Colorimetric and Fluorometric Detection of Nucleic Acids Using Cationic Polythiophene Derivatives

Abstract

Simple and reliable sequence-specific methods are needed for the rapid detection of oligonucleotides, to diagnose infections and various genetic diseases. In this regard, interesting optical and electrochemical DNA-hybridization sensors have been proposed.[1±5] The recognition capabilities of DNA are well established but, to transduce the recognition event into a physically measurable value, a fluorescent or electroactive tag is often bound to the analyte. Electrochemical and optical sensors based on conjugated polymers have also been reported[6±9] and some oligonucleotide-functionalized conjugated polymers can also transduce hybridization events into an electrical signal without labeling of the oligonucleotide target.[10±12] The detection relies on a modTo our knowledge this is the first report on the use of singlemolecule atomic-force spectroscopy to study the reduction pathway of multiple disulfide bonds in proteins and to evaluate the distributions of intermediates obtained under different reducing conditions without separating them and without any blocking and fractionation steps. The characterization of these intermediates has so far been accomplished by first blocking them with reagents such as alkylalkanethiosulfonates and then by fractionation by ion-exchange chromatography, 2D or capillary gel electrophoresis, or gel filtration.[11] The determination of thiol groups and disulfide bonds in a polythiol systems has always been a very challenging problem.[12] The single-molecule force-spectroscopy data presented here show: 1) how a redox environment can modulate the mechanical properties of angiostatin; 2) how this modulation relies, at the single-molecule level, on the extent of reduction of the disulfide bonds; and 3) how, at the level of a large sample of molecules, the distribution of the different thiol/ disulfide intermediates after reduction can be estimated by statistical analysis of the force curves.