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 i 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 ii 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 LITERATURE REVIEW .1 1.1 Hybridization and Detection of Nucleic Acids 1.1.1 Basic of nucleic acid hybridization .1 1.1.2 Label-free detection of nucleic acids 1.1.2.1 Electrochemical 1.1.2.2 Optical 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 iii CHAPTER 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 (SAbeads) .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 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 iv CHAPTER 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 ELECTROCHEMICAL DETECTION OF LEUKEMIA ONCOGENES AT ATTOMOLES LEVELS WITH A CARBON NANOTUBESBASED 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 CNTbased labels 167 v 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 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 CONCLUDING REMARKS AND FUTURE WORKS 200 vi 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 BCRABL 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 vii 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 10-12 M (60 10-18 mol in 60 L) 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 oaminophenol 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 = %; 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 ì 10-18 mol in 60 àL) targets oligonuecleotides and a 4-order wide dynamic range of target concentration The resulting assay allowed a robust discrimination between the viii perfect match and a three-base mismatch sequence When subjected to full-length oncogene (491 bp), the approach demonstrated a detection limit of 10-16 mol in 60 L 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 10-15 M (1 10-19 mol in 100 L) 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 ix (A) (B) b b a a 2E-07 (C) (D) 0E-07 2.0E-07 6E-07 8.0E-08 Current (A) Current (A) b 6.0E-08 4.0E-08 y = 2E-05x R = 0.9935 2E-07 8.0E-08 4.0E-08 2.0E-08 a 0.0E+00 0.0E+00 Substrate Concentration (m M) 0.002 0.004 0.006 0.008 ALP concentration (U/m L) 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 in which curves a = 2.5 mM substrate and curves b = 0.025 U mL-1 ALP in 2.5 mM substrate; scanning conditions for CV: potential scanning = 0.1 V s-1; SWV: step potential = 0.004 V, amplitude = 0.025 V and frequency = 25 Hz (C) Optimization of substrate concentration ranged from 0.01 to mM Curves a and b represent substrate solution in the absence and presence of 0.1 U mL-1 ALP, respectively (D) The linearplot of SWV detection of ALP All enzymatic reactions were allowed to preceed for 10 minutes A detection limit of 0.00017 U mL-1 (corresponds to 87 fM or 4.8 in 55 10-15 mol of ALP L of total assay solution) was estimated based on the basis of SD of determination of the zero standards, i.e blank substrate solution in connection with the 10-minutes enzymatic reaction 189 0.01 6.3.3 bDNA hybridization assay of p185-ssDNA standards In recent years, the bDNA has been explored as an alternative to the RQ-PCR for the quantitative analysis of genetic materials In both cases, it is critical to choose the right control gene and to prepare a standard for reliable quantitative or semiquantitative analysis of the target genes The p185-ssDNA derived from the target mRNA template was used as standards to evaluate the performance of the electrochemical bDNA assay for p185 BCR-ABL gene Figure 6.3A displays the typical electrochemical response with increasing concentrations of standard Well defined voltammetric peaks are observed with low concentration of standards The peak currents increased with the increase of standard concentrations A negligible signal was observed in the negative control sample (in the absence of p185-ssDNA) The resulting calibration plot of the currents versus standard concentrations (Figure 6.3B) is linear over the range of 2.2 × 105 to 1.6 × 108 copies of targets and is suitable for quantitative analysis The log linear range is comparable to the performance achieved by luminescence detection (> 3.5 log as indicated by the manufacturer) A detection limit of 6.1 104 copies was achieved based on the basis of SD of determination of the zero standards This detection limit corresponds to fM (1 10-19 mol of target nucleic acids in the 100- L sample), or approximately 17 fg, which is comparable to that reported earlier for detecting the breast cancer susceptibility gene (tumor protein p53, 1182 bp) with a detection limit of 0.5 fM.20 190 (A) 1.0E-06 (B) 8.0E-07 Current (A) 6.0E-07 4.0E-07 2.0E-07 0.0E+00 0.0E+00 4.0E+07 8.0E+07 1.2E+08 1.6E+08 2.0E+08 Standard (copies) 1.6E-07 (C) y = 8E-15x + 3E-10 R2 = 0.9981 Current (A) 1.2E-07 8.0E-08 4.0E-08 0.0E+00 0.0E+00 5.0E+06 1.0E+07 1.5E+07 2.0E+07 Standard (copies) Figure 6.3 (A) Typical SWV responses with increasing standard concentrations From bottom to top: 0, 2.2 × 105, 6.7 × 105, 2.0 × 106, 6.0 × 106, 1.8 × 107, 5.4 × 107, 1.6 × 108 copies The inset shows the enlarged voltammograms of – 6.7 × 105 copies (B) The resulting calibration plot with the lower left portion being enlarged as in (C) SWV measurements were performed with mM substrate and 30 minutes enzymatic reaction Other conditions of SWV analysis and bDNA hybridization assay were described in the experimental Section 6.2.2 and 6.2.5 Error bars are based on standard deviation with n = 191 To our best knowledge, this is the lowest reported amount of full length genetic targets detected by electrochemical bDNA assay The detection limit of bDNA assay could vary dependent on the length of target nucleic acids A longer target of nucleic acid enables more bindng of bDNA amplifiers through the LEs This implies that the selection and preparation of ssDNA standard of similar or identical length and sequence is crucial for accurate quantitative bDNA assay The high sensitivity and specificity exhibited by the proposed assay were coupled with good reproducibility A series of six measurements of standard with 106 copies of standard ssDNA yielded reproducible signals with a RSD of 6.9 % 6.3.4 Detection of p185 BCR-ABL mRNA fusion transcript extracted from cell line Figure 6.4A shows the PCR-free electrochemical detection of mRNA targets and the excellent specificity of the proposed assay In this study, mRNA targets were extracted from the SUP-B15 cell line whereas those obtained from the HL-60 cell line were used as a negative control The current responses increased unambiguously with increasing concentration of the mRNA target (from 4.6 to 53.8 ng) The resulting calibration (Figure 6.4B) indicates that the assay responds linearly with the amount of mRNA target In contrast, we observed the null response to the negative control mRNA samples in spite of the increasing amount added to the assay Similarly, a null response was observed in the blank control (0 ng of mRNA) Such performance is attributed to the effective blocking by the blocking reagents and BLs Furthermore, the cooperative hybridization of both CEs and LEs ensured the specific recognition to the fusion transcripts containing both p185 BCL-ABL sequences, which in turn eliminated the binding of any other sequences A detection limit of ca ng of total 192 mRNA population was achieved based on the basis of SD of determination of the zero standards A comparative study was carried out to evaluate the performance of electrochemical assay as compared to that of conventional chemiluminescent detection Figure 6.5 presents the chemiluminescent responses of the mRNA target performed under the identical conditions The assay achieved a detection limit of ca 2.4 ng of total mRNA population, which is in the same order of magnitude achieved by our electrochemical analysis Our assay unambiguously detects the target gene from nanograms amount of mRNA samples, which is an amount of at least 200-fold less than that required for Northern blot analysis (0.5-20 g of mRNA).21 Such an amount is also comparable to 5-50 ng of mRNA required for a two-step RT-PCR analysis by using Superscript First-Strand Synthesis kit (Invirogen’s recommendation: 50-500 ng of mRNA for RT reaction followed by the PCR using 10 % of the resulting RT reaction product).22 The current assay could be applied for gene expression analysis by incorporating the house-keeping gene for normalizations In gene expression studies, it has been a challenge to discriminate the gene expression that differs less than 2-fold Our approach overcomes such limitation, which was evidenced by the well-resolved current responses between 2-fold differences in the mRNA target (Figure 6.4B) 193 6.0E-08 (A) leukemia cells 5.0E-08 normal cells Current (A) 4.0E-08 3.0E-08 2.0E-08 0E-08 0.0E+00 substrate ng 4.6ng 9.2ng 6.6ng 29.9ng 53.8ng Samples 6.0E-08 (B) leukemia cells 5.0E-08 normal cells Current (A) 4.0E-08 3.0E-08 2.0E-08 0E-08 0.0E+00 20 30 40 50 60 mRNA (ng) 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 Conditions as for Figure 6.3 The data point with ng represents the control sample Error bars are based on standard deviation with n = 194 2500 2000 RLU 500 000 500 substrate ng 0.8ng 2.4ng 7.2ng Samples Figure 6.5 Chemiluminescent measurement of the p185 BCR-ABL mRNA fusion transcript The data point with ng represents the control sample The luminescent detection was performed using the Lumingen® APS-5 substrate by following the manufacturer’s instruction in the bDNA assay kit Error bars are based on standard deviation with n = 6.3.5 Quantitative mRNA assay: electrochemical bDNA assay vs fluorescent RQ-PCR An mRNA population may contain thousands genes A specific gene may comprise only 0.01 to % of the entire mRNA population.23 In this study, the actual copies of the target gene in the mRNA population were determined by using an electrochemical bDNA assay in comparison to that of a conventional fluorescent RQ-PCR Most of the genetic analyses reported to date are semi-quantitative in which the gene expression is quantified in terms of the fold of change relative to that of a housekeeping gene Unlike the semi-quantitative method, the absolute quantification assay depends on knowing the absolute quantities of the nucleic acid standards Collins et al proposed a method to generate a trustworthy standard for bDNA assay.24 We demonstrated herein a standard curve method for absolute quantification of the target gene in the mRNA population The p185-ssDNA bearing an identical sequence of the target gene was prepared from the biotinylated double-strand PCR product and used as a standard in the electrochemically bDNA assay This p185-ssDNA standard 195 was quantified by spectrophotometry and RQ-PCR Both techniques provided similar quantities in which absorbance value was ca 37 % higher than that of the RQ-PCR The value obtained by RQ-PCR was used in the subsequent tests 1.2E-07 Current (A) 1.0E-07 8.0E-08 6.0E-08 4.0E-08 2.0E-08 0.0E+00 0.0E+00 2.0E+06 4.0E+06 6.0E+06 8.0E+06 1.0E+07 Copies number 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 p185ssDNA standards (blue) Conditions as for Figure 6.3 Error bars are based on standard deviation with n = Figure 6.6 shows the electrochemical bDNA assay for the absolute quantitative analysis of the p185 BCR-ABL mRNA The mRNA samples were diluted in nuclease-free water to three different concentrations with each run in triplicate The corresponding average current responses were correlated to those of p185-ssDNA standards Our assay determined that the mRNA sample contained an average of 62900 copies of p185 BCR-ABL per ng of mRNA Considering the fact that the expression level of a specific gene could vary between batches of culture, the same batch of mRNA was reversed transcribed to cDNA followed by the RQ-PCR which yielded an average of 1240 copies per ng of mRNA template The amount of target gene contained in the mRNA sample determined by our proposed assay was at least 196 50-fold higher than that of the RQ-PCR analysis This result is consistent with the finding reported earlier16, 18 which suggests that the RQ-PCR method might underestimate the actual amount of target gene due to the drawbacks inherent in the RT reaction (i.e., incomplete RT from mRNA to cDNA) and bias introduced during the thermal cycles The underestimation of target contents in patient samples could lead to false negative diagnosis and delays in the treatment The current assay demonstrated a reliable alternative to the RQ-PCR method for clinical diagnosis 6.4 Conclusions We have demonstrated a novel electrochemical bDNA assay for simple, sensitive, accurate and quantitative detection of genetic marker in the mRNA population without RT and PCR amplification The use of bDNA (QuantiGene 2.0) for signal amplification showed a 103–fold improvement in assay sensitivity, which enables the detection of full-length p185 BCR-ABL transcripts at femtomolar levels from as little as nanograms of total mRNA population The electrochemical analysis is consistently comparable to the chemiluminescent detection and can be readily integrated for the development of POC systems The electrochemical responses were unambiguously resolved for less than 2-fold difference in gene expression level In combination of the use of a well-quantified standard, the proposed assay indicated that RQ-PCR could underestimate the target gene by at least 50-fold Our approach has addressed the shortcomings of the RQ-PCR analysis by the reduction of the assay complexity and minimization of assay bias caused by non-ideal RT efficiency The developed assay therefore provides a new approach for disease diagnosis 197 6.5 References Zhou L, Otulakowski G, Lau CY Use of quantitative polymerase chain reaction to study cellular retinoic acid-binding protein-II mRNA expression in human skin Meth Enzymol 1997;282:64-76 Capaldi S, Getts RC, Jayasena SD Signal amplification through nucleotide extension and excision on a dendritic DNA platform Nucleic Acids Res 2000;28:e21 Collins ML, Irvine B, Tyner D, Fine E, Zayati C, Chang CA, Horn T, Ahle D, Detmer J, Shen LP, Kolberg J, Bushnell S, Urdea MS, Ho DD A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml Nucleic Acids Res 1997;25:2979-2984 Wang J, Polsky R, Merkoci A, Turner KL "Electroactive beads" for ultrasensitive DNA detection Langmuir 2003;19:989-991 Liu G, Wang J, Wu H, Lin Y Versatile apoferritin nanoparticle labels for assay of protein Anal Chem 2006;78:7417-7423 Lee AC, Ye JS, Tan SN, Poenar DP, Sheu FS, Heng CK, Lim TM Carbon nanotube-based labels for highly sensitive colorimetric and aggregation-based visual detection of nucleic acids Nanotechnology 2007;18:455102 Wang J, Liu G, Rasul Jan M Ultrasensitive electrical biosensing of proteins and DNA: Carbon-nanotube derived amplification of the recognition and transduction events J Am Chem Soc 2004;126:3010-3011 Munge B, Liu G, Collins G, Wang J Multiple enzyme layers on carbon nanotubes for electrochemical detection down to 80 DNA copies Anal Chem 2005;77:4662-4666 Patolsky F, Lichtenstein A, Kotler M, Willner I Electronic transduction of polymerase or reverse transcriptase induced replication processes on surfaces: Highly sensitive and specific detection of viral genomes Angew Chem Int Ed 2001;40:2261-2265 10 Patolsky F, Lichtenstein A, Willner I Highly sensitive amplified electronic detection of DNA by biocatalyzed precipitation of an insoluble product onto electrodes Chem Eur J 2003;9:1137-1145 11 Nilsen TW, Grayzel J, Prensky W Dendritic nucleic acid structures J Theor Biol 1997;187:273-284 12 Demidov VV DNA diagnostics in the fifty-year retrospect Expert Rev Mol Diagn 2003;3:121-124 198 13 Detmer J, Lagier R, Flynn J, Zayati C, Kolberg J, Collins M, Urdea M, Sánchez-Pescador R Accurate quantification of Hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology J Clin Microbiol 1996;34:901-907 14 Hendricks DA, Stowe BJ, Hoo BS, Kolberg J, Irvine BD, Neuwald PD, Urdea MS, Perrillo RP Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay Am J Clin Pathol 1995;104:537546 15 Zaaijer HL, ter Borg F, Cuypers HT, Hermus MC, Lelie PN Comparison of methods for detection of Hepatitis B virus DNA J Clin Microbiol 1994;32:2088-2091 16 Grimes RM, Lewis ST, Visnegarwala F, Goodly J, Sutton R, RodriguezBarradas M Use of bDNA testing in the immunologically nonresponding patient who has a low or undetectable viral load by RT-PCR testing HIV Clin Trials 2003;4:92-98 17 Pasquier C, Sandres K, Salama G, Puel J, Izopet J Using RT-PCR and bDNA assays to measure non-clade B HIV-1 subtype RNA J Virol Meth 1999;81:123-129 18 Nolte FS, Boysza J, Thurmond C, Clark WS, Lennox JL Clinical comparison of an enhanced-sensitivity branched-DNA assay and reverse transcriptionPCR for quantitation of human immunodeficiency virus type RNA in plasma J Clin Microbiol 1998;36:716-720 19 Wu SJ, Spink DC, Spink BC, Kaminsky LS Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection Anal Biochem 2003;312:162-166 20 Xie H, Yu YH, Xie F, Lao YZ, Gao Z A nucleic acid biosensor for gene expression analysis in nanograms of mRNA Anal Chem 2004;76:4023-4029 21 Zhang N, Harrex AL, Holland BR, Fenton LE, Cannon R, Schmid H Six alleles of the ALS7 open reading frame in Candida albicans:ALS7 is a hypermutable contingency locus Genome Res 2003;13:2005-2017 22 In SuperScript first-strand synthesis system for RT-PCR instruction manual, Version D, Califormia: Invitrogen, 2003:27 23 In Oligotex® handbook, 2nd ed California: Qiagen, 2002:94 24 Collins ML, Zayati C, Detmer JJ, Daly B, Kolberg JA, Cha TA, Irvine BD, Tucker J, Urdea MS Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays Anal Biochem 1995;226:120-129 199 CHAPTER CONCLUDING REMARKS AND FUTURE WORKS The current research has successfully developed ultrasensitive nucleic acids assays involving a novel CNT-derived label or a bDNA amplifier for the dramatic amplification of nucleic acids hybridization signal Both signal amplified assays are capable of PCR-free detection and quantification of a human acute lymphocytic leukaemia (ALL) oncogene, namely, p185 BCR-ABL fusion transcript in samples of mRNA population A pure and well-quantified ssDNA standard bearing the identical sequence and size of the p185 BCR-ABL target gene was also created for use in PCRfree quantitative assays Significantly, a novel multiple HRP enzymes conjugated CNT-based label was developed which was capable of enhancing the nucleic acids assay sensitivity by at least 1000 times compared to that of conventional labels used in enzyme-linked oligosorbent assay (ELOSA) The label was used in the detection of p185 BCR-ABL fusion transcript target by measuring the signal of target-associated HRP enzymes using optical (i.e colorimetric) and electrochemical method The colorimetric nucleic acid assay amplified by CNT-based labels demonstrated an excellent detection limit of oligonucleotide target in 60 10-12 M (60 10-18 mol of L) and a 4-order wide dynamic range of target concentration Interestingly, hybridizations using the CNT-based labels could lead to a concentration-dependent formation of visible dark aggregates, which in turn allows for the visual detection of target DNA Further studies will be needed to determine the appropriate ratio of CNT labels to target-bound magnetic beads, bead concentration as well as the corresponding detection probes and capture probes densities required for controllable target-specific aggregation 200 The electrochemical detection of target oncogene amplified by CNT-based labels was demonstrated based on the monitoring of target-associated HRP activities using SWV analysis at a disposable screen-printed carbon electrode The analysis measured the electroactive 3-APZ enzymatic product in the presence of o-AP and H2O2 substrate solution The enzymatic product of o-AP exhibited a well-separated peak compared to the blank substrate This characteristic offers a better specificity for the HRP assay compared to other electrochemical substrates However, it was found that in the absence of HRP enzymes, the o-AP substrate tends to exhibit a significant level of background signal formed by its electro-oxidation at a potential greater than V (vs Ag/AgCl) Subsequent efforts on the optimization of o-AP substrate concentration and the initial scan potential of SWV analysis minimized the background response which in turn significantly improved the HRP assay sensitivity (detection limit = 1.25 ì 10-15 mol of HRP in 25 àL) The resulting HRP assay was coupled to the nucleic acids assay amplified by the CNT-based labels The signal-amplified assay achieved a detection limit of 83 × 10-15 M (5 × 10-18 mol of oligonucleotide target in 60 µL) and a 4-order wide dynamic range of target concentration Such limit was 12-fold higher than that of colorimetric assay The resulting assay also allowed for a robust discrimination between the perfect match targets from a 3-base mismatch sequence and the non-complementary sequences present as pure oligonucleotides or mRNA population extracted from negative ALL cell lines HL-60 When subjected to full-length oncogene (491 bp), the approach demonstrated a detection limit of 1.67 × 10-12 M (1 10-16 mol in 60 L) which corresponded to approximately 33 pg of the target gene Without relying on PCR target amplification, the CNT labels demonstrated for the first time, a direct detection of p185 BCR-ABL mRNA fusion transcript in as little as 65 ng of mRNA 201 population extracted from positive ALL cell lines SUP-B15, in comparison to those obtained from negative cell lines HL-60 In the effort to minimize non-specific background signal of nucleic acids assay, the composition of CNT-based labels was also studied and optimized The HRP loading on a CNT carrier showed a profound influence on the detection S/N A higher HRP loading beyond the optimum will compromise the S/N of nucleic acids detection due to an increase of background level and decrease of target response In addition, a novel bDNA-amplified electrochemical nucleic acids assay was demonstrated for the first time In cooperation with Panomic Inc., a bDNA amplifier carrying high loading of ALP enzymes and probes designed specifically to hybridize with the p185 BCR-ABL fusion transcript was prepared and employed for the PCRfree target detection in mRNA samples In contrast to the conventional chemiluminescent measurement, the activity of target-associated ALP enzymes was monitored by SWV analysis measuring the electroactive enzymatic product in the presence of 1-napthyl-phosphate A detection limit 10-15 M (1 10-19 mol of full- length oncogene in 100 L) and a 3-order wide dynamic range of concentration were achieved Such limit corresponded to approximately 17 fg of the p185 BCR-ABL The electrochemical analysis is consistently comparable to the chemiluminescent detection and can be readily integrated for the development of point-of-care (POC) systems The assay also enabled direct detection of target transcript in as little as 4.6 ng of mRNA without PCR amplification In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a quantitative analysis of target transcript in total mRNA population The proposed quantitative assay also indicated that RQ-PCR could underestimate the target gene by at least 50fold, which is in agreement with earlier studies The approach used in this study has 202 addressed the shortcomings of the RQ-PCR analysis by the reduction of the assay complexity and minimization of assay bias caused by non-ideal RT efficiency The newly developed assay provides a new approach for disease diagnosis Overall, the proposed signal amplified nucleic acids assays hold promise for simple, low cost and ultrasensitive electrochemical nucleic acids detection in portable devices, POC and early disease diagnostic applications In combination with the use of a well-quantified standard, these approaches could provide a simple and sensitive quantitative tool alternative to the RQ-PCR Gene expression analysis could also be realized by incorporating the house-keeping gene as an internal standard The enzymes-tagged CNT and bDNA labels can also be readily detected by various methods such as electrochemical, fluorescent and luminescent modes Such extension could pave the way for ultrasensitive in situ hybridization and imaging of specific sequences in the cells 203 ... 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. .. quantitative tool alternate to the RQPCR 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... 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