Development of polymeric matrices and fluorescent nanoparticles for immuno-based detection of pathogenic bacteria

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DEVELOPMENT OF POLYMERIC MATRICES AND FLUORESCENT NANOPARTICLES FOR THE

IMMUNO-BASED DETECTION OF PATHOGENIC BACTERIA

By

SWATI JAIN

CENTER FOR BIOMEDICAL ENGINEERING

Thesis Submitted

In fulfillment of the requirements of the degree of

DOCTOR OF PHILOSOPHY

to the

INDIAN INSTITUTE OF TECHNOLOGY, DELHI

DECEMBER, 2010

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© Indian Institute of Technology, Delhi (IIT D), New Delhi, 2010

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CERTIFICATE

This is to certify that the thesis entitled "Development of Polymeric Matrices and Fluorescent Nanoparticles for the Immuno-based Detection of Pathogenic Bacteria" submitted by Ms. Swati Jain to the Indian Institute of Technology, Delhi for the award of degree of Doctor of Philosophy, in Biomedical Engineering is a record of bonafide research work carried out by her. Ms. Swati Jain has worked under my guidance and supervision and has fulfilled the requirement for the submission of this thesis.

The results contained in this thesis are original and have not been submitted in partial or full, to any other university or institute for the award of any degree or diploma.

(Dr. Harpal Singh) Professor

Centre for Biomedical Engineering Indian Institute of Technology, Delhi

Hauz Khas, New Delhi -110016

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This thesis is a result four years of work which would not have been possi6Ce without the guidance and help of severa(individua(s who have exfended their va(ua6Ce suggestions and assistance for completion of this work. I have great pleasure to express my gratitude to all of them.

Foremost, I wish to record a deep sense of utmost gratitude and feeling of reverence to my supervisor Prof fl-rarpa(Singh, Indian Institute of Technology..ifis unwavering guidance, support and helping nature, created a comforta6Ce research and working environment..lfis vast knowledge, scientific wisdom, visionary approach and infectious enthusiasm for learning have improved my concepts. A forma(gesture wi(C6e inadequate for his inva(ua6Ce concern and consideration for my work I am sure this rewarding and enriching experience wi(C6enfit me in persona(andprofessiona((ife.

I feel deeply thanifu( toOr. Joseman Jaco6, Cc'SE, IITDi and Mr. S.MLK 12ahman, CB!1E, IITDi for their generous advice and counsel from time to time. I owe deepest gratitude to Prof A.12., 12ay, .Mead CB ME for his kind attitude toward my academic requirements and approval of my thesis. I take the opportunity to a(so thank all the faculty members of C.B. M.E. for their 6eneficia(guidance..

No words will be a6Ce to do justice in acknowledgingOr. Sruti Chattopadhyay for her continuous support. Yfer vast knowledge, analytical bent of mind and experience has helped in more than one way. I a(so sincerely thankall my he(pfu(seniorsOr. Charu, /Jr.

geeta, /Jr. Seema,Or. vinay,Or. Lomas andOr. 9VirmaC for their concern and co-

operation. Any journey is fu(( of hiccups, hurdles, dead-ends, final triumph and tribulations flis ride was incomplete without Or 12icha Jackeray and Jr. Zaniu(46id.

My affectionate thanks are expressed for providing me surplus immunoreagents to 1Or.

12icha and software analysis c computer operation a6i(ity to /Jr. A6id. I wi(C always treasure my beautiful memories of creative discussions, as wed as lively interactions in

IITc/J with them.

I had a great pleasure in working with my Ca6mates and friends at IITI~i . I express my appreciation and thanks to Vr. 9Vavdeep, Vr. Cgurpa( Singh, Vs Vanu 'Yadav, Vr.

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Manoj, Mrs Avneet, Mr. 12aj, Mr. Manu Da(e(a, Mr. vivek and my other co(Ceagues I a(so eytend my thanks to al( the staff members of C(91E specialCy Mr. Sitaram and 11r.

12ajesh for their prompt assistance during grueling working hours. My heartfelt thanks are due to Mrs Jyati, tor. Chattar Singhi, Mr. Sharma (SEW) and Mr. Munna(a( (WM I

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for their help during this investigationa(study.

I take this space to thank my friends Ms. Divya, Ms Isha, Mrs Anju(a and 1Dr. Shivani for always cheering, Helping and encouraging me throughout my life. their presence has boosted my confidence andgiven me courage. I also wish to sincerely thankmy schooland co(Cege teachersfor introducing adisciplined and met hodica(approach in work

I gratefully acknowledge ?University Grant Commission, Indict' Department of cBiotechno(ogy, Indic, and Lockheed Martin ZJSA for their inestima6Ce financia(aid I am deeply indebted to IIT'e(hi for providing a superb working environment, infrastructure and instrumenta(faci(ities to smoothly carry out research.

It is an honour for me to thank my parents for their blessings, unconditional Cove and support. My thesis would never have been possible without their encouragement, patience and affection. I also thank my Cooing brother ir. Ankit, whose constant help and criticism sailed me through bitter-sweet times. Word "thanks" would not encompass alt my feelings andgratitude for my family.

Above a(C, I would dedicate this thesis to the omnipresent Cod I thank the creator and sustainer of life for everything. I see this day of academic pursuit because of his mercy and grace. This workis his onCy.

Swati ,lain

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Contents

Topic Page no.

Chapter I: Introduction and Literature Review 1

1.1 Introduction 1

1.2 Pathogenic bacteria and diseases 2

1.2.1 Salmonella species and S. typhi 4

1.2.2 S. typhi and pathogenesis 5

1.2.3 Typhoid fever 6

1.3 Diagnosis methodologies for pathogenic bacteria 7 1.3.1 Conventional microbiological identification method 7

1.3.2 Nucleic acid based method 8

1.3.3 Bacteria identification through instruments 8 1.3.4 Antibody based techniques-immunoassays 9 1.3.4.1 Antibody based immunoassays for S. typhi 11 1.3.4.2 Commercially available kits 12 1.4 Immunoassays and their Classification based on label 15

1.4.1 Radioimmunoassay 15

1.4.2 Immunofluorescence 16

1.4.3 Enzyme immunoassays (EIA) 17

1.4.3.1 Chemiluminescence assay (CLIA) 17 1.4.3.2 Enzyme linked immunosorbent assay (ELISA) 18

1.5 Sandwich ELISA 18

1.5.1 Immobilization Techniques 19

1.5.1.5 Physical immobilization 19

1.5.1.6 Covalent immobilization 19

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1.5.2 Solid Phase for immunoassay 21 1.5.2.1 Conventional microtiter plate for ELISA 22 1.5.2.2 Polymeric microspheres, beads and membrane for

ELISA 23

1.5.3 Modification of polymeric solid support 24 1.6 Fluorescent based detection and Nanotechnology 26

1.6.1 Gold nanoparticles 28

1.6.2 Nanocrystals or Quantum Dots (QDs) 29

1.6.3 Silica nanoparticles 30

1.6.4 Polymeric nanoparticles 31

1.6.4.1 Immune-based detection on functionalized polymeric

nanoparticles 33

1.6.4.2 Immunofluorescent detection of analyte using dye

doped NPs 33

1.7 Rationale and Objective of work 34

References 38

Chapter II: Surface modification of polyacrylonitrile fibers and

immobilization of antibodies 46

2.1 Introduction 47

2.2 Experimental 50

2.2.1 Materials 51

2.2.2 Reduction of PAN fibers 51

2.2.3 Attachment of antibodies onto modified PAN fibers 51 2.2.3.1 Activation of aminated fibers 51

2.2.3.2 Immobilization of IgGs 51

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2.2.4 Characterization 52

2.2.4.1 Amine content 52

2.2.4.2 ATR-FTIR Spectroscopy 52

2.2.4.3 Scanning Electron Microscopy 52 2.2.4.4 Diffrential Scanning Microscopy 53 2.2.4.5 Confocal Laser scanning Microscopy 53 2.2.5 Evaluation of modified fibers for the detection of analyte (RAG-

IgG) 53

2.2.5.1 Optimization of parameter by checkerboard ELISA 53 2.2.5.2 Evaluation of analytical sensitivity of the developed

PAN-ELISA 54

2.2.6 Detection human blood antibodies 55 2.2.6.1 Specimen of blood samples and preparation of

Elutes 55

2.2.6.2 Detection of human blood IgG's using

PAN-ELISA Sensitivity, Specificity and comparison

with conventional method 55

2.3 Results and Discussion 56

2.3.1 Amination of polyacrylonitrile fibers 56

2.3.2 Antibody immobilization 57

2.3.2.1 Activation and immobilization of IgGs

2.3.2.2 Activity of enzyme conjugate immobilized on modified

PAN fibers 58

2.3.3. Characterization 59

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2.3.3.1 Evaluation of amine content 59

2.3.3.2 ATR-FTIR spectroscopy 60

2.3.3.3 Differential Scanning Calorimetry 62 2.3.3.4 Scanning Electron Microscopy 64 2.3.3.5 Confocal Laser Scanning Microscope 66 2.3.4 Evaluation of the modified fibers for the detection of analyte

(RAG) by performing checkerboard ELSIA 67

2.3.5 Detection of Human blood IgG's 70

References 72

Chapter III Modified PAN fiber-ELISA for detection of antigen 74

3.1 Introduction 75

Part A detection of S. typhi bacteria using modified PAN fibers 79

3.2 Experimental 79

3.2.1 Material and Reagents 79

3.2.2 Attachment of antibodies onto modified Pan fibers 80 3.2.3 Evaluation of the modified fibers for the detection of

bacteria S. typhi 80

3.3 Results and Discussions 81

3.3.1 Attachment of antibodies on modified PAN fibers 81 3.3.2 Colorimeteric detection of S. typhi bacteria by

modified PAN-ELISA 82

Part B Detection of anti-tetanus toxoid antibody on modified PAN fibers 86

3.4 Experimental 86

3.4.1 Materials and Reagents 86

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3.4.2 Attachment of tetanus toxoid onto modified

PAN fibers 87

3.4.1.1 Activation of the aminated fibers 87 3.4.2.2 Immobilization of tetanus toxoid 87

3.4.3.1 ELISA plate reader 87

3.4.3.2 Scanning Electron Microscope 87 3.4.4 Detection of Tetanus toxoid antibody using modified PAN

fibers 88

3.4.5 Detection of HAT antibody spiked in human blood 89 3.4.6 Evaluation of the immunity of patients against tetanus 90

3.5 Results and Discussion 90

3.5.1 Immobilization of tetanus toxoid 90

3.5.2 Scanning Electron Microscope 91

3.5.3 Detection of HAT tetanus toxoid antibody 92 3.5.3.1 Optimization of enzyme conjugate dilution 92 3.5.3.2 Evaluation of analytical sensitivity, specificity

and reproducibility of the modified PAN-ELISA system 94 3.5.3.3 Comparison with conventional microtiter plate 96 3.5.4 Detection of HAT antibody in human blood sera 97 3.5.5 Evaluating the immunity of patients against tetanus 98

References 100

Chapter IV Development of modified polycarbonate membrane-ELISA for the

detection of S. typhi 102

4.1 Introduction 103

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Part A Surface functionalization of polycarbonate membrane 106

4.2 Experimental 106

4.2.1 Materials 106

4.2.2 Amination of polycarbonate membrane 106 4.2.3 Activation of PC by Glutaraldehyde 106 4.2.4 Characterization of functionalized PC 107 4.2.4.1 Evaluation of surface amine density 107

4.2.4.2 Contact angle 107

4.2.4.4 Scanning electron microscopy 108

4.3 results and discussion 108

4.3.1 Chemical derivatization of PC membranes 108 4.3.1.1 Generation of amino groups on PC membranes 108

4.3.1.2 Activation of mPC 109

4.3.2 Characterization of modified mPC 110 4.3.2.1 Evaluation of amine content 110

4.3.2.2 Contact angle 112

4.3.2.3 ATR-FTIR Spectroscopy 114

4.3.2.4 Scanning electron microscope 115 Part B Evaluation of modified mPC for detection of S. typhi 116

4.4.1 Materials and reagents 116

4.4.2 Attachment of S. typhi Ab and activity of immobilized Ab 117

4.4.3.1 Scanning electron microscope 117

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4.4.3.2 Confocal laser scanning microscopy 118 4.4.4 Evaluation of modified PC membrane for the colorimetric detection of S. typhi in sandwich ELISA format 118 4.4.4.1 Optimization of S. typhi-Ab 118 4.4.4.2 Immobilization Efficiency 119 4.4.5 Modified PC membrane based immunoassay 120

4.4.5.1 Sensitivity 120

4.4.5.2 Specificity 120

4.4.5.3 Precision 121

4.4.5.4 Binding efficiency of S. typhi antigen on mPC 121 4.4.5.5 Comparison with PS microtiter plate-ELISA 122

4.6 Results and discussion 122

4.6.1 Antibody Immobilization 122

4.6.2.1 Scanning electron microscopy 124 4.6.2.2 Confocal laser scanning microscopy 126 4.6.3 Evaluation of mPC for the detection of S. typhi

and development of sandwich immunoassay 126 4.6.3.1 Optimization of S. typhi Ab 126 4.6.3.2 Immobilization efficiency 128

4.6.4 Modified PC based immunoassay 129

4.6.4.1 Dose response/Sensitivity 129

4.6.4.2 Specificity 130

4.6.4.3 Precision 130

4.6.4.4 Binding studies 131

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4.6.4.5 Conventional PS microtiter plate-ELISA 132

References 134

Chapter V Development of bioconjugated fluorescent polymeric nanoparticles

for detection of S. typhi 137

5.1 Introduction 138

Part A Synthesis of novel functionalized fluorescent polymeric nanoparticles 140

5.2.1 Materials and Reagents 140

5.2.2 Synthesis of fluorescent nanoparticles 140 5.2.3 Characterization of synthesized FPNPs 142

5.2.3.1 Solid content 142

5.2.3.2 Particle size analysis 142

5.2.3.3 ATRFTIR Spectroscopy 143

5.2.3.4 Scanning Electron Microscopy 143

5.2.3.5 Transmission Electron Microscopy 143 5.2.3.6 Confocal Laser scanning Microscopy 143

5.2.3.7 Fluorescence Spectroscopy 144

5.2.3.7.1 Effect of particle size on fluorescent intensity 144 5.2.3.7.2 Effect of concentration of nanoparticles on

fluorescent intensity 145

5.2.3.7.3 Effect of pH on fluorescent intensity 145 5.2.3.7.4 Effect of ionic strength on fluorescent intensity 145 5.2.3.7.5 Effect of time on fluorescent intensity 146

5.2.4 Attachment of antibodies 146

5.2.4.1 Activity of GAR-HRP immobilized on FPNP 146

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5.2.4.2 Confocal laser scanning microscopy 147

5.3 Results and discussion 147

5.3.1 Synthesis of nanoparticles 147

5.3.2 Characterization of Nps 149

5.3.2.1 Solid content 149

5.3.2.2 Particle size and polydispersity index 149

5.3.2.4 Scanning Electron Microscopy 151 5.3.2.5 Transmission Electron Microscope 152 5.3.2.6 Confocal Laser Scanning Microscopy 153

5.3.2.7 Fluorescence spectroscopy 154

5.3.2.7.1 Effect of particle size on fluorescent intensity155 5.3.2.7.2 Effect of nanoparticle concentration on

fluorescent intensity 157

5.3.2.7.3 Effect of pH on fluorescent intensity 158 5.3.2.7.4 Effect of ionic strength on fluorescent

intensity 159

5.3.2.7.5 Effect of time on fluorescent intensity 160

5.3.3 Antibody Immobilization 161

5.3.3.1 Activity of immobilized GAR-HRP 161 5.3.3.2 Fluorescence of FPNP-GAR-FITC 164 Part B Development of fluoroimmunoassay for the detection of S. typhi

using bioconjugated FPNPs 165

5.4.1 Materials and reagents 165

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5.4.2 Preparation of Bioconjugated Fluorescent probe 165 5.4.3 Characterization of FPNP-S. typhi Ab 165 5.4.3.1 Fluorescence spectroscopic studies of bioconjugated

FPNP 165

5.4.3.2 Estimation of immobilized Ab concentration on

FPNP 166

5.4.3.3 ATR-FTIR spectroscopy 166

5.4.3.4 Scanning electron microscopy 167 5.4.3.5 Transmission Electron Microscopy 167 5.4.4 Development of fluorescence based assay using mPC

and FPNPs 167

5.5 Results and discussions 168

5.5.1 Preparation of S. typhi-IgG immobilized fluorescent

probe 168

5.5.2 Characterization of FPNP-S. typhi Ab168

5.5.2.1 Spectroscopic characterization of bioconjugate

probe 169

5.5.2.2 Quantification of S. typhi Ab immobilized FPNP 170

5.5.2.4 Scanning electron microscopy 172 5.5.2.5 Transmission Electron microscope 173 5.5.3 Determination of S. typhi antigen using mPC membrane and

bioconjugated fluorescent probe 174

References 175

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Chapter VI-Summary and future scope of work

6.1 Summary 178

6.2 Future scope of work 185