Electrochemical detection of PCR amplicons using electroconductive polymer modified electrode and multiple nanoparticle labels

We report the utilization of an oligonucleotide-functionalized electroconducting polymer, coupled with a nanoparticle-based hybridization indicator for a sensitive detection of multi-labeled PCR amplicons. The electroconducting polymer permits the immobilization of an active probe onto the transducer surface. In this work, 2-aminobenzoic acid is electropolymerized onto an indium tin oxide electrode. The coated film has carboxyl groups for the immobilization of amino-terminated probe, mediated by coupling agents of water-soluble carbodiimide and N-hydroxysulfosuccinimide. This coupling chemistry has minimal nonspecific adsorption of the gold nanoparticle indicator, leading to a low background signal allowing a more sensitive hybridization detection. Signal transduction of the hybridization event is achieved through a series of steps: i) the binding of the gold nanoparticle label to the hybridized target based on streptavidin-biotin interaction, ii) the catalytic silver deposition onto the gold label (i.e., the silver enhancement) and iii) potentiometric stripping analysis of the deposited silver. In addition to signal amplification by silver enhancement, multiple-labeling of the amplicons by the incorporation of biotin-16-dUTP is employed to further enhance the signal to background ratio. The sequence-specific identification of both symmetric and asymmetric PCR amplicons is also demonstrated.