Anal. Chem. 2006, 78, 7054-7057
Correspondence
Electroenzymatic Polypyrrole-intercalator Sensor for the Determination of West Nile Virus cDNA Serge Cosnier,*,† Rodica E. Ionescu,† Sebastien Herrmann,‡ Laurent Bouffier,†,§ Martine Demeunynck,§ and Robert S. Marks*,‡
LEOPR (CNRS UMR 5630), Institut de Chimie Mole´ culaire de Grenoble FR CNRS 2607, Universite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France, Department of Biotechnology Engineering and National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, 84105 Beer-Sheva, Israel, and LEDSS UMR 5616 & ICMG-FR 2607, Universite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
The chemical binding of a redox acridone derivative onto a polypyrrole film functionalized by N-hydroxysuccinimide groups provided an electrode capable of anchoring DNA duplex by simple insertion of the grafted acridone intercalator into the dsDNA solution. This electrode was applied for the detection of a ssDNA derived from a West Nile virus sequence. The latter was thus amperometrically detected after its hybridization in solution with a biotinylated complementary oligonucleotide followed by its anchoring and labeling by a glucose oxidase at 1 pg/mL. In a time when dangerous viruses (such as AIDS, SARS, West Nile virus (WNV)) are afflicting more and more humans, early detection is believed to be an essential tool to help to fight them. One such direction that has gained considerable interest for a rapid and inexpensive diagnostic of an illness is one based on the development of DNA sensors (or genosensors) for the detection of the specific DNA sequences related to these diseases via a hybridization process. Basically, DNA sensors consist of the immobilization of an oligonucleotide probe onto a physical transducer generating directly or indirectly a signal upon hybridization with the DNA target. Over the past decade, enormous progress has been made toward the development of electrochemical DNA biosensors.1-4 Among the conventional methods for detecting a DNA hybridization event, the electrochemical transduction indeed ensured a faster, easier, and less expensive detection than conventional optical or gravimetric transduction. In general, the electrochemical detection of a hybridization event between a single strand of DNA (ssDNA) (target) present in a complex aqueous mixture of ssDNAs and an immobilized comple* Corresponding authors. Tel: 33 4 76 51 49 98. Fax: 33 4 76 51 42 67. E-mail:
[email protected] and
[email protected]. † LEOPR, Universite ´ Joseph Fourier. ‡ Ben-Gurion University of the Negev. § LEDSS, Universite ´ Joseph Fourier. (1) Chiti, G.; Marrazza, G.; Mascini, M. Anal. Chim. Acta 2001, 427, 155164. (2) Wang, J. Anal. Chim. Acta 2002, 469, 63-71. (3) Gooding, J. J. Electroanalysis 2002, 14, 1149-1156. (4) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192-1199.
7054 Analytical Chemistry, Vol. 78, No. 19, October 1, 2006
mentary oligonucleotide (probe) is performed via the use of external electroactive or catalytic labels such as metal complexes, organic redox markers, enzymes, or nanoparticles.5-10 These labels are anchored by covalent binding or affinity interactions to a ssDNA or intercalated into a double-stranded DNA (dsDNA). However, it should be noted that each target requires the chemical modification of the sensor surface by a specific oligonucleotide probe. In addition, the oligonucleotide attachment on the electrode surface must preserve the accessibility and molecular orientation of the DNA probe. Taking in mind the intercalation process, we report here an original concept of DNA sequence detection that consists of first adding the complementary oligonucleotide into an aqueous solution of ssDNAs and then “fishing” the resulting duplex with an electrochemical sensor. The latter will be constituted of a synthetic planar intercalator, a redox acridone derivative (RAD),11 chemically grafted onto the electrode surface via a new electropolymerized polypyrrole N-substituted by N-hydroxysuccinimide groups film (Figure 1). The grafted RAD will then be used to bind by intercalation the dsDNA on the electrode surface. Because of the rising epidemy of WNV infections with no good antivirals or vaccines in sight, a greater reliance on early WNV diagnostics is needed.12 First isolated in Uganda in 1937, WNV is a single-stranded RNA flavivirus transmitted to humans by an infected mosquito bite. Human-to-human transmission via mosquito bites does not occur; however, WNV can be transmitted from one person to another via blood transfusion, organ transplantation, breast-feeding, and transfer across the placenta.13 (5) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2001, 123, 51945205. (6) Yang, J.; Zhang, Z.; Rusling, J. F. Electroanalysis, 2002, 14, 1494-1500. (7) Wang, J.; Liu, G.; Merkoc¸ i, A. J. Am. Chem. Soc. 2003, 125, 3214-3215. (8) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010-3011. (9) Davis, F.; Nabok, A. V.; Higson, S. P. J. Biosens. Bioelectron. 2005, 20, 1531-1538. (10) Peng, H.; Soeller, C.; Cannell, M. B.; Bowmaker, G. A.; Cooney, R. P.; TravasSejdic, J. Biosens. Bioelectron. 2006, 21, 1727-1736. (11) Bouffier, L.; Demeunynck, M.; Millet, A.; Dumy, P. J. Org. Chem. 2004, 69, 8144-8147. (12) Shirato, K.; Miyoshi, H.; Kariwa, H.; Takashima, I. J. Virol. Methods 2005, 126, 119-125. (13) Prince, H. E.; Hogrefe, W. R. Clin. Appl. Immunol. Rev. 2005, 5, 45-63. 10.1021/ac060926a CCC: $33.50
© 2006 American Chemical Society Published on Web 08/29/2006
Figure 1. Structures of the redox acridone derivative (RAD) (a) and the pyrrole N-substituted by N-hydroxysuccinimide group 1 (b).
Before 1996, WNV was known to be endemic in Africa, the Middle East, and western Asia, where it caused sporadic outbreaks associated with mild illness. Since then, epidemics of WNV infection associated with increased rates of neurological complications and death have occurred in Romania, Russia, and Israel, as well as the United States and Canada. Although most WNVinfected persons are asymptomatic, 10-20% develop symptoms such as fever, headache, rash, and malaise. A small number (
