Enzyme Affinity Assays Involving a Single-Use Electrochemical Sensor. Applications to the Enzyme Immunoassay of Human Chorionic Gonadotropin Hormone and Nucleic Acid Hybridization of Human Cytomegalovirus DNA

A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode adapted to the bottom of a polystyrene microwell has been developed and applied to two bioaffinity assays, i.e., a two-site heterogeneous enzyme immunoassay of human chorionic gonadotropin hormone (hCG) and a DNA enzyme hybridization assay of an oligonucleotide sequence related to the human cytomegalovirus (hCMV). In each case, the alkaline phosphatase label was used to hydrolyze the monoester phosphate salt of [(4-hydroxyphenyl)aminocarbonyl]-cobaltocenium. This anionic substrate is transformed into a cationic electroactive product, which is then accumulated by ion-exchange at the electrode surface to give an amplified electrochemical response. Detection limits of 100 mIU mL–1 and 10 amol mL–1 were thus achieved for hCG and amplified hCMV DNA, respectively. The electrochemical approach competes favorably with the conventional absorption spectrophotometry using p-nitrophenyl phosphate as the substrate, since it was 10 to 40-fold more sensitive.