Surfaces B Biointerfaces. 2007; 59:67 73. [115] Okochi M, Ogawa M, Kaga C, Sugita T, Tomita Y, Kato R, et al. Screening of peptides with a high affinity for ZnO using spot synthesized peptide arrays and computational analysis. Acta Biomater. 2010;6:2301 6. [116] Golec P, Karczewska Golec J, Los M, Wegrzyn G. Novel ZnO binding peptides obtained by the screening of a phage display peptide library. J Nanopart Res. 2012;14:1218. [117] Willem Smit HNS. Electroosmotic zeta potential measurements on single crystals. Journal of Colloid and Interf ace Science. 1977;60:299 307. [118] Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984;309:30 3.

PAGE 165

165 [119] Hwang NS, Varghese S, Zhang Z, Elisseeff J. Chondrogenic differentiation of human embryonic stem cell derived cells in arginine glycine aspartate modified hydrogels. Tissue Eng. 2006;12:2695 706. [120] Salinas CN, Cole BB, Kasko AM, Anseth KS. Chondrogenic differentiation potential of human mesenchymal stem cel ls photoencapsulated within poly(ethylene glycol) arginine glycine aspartic acid serine thiol methacrylate mixed mode networks. Tissue Eng. 2007;13:1025 34. [121] Itoh D, Yoneda S, Kuroda S, Kondo H, Umezawa A, Ohya K, et al. Enhancement of osteogenesis on hydroxyapatite surface coated with synthetic peptide (EEEEEEEPRGDT) in vitro. J Biomed Mater Res. 2002;62:292 8. [122] Rezania A, Healy KE. The effect of peptide surface density on mineralization of a matrix deposited by osteogenic cells. J Biomed Mater R es. 2000;52:595 600. [123] Gunn JW, Turner SD, Mann BK. Adhesive and mechanical properties of hydrogels influence neurite extension. J Biomed Mater Res A. 2005;72:91 7. [124] Patel S, Tsang J, Harbers GM, Healy KE, Li S. Regulation of endothelial cell func tion by GRGDSP peptide grafted on interpenetrating polymers. J Biomed Mater Res A. 2007;83:423 33. [125] Massia SP, Hubbell JA. Covalent surface immobilization of Arg Gly Asp and Tyr Ile Gly Ser Arg containing peptides to obtain well defined cell adhesive substrates. Anal Biochem. 1990;187:292 301. [126] Schense JC, Hubbell JA. Three dimensional Migration of Neurites Is Mediated by Adhesion Site Density and Affinity. The journal of Biological Chemistry. 2000. [127] Alireza Rezania KEH. Biomimetic Peptide S urfaces That Regulate Adhesion, Spreading, Cytoskeletal Organization, and Mineralization of the Matrix Deposited by Osteoblast like Cells. Biotechnology Progress. 2008;15:19 32. [128] Kay C. Dee TTA, Rena Bizios. Design and function of novel osteoblast a dhesive peptides for chemical modification of biomaterials. Journal of Biomedical Materials Research. 1998;40:371 7. [129] Hubbell JA, Massia SP, Desai NP, Drumheller PD. Endothelial Cell Selective Materials for Tissue Engineering in the Vascular Graft Via a New Receptor. Nature Biotechnology. 1991;9:568 72. [130] Oren EE, Notman R, Kim IW, Evans JS, Walsh TR, Samudrala R, et al. Probing the Molecular Mechanisms of Quartz Binding Peptides. Langmuir. 2010;26:11003 9. [131] Slocik JM, Naik RR. Probing peptide nanomaterial interactions. Chemical Society Reviews. 2010;39:3454 63.

PAGE 166

166 [132] Kelly SM, Price NC. The use of circular dichroism in the investigation of protein structure and function. Curr Protein Pept Sci. 2000;1:349 84. [133] Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nature Protocols. 2007;1:2876 90. [134] Seker UOS, Wilson B, Sahin D, Tamerler C, Sarikaya M. Quantitative Affinity of Genetically Engineered Repeating Polypeptides to Inorganic Surfaces. Biomacro molecules. 2009;10:250 7. [135] Raynor JE, Petrie TA, Fears KP, Latour RA, Garca AJ, Collard DM. Saccharide Polymer Brushes To Control Protein and Cell Adhesion to Titanium. Biomacromolecules. 2009. [136] So CR, Tamerler C, Sarikaya M. Adsorption, diffusi on, and self assembly of an engineered gold binding peptide on Au(111) investigated by atomic force microscopy. Angew Chem Int Ed Engl. 2009;48:5174 7. Terminal Mineral Modification Sequence from the Mollusk Shell Protein Asprich. Crystal Growth & Design. 2006;6:839 42. [138] Kim W, Collino S, Morse DE, Evans JS. A Crystal Modulating Protein from Molluscan Nacre That Limits the Growth of Calcite in Vitro. Crystal Growth & Design. 2006;6:1078 82. [139] Whitmore L, Wallace BA. Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers. 2008;89:392 400.

PAGE 167

167 BIOGRAPHICAL SKETCH Ya Wen Yeh received her Ph.D. the Department of Materials Science and Engineering at University of F lorida. Her research interest was in molecular recognition in organic inorganic hybrid materials. She received her M.S degree in b iomedical e ngineering from University of Florida in 2009 and B.S in b ioengineering from Tatung University in Taiwan in 2007.