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WO 2008/097190 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date PCT 14 August 2008 (14.08.2008) WO 2008/097190 Al (51) International Patent Classification: (74) Agent: SCHIWECK, Wolfram; Viering, Jentschura & C12Q 1/26 (2006.01) C12Q 1/28 (2006.01) Partner, P.O. Box 1088, Rochor Post Office, Rochor Road, C12Q 1/68 (2006.01) GOlN 27/327 (2006.01) Singapore 911833 (SG). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/SG2007/000037 kind of national protection available): AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN, (22) International Filing Date: 5 February 2007 (05.02.2007) CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, (25) Filing Language: English JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LV,LY, MA, MD, MG, MK, MN, MW, MX, MY, (26) Publication Language: English MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN, (71) Applicant (for all designated States except US): TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH [SG/SG]; 20 Biopolis Way #07-01, Centres, (84) Designated States (unless otherwise indicated, for every Singapore 138668 (SG). kind of regional protection available): ARIPO (BW, GH, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (72) Inventors; and ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), (75) Inventors/Applicants (for US only): GAO, Zhiqiang European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, [SG/SG]; Institute of Microelectronics, 11 Science Park FR, GB, GR, HU, IE, IS, IT, LT, LU, LV,MC, NL, PL, PT, Road, Singapore Science Park II, Singapore 117685 RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, (SG). FAN, Yi [CN/SG]; Institute of Microelectronics, 11 GN, GQ, GW, ML, MR, NE, SN, TD, TG). Science Park Road, Singapore Science Park II, Singapore 117685 (SG). CHEN, Xiantong [CN/SG]; Institute of Published: Microelectronics, 11 Science Park Road, Singapore Sci — with international search report ence Park II, Singapore 117685 (SG). KONG, Jinming — with sequence listing part of description published sep a [CN/SG]; Institute of Microelectronics, 11 Science Park rately in electronic form and available upon request from Road, Singapore Science Park II, Singaproe 117685 (SG). the International Bureau (54) Title: METHODS OF ELECTRICALLY DETECTING A NUCLEIC ACID BY MEANS OF AN ELECTRODE PAIR (57) Abstract: Methods of electrically detecting a target nucleic acid molecule. A pair of electrodes is arranged at a distance from one another and within a sensing zone. A PNA capture molecule, with a nucleotide sequence that is at least partially complementary to a portion of the target nucleic acid, is immobilised upon an immobilisation unit. The target nucleic acid molecule hybridises and forms a complex with this capture molecule. A polymerisable positively chargeable precursor with an electrostatic net charge complementary to the target nucleic acid is then added. These precursor associates to the complex formed between the PNA capture molecule and the target nucleic acid. Upon addition of a suitable reactant molecule polymerisation is initiated. The electroconductive polymer that is formed influences the electrical characteristics of the region between the electrodes allowing detection of the presence of the target nucleic acid molecule. METHODS OF ELECTRICALLY DETECTING A NUCLEIC ACID BY MEANS OF AN ELECTRODE PAIR [0001] The present invention relates to methods of electrically detecting a target nucleic acid molecule by means of an electrode pair. [0002] The detection and quantification of nucleic acids is a fundamental method not only in analytical chemistry but also in biochemistry, food technology or medicine. The most frequently used methods for determining the presence and concentration of nucleic acids include the detection by autoradiography, fluorescence, chemiluminescence or bioluminescence as well as electrochemical and electrical techniques. [0003] Different from electrochemical detection methods, electrical detection of nucleic acid molecules relies on the conversion of the base pairing events into detectable electrical signals such as capacitance (Moreno-Hagelsieb, L., et al., Sens. Actuat. B-Chem. (2004) 98, 269-274) and resistance (Park, S. J., et al., Science (2002) 295, 1503-1506). Benefited from the inherent superiorities of electrical transduction methods, nucleic acid biosensors based on the electrical detection are able to provide high performance in terms of accuracy and sensitivity as well as low-cost, miniaturised readout unit and thus exempt from the problems encountered in the optical detection system hi addition, combining with microfabrication technology and microelectronics, microscale electrical detection-based biosensors can be easily constructed, such as silicon nanowire biosensors 1. Mirkin and co- workers also put forward a successful sample of electrical detection of DNAs with paired microelectrodes separated by a microgap and gold nanoparticle-conjugated oligonucleotide detection probes (Park et al., 2002, supra). The hybridisation of the detection probes to target DNAs led to the aggregation of Au nanoparticles in the gap between the electrodes. A further deposition of silver facilitated by the gold nanoparticles bridged the gap and produced measurable conductivity changes. It was demonstrated that the target DNAs are detected at 500 fM. [0004] The deposition of electrically active materials on biomacromolecules such as DNA has led to impressive results such as nano-scaled biofunctional matrices as well as highly selective biosensors. An example is the DNA templated deposition of conducting nanoparticles (Torimoto, T., et al., J. Phys. Chem. B (1999) 103, 8799-8803). Benefited from enzymatic polymerisation methods, which provide mild and controllable reaction conditions and allow using delicate biomolecules as templates, electroconducive polymers such as polyaniline have also been fabricated along oligonucleotides and λ-DNA to form nanowires (Ma, Y., et al., J. Am. Chem. Soc. (2004) 126, 7097-7101), where the phosphate groups of the nucleic acid molecules served as templates, guiding the deposition of polyaniline. An increase of conductivity of the polyaniline could be achieved by a simple doping process with HCl vapour. [0005] A technique for the specific detection of a selected nucleic acid well established in the art is based on the hybridisation between a nucleic acid capture probe and a target nucleic acid. Typically the respective nucleic acid capture probe is immobilised onto a solid support, and subsequently one of the above mentioned detection methods is employed. [0006] Sensitivity and selectivity are the two important issues being constantly addressed in the evaluation of nucleic acid detection systems. The most popular methods are performed using fluorescent tags in array formats in conjunction with solution phase (off- chip) pre-amplification/labelling approaches employing polymerase chain reactions. They offer the highest degree of sensitivity, the highest throughput, and the widest dynamic range. However, the amplification power of polymerase chain reactions may be dramatically affected by small variations in experimental conditions and sample compositions. Optimisation of the complicated primers and experimental conditions for each specific gene is a formidable task. Often, the finalised amplification protocols are not optimal for many genes being studied. PCR-based amplification methods therefore do not faithfully reproduce the relative concentrations of genes in complex matrixes, due to selective and nonlinear target amplifications (Ho, H.A., et al., J. Am. Chem. Soc. (2005) 127, 12673-12676). Moreover, optical transduction engages not only highly precise and expensive equipment but also complex algorithms to interpret the data. Furthermore, off-chip target amplifications also significantly increase the cost of the procedures and often lead to sequence-dependent quantification bias. [0007] Consequently, in situ signal amplification strategies, such as rolling circle amplification (RCA), T7 DNA polymerase, branched DNA technology, catalysed reporter deposition, dendritic tags, enzymatic amplification and chemical amplification, have been proposed for both optical and electrochemical systems. Among them, the inherent miniaturisation of electrochemical devices, excellent compatibility with advanced semiconductor technology, and low cost make electronic transduction-based biosensors very promising candidates for analysing nucleic acids with reduced cost and size of the read-out unit in comparison with the more conventional optical systems. It has been shown that the sensitivity of those assays is comparable to that of PCR-based fluorescent assays. As few as 103 copies of genes can be directly detected (Zhang, Y., et al., Anal. Chem. (2003) 75, 3267- 3269; Xie, H., et al., Anal. Chem. (2004) 76, 1611-1617). While such improvements may enhance the sensitivity of nucleic acid detection, inherent disadvantages are additional costs for amplification reagents such as primers, as well as the additional time required for signal amplification. [0008] Thus, there remains a need for an alternative method for the detection of nudeic acids, which overcomes the above
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