ZUSCHRIFTEN Angew. Chem. 2001, 113, Nr. 12  WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001 0044-8249/01/11312-2321 $ 17.50+.50/0 2321 Electronic Transduction of Polymerase or Reverse Transcriptase Induced Replication Processes on Surfaces: Highly Sensitive and Specific Detection of Viral Genomes** Fernando Patolsky, Amir Lichtenstein, Moshe Kotler, and Itamar Willner* Dedicated to Professor AndreÂM. Braun on the occasion of his 60th birthday The detection of pathogens or autosomal recessive diseases is one of the future challenges of medicine and diagnostics. [1] Sensitive gene detection is accomplished by the polymerase chain reaction (PCR) amplification or secondary signal amplification routes. [2, 3] Major goals in future gene analysis include the parallel detection of a variety of pathogens and their quantitative assay. Inherent limitations of PCR prohibit the application of the method for quantitative and parallel high-throughput analyses. Microarrays of DNA have attract- ed substantial research efforts for the simultaneous analysis of genetic materials. In most of these systems the analyte samples are amplified by PCR cycles, and the microarrays act as a sensing interface that lacks amplification capabil- ities. [4, 5] Herein we address the development of ultrasensitive DNA-detection methods where in situ amplification proceeds on functionalized surfaces (electrodes or piezoelectric crys- tals) and the detection process is electronically transduced. The method enables the quantitative analysis of viral DNA and may be adopted for parallel analyses on arrays. Previous reports addressed the electrochemical [6±9] or microgravimet- ric, [10] quartz-crystal microbalance detection (QCM) of DNA. Several recent studies reported attempts to amplify DNA sensing processes: Dendritic, hyperbranched, oligonucleoti- des were employed to enhance the binding of DNA to electrodes. [11] The biocatalyzed precipitation of an insoluble product on the electronic transducer, that follows the primary hybridization between the analyte DNA and the probe oligonucleotide, was used to amplify the sensing event. [12] Also, labeled liposomes were employed as micromembrane interfaces that amplify the primary DNA-sensing events by their association to the probe-oligonucleotide/DNA-analyte complex generated on the transducer. [13, 14] Similarly, dendrit- ic-type amplification of the analysis of a target DNA was accomplished by the use of oligonucleotide-functionalized Au-nanoparticles. [15] Faradaic impedance spectroscopy or frequency changes of the piezoelectric crystal, were used to transduce the different amplified sensing processes. Here we report a novel ultrasensitive method for the electronic transduction of the detection of viral nucleic acids. We demonstrate the surface polymerase-induced or reverse tran- scriptase stimulated formation of double-stranded DNA or RNA on the transducer, and the secondary amplification of the sensing process by the biocatalyzed precipitation of an insoluble product. Electrochemical and microgravimetric QCM methods are used as electronic transduction means for the DNA detection. The process is exemplified by the analysis of the M13 mp8 (M13 f) DNA (ca. 300 copies per 10 mL) and of the RNA of vesicular stomatitis virus (VSV; ca. 60 copies per 10 mL). The method for analysis of the target is depicted in Scheme 1. The primer thiolated oligonucleotide 1, comple- mentary to a segment of the target M13 mp8 DNA, is assembled on an Au-electrode or an Au-quartz crystal through a thiol functional group. [16, 17] The sensing interface is then treated with the analyte DNA of M13 mp8 () strand, and the resulting complex on the transducer is treated with dATP, dGTP, dTTP, dCTP, and biotinylated-dCTP (ratio 1:1:1:2/3:1/3, nucleotides concentration of 1mm) in the presence of DNA polymerase I, Klenow fragment (20 U mL 1 ). [18] Polymerization and the formation of a dou- ble-stranded assembly with the target DNA is anticipated to provide the first amplification step of the analysis of the viral DNA. Polymerase introduces biotin tags to the double- stranded assembly, thus providing a high number of docking sites for the binding of the avidin ± alkaline-phosphatase conjugate. The associated enzyme biocatalyzes the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate (2) to form the insoluble indigo product 3, that precipitates on the transducer, thus providing a second amplification step for the analysis of the target DNA. [19] The synthesized strand on the electrode is anticipated to attract a positively charged redox label that can be assayed by chronocoulometry. [20] This approach enables us to monitor the polymerization process continuously. The negatively charged double-stranded assem- [21] A. C. Templeton, S. Chen, S. M. Gross, R. W. Murray, Langmuir 1999, 15, 66 ± 76. [22] J. Liu, W. Ong, E. Roma Ân, M. J. Lynn, A. E. Kaifer, Langmuir 2000, 16, 3000 ± 3002. [23] C. S. Weisbecker, M. V. Merritt, G. M. Whitesides, Langmuir 1996, 12, 3763 ± 3772. [24] B. T. Houseman, M. Mrksich, Angew. Chem. 1999, 111, 876 ± 880 ; Angew. Chem. Int. Ed. 1999, 38, 782 ± 785. [25] J. Jime Ânez-Barbero, E. Junquera, M. Martín-Pastor, S. Sharma, C. Vicent, S. Penade  s, J. Am. Chem. Soc. 1995, 117 , 11 198 ± 11 204. [26] J.C. Morales, D. Zurita, S. Penade  s, J. Org. Chem. 1998, 63, 9212 ± 9222. [27] J. J. Distler, G. W. Jourdian, J. Biol. Chem. 1973, 248, 6772 ± 6780. [28] C. Tromas, J. Rojo, J.M. de la Fuente, A.G. Barrientos, R. Garciµ, S. PenadØs, unpublished results. [29] H. H. Riese, A. Bernad, J. Rojo, A. G. Barrientos, J. M. de la Fuente, S. PenadØs, unpublished results. [30] The existence of this interaction has also been shown by using NMR spectroscopy: A. Geyer, C. Gege, R. R. Schmidt, Angew. Chem. 1999, 111, 1569 ± 1571; Angew. Chem. Int. Ed. 1999, 38, 1466 ± 1468. [*] Prof. I. Willner, F. Patolsky, A. Lichtenstein Institute of Chemistry The Hebrew University of Jerusalem Jerusalem 91904 (Israel) Fax: ( 972) 2-6527715 E-mail: willnea@vms.huji.ac.il Prof. M. Kotler Department of Experimental Pathology, The Hebrew University, Hadassah Medical School Jerusalem 91120 (Israel) [**] Parts of this research are supported by the Israel Ministry of Science as an Infrastructure Project in Biomicroelectronics and as an Israel ± Japan cooperation. M.K. acknowledges the support of the American Foundation for Aids Research, AmfAR (grant No. 02730-28-RG).