Electrochemical DNA sensor for detection of environmental pathogens

ELECTROCHEMICAL DNA SENSOR FOR DETECTION OF ENVIRONMENTAL PATHOGENS VARUN RAI (M.Sc Chemistry Indian Institute of Technology Bombay) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2013 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of Asst Prof Toh Chee Seng Chemistry Department, National University of Singapore, between 07-01-2009 to 30-07-2010 and Asst Prof Toh Chee Seng Division of Chemistry and Biological Chemistry, Nanyang Technological University between 01-08-2010 to till now I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously The content of the thesis has been partly published in: (1) PLoS ONE, 2012 7(8): p e42346 (2) Talanta, 2012 98 : 112-117 (3) Biosensors and Bioelectronics, 2012.32(1): p 133-140 (4) Nanosciene and Nanotechnology letters, 2013 (5): 613-623 Name Signature i Date Acknowledgements I heartily express my deep sense of thanks to my supervisor, Assistant Professor Toh Chee-Seng for his throughout guidance, motivation and continual support It has been my privilege to get an opportunity to work under him, who has been very inspiring and modest to me in all scientific discussions and pursuing research as career I also thanks to Prof Li Fong Yau Sam for being my co-supervisor I gratefully thanks to Dr Ng Lee Ching, Dr Chanditha and her group, Environmental Health Institute, National Environment Agency, for providing real life samples of Legionella sp for my electrochemical sensing experiments I also thanks to Dr Yee Sin Leo and Ms Siew Hwa SOH, Communicable Disease Centre, Tan Tock Seng Hospital, Singapore for providing DENV infected serum sample for validation of electrochemical sensor in challenging with real life sample I gratefully acknowledge MOE- NUS research scholarship for my graduate studies in Singapore I would also like to thanks to Ms Tang Chui Ngoh and Mr Lee Ka Yau, Department of Chemistry, National University of Singapore, for their assistance in SEM studies I express my thanks & obligation to Professor Hiroaki Ogawa Iyehara, Assistant Professor Toshinari Maeda, Saiki and their group members, Graduate School of Life Science and System Engineering, Kyushu Institute of Technology, for supporting me in doing my JENESYS project I would like to thank my all co-workers in my lab Dr Nguyen Thi Thanh Binh, Ms Yin Thu Nyine, Ms Wong Lai Peng, Deng Jia Jia, Cheng Ming Soon, Yan Yan, Peh En Kai Alister, Dr Kamalakanta Behera for their day to day help and support and other many students who visited our lab for research project in my lab Fatima, Saurav K Guin, Chumpu and many more I sincerely thanks to all my friends and hostel mates who remained helping throughout my academic to ii personal life Lastly I would like to express my deepest gratitude and love to my parents, bade papa (uncle), amma (aunt), brothers, sisters and my all caring family members for their support throughout my study iii Table of contents Chapter Introduction and Literature review 1.1 Biosensors .1 1.1.1 Resonant biosensors 1.1.2 Optical detection biosensors 1.1.3 Thermal detection biosensors 1.1.4 Piezoelectric transduction biosensors 1.1.5 Electrochemical biosensors 1.1.5.1 Amperometric biosensors .8 1.1.5.2 Potentiometric biosensors .10 1.1.5.3 Impedimetric biosensors .11 1.1.5.4 Capacitance biosensors 12 1.1.5.5 Conductimetric biosensors 13 1.2 Electrochemical DNA Sensors 14 1.2.1 Electrochemical amplification strategies 16 1.2.2 Intercalation reagents .21 1.2.3 Primary reporter probe using electrochemical molecular beacon 23 1.2.4 Secondary reporter probe 24 1.2.5 Amplification methods using nanopores and nanochannels 26 1.3 Scope of study .30 References 31 Chapter Electrochemical Techniques 2.1 Cyclic voltammetry CV 41 2.2 Differential pulse voltammetry DPV 49 2.3 Electrochemical Impedance Spectroscopy EIS 54 2.4 SEM 59 References 64 Chapter Molecular beacon biosensor based on electrochemical signal amplification 3.1 Introduction 66 iv 3.1.1 Electrochemical signal amplification and electrochemical signal amplification based DNA biosensor .69 3.2 Materials and Methods 73 3.2.1 Reagents 73 3.2.2 Construction of EAMB biosensor 74 3.2.3 Design of hairpin DNA-ferrocene probe 76 3.2.4 Procedure for analyses of DNA targets 78 3.2.5 Electrode surface characterization by Atomic Force Microscopy…………….80 3.2.6 Transmission Electron Microscopy (TEM) image of DNA on Cu grid…… 81 3.3 Results and discussion 82 3.3.1 Electrochemical signal amplification using gold electrode towards charged redox species ferrocene (Fc) and ferrocyanide Fe (CN)64- for selective faradic response 82 3.3.2 The electrochemically amplified molecular beacon biosensor (EAMB) 84 3.3.3 Biosensor signals derived from differential pulse voltammetry 88 3.3.4 Simple model of voltammetric potential shift during complementary DNA binding of EAMB biosensor .92 3.3.5 Biosensing mechanism .94 3.3.6 Analytical performance 96 3.3.7 Specific response towards one and three base-pair mismatches 97 3.3.8 Detection of Legionella pneumophila genomic DNA .99 3.4 Conclusions .102 References .102 Chapter Electrochemical Nanoporous Alumina Membrane based Label free DNA Biosensor for Detection of Legionella Pneumophila 4.1 Introduction .110 4.2 Materials and Methods 116 2.1 Reagents 116 4.2.2 Procedure for analyses of DNA targets 117 4.2.3 Construction of Nanoporous alumina membrane-based DNA biosensor 118 4.2.4 Electrochemical Measurements .122 4.3 Results and discussion 123 v 4.3.1 Electrochemical characterization of nanoporous alumina membrane based DNA biosensor from cyclic voltammetry (CV) 123 4.3.2 Nanoporous alumina membrane based DNA biosensor signal derived from differential pulse voltammetry (DPV) 125 4.3.3 Analytical performance 129 4.3.4 Specific response towards target with single base mismatch (MM1) and triple bases mismatch (MM3) 129 4.3.5 Detection of Legionella pneumophila genomic DNA 131 4.4 Conclusions 133 References 134 Chapter Electrochemical Nanoporous Alumina Membrane-based Biosensor for Ultrasensitive cDNA detection of Dengue Virus RNA 5.1 Introduction .139 5.2 Materials and Methods 143 5.2.1 Reagents 143 5.2.2 Procedure for analyses of DNA targets 144 5.2.3 Fabrication of Nanoporous membrane-based DNA biosensor 146 5.2.4 Electrochemical Measurements .147 5.3 Results and Discussion .147 5.3.1 Biosensing mechanism and Sensing signals derived from differential pulse voltammetry (DPV) 147 5.3.2 Analytical performance 149 5.3.3 Specific response towards one base-pair mismatch of DENV3 sequence .151 5.3.4 Regeneration of biosensors with subsequent heating 152 5.3.5 Detection of PCR amplicons DNA sample derived from DENV1 genomic RNA 154 5.4 Conclusions .156 References .156 Chapter Conclusions Conclusions and future perspective 160 6.2 Comparative performances table 168 vi References .170 vii List of Tables Table 3.1 Sequences for the 157 bp target of Legionella pneumophila, two primers and the probe DNA for EAMB 101 Table 4.1 Sequences for the 157 bp target of Legionella pneumophila, two primers and the probe DNA for nanoporous alumina membrane based DNA sensor 133 Table 5.1 Sequences of the 183 bp target of DENV 1, two primers and the probe DNA……………………………………………………………………………… 145 Table 6.2 Comparative performances table 168 viii List of Figures Figure 2.1 (A) Cyclic voltammetry potential waveform (B) and Cyclic voltamm ogram of gold electrode immersed in solution of 1mM Fe(CN)64- in 1X PBS 43 Figure 2.2 Potential wave form for differential pulse voltammetry (A) descriptive view of potential waveform (B) and Differential pulse voltammogram of gold electrode immersed in solution of 1mM Fe(CN)64- in 1X PBS (C) 51 Figure 2.3 Differential pulse voltammetry measurement across working electrode (nanoporous alumina membrane based electrochemical biosensor) immersed along with platinum gauge (counter electrode) and Ag/AgCl in 1.0 M KCl electrode (reference electrode) in solution of 1mM Fe(CN)64- in 1X PBS 53 Figure 2.4 Sinusoidal current response in a linear system………………………….55 Figure 2.5 Nyquist plot with impedance vector from the R C circuit………………57 Figure 2.6 The Bode plot for the RC circuit……………………………………… 58 Figure 2.7 SEM micrograph of nanoporous alumina materials anodized on single Pt wire of 76 µm diameter (A) and EDS plot of the sample (B)…………………….…60 Figure 3.1 Schematic of (A) EAMB Biosensor preparation procedure (B) Analytical procedure of analyte DNA sample and (C) Electrochemical analytical cell set up 76 Figure 3.2 DNA Hairpin Probe sequence immobilized on the gold electrode 77 Figure 3.3 Control experiment to investigate the effect of number of heating cycles (70 oC, 25 per cycle) on the reproducibility of the ‘blank’ biosensor response in the absence of DNA targets Average error bars are derived from three consecutive DPV signals after each regeneration step 79 Figure 3.4 Schematic of electrochemical signal amplification of modified gold electrode with ferrocenehexanethiol and mercaptohexanol (MCH) in presence of sacrificial electron donor Fe(CN)64-…………………………………………………83 Figure 3.5 Cyclic voltammograms of gold electrode immobilized with ferrocenehexanethiol and mercaptohexanol (MCH) in absence (A) and in presence (B = 1mM) and (C= 10 mM) of Fe (CN)64- solution in 1X pH 7.4 PBS……………83 Figure 3.6 (A) Cyclic voltammogram of bare gold electrode ( -) and gold electrode immobilized with hairpin DNA without ferrocene (Fc) and MCH ( ) in presence of 1mM Fe(CN)64- (B) Cyclic voltammogram of gold electrode immobilized with hairpin DNA probes in the absence ( ) and presence ( -) of 1mM Fe(CN)64- ix formation of kink at the mismatched position which disrupts complementary binding of the remaining bases in the same sequence It is clear that the complementary target and sequence with one nucleotide mismatch can be easily differentiated Three nucleotide mismatches at terminal positions 1, and 21 were incorporated in target analyte to test the limit of this method for further high specificity detection Mismatch of base-pairs at terminal positions form dangling ends instead of kinks, such 3-points mismatch sequence has a much closer Tm (57 ◦C) to the complementary sequence, thus making more difficult to distinguish More interestingly, at higher hybridization temperature of 55◦C, the base-pair mismatch sequence shows negligible change in biosensor signal response and can be clearly differentiated from the complementary target Previous study on terminal mismatch at proximal end of surface tethered DNA sequences indicate enhanced flexibility of the DNA-linker region, likely due to fraying of the strands at the terminal mismatch position Such orientation changes may affect the kinetics of hybridization and further influence the selectivity against the mismatch sequence Consequently, sequences with one and three mismatches have lower Tm than the hybridization temperature 55 ◦C and not hybridize well with the probe sequence, giving significant differences in the normalized DPV peak currents and absolute peak potential values EAMB has been also challenged with PCR amplicons for Legionella genomic DNA in order to test the applicability of the EAMB biosensor in real environmental water sample analysis As environmental water sample contaminated with Legionella sp is used for isolation of bacteria for amplification of genomic sequences A 157-bp region between position 58 and 78 in the genomic DNA is selected as the target (see Table 3.1) with complementary to loop sequence in the hairpin DNA-ferrocene probe 162 of the biosensor Legionella pneumophila is cultured overnight, and its genomic DNA sequences are isolated using a bacteria genomic DNA isolation kit (Cat#51306 QIAamp DNA Mini Kit) PCR amplification is performed in a PCR cycler (Applied Bio systems Gene Amp PCR system 2700) A pair of asymmetric primers (10 Primer1/1 Primer2) is employed in order to generate the ss-DNA target The amplification protocol comprises an initial heating at 95 ◦C followed by 35 cycles of 95 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 30 s The reaction system is further incubated for at 72 ◦C to extend any incomplete products The PCR products are subsequently diluted by 10-fold successively up to four serial dilutions for detection by the biosensor and the biosensor signal response is recorded towards serially diluted PCR amplicon samples of the isolated target sequence The biosensor can be regenerated after exposure to the series of diluted PCR amplicon samples using 75 ◦C, 30 heating cycle Since this EAMB system is useful in understanding fundamental insights of electrochemical signal amplification and extending its application for DNA detection However application of EAMB for DNA detection is fairly limited to scientific domain and more optimization and fabrication of sensing surfaces are required to extend this method for clinical applicability Ultrasensitive detection of a label-free 21-mer complementary DNA sequence of L pneumophila at (∼20) fM level can be achieved The EAMB biosensor selectively differentiates between L pneumophila and two other species and shows exceptionally wide linear range derived from DPV current signal from 10−14 to 10−6 M The equally responsive biosensor peak potential provides additionally useful quantitative and selective analytical data about complementary DNA analyte within the linear range from 10−13 to 10−6 M 163 the Nanoscale material can also be used in a variety of electrochemical biosensing schemes to improve the sensitivity and specificity of diagnostic tools as well as miniaturizing these devices for onsite applications Thus utilization of nanoscale materials has achieved significant research interest in electrochemical sensing devices because of the unique physical, chemical properties and electron transport properties The large surface to volume ratio of nanoscale materials offers more surface area for immobilization of (bio element) probe molecules and number density of probe molecules are important parameter to improve the sensitivity and performance of sensing device Alumina nanoporous membrane structure is synthesized by electrochemical anodization method to develop electrochemical nanoporous alumina membrane based label free DNA biosensor for detection of Legionella sp and complementary DNA (cDNA) of dengue RNA An electrochemical nanoporous alumina membrane-based label free DNA biosensor is developed using 5'-aminated DNA probes, immobilized into the nanochannels of alumina to detect DNA sequences of Legionella Alumina nanoporous membranelike structure is carved over platinum wire electrode of 76 μm diameter dimension by electrochemical anodization The hybridization of complementary target DNA with probe DNA molecules attached inside the nanochannels influences the pore size and ionic conductivity Electrochemical biosensing signal is derived from only redox species Fe(CN)64- across single wire Pt electrode and monitored using cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy However DPV has been widely used in ultrasensitive detection as capacitive current is associated with CV that limits the ultrasensitive selective differentiation and in EIS the most challenging problem is modelling of the electrode processes, where to some extent the quantitative problems and errors arise The 164 biosensors sensing mechanism relies on the monitoring of electrode’s faradic current response towards redox species, Fe(CN)64-, which is sensitive towards the hybridization of complementary target with probe DNA immobilized into the alumina nanochannels The biosensor demonstrates wide linear range over orders of magnitude with ultrasensitive detection limit 3.13 ×10-13 M for the quantification of ss21 mer DNA sequence and selectively differentiates the complementary sequence from target sequences with single base mismatch (MM1) and triple bases mismatch (MM3) of different strain of Legionella sp Its applicability has been also challenged against PCR amplicons sample derived from Legionella pneumophila genomic DNA using asymmetric PCR method The construction of nanoporous alumina membrane based DNA biosensor is very simple and relatively easier to carve nanoporous structure by electrochemical anodization than conventional lithography e.g electron beam or focussed ion beam and sample analysis time is around ~ 45 Electrochemical biosensing signal is derived only from Fe(CN)64across single wire Pt electrode in contrast to label DNA sensor and amplified DNA sensor where redox active or label is attached in the probe or target DNA which requires additional synthetic and purification steps Thus detection of DNA sequences of Legionella using electrochemical nanoporous alumina membrane-based biosensor shows fairly comparable sensitivity with electrochemical amplified molecular beacon biosensor Anodized alumina nanoporous material based biosensor is also used to detect the complementary DNA (cDNA) of dengue virus (DENV I) to extend this concept further ahead in detection and understanding insight fundamental electrochemistry about biosensing inside the nanochannels Dengue virus (DENV), a single-stranded RNA positive-strand mosquito-borne virus is of the genus flavivirus DENV is 165 highly infectious and widespread in tropical and subtropical region with epidemic challenge There are four antigenically different serotypes of the virus (DENV1-4) To mitigate the effect of epidemic spread of dengue infection, its early detection is deemed necessary with follow up vector control measures and a responsive medical support system Therefore a nanoporous alumina membrane based ultrasensitive DNA biosensor for detection of cDNA of DENV1 RNA is constructed using 5’aminated DNA probes immobilized onto the alumina nanochannel walls Probe DNA molecules are covalently attached into the alumina nano channels which selectively bind to 31 mer specific DENV1 DNA target sequence The hybridization of complementary target DNA with probe DNA molecules attached inside the pores affects ionic conductivity of redox species Fe(CN)64- Binding of target complementary DNA to probe inside nanochannels causes changes in mass transfer of redox species Fe(CN)64- through it due to blocking of the pores Mass transfer changes through alumina nanopores are translated into electrochemical signal using differential pulse voltammetric technique (DPV) DPV oxidative peak current of Fe(CN)64- successively drops with increase in target complementary DNA concentration The biosensor demonstrates linear range over orders of magnitude with ultrasensitive detection limit of 9.55× 10-12 M for the quantification of ss-31 mer DNA sequence Its applicability is challenged against cDNA PCR amplicons sample of dengue virus serotype1 derived from asymmetric PCR Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated Thus we summarize development of DNA biosensors for detection of Legionella sp and dengue virus based on electrochemical amplification strategy and utilizing nanoporous alumina membrane based materials However in electrochemical analysis, reproducibility is a bit minor issue in signal recording 166 across electrodic interface because of some fouling of sensing surface in case of homemade nanoporous alumina electrode and sensing surface defects like non uniformity of molecular assembly on the gold electrode This concept of nanoporous anodized alumina membrane based DNA biosensors is being extended to replace anodized sputter coated alumina with multichannel alumina membrane of nanosize range This multi nanochannels alumina membrane can be used as template to develop DNA biosensors utilizing two chamber electrochemical cell setup to study electro osmotic and fluidics of redox species corresponding to selective hybridization event In this method multi nanochannels based alumina membranes are sputter coated with platinum to make electrical contacts with extension wires of potentiostat and this Pt sputter coated multi nanochannels alumina membrane are utilized to immobilize probe ss DNA inside the nanochannels and results into biosensing surface inside the nanochannels of alumina membrane With subsequent addition of complementary target DNA, it binds to probe DNA immobilized inside the nanochannels and influences the ionic redox species movement through the nanochannels of alumina membrane Successful hybridization event inside the nanochannels of alumina membrane modulates the ionic current associated with redox species and supporting electrolyte Thus monitoring of ionic current passing through alumina nanochannels can be used as electrochemical parameter to detect and validate selective DNA hybridization event inside the multi nanochannels of alumina membrane Future perspective include coupling of amplification strategy with nanoscale based materials to improvise sensitivity many folds and extending this sensing signal off strategy detection to signal on where successful target binding with probe may lead to increase in electrochemical signal with increase in target concentration More 167 optimization and trials with actual human serum sample and environmental samples are necessary to extend electrochemical sensing concept from scientific domain to clinical domain 6.2 Comparative performances table: Comparison of the limit of detection (LOD) of various recent biosensors system for detection of target DNA and SNP differentiation Detection Methods Transducer/ strategy Detection limit SNP Sensor differentiation Mode References Electrochemical amplified molecular beacon DNA biosensor Enzyme-based electrochemical DNA sensor Electrochemical, Electrochemical signal amplification Electrochemical, enzyme based signal amplification Electrochemical, without amplification 10-14M good turn-off [1] 10 fM good turn-on [2] 10 pM not reported turn-off [3] Electrochemical DNA sensor involving artificial DNAPEG-DNA triblock probe Electrochemical Sandwich strategy 200 pM not reported turn-on [4] Electrochemical DNA sensor involving MB-tagged DNA pseudo knot probe Electrochemical, Target induced Conformational changes in the probe Electrochemical, involving capturing probe, signalling probe tagged with methylene blue, target strand nM good turn-on [5] 400 fM not reported turn-on [6] Ligase – mediated electrochemical DNA sensor Electrochemical, based on the combined use of ligase and reverse molecular beacon strategy pM good turn-on [7] Stem-loop-based optical detection with electrical Fluorescent, based on scanning nM good turn-on [8] Electrochemical DNA sensor involving ferrocenetagged DNA stem-loop probe molecule Displacement based electrochemical DNA sensor 168 potential control potential hairpin denaturation Surface immobilized molecular beacons based DNA biosensor Fluorescent, based on unquenching of fluorphore Electrochemical, based on impedance Electrochemical based on impedance Electrochemical, based on DNAzyme amplification Electrochemical, based on electrochemically active inactive switching Electrochemical, based on nanoscale materials 10 nM good 100 pM good [11] 100 pm good [12] Electrochemical, based on dynamic polymeraseextending hybridization Optical 0.5nM 0.3 nM not reported Optical, based on reflectivity measurement Fluorescence, 2nmol/cm2 not reported Surface-enhanced raman scattering (SERS) spectroscopy Surface Plasmon Resonance 20 fM good [21] 10-18 M good [22] Electrochemical DNA sensor Electrochemical DNA sensor involving unlabeled hairpin DNA probe Electrochemical DNA biosensor based on proximity-dependent DNA ligation assays Electrochemical molecular beacon DNA biosensor Nanoporous alumina membrane based electrochemical DNA biosensor Aluminium anodized oxide (AAO) membranes based DNA biosensor DNA biosensor involving Nanoporous Alumina Filters Interferometric DNA sensing using Nanoporous Aluminium Oxide Templates Multiplexed biosensing using biological chips DNA sensing using nanoparticles with raman spectroscopic fingerprints Nanoparticle-enhanced surface plasmon resonance imaging (SPRI) DNA sensor 100 fM [9, 10] [13] 30 nM good turn-on [14] 10-13M good turn off [15, 16] turn-off [17] turn-on [18] 1pM 169 turn-on [19] turn-on [20] References Rai, V.;Nyine, Y T.;Hapuarachchi, H C.;Yap, H M.;Ng, L C.;Toh, C S., Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp Biosensors and Bioelectronics, 2012 32(1): p 133-140 Liu, G.;Wan, Y.;Gau, V.;Zhang, J.;Wang, L.;Song, S.;Fan, C., An EnzymeBased E-DNA Sensor for Sequence-Specific Detection of Femtomolar DNA Targets Journal of the American Chemical Society, 2008 130: p 6820-6825 Fan, C.;Plaxco, K W.;Heeger, A J., Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA Proceedings of the National Academy of Sciences of the United States of America, 2003 100(16): p 9134-9137 Immoos, C E.;Lee, S J.;Grinstaff, M W., DNA-PEG-DNA triblock macromolecules for reagentless DNA detection Journal of the American Chemical Society, 2004 126(35): p 10814-10815 Xiao, Y.;Qu, X.;Plaxco, K W.;Heeger, A J., Label-free electrochemical detection of DNA in blood serum via target-induced resolution of an electrode-bound DNA pseudoknot Journal of the American Chemical Society, 2007 129(39): p 11896-11897 Xiao, Y.;Lubin, A A.;Baker, B R.;Plaxco, K W.;Heeger, A J., Single-step electronic detection of femtomolar DNA by target-induced strand 170 displacement in an electrode-bound duplex Proceedings of the National Academy of Sciences of the United States of America, 2006 103(45): p 16677-16680 Wu, Z S.;Jiang, J H.;Shen, G L.;Yu, R Q., Highly sensitive DNA detection and point mutation identification: An electrochemical approach based on the combined use of ligase and reverse molecular beacon Human Mutation, 2007 28(6): p 630-637 Wei, F.;Chen, C.;Zhai, L.;Zhang, N.;Xin, S Z., Recognition of single nucleotide polymorphisms using scanning potential hairpin denaturation Journal of the American Chemical Society, 2005 127(15): p 5306-5307 Du, H.;Disney, M D.;Miller, B L.;Krauss, T D., Hybridization-based unquenching of DNA hairpins on Au surfaces: Prototypical "molecular beacon" biosensors Journal of the American Chemical Society, 2003 125(14): p 4012-4013 10 Du, H.;Strohsahl, C M.;Camera, J.;Miller, B L.;Krauss, T D., Sensitivity and Specificity of Metal Surface-Immobilized "Molecular Beacon" Biosensors Journal of the American Chemical Society, 2005 127(21): p 7932-7940 11 Long, Y T.;Li, C Z.;Sutherland, T C.;Kraatz, H B.;Lee, J S., Electrochemical detection of single-nucleotide mismatches: Application of MDNA Analytical Chemistry, 2004 76(14): p 4059-4065 12 Gong, H.;Zhong, T.;Gao, L.;Li, X.;Bi, L.;Kraatz, H B., Unlabeled hairpin DNA probe for electrochemical detection of single-nucleotide mismatches based on MutS-DNA interactions Analytical Chemistry, 2009 81(20): p 8639-8643 171 13 Sun, C.;Zhang, L.;Jiang, J.;Shen, G.;Yu, R., Electrochemical DNA biosensor based on proximity-dependent DNA ligation assays with DNAzyme amplification of hairpin substrate signal Biosensors and Bioelectronics, 2010 25(11): p 2483-2489 14 Wu, J.;Huang, C.;Cheng, G.;Zhang, F.;He, P.;Fang, Y., Electrochemically active-inactive switching molecular beacon for direct detection of DNA in homogenous solution Electrochemistry Communications, 2009 11(1): p 177-180 15 Rai, V.;Hapuarachchi, H C.;Ng, L C.;Soh, S H.;Leo, Y S.;Toh, C S., Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor PLoS ONE, 2012 7(8) 16 Rai, V.;Deng, J.;Toh, C S., Electrochemical nanoporous alumina membranebased label-free DNA biosensor for the detection of Legionella sp Talanta, 2012 17 Wang, L.;Liu, Q.;Hu, Z.;Zhang, Y.;Wu, C.;Yang, M.;Wang, P., A novel electrochemical biosensor based on dynamic polymerase-extending hybridization for E coli O157:H7 DNA detection Talanta, 2009 78(3): p 647-652 18 Vlassiouk, I.;Krasnoslobodtsev, A.;Smirnov, S.;Germann, M., "Direct" detection and separation of DNA using nanoporous alumina filters Langmuir, 2004 20(23): p 9913-9915 19 Pan, S.;Rothberg, L J., Interferometric sensing of biomolecular binding using nanoporous aluminium oxide templates Nano Letters, 2003 3(6): p 811814 172 20 Fodor, S P.;Rava, R P.;Huang, X C.;Pease, A C.;Holmes, C P.;Adams, C L., Multiplexed biochemical assays with biological chips Nature, 1993 364(6437): p 555-556 21 Cao, Y C.;Jin, R.;Mirkin, C A., Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection Science, 2002 297(5586): p 15361540 22 D'Agata, R.;Breveglieri, G.;Zanoli, L M.;Borgatti, M.;Spoto, G.;Gambari, R., Direct detection of point mutations in nonamplified human genomic DNA Analytical Chemistry, 2012 83(22): p 8711-8717 173 PUBLICATIONS Rai, V.;Hapuarachchi, H C.;Ng, L C.;Soh, S H.;Leo, Y S.;Toh, C.-S., Ultrasensitive cDNA Detection of Dengue Virus RNA Using Electrochemical Nanoporous Membrane-Based Biosensor PLoS ONE, 2012 7(8): p e42346 Rai, V.;Deng, J.;Toh, C S., Electrochemical nanoporous alumina membrane-based label-free DNA biosensor for the detection of Legionella sp Talanta, 2012 98 : 112117 Rai, V.;Nyine, Y T.; Hapuarachchi, H C.;Yap, H M.;Ng, L C.;Toh, C S.,Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp Biosensors and Bioelectronics, 2012 32(1): p 133-140 Rai, V; Toh, C.S “ SI Electrochemical amplification strategies in DNA nanosensors” Review article, Nanosciene and Nanotechnology letters, American Scientific Publishers,2013 (5): 613-623 Nanoporos titania based covalently immobilzed cyto-c bioelectorchemical system for electron transfer study and detection of H2O2 Varun Rai, Chee-Seng Toh, Saurav K Guin and Suresh K Aggarwal under revisoin Manuscripts under preparation Electrochemical alumina membrane DNA sensor on measuement of ionic current blockage using two chamber electrochemical cell Varun Rai , Chee Seng Toh 174 PROCEEDINGS AND POSTER PRESENTATION Varun Rai, Chee Seng Toh Electrochemical Nanoporous Membrane-Based Biosensor for ultrasensitive cDNA detection of Dengue Virus RNA Singapore International Conference on Dengue and Emerging Infections, 21-23 November 2012, Singapore Varun Rai, Chee-Seng Toh Electrochemical DNA Sensor for Ultrasensitive DNA Sequence Specific Detection of Legionella sp and Dengue 18th Australian Electrochemistry Symposium (AES) April 15th 2012, Curtin University, Perth, Australia, p-56 Varun Rai, Chee-Seng Toh Electrochemical DNA Sensor for Ultrasensitive DNA Sequence Specific Detection of Legionella sp and Dengue 10th Spring Meeting of the International Society of Electrochemistry from 15 - 18 April 2012, Perth, Australia Varun Rai, Yin Thu Nyine, Chee-Seng Toh Electrochemical Hairpin DNA Sensor for Ultrasensitive Detection of Sequence-Specific Legionella sp Regional Electrochemistry Meeting of South-East Asia (REMSEA 2010) Bangkok, Thailand from November 16 – 19, 2010 Varun Rai, Clara Ang, Chee-Seng Toh Electrochemical hairpin DNA sensor for Legionella pneumophila Keystone Symposia Developmental Origins and Epigenesis in Human Health and Disease, Singapore, April 26 - 30, 2010 175 176 ... technique of DNA detection require minimal sample preparation step for analyte detection and show very low detection limit Among non pcr based DNA detection, the focus of the study is only based on electrochemical. .. sensitive electrochemical impedimetric DNA biosensor for detection of even single nucleotide mismatches [24] In this method EIS was used to study the electrochemical behaviour of probe ss DNA attached... nm for the detection of whole virus particle [73] and E coli cells [74] More recently, the same nanoporous alumina membrane nanobiosensor has been extended for the detection of ssDNA target of