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

• Published in: Analytica chimica acta 2009-05, Vol.641 (1), p.1-6
• Main Authors: Laczka, Olivier, Ferraz, Rosa María, Ferrer-Miralles, Neus, Villaverde, Antonio, Muñoz, Francesc Xavier, Campo, F. Javier del
• Format: Article
• Language: English
• Subjects: Allosteric enzymes; Allosteric Site; Aminophenols - analysis; Aminophenols - chemistry; Amperometry of 4-aminophenol; Analytical chemistry; Anti-HIV antibodies; Antibodies, immunoglobulins; Beta-galactosidase; beta-Galactosidase - metabolism; Biological and medical sciences; Catalytic Domain; Chemistry; Coulometry; Disk microelectrode arrays; Electrochemical methods; Electrochemistry - methods; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Galactosides - chemistry; HIV Antibodies - blood; HIV Antibodies - immunology; HIV Envelope Protein gp41 - immunology; Human immunodeficiency virus; Microelectrodes; Molecular immunology
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2009.03.008

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 β-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 β- 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 β-galactosidase. In the present case, photolithographically produced microelectrode arrays resulted in a detection limit of 4 μM 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.