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Article

Characterization and Optimization of a Real-Time, Parallel, Label-Free, Polypyrrole-Based DNA Sensor by Surface Plasmon Resonance Imaging

Philippe Guedon,^† Thierry Livache,^‡ Françoise Martin,^† Frédéric Lesbre,^‡ André Roget,^‡ Gérard Bidan,^‡ and Yves Levy*^†

Laboratoire Charles Fabry de l'Institut d'Optique Thorique et Applique, UMR8501, Bat. 503, BP147, 91403 Orsay Cedex, France, and Dpartement de Recherche sur la Matire Condense, LEMSI, UMR 5819 (CEA, CNRS, Universit J. Fourier), CEA-Grenoble 17 Rue des martyrs, 38054 Grenoble Cedex 09, France

Anal. Chem., 2000, 72 (24), pp 6003–6009

DOI: 10.1021/ac000122+

Publication Date (Web): November 14, 2000

†

Laboratoire Charles Fabry de l'Institut d'Optique Théorique et Appliquée.

‡

CEA, CNRS, Université J. Fourier.

To whom correspondence should be addressed: (phone) 33-1 69 35 87 95; (fax) 33-1 69 35 87 00; (e-mail) yves.levy@iota.u-psud.fr.

Abstract

We describe in this paper a methodology to quantify multispot parallel DNA hybridizations and denaturations on gold surfaces by using, on one hand, a polypyrrole-based surface functionalization based on an electrospotting process and, on the other hand, surface plasmon resonance imaging allowing real-time measurements on several DNA spots at a time. Two characterization steps were performed in order to optimize the immobilization of oligonucleotide probes and, thus, to increase the signal-to-noise ratio of monitored hybridization signals: the first step consisted of characterizing the signal dependence upon the density of immobilized 15-mer probes, and, the second step, in analyzing the hybridization response versus spot thickness. We further demonstrated that a surface density of polypyrrole/DNA probes of 130 fmol/mm² (590 pg/mm²) optimizes the hybridization signal that can be detected directly. Optimal thickness of the spot was found to be close to 11 nm. Specificity and regeneration steps on each spot have also been demonstrated successfully, showing this method to be very competitive and convenient in use.