Hybridization assay at a disposable electrochemical biosensor for the attomole detection of amplified human cytomegalovirus DNA

A disposable electrochemical biosensor for the detection of DNA sequences r elated to the human cytomegalovirus (HCMV) is described, The sensor relies on the adsorption of an amplified human cytomegalovirus DNA strand onto the sensing surface of a screen-printed carbon electrode, and to its hybridiza tion to a complementary single-stranded biotinylated DNA probe. The extent of hybrids formed was determined with streptavidin conjugated to horseradis h peroxidase. The peroxidase label was indirectly quantified by measuring t he amount of the chromophore and electroactive product 2,2'-diaminoazobenze ne generated from the o-phenylenediamine substrate, The intensity of differ ential pulse voltammetric peak currents resulting from the reduction of the enzyme-generated product was related to the number of target HCMV-amplifie d DNA molecules present in the sample, and the results were compared to tho se obtained with standard methods, i,e,, agarose gel electrophoresis quanti fication and colorimetric hybridization assay in a microtiter plate. A dete ction limit of 0.6 amol/ml of HCMV-amplified DNA fragment was obtained with the electrochemical DNA biosensor. The electrochemical method was 23,000-f old more sensitive than the gel electrophoresis technique and 83-fold more sensitive than the colorimetric hybridization assay in a microtiter plate. (C) 2000 Academic Press.