Carbon nanotubes and branched DNA based nucleic acid assays towards a PCR free detection and quantification of nucleic acids

Electrochemical and optical nucleic acids assays based on I a novel carbon nanotube CNT-based label, II a branched DNA bDNA amplifier have been developed for the significant amplificatio

CARBON NANOTUBES AND BRANCHED-DNA BASED

NUCLEIC ACIDS ASSAYS:

TOWARDS A PCR-FREE DETECTION AND

QUANTIFICATION OF NUCLEIC ACIDS

LEE AI CHENG

(M.Sc., NTU, Singapore)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE

2009

ACKNOWLEDGEMENTS

I would like to extend my sincere gratitude to all those who made this thesis possible Special thanks go to my supervisor A/P Lim Tit Meng (Dept of Biol Sci, NUS) and co-supervisors A/P Heng Chew Kiat (Dept of Paed., NUS) and A/P Tan Swee Ngin (Nat Sci & Sci Edu., NTU) who offered their expert guidances, invaluable counsel and constant encouragements

The scope of this research is multidisciplinary; many have contributed their insights, their expertise and their precious time to help me Thanks to A/P Allen Yeoh (Dept of Paed., NUH & NUS) and Ms Chen Siew Peng for providing invaluable information on leukaemia disease Thanks to Dr Ye Jian-Shan (NUS) for the many fruitful discussions on carbon nanotubes and biosensors I want to thank A/P Sheu Fwu-Shan (NUS) and A/P Poenar D Puiu (NTU) for their guidances Special thanks

to Dr Eishi Igata (NTU) for sharing his insights and experiences as a "veteran researcher"

The support of my collaborators has allowed me to obtain excellent results for publications Special thanks to Dr Lin Yuehe for the opportunities to collaborate with his team (Dr Chen Baowei, Dr Liu Guodong, Dr Wangjun, and Ms Wu Hong) at Pacific Northwest National Laboratory (PNNL) I would like to thank Dr Zhang Aiguo (Panomics, Inc) for the collaboration on using bDNA technology I am grateful

to Dr Dai Ziyu (PNNL) for his technical helps on preparing the p185-ssDNA standard for quantitative assay and real-time PCR studies Thanks to Prof John Wang (Dept of Mat Sci., NUS) and Ms Agnes Lim Mui Keow for their assistances during the AFM studies I would like to acknowledge Ms Karen Lee Siang Ling and Mr Yan Tie for their dedicated supports in our research laboratories I would like to thank

my fellow colleagues who have helped me in this project in one way or another

The works described in this thesis were partially performed at the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S Department of Energy's (DOE's) Office of Biological and Environmental Research and the core R&D Laboratory of Fungal Biotechnology funded by the DOE Biomass Program, which are located at PNNL, USA I gratefully acknowledge the awards of NUS Postgraduate Research Scholarship and PNNL Fellowship as well as the financial supports from A*STAR, Singapore (Grant No 022

107 0008, project 'BioMEMS for Cell Characterization')

Lastly, I am grateful to my husband, parents and other family members for their encouragements and patience throughout my research

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS iii

SUMMARY vii

LIST OF PUBLICATIONS xi

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xvii

CHAPTER 1 LITERATURE REVIEW 1

1.1 Hybridization and Detection of Nucleic Acids 1

1.1.1 Basic of nucleic acid hybridization 1

1.1.2 Label-free detection of nucleic acids 2

1.1.2.1 Electrochemical 3

1.1.2.2 Optical 9

1.1.2.3 Piezoelectric 10

1.1.3 Label-based detection of nucleic acids 11

1.1.3.1 Redox-active molecules 11

1.1.3.2 Enzymes 15

1.1.3.3 Nanoparticles 19

1.1.3.4 Multiple labelling for signal amplification 27

1.2 Carbon Nanotubes (CNTs) 27

1.2.1 Introduction 27

1.2.2 Structural and physical properties of CNTs 28

1.2.3 Functionalization of CNTs 29

1.2.3.1 Covalent 29

1.2.3.2 Non-covalent 32

1.2.4 Biological applications of CNTs 34

1.2.4.1 Delivery vectors 35

1.2.4.2 CNTs-based chemical and biosensors 37

1.3 Leukemia Models 44

1.3.1 Types of leukemia and classification 44

1.3.2 Conventional methods for the diagnosis of leukemia 45

1.3.3 The Philadelphia (Ph) chromosome and BCR-ABL variants 49

1.3.3.1 BCR-ABL variants 49

1.3.4 Detection of Ph chromosome and BCR-ABL oncogenes 52

1.4 Aims of the Study 54

1.5 References 56

CHAPTER 2 CARBON NANOTUBE-BASED LABELS FOR HIGHLY

SENSITIVE COLORIMETRIC AND AGGREGATION-BASED VISUAL

DETECTION OF NUCLEIC ACIDS 74

2.1 Introduction 74

2.2 Materials and Methods 77

2.2.1 Reagents 77

2.2.2 Preparation of conventional and CNT-based labels 79

2.2.3 Atomic force microscope (AFM) characterization and sample preparation .79

2.2.4 Sandwich hybridization 80

2.2.4.1 Immobilization of capture probes to streptavidin-coated beads (SA-beads) 80

2.2.4.2 Immobilization of capture probes to carboxylic acid functionalized beads (COOH-beads) 80

2.2.4.3 Determination of CP density on SA-beads and COOH-beads 81

2.2.4.4 Hybridization with TG and NTG 81

2.2.4.5 Hybridization with conventional and CNT-based labels 82

2.2.5 Colorimetric detection 82

2.3 Results and Discussion 83

2.3.1 Synthesis and AFM characterization of the CNT-based labels 83

2.3.2 Detection and signal amplification principles 86

2.3.3 Specificity of bead conjugates on target detection using CNT-based labels .90

2.3.3.1 Types of bead 90

2.3.3.2 Blocking of bead surfaces 92

2.3.4 Analytical performance of CNT-based labels 95

2.3.5 Target-specific aggregation of CNT labels and beads 97

2.4 Conclusions 101

2.5 References 102

CHAPTER 3 SENSITIVE ELECTROCHEMICAL DETECTION OF HORSERADISH PEROXIDASE AT a DISPOSABLE SCREEN-PRINTED CARBON ELECTRODE 106

3.1 Introduction 106

3.2 Materials and Methods 109

3.2.1 Reagents 109

3.2.2 Voltammetric and amperometric measurements 109

3.3 Results and Discussion 111

3.3.1 Cyclic voltammetric characteristics of HRP-oAP–H2O2 at the SPCE 111

3.3.2 Comparison of voltammetric and amperometric detection of the enzymatic product 113

3.3.3 Factors affecting SWV analysis of HRP-o-AP-H2O2 enzyme-substrate

system 116

3.3.3.1 Initial scanning potential 116

3.3.3.2 Working concentration of o-AP 118

3.3.4 Analytical characteristics of SWV detection of HRP 119

3.4 Conclusions 121

3.5 References 122

CHAPTER 4 PREPARATION OF SINGLE-STRAND DNA STANDARD FOR

THE QUANTITATIVE ASSAYS OF P185 BCR-ABL ONCOGENE 125

4.1 Introduction 125

4.2 Materials and Methods 127

4.2.1 Reagents 127

4.2.2 Cell cultures 129

4.2.3 mRNA extraction 129

4.2.4 RNA handling 130

4.2.5 Absorbance measurements 130

4.2.6 Synthesis of full-length p185 BCR-ABL single-strand DNA (p185-ssDNA) .131

4.2.6.1 Synthesis of the biotinylated double-strand DNA (dsDNA) 131

4.2.6.2 Purification of biotinylated dsDNA and gel electrophoresis 132

4.2.6.3 Purification of sense p185-ssDNA 133

4.2.7 Real-time quantitative PCR (RQ-PCR) 134

4.3 Results and Discussion 137

4.3.1 Principle of the preparation of p185-ssDNA standard 137

4.3.2 Gel electrophoresis 140

4.3.3 Quantification of mRNA 141

4.3.4 Quantification of p85-ssDNA standard 143

4.3.5 Functionality of p185-ssDNA 144

4.4 Conclusions 146

4.5 References 147

CHAPTER 5 ELECTROCHEMICAL DETECTION OF LEUKEMIA ONCOGENES AT ATTOMOLES LEVELS WITH A CARBON NANOTUBES-BASED LABEL 149

5.1 Introduction 149

5.2 Materials and Methods 153

5.2.1 Reagents 153

5.2.2 Preparation of CNT-based labels 154

5.2.3 UV-vis absorption spectroscopy 155

5.2.4 Cell cultures and mRNA extraction 155

5.2.5 Synthesis of full-length p185-ssDNA 156

5.2.6 Sandwich hybridization 156

5.2.6.1 Immobilization of CP-2 to COOH-beads 156

5.2.6.2 Targets hybridization 157

5.2.6.3 Hybridization with CNT-based labels 157

5.2.7 Samples assay 157

5.2.7.1 HRP enzymatic reaction 157

5.2.7.2 Electrochemical detection 158

5.3 Results and Discussion 159

5.3.1 Characteristics of the CNT-based labels 159

5.3.2 Electrochemical detection and signal amplification principles 160

5.3.3 Composition of CNT-based Labels 162

5.3.3.1 Effect of DP loading 162

5.3.3.2 Effect of HRP loading 164

5.3.4 Amount of CNT-based labels 166

5.3.5 SWV detection of leukemic oligonucleotide targets amplified with CNT-based labels 167

5.3.6 Discrimination of non-complementary and mismatched sequences 170

5.3.7 Detection of p185-ssDNA targets 172

5.3.8 Detection of p185 BCR-ABL mRNA fusion transcript extracted from cell line 173

5.4 Conclusions 175

5.5 References 176

CHAPTER 6 ELECTROCHEMICAL BRANCHED-DNA ASSAY FOR PCR-FREE DETECTION AND QUANTIFICATION OF ONCOGENES IN MESSENGER RNA .179

6.1 Introduction 179

6.2 Materials and Methods 182

6.2.1 Reagents 182

6.2.2 Electrochemical measurements for ALP enzymes 183

6.2.3 Cell cultures and mRNA extraction 184

6.2.4 Preparation of sense p185-ssDNA 184

6.2.5 bDNA hybridization assay 184

6.3 Results and Discussion 185

6.3.1 Detection and signal amplification principles 185

6.3.2 Electrochemical Measurements of ALP on the SPCE 187

6.3.3 bDNA hybridization assay of p185-ssDNA standards 190

6.3.4 Detection of p185 BCR-ABL mRNA fusion transcript extracted from cell line 192

6.3.5 Quantitative mRNA assay: electrochemical bDNA assay vs fluorescent

RQ-PCR 195

6.4 Conclusions 197

6.5 References 198

CHAPTER 7 CONCLUDING REMARKS AND FUTURE WORKS 200

SUMMARY

The research effort is directed towards the development of polymerase chain reaction (PCR)-free ultrasensitive nucleic acids detection based on signal amplification approach Electrochemical and optical nucleic acids assays based on (I) a novel carbon nanotube (CNT)-based label, (II) a branched DNA (bDNA) amplifier have been developed for the significant amplification of nucleic acids hybridization signal, which in turn improves the assay sensitivity, and (III) a single-strand DNA (ssDNA) standard bearing the same sequence and length as of the target gene was created and accurately quantified for use in the PCR-free quantitative assays These assays have

been successfully validated for PCR-free detection and quantification of p185

BCR-ABL fusion transcript in the messenger RNA (mRNA) population extracted from

human acute lymphocytic (ALL) leukemia cell line SUP-B15

The details of various sections are as follows:

(I) CNT-based label

A novel multiple enzymes conjugated CNT-based label has been developed for highly

sensitive optical and electrochemical detection of human ALL related p185 BCR-ABL

fusion transcript The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) tracers and DNA detection probes (DP) via diimide-activated amidation The constructs were used as label for a magnetic bead-based DNA hybridization assay The labels were attached to the magnetic beads surface via specific hybridization of DNA detection probes to the captured targets The amount of targets was quantified by measuring the activity of captured HRP

The hybridization assay amplified by CNT-based label was first demonstrated via colorimetric measurements by measuring the absorbance of product The resulting CNT labels significantly enhanced the nucleic acids assay sensitivity by at least 1000 times compared to that of conventional labels used in enzyme-linked oligosorbent assay (ELOSA) An excellent detection limit of 1 × 10-12 M (60 × 10-18 mol in 60 μL) and a 4-order wide dynamic range of target concentration were achieved Such approach makes the sensitivity of conventional colorimetric ELOSA of DNA comparable to that of fluorescent and luminescent techniques Hybridizations using these labels were coupled to a concentration-dependent formation of visible dark aggregates Targets can thus be detected simply with visual inspection, eliminating the need for expensive and sophisticated detection systems

A rapid and simple electrochemical assay of HRP performed on disposable screen-printed carbon electrode was developed HRP activities were monitored by

SWV measuring the electroactive enzymatic product in the presence of

o-aminophenol and hydrogen peroxide substrate solution The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were optimized With optimized conditions, a linear response for HRP from 0.003 - 0.1 U mL-1 and a detection limit of 1.25 × 10-15 mol (in 25 µL) were obtained with a good precision (relative standard deviation = 8 %; n = 6) The resulting HRP assay was coupled to the nucleic acids assay amplified by the CNT-based labels The effect of DP and HRP loading of the labels on the signal-to-noise ratio of electrochemical detection was studied systematically With optimized conditions, the signal-amplified assay achieved a detection limit of 83 × 10-15 M (5 × 10-18 mol in 60 µL) targets oligonuecleotides and a 4-order wide dynamic range of target

perfect match and a three-base mismatch sequence When subjected to full-length oncogene (491 bp), the approach demonstrated a detection limit of 1 × 10-16

mol in 60

μL which corresponded to approximately 33 pg of the target gene The high sensitivity and specificity of assay enabled PCR-free detection of target transcripts in approximately 65 ng of mRNA extracted from positive ALL cell lines SUP-B15, in comparison to those obtained from negative cell lines HL-60 The approach holds promise for simple, low cost and ultrasensitive electrochemical nucleic acids detection in portable devices, POC and early disease diagnostic applications

(II) bDNA-amplified electrochemical nucleic acids assay

A novel electrochemical bDNA assay has been developed for PCR-free detection and

quantification of p185 BCR-ABL leukemia fusion transcript in the population of

mRNA extracted from cell lines The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA The activity of captured ALP was monitored by SWV analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized A detection limit of 1 × 10-15 M (1 × 10-19 mol in 100 μL) of full-length oncogene and a 3-order wide dynamic range of concentration were achieved Such limit corresponded

to approximately 17 fg of the p185 BCR-ABL The specificity and sensitivity of assay

enabled direct detection of target transcript in as little as 4.6 nanograms mRNA without PCR amplification In combination with the use of a well-quantified standard (III), the electrochemical bDNA assay was capable of direct use for a quantitative

analysis of target transcript in total mRNA population The assay could detect 62900

copies of p185 BCR-ABL in one nanogram mRNA population, which was at least

50-fold higher than that of real-time quantitative PCR (RQ-PCR) The finding was consistent with the underestimation of targets by RQ-PCR reported earlier The approach thus provides a simple, sensitive and quantitative tool alternate to the RQ-PCR for early disease diagnosis

(III) Preparation of ssDNA standard for quantitative assays

A ssDNA standard bearing the same sequence and length as of the target, i.e p185

BCR-ABL (denotes p185-ssDNA) was created First, biotinylated reverse primer was

used for full-length reverse transcription (RT)-PCR of p185 BCR-ABL to produce

biotinylated double-strand PCR amplicons The resulting amplicons were purified by gel electrophoresis followed by agarose digestion, phenol-chloroform extraction and ultrafiltration The purified biotin-amplicons were immobilized on the streptavidin-coated magnetic beads The non-biotinylated sense-strand was separated from the undesirable bead-bound antisense-strands by elution with Tris-HCl buffer at 95 oC for

5 min The eluate containing pure sense strands p185-ssDNA was collected The purified p185-ssDNA was quantified by UV absorbance at 260 nm and bench-marked

by the RQ-PCR Successful detection of the p185-ssDNA by both CNT-based label and bDNA hybridization assays indicated good integrity and functionality of the standard The approach offers a means to prepare standard suitable for non-PCR based quantitative nucleic acid assays

LIST OF PUBLICATIONS

Journal papers:

1 Lee AC, Dai Z, Chen B, Wu H, Wang J, Zhang A, Zhang L, Lim TM, Lin Y Electrochemical branched-DNA assay for polymerase chain reaction-free detection

and quantification of oncogenes in messenger RNA Anal Chem 2008;80:9402-9410

2 Lee AC, Liu G, Heng CK, Tan SN, Lim TM, Lin Y Sensitive electrochemical detection of horseradish peroxidase at disposable screen-printed carbon electrode

4 Lee AC, Ye JS, Tan SN, Poenar DP, Sheu FS, Heng CK, Lim TM, Carbon

nanotube-based labels for sensitive nucleic acids detection Nanotech 2006; 2: 232-

Conference papers:

1 Lee AC, Liu G, Lin Y, Lim TM, Heng CK, Tan SN, Wang J, Wu H, Lin YY Highly sensitive electrochemical nucleic acids detection based on carbon nanotubes-

derived labels 62 nd Northwest Regional meeting (NORM 2007), 17-20 June 2007,

Boise, Idaho, USA

2 Lee AC, Liu G, Lin Y, Heng CK, Tan SN, Wang J, Wu H, Lim TM, Lin Y Highly sensitive electrochemical nucleic acids biosensor based on carbon-nanotubes-derived

labels for leukemia diagnosis Medicine-infectious Diseases Continuing Medical

Education Conference: What’s New in Medicine 2007, 8 & 9 June 2007, Kennewick,

WA, USA

3 Lee AC, Ye JS, Tan SN, Poenar PD, Sheu FS, Heng CK, Lim TM Carbon

nanotube-based labels for sensitive nucleic acids detection 2006 NSTI Nanotechology

conference and Trade Show, 7-11 May 2006, Boston, MA, USA

LIST OF TABLES

Table 1.1 Techniques used for label-free detection of nucleic acids 3

Table 1.2 Redox-active labels used in electrochemical nucleic acid assays 12

Table 1.3 Enzyme labels used in electrochemical nucleic acid assays 16

Table 1.4 Nanoparticles labels used in electrochemical nucleic acid assays 26

Table 1.5 Methods for detecting phenotypic and genotypic features of leukemic cells .46

Table 2.1 Sequences of oligonucleotides used in hybridization assay 78

Table 2.2 Parameters of CP-bound COOH-beads and SA-beads used for each hybridization reaction 91

Table 4.1 Sequences of primers used in general PCR, RT-PCR and RQ-PCR .128

Table 4.2 Typical yields of mRNA extracted from individual batches of (A) SUP-B15 and (B) HL-60 cell cultures 142

Table 4.3 Typical concentration and purity of p185-ssDNA standard .144

LIST OF FIGURES

Figure 1.1 Basic principle of nucleic acids hybridization and detection on a solid

surface 2

Figure 1.2 Direct electrochemistry DNA detection based on the oxidation of guanine base 5

Figure 1.3 Labeling of nucleic acids with electroactive labels .13

Figure 1.4 An enzymatic amplification of DNA hybridization signal measured by EIS method 19

Figure 1.5 Metal nanoparticle-based electrochemical detection of DNA 20

Figure 1.6 Detection of DNA based on conductivity measurement 25

Figure 1.7 Schematic representation of (A) a MWNT and (B) a SWNT 28

Figure 1.8 Surface groups bonded to aromatic rings on CNTs 30

Figure 1.9 Carboxylic acid derivatization of CNTs 31

Figure 1.10 Configuration of CNTs-modified electrochemical electrodes (A) randomly oriented CNTs, (B) vertically aligned CNTs array, (C) field effect transistor (FET) 38

Figure 1.11 The translocation of BCR-ABL The structures of BCR and ABL are shown in (A) and (B), respectively Boxes denote exons and lines refer to introns (C) Depending on the location of breakpoints (arrows) in BCR, the translocation results in the formation of three variants of the BCR-ABL gene, i.e p185, p210 and p230 53

Figure 2.1 Diimide-activated amidation for conjugation of SWNT–HRP–DP Horseradish peroxidase (HRP) enzyme-labeled detection probes (DPs) were covalently conjugated to the carboxylated SWNTs in the presence of the cross-linker N-(3-dimethylaminopropyl)-N’ -ethylcarbodiimide hydrochloride (EDC) 83

Figure 2.2 Height images from the AFM analysis of (A) a bare SWNT (average height = 1.0 nm), (B) a SWNT-HRP-DP conjugate (a, b, c, d = 1.3, 0.8, 3.3 and 3.1 nm respectively), (C) the conventional HRP-DP labeling reaction mixture (e, f, g = 2.5, 4.3, 3.0 nm respectively) on mica substrate 85

Figure 2.3 Schematic representation of signal amplification for sandwich hybridization assay performed on magnetic beads The signal-to-single hybridization event ratio of (A) the conventional HRP label is amplified by (B) multiple HRP and DP-conjugated CNT-based labels 87

Figure 2.4 Concentration of CPs in solution collected during CP-SA-beads immobilization reaction analyzed by NanoDrop ND-1000 spectrophotometer .88

Figure 2.5 Concentration of CPs in solution collected during CP-COOH-beads

immobilization reaction analyzed by NanoDrop ND-1000 spectrophotometer .89

Figure 2.6 Carboxylic acid functionalized beads (COOH-beads) and

streptavidin-coated beads (SA-beads) for the hybridization of 1 × 10−8 M of target (gray bars) and negative control samples (white bars) using CNT-based labels .91

Figure 2.7 Effect of blocking in the hybridization of 1 × 10−8 M of target (gray bars) and negative control samples (white bars) using CNT-based labels 93

Figure 2.8 The effects of specificity of bead conjugates on the performance of TG

detection using CNT-based labels (A) SA-beads (B) COOH-beads 94

Figure 2.9 Analytical performance of CNT-based labels (A) responses to TG

samples of the hybridization assay performed on COOH-beads with 8 % blocking The bars for control, 1E-12M and 1E-11M of TG in (A), have been enlarged in the inset at the upper left (B) the corresponding log–log graph of the dynamic detection range plotted with values after control subtraction 96

Figure 2.10 The appearance of magnetic beads (SA-beads) in wash buffer solution

after hybridization in the assay using CNT-based labels 100

Figure 3.1 (A) Electro-oxidation of o-AP in acidic solution produces

3-aminophenoxazone (3-APZ) as the major product (B) The redox reaction of 3-APZ 108

Figure 3.2 Cyclic voltammograms of o-AP in the absence and presence of HRP using

the SPCE in a pH5.7 BR buffer solution containing 0.04 M each of H3PO4, HOAc and

H3BO3: (a) 1.2 × 10-5 M o-AP + 2 × 10-4

M H2O2; (b) as in (a) + 0.25 U mL-1 HRP 112

Figure 3.3 Responses at the SPCE to o-AP enzymatic product catalyzed by HRP in

BR buffer recorded using (a) SWV, (b) DPV and (c) amperometric techniques 115

Figure 3.4 Effect of initial scanning potential on SWV responses of enzymatic

product (grey bars) and blank substrate (white bars) solution 117

Figure 3.5 Optimization of substrate concentration 119

Figure 3.6 Typical SWV responses of increasing HRP concentration in PBS buffer

From bottom to top, the concentration of HRP in PBS buffer is 0.1, 0.05, 0.025, 0.012, 0.006, 0.003 and 0 U mL-1, respectively Insert shows the calibration curve of HRP 120

Figure 4.1 (A) pGEM®-T Vector circle map Multiple cloning site (MCS) is located

at 10 to 113 This vector has been linearized with EcoR V at base 51 from that of the circular pGEM®-5Zf(+) vector and a T is added to both 3´-ends The p185 BCR-ABL

Figure 4.2 Schematic representation of the synthesis of biotinylated p185 dsDNA

from p185 BCR-ABL mRNA template 138

Figure 4.3 Schematic representation of the purification of sense p185-ssDNA from

biotinylated p185 dsDNA 139

Figure 4.4 Agarose gel electrophoretic image of the double-stranded (Lane 2) and

single-stranded DNA (Lane 3) for the p185 BCR-ABL gene 140

Figure 4.5 Validation of p185-ssDNA standards based on electrochemical

hybridization assays amplified by (A) CNT-based labels and (B) bDNA amplifiers 145

Figure 5.1 Visible absorption spectra of (a) horseradish-peroxidase enzymes (HRPs),

(b) single-stranded DNA detection probes (DPs), (c) SWNT-DP-HRP conjugates and (d) single-walled carbon nanotubes (SWNTs) in ultrapure water .160

Figure 5.2 Schematic representation of signal-amplified electrochemical detection of

nucleic acid targets based on CNT-based labels (A) Sandwich hybridization assay performed on magnetic beads (B) The targets were quantified by measuring the electroactive enzymatic product, formed by the HRP catalyzed reaction with o-AP-

H2O2 substrate solution using a screen-printed electrode (C) The resulting SWV voltammogram indicates a well-defined reduction peak for TG/p185-ssDNA/mRNA targets 161

Figure 5.3 Influence of the composition of CNT-based labels on TG detection (A)

The effect of DP loading per gram of CNT added to the label synthesis mixture (B) The effect of HRP loading .165

Figure 5.4 Optimal amount of CNT-based labels required for each hybridization

reaction The labels were synthesized at optimum reaction composition of 2.5 µmol/g

of CNT and 35 % v/v HRP 167

Figure 5.5 Calibration curves for the electrochemical quantitation of leukemic DNA

target amplified by the CNT-based labels The dynamic detection range constitutes (A) the high-range linear plot (left) and the corresponding square-wave voltammograms (right), and (B) the enlarged low-range linear (left) and the corresponding voltammograms (right) 169

Figure 5.6 Specificity of the electrochemical detection of leukemic DNA targets

amplified by CNT-based labels (A) Discrimination between complementary target (TG) and non-complementary target (NTG) (B) Discrimination between TG and mismatched sequences 171

Figure 5.7 Typical SWV responses with increasing amounts of p185-ssDNA From

top to bottom, the amounts of target is 0, 4.8 × 10-16, 1.4 × 10-15, 2.9 × 10-15, 5.7 × 10

-15

and 1.2 × 10-14 mol, respectively Inset shows the resulting calibration plot 173

Figure 5.8 Direct electrochemical measurement of p185 BCR-ABL mRNA fusion

transcript extracted from the positive leukemia cell line SUP-B15 and negative cell line HL-60 174

Figure 6.1 Schematic illustration of the branched DNA (bDNA)-amplified

electrochemical detection and quantitation of human leukemia BCR-ABL mRNA

fusion transcript 187

Figure 6.2 (A) CV and (B) SWV voltammograms of 1-napthyl-phosphate substrate

solution in the absence (curves a) and presence (curves b) of ALP using the SPE (C) Optimization of substrate concentration ranged from 0.01 to 5 mM (D) The linear-plot of SWV detection of ALP 189

Figure 6.3 (A) Typical SWV responses with increasing standard concentrations (B)

The resulting calibration plot with the lower left portion being enlarged as in (C) 191

Figure 6.4 (A) Direct electrochemical measurement of p185 BCR-ABL mRNA fusion

transcript The mRNA samples were extracted from the positive leukemia cell line SUP-B15 and negative cell line HL-60 (B) The corresponding linear response and null response of SWV to the mRNA samples extracted positive and negative cell lines, respectively 194

Figure 6.5 Chemiluminescent measurement of the p185 BCR-ABL mRNA fusion

transcript 195

Figure 6.6 Electrochemical bDNA assay for quantification of target genes in the

mRNA samples The unknown quantities of the target genes on three different amounts of mRNA sample (pink) were determined using a set of 5-points p185-ssDNA standards (blue) .196

LIST OF ABBREVIATIONS

3-APZ 3-aminophenoxazone

ABTS 2’azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid

ALL acute lymphocytic leukemia

AML acute myelogenous leukemia

ASV adsorption stripping voltammetry

ATCC american type culture collection

BCR breakpoint cluster region

bpy bipyridine

BR britton-robinson

BRCA1 breast cancer 1 gene

BSA bovine serum albumin

BVRB biliverdin IXß reductase enzyme

CE capture extender

CLL chronic lymphocytic leukemia

CML chronic myelogenous leukemia

CML-N neutrophilic-chronic myeloid leukemia

CNNFET CNTs network FET

ECL electrogenerated chemiluminescent

EDTA ethylenediamine tetraacetic

EIS electrochemical impedance spectroscopy

ELOSA enzyme-linked oligosorbent assay

FBS fetal bovine serum

FISH fluorescence in situ hybridization

FND ferrocenyl naphthalene diimide

FET field effect transistor

GCE glassy carbon electrode

GMA gold microelectrode array

HER-2 human epidermal growth factor receptor 2

HMDE hanging mercury drop electrode

HOPGE highly oriented pyrolytic graphite electrode

IMDM Iscove’s modified dulbecco’s medium

ITO indium tin oxide

m-bcr minor breakpoint cluster

MES 2-(N-morpholino) ethanesulfonic acid

MIC morphologic immunologic cytogenetic

MRD minimal residue disease

NaDDS sodium dodecylbenzenesulfonate

NADH nicotinamide adenine dinucleotide hydrogen (reduced)

Os,biby osmium textroxide with 2,2’-bipyridine

PBS phosphate buffer saline

PCR polymerase chain reaction

PGE pyrolytic graphite electrode

PIND-Os Os(2,2’-bipyridine)2Cl+ pendants

PNA peptide nucleic acid

QCM quartz crystal microbalance

Ret electron transfer resistance

SA-beads streptavidin-coated beads

SAM self assembled monolayer

SDS sodium dodecyl sulfate

SNP single nucleotide polymorphism

SPCE screen-printed carbon electrode

SPR surface plasmon resonance

STM scanning tunnelling microscopy

Tween-20 polyethylene glycol sorbitan monolaurate

-bcr micro-bcr

UV ultra violet

WHO World Health Organization

CHAPTER 1 LITERATURE REVIEW

1.1 Hybridization and Detection of Nucleic Acids

1.1.1 Basic of nucleic acid hybridization

The nucleic acid is a polymer consists of monomers called nucleotides Each nucleotide composes of a nitrogenous base, a pentose sugar and a phosphate group In deoxyribonucleic acid (DNA), the bases are the purine adenine (A) and guanine (G)

as well as the pyrimidines cytosine (C) and thymine (T) In deoxyribonucleic acid (RNA), T is replaced by uracil (U) Nucleic acid hybridization relies on the Watson-Crick base-pairing properties of DNA The base pairing is specific in which G is paired to C and A to T (A to U in RNA:DNA hybrids) by hydrogen bonding

Most of the nucleic acid hybridization assays are performed on a solid surface These solid surfaces can be the membrane used in conventional Southern1 and Northern2 blots (for the detection of DNA and RNA respectively), glass or silicon in microarray,3 electrode in electrochemical sensors4 - 6 and microsphere beads.7 - 9

The hybridization assays rely mainly on the specific hybridization of a surface-bound single-stranded oligonuecleotides sequence (i.e the capture probe) to the complementary region of the target nucleic acid.4 This molecular recognition event triggers a usable signal, which can be measured by means of optical, electrochemical or piezoelectric readout depending on the methods of signal transduction (Figure 1.1) Among them, electrochemical nucleic acids biosensors have gained considerable attention in recent years due to its low-cost and simplicity suitable for the development of portable devices Several comprehensive reviews on electrochemical DNA biosensors have also been published.4 - 6, 10

Figure 1.1 Basic principle of nucleic acids hybridization and detection on a solid

surface

The signal transduction strategies of nucleic acids hybridization can be broadly divided into two main categories: label-free and label-based approaches Major considerations in the nucleic acids sensing are the detection limit and specificity of the detection An ideal approach should be capable of discriminating the perfectly matched target nucleic acids from those with a single base-pair mismatch without requiring amplification of the target samples Unfortunately, in many cases, each technique has their pros and cons

The following sections aim to introduce the label-free (Section 1.1.2) and label-based (Section 1.1.3) approaches and the efforts towards improving the sensitivity and specificity of assays with an emphasis on the electrochemical nucleic acids detection

1.1.2 Label-free detection of nucleic acids

There are attempts to measure the hybridization signal directly in the absence of a secondary reporting molecule, i.e label The common label-free techniques used to transduce the hybridization signal to a measurable read-out are listed in Table 1.1

Target Capture

range of nM to sub-pM.5, 10 Further efforts have been directed towards label or mediator-assisted signal amplification to increase assay sensitivity (Table 1.1)

Table 1.1 Techniques used for label-free detection of nucleic acids

Detection Limits [Ref.]

Signal Transduction

Methods

Signal

Label-free Label-assisted Electrochemical

Voltammetric

Voltammetric

EIS

Guanine oxidation current Adenine oxidation current Electron transfer resistance (Ret)

77 nM [12]; 51 pM [13]; 17 nM [14]

1.1.2.1.1 Direct electrochemistry of guanine

Of the nucleic acid components, only the bases undergo redox processes whereas the sugar and phosphate group are electrochemically inactive Among the four nucleic acid bases, the guanine and adenine are the most easily oxidized species Their oxidation signals on carbon electrodes was being observed at ca +1.00 V and +1.30 V (vs Ag/AgCl) in 0.50 M acetate buffer solution (pH 4.80), respectively, as reported

by Jelen et al 11 Monitoring the changes of these oxidative signals at the respective potential upon duplex formation enabled the detection of hybridization Such procedure eliminated the use of external labels

Figure 1.2 shows the basic principle of label-free nucleic acids detection based

on the monitoring of guanine signal The capture probes were immobilized onto an electrode; guanine bases in these capture probe sequences were substituted by inosine bases Like guanine, the inosine also binds specifically to cytosine bases but its oxidation signal is well separated from that of guanine Thus, no guanine signal is observed prior to hybridization with the target sequence Once the target strand is hybridized to the immobilized inosine-containing probe, the guanine peaks from the target nucleic acids can be detected using constant current chronopotentiometry

Using such approach, Wang et al reported a detection limit of 77 nM for an

oligo(dG)20 target using a renewable pencil electrode.12 Inosine-substituted probes for

nucleic acids biosensors were applied onto a carbon paste electrode by Ozkan et al 13

A differential pulse voltammetry (DPV) measurement was used to monitor the hybridization with a detection limit of 51 pM for a 23-mer DNA target (contaning 7 guanine residues) related to the allele-specific factor V Leiden mutation.13

Figure 1.2 Direct electrochemistry DNA detection based on the oxidation of guanine

base

To minimize the nonspecific adsorption (NSA) of unhybridized nucleic acids

at the probes-immobilized electrode surface, the hybridization and chronopotentiometry transduction steps were carried out at different surfaces, i.e magnetic beads and unmodified electrodes, respectively The approach demonstrated

a detection limit of 17 nM for a 19-mer DNA target related to the BRCA1 breast

cancer gene containing 4 guanine residues using a pencil electrode.14 In the presence

of copper ions, an analogous stripping hybridization measurement yielded a detection limit of 0.85 nM.15 The 20-fold improvement of the detection limit was attributed to the presence of copper ions which greatly enhanced the anodic response of free guanide via increased adsorption of the copper (I)–purine complexes at electrode surface.16

Guanine oxidation potential

Current

Potential

Current

Potential

1.1.2.1.2 Indirect electrochemistry of guanine

The electrocatalytic oxidation of guanine moieties within DNA or RNA mediated by Ru(2,2’-bipyridine)32+ [Ru(bpy)32+] was explored by Thorp’s group.17, 18 This approach involves the shuttling of electrons from surface-bound nucleic acids to solid electrodes using soluble redox-active mediators with potentials matched to that of guanine The inorganic metal complex Ru(bpy)33+ has a reduction potential of +1.06

V (vs Ag/AgCl), and is therefore commonly used as the redox-active mediator to oxidize guanine according to the following catalytic cycle:

The 1497-bp DNA targets molecules related to the HER-2 gene were adsorbed

onto indium tin oxide (ITO) electrodes followed by cyclic voltammetry (CV) measurements in the presence of Ru(bpy)32+ The mediated catalysis of the guanine oxidation gives rise to the enhanced current response and yielded a detection limit of

550 amol (ca 3 × 108 molecules) of adsorbed target strands.17

1.1.2.1.3 Direct electrochemistry of adenine

Detection of DNA hybridization based on the redox current of adenine was also been explored.19, 20 Palecek et al described a label-free detection of DNA based on the

determination of adenine by cathodic stripping voltammetry (CSV) at a hanging mercury drop electrode (HMDE).20 The target DNAs are specifically captured at Dynabeads Oligo(dT)25 magnetic beads The adenine moieties were subsequently released from the captured DNA by acid treatment Using the CSV, acid treated DNA could be easily determined at ppb level.20

de-los-Santos-Alvarez et al demonstrated that the oxidation of the adenine

moieties produces oxidation products that are electroactive at low potentials, i.e ca 0

V (vs Ag/AgCl) in alkaline media (pH 10.0).19 In this work, the electro-oxidation of the adenine moieties of oligonucleotides that were adsorbed on a pyrolytic graphite electrode (PGE) occurred at high potential, i.e ca +0.80 V (vs Ag/AgCl) in 0.1 M phosphate buffer (pH 10.0) The oxidation gives rise to electrochemically active products which were strongly adsorbed on the electrode surface The oxidation of adenine within DNA could be used to detect deoxyadenylic acid icosanucleotide (dA)20 at a potential ca 0 V with a detection limit of of 32 nM.19

1.1.2.1.4 Electrical interfacial properties

Among the commonly used electrochemical techniques, EIS has been shown to be an effective method to probe the interfacial changes originating from biorecognition events at electrode surfaces.21 The EIS detects changes in the interfacial properties, e.g Ret22 - 24 and capacitance25 at the electrode-solution interfaces

The principle of EIS sensing of nucleic acids hybridization is based on the formation of duplexes (i.e single-stranded DNA (ssDNA) capture probe-target hybrids) at an electrode surface which alters the interfacial properties As nucleic acids are oligoanionic polymers, their immobilization on surfaces generates a negatively charged interface that repels negatively charged redox labels such as [Fe(CN)6]3-/4- The repulsion of the redox labels from the electrode introduces a barrier for electron transfer thus leads to an enhanced Ret Thus, Ret will increase with the increasing amount of hybridized target strands

Liu et al employed the EIS in combination with peptide nucleic acid (PNA)

to monitor the in-situ hybridization kinetics of PNA-DNA duplex on a gold

electrode.22 The PNA is a synthetic oligomer that has a neutral peptide-like backbone with nucleobases that allows for the molecules to hybridize to complementary DNA (cDNA) strands with high affinity and specificity.26 The immobilized PNA probes on the sensor surface are uncharged, and hence, do not affect the charge transfer from the redox indicator [Fe(CN)6]3-/4- to the electrode Hybridization of DNA targets to PNA increased the charge density at the sensor surface, and led to an enhanced Ret This method enabled the detection of cDNA targets at a concentration of 1 nM.26

The label-free EIS approach was further improved by using additional charged molecules such as biotin-avidin24 and nanoparticles23 to amplify changes in Ret that

signals the hybridization event Bardea et al amplified the DNA sensing process by

the biotin–avidin complex at the sensing interface.24 A gold electrode was immobilized with capture probes for a specific binding with the target Tay–Sachs (TS)13 oligonucleotide mutant The Ret for the bare gold electrode was ca 60 Ω and it

increased to ca 80 Ω and ca 105 Ω upon immobilization of the surface with capture

hybridization signal was further amplified by binding the resulting duplexes with the

biotinylated oligonucleotides and avidin which increased the Ret further to ca 134 Ω and 340 Ω, respectively This method could achieve a detection limit of 3.5 nM

Xu et al demonstrated an impedimetric detection of target DNA based on the

use of cadmium sulfide nanoparticles. 23 In this approach, the amine-modified ss-DNA capture probes were covalently immobilized onto a self assembled monolayer (SAM)-modified gold electrode After hybridization with target DNA, a double helix film formed on the electrode, EIS was used to detect the change of interfacial Ret of the redox marker, [Fe(CN)6]4-/3-, from solution to transducer surface It was demonstrated that without labeling the target DNA, the EIS method achieved only a detection limit

of 0.14 nM of target However, a remarkably increased Ret value was detected upon hybridization with target DNA tagged with cadmium sulfide nanoparticles, which yielded a detection limit of 4.5 pM.23 The increased Ret was attributed to the more negative charges, space resistance and the semiconductor property of cadmium sulfide nanoparticles

1.1.2.2 Optical

Surface plasmon resonance (SPR) spectroscopy is an optical technique that detects changes in the refractive index of thin films assembled on a noble-metal surface The measured signals are proportional to the molecular weight of the adsorbed materials, and thus provide a means to quantify the amount of adsorbed molecules It has been used to quantitatively detect the DNA molecules, by measuring the changes in the refractive index of a gold thin film upon hybridization of complementary targets DNA with the immobilized oligonucleotide probes For the hybridization adsorption of

DNA targets, the detection limits of 9.2 nM and 10 nM were reported.27, 28 It was notably that the use of labels in conjunction with SPR measurement could offer a greater sensitivity The detection limit of 10 pM or 1.38 fM was demonstrated when aided by signal amplification through gold nanoparticles labels.29, 30

1.1.2.3 Piezoelectric

Piezoelectric devices such as quartz crystal microbalances (QCMs) monitor mass changes to the immobilized recognition layer on the sensing surfaces A QCM measures a mass per unit area by monitoring the change in frequency of a quartz crystal resonator This frequency change is proportional to the change in mass The

use of QCM transducer offers an in situ monitoring and detection of DNA

hybridization events, without the need for labels.31, 32

Livache et al reported the monitoring of DNA hybridization using a QCM

The recognition layer on quartz surface was prepared by electro-controlled copolymerization of a pyrrole-modified oligonucleotide probe and pyrrole The hybridization of DNA with the immobilized probes resulted in a mass change which was detected by the piezoelectric transducer The approach demonstrated a detection limit of 250 nM for a short oligonucleotide target.31

Wang et al described a QCM hybridization biosensor, based on derivatized PNA capturing probes The QCM enabled in situ discrimination of a

thiol-single-base mismatch in a 15-mer DNA segment related to a common point mutation

in the p53 gene The approach showed a detection limit of 220 nM target DNA The greater specificity of the approach over those of analogous PNA-based carbon

electrodes was attributed to the formation of a PNA monolayer and the use of a hydrophilic ethylene glycol linker.32

Amplification of the mass change on the piezoelectric crystals is usually performed to improve the QCM sensitivity of gene detection Recently, gold nanoparticles have been used in QCM detection of DNA to increase the mass changes associated with the target hybridization Two layers of gold nanoparticles were linked

to the captured target DNA (18mer) with an improved detection limit of 10 fM.33

1.1.3 Label-based detection of nucleic acids

Nucleic acid does not have intrinsic properties sufficient for direct high-sensitivity detection Many types of nucleic acid assays developed thus require a label, which leads to the amplification of hybridization signal.34

Each of the existing labels has their attributes for the detection and quantification of analytes of interest The most commonly used labels for electrochemical nucleic acids sensing are for examples, the redox active molecules

(Section 1.1.3.1), enzymes (Section 1.1.3.2) and nanoparticles (Section 1.1.3.3)

1.1.3.1 Redox-active molecules

These labels are electrochemically active in which the redox current associated with the hybridization of target strands are detected by electrochemical methods The redox label should posses a well-defined, low-potential, voltammetric response Electrochemical techniques such as voltammetry35-38 or constant-current chronopotentiometry39 can be used to detect the presence of the redox indicator

associated with the target nucleic acids Table 1.2 lists the commonly used redox

labels in electrochemical nucleic acid assays

Table 1.2 Redox-active labels used in electrochemical nucleic acid assays

PGE = pyrolytic graphite electrode; HOPGE = highly oriented pyrolytic graphite electrode; SPCE = Screen printed carbon electrode; GMA = Gold microelectrode array; Os, bipy = osmium tetroxide with 2,2 -bipyridine; FND = Ferrocenyl naphthalene diimide

Direct labelling of oligonucleotides with electroactive labels, such as ferrocene,35 osmium tetroxide with 2,2 -bipyridine (Os, bipy)36 or anthraquinone40tags were reported These labels were usually conjugated to the target strand36,40(Figure 1.3A) or a detection probe which was then hybridized to the target in sandwich hybridization35 (Figure 1.3B) Yu et al employed the eSensorTM DNA

Ferrocene Alternating current

voltammetry

vs Ag/AgCl

Not reported [35]

Os, bipy Adsorptive stripping

voltammetry

PGE and HOPGE

-0.65 V

vs Ag/AgCl

4.5 fmol (~ 0.64 nM)[36] Daunomycin Chronopotentiometry SPCE +0.40 V

vs Ag/AgCl

0.2 mg L-1 (~ 31 nM) [39] Methylene blue Chronocoulometry GMA -0.35 V

voltammetry

vs Ag/AgCl

10-13 g mL-1 (~66 aM) [38]

Co(bpy)32+/3+ Cyclic voltammetry Carbon

paste

+0.22V

vs Ag/AgCl

10-8 g mL-1 [43]

clinical samples.35 This detection platform comprised a gold electrode spotted with capture probes for capturing the complementary HPV DNA The duplexes were then

labelled via the hybridization with a ferrocene-modified detection probe Fojta et al

labelled the target DNA sequences with a complex of Os, bipy.36 The labelled target was captured to magnetic beads and then detected by adsorptive transfer stripping voltammetry at a carbon electrode Voltammetric detection of the hybridization of target DNA labelled with electroactive Os, bipy allowed for the detection of 10-10 M

of both short (71-mer oligonucleotide) and long (3-kb plasmid DNA) target DNAs.36

Figure 1.3 Labeling of nucleic acids with electroactive labels (A) Label is directly

conjugated to the target strand, or (B) conjugated to a detection probe which can then hybridize to a specific portion of the target sequence during a sandwich hybridization assay; (C) the duplex-specific indicators are intercalated between the base pairs of a capture probe-target duplex

(A)

(B)

Labelled target

Unlabelled target

Labelled detection probe

One of the most common electrochemical strategies for detecting the hybridization of unlabeled DNA depends on the redox-active hybridization indicators that bind more strongly to DNA duplexes than to single-stranded DNA, i.e duplex-specific indicators When hybridization occurs, the duplex-specific indicators bind to the resulting capture probe-target duplex and the local concentration of the indicator

at the electrode surface is greatly increased (Figure 1.3C) An increase of current is produced due to the accumulation of redox indicators The duplex-specific indicators include the intercalators such as methylene blue (MB),41 daunomycin,39,42 ferrocenylnapthalene diimide (FND)37 which intercalate between the Watson-Crick base pairs of a DNA duplex, groove binders38 and cationic metal complexes.43

Boon et al developed a DNA assay based on the electrocatalytic reduction of

ferricyanide by MB.41 The MB intercalated into the duplex DNA upon hybridization

of target to the capture probe-modified electrode Electrons flow from the electrode surface to the intercalated MB in a DNA-mediated reaction In the case of a DNA film containing a mismatch, the bound MB is not catalytically active and the electrochemical signal is greatly attenuated Using this assay, the possible single-base mismatches have been readily detected The reaction has also been carried out on microelectrode array containing 18 separately addressable gold electrodes The hybridization reaction on the electrode surfaces was investigated by chronocoulometry at -0.35 V in the presence of MB and [Fe(CN)6]3-/4- At a 30-μm electrode, as few as ca 108 duplexes (corresponding to 10-16 mol of target DNA), were detected A detection limit of fM (corresponding to 10-20 mol of DNA in a

sample) was obtained by Takenaka et al using a threading intercalator FND.37 This threading intercalator binds to the DNA duplex more tightly than usual intercalators

probe was chemisorbed onto gold electrodes through a thiol anchor Targets were detected based on DPV monitoring of the hybridization event

Hashimoto et al demonstrated a gene detection method using a gold electrode

modified with DNA probes and Hoechst 33258 as a hybridization indicator.38 The

DNA probe was immobilized on the gold electrode at a site of the 5’ end through a

mercaptohexyl group Hoechst 33258 is a DNA minor groove binder and it selectively binds to the dsDNAs, thereby is pre-concentrated on the electrode surface upon hybridization event This method allowed for the detection of 10-13 g mL-1 of target DNA (corresponding to 4 × l04

copies mL-1 or 66 aM).38

Mikkelsen et al detected DNA on carbon electrodes by immobilizing the

DNA onto the electrode The cationic metal complex, Co(bpy)32+/3+ was used as an indicator.43 An enhanced faradaic current response of Co(bpy)32+/3+ was measured in the presence of the surface attached target DNA The detection limit of this system

was estimated to be 250 pg of a 400-bp polymerase chain reaction (PCR) product

1.1.3.2 Enzymes

Similarly, enzyme labels have been conjugated directly to the target DNA (Figure 1.3A) or detection probes (Figure 1.3B) Alternatively, the hybridization can be performed on a different surface, such as magnetic beads,7-9 membranes,44 and silicon-based microchip surfaces,31 followed by measurement using an external transducer

Enzyme labels can generate colorimetric, fluorescent, chemiluminescent or electrochemical signals given the appropriate substrates for the enzyme reaction Bioluminescence and chemiluminescence enables detection of a few molecules and

achieves detection limits down to 10-18 to 10-21 moles,45 which are generally 100 to

1000 times more sensitive than common spectroscopy or colorimetric method.46 The luminescent-based enzyme labels included acetate kinase coupled with bioluminescent assay and the bioluminescent luciferase class of enzymes.34

Table 1.3 lists the common enzymes and substrates used in electrochemical nucleic acid assays The presence and amount of enzymes are monitored via voltammetric, amperometric or impedimetric signal of enzymatic product Among the enzyme labels, horseradish peroxidise (HRP) and alkaline phosphatase (ALP) are two

of the most commonly used enzyme labels for affinity biosensors

Table 1.3 Enzyme labels used in electrochemical nucleic acid assays

Enzymes Transduction modes Substrates Detection limits

Chronopotentiometry α-naphthyl

phosphate

7.1 fmol in 50 μL (~0.14 nM) [50]

Zhang et al reported a HRP-amplified amperometric detection of DNA

hybridization.48 A detection limit of 3000 copies in a 10 µL droplet at a concentration

of 0.5 fM was demonstrated Azek et al described a disposable electrochemical

biosensor for the detection of target DNA sequences related to the human cytomegalovirus (HCMV).49 The target DNA strands were adsorbed onto the sensing surface of a screen printed carbon electrode (SPCE), followed by its hybridization to a complementary single-stranded biotinylated DNA probe The extent of hybrids formed was determined with streptavidin conjugated to HRP The HRP label was quantified by DPV technique measuring the amount of the electroactive enzymatic

product 2,2’-diaminoazobenzene generated from the o-phenylenediamine

dihyrochloride (OPD) substrate The intensity of DPV peak currents resulting from the reduction of the enzyme-generated product was related to the number of target DNA molecules present in the sample A detection limit of 0.6 fM of target DNA fragment was obtained The resulting electrochemical method was 23,000-fold more sensitive than the gel electrophoresis technique and 83-fold more sensitive than the colorimetric hybridization assay in a microtiter plate Other common substrates and signal transduction modes used for electrochemical HRP–labelled bioassays are also discussed in Section 3.1

The ALP enzyme has been widely employed in electrochemical DNA

biosensors based on various signal transduction modes Wang et al described a

protocol that combined an enzyme-linked sandwich hybridization, with a particle-labelled probe hybridizing to a biotinylated DNA target that captured a streptavidin-ALP. 7 Using the α-naphthyl phosphate as a substrate, the product of the enzymatic reaction (i.e α-naphthol) was quantified through a low-potential (+0.10 V

magnetic-vs Ag/AgCl) DPV peak current at the disposable screen printed electrode.7 A