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

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 approximately 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.

Versatile analysis of multiple macromolecular interactions by SPR imaging: application to p53 and DNA interaction

“…Surface plasmon resonance (SPR) imaging is a label-free, surface-sensitive spectroscopic technique that can be used to detect the binding of DNA, RNA, and proteins onto arrays of probe biomolecules covalently attached to chemically modified gold surfaces (Rich and Myszka, 2000). Our previous work focused on the creation and characterization of robust surface chemistries for the attachment of various biomolecules onto chemically modified gold surfaces with a novel methodology based on polypyrrole-based surface functionalization (Guedon et al, 2000). We also showed that multispot parallel DNA hybridization and denaturation could also be detected (Livache et al, 2003), unlike the commercially available instruments that are limited to four channel detection.…”

“…In step 1, the gold-coated prism is functionalized at different specific locations in the form of circular spots. On each spot, the specific single-strand zip-code oligonucleotide linked to a pyrrole residue (the probe) is electropolymerized on the gold surface by the methods described by Guedon et al (2000) leading to covalent binding on the chips. Some spots were created with free pyrrole used as negative control.…”

“…The chips were made by electrochemical copolymerization of each pyrrole-functionalized zip-code (Zip-Pyr) using the electrospotting method as previously described (Guedon et al, 2000). Briefly, the electrochemical cell, that is, a pipette tip with a platinum wire, was filled with a solution containing 20 mM pyrrole in a saline buffer and various concentrations of Zip-Pyr.…”

Plasmonic Conducting Polymers for Heavy Metal Sensing

“…Guedon et al, [20], reported a DNA hybridization sensor based on ppy-ODN matrix coupled with SPR imaging. They prepared SPR chip by electrospotting pyrrole and pyrrole linked to an oligonucleotide (ODN).…”

Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

“…SPRI detects the specific binding of unlabeled biomolecules to arrayed capture molecules attached to a modified gold thin film by measuring changes in the local index of refraction upon adsorption (11). It has been proven to be a valuable tool for investigating molecular interactions without the use of any fluorescent or radiochemical labels (4,(6)(7)(8). Use of the SPR principle can be further expanded to the detection of microbial cells with immobilized antibodies or protein ligands on an SPR sensor chip (9,12).…”