Fast electrochemical detection of anti-HIV antibodies: coupling allosteric enzymes and disk microelectrode arrays

Here a novel electrochemical method for the rapid detection of anti-HIV antibodies in serum is presented. The novelty lies in the combination of allosteric enzymes and coulometry to yield a fast, simple and reliable HIV diagnostic method. We have used a previously developed beta-galactosidase enzyme that is efficiently activated by anti-HIV antibodies directed against a major B-cell epitope of the gp41 glycoprotein. When these antibodies bind the enzyme, the 3D conformation changes positively affecting the performance of the active site and, consequently, the enzyme activity is stimulated. Using 4-aminophenyl beta-D-galactopyranoside (PAPG) as substrate yields p-aminophenol (PAP), which is reversibly oxidised at a very mild potential, ca. 0.37 V vs. Ag/AgCl over a range of electrode materials within the working pH range of beta-galactosidase. In the present case, photolithographically produced microelectrode arrays resulted in a detection limit of 4 microM for 4-aminophenol (PAP). The presence of anti-HIV antibodies results in enzyme activity increases above 50% which, combined with the sensitivity and response time afforded by the microelectrode arrays, allowed for the diagnosis of HIV in sera samples within an hour.