Impedance method for detecting HIV-1 protease and screening for its inhibitors using ferrocene-peptide conjugate/Au nanoparticle/single-walled carbon nanotube modified electrode

A highly sensitive screening assay based on electrochemical impedance spectroscopy (EIS) has been developed for detecting HIV-1 protease (PR) and subsequent evaluation of its corresponding inhibitors at picomolar levels. The assay format was based on the immobilization of the thiol terminated ferrocene(Fc)-pepstatin conjugate on a single-walled carbon nanotube/gold nanoparticle (SWCNT/AuNP) modified gold electrode. The alteration of the interfacial properties of electrodes upon HIV-1 PR and Fc-pepstatin conjugate interaction was traced by EIS. On the basis of the charge transfer resistance data obtained and using a mixed kinetic and diffusion model, this procedure was capable of detecting picomolar HIV-1 PR owing to the specific binding of this enzyme to Fc modified pepstatin. A competitive inhibition assay format was then performed using four potent HIV-1 PR inhibitors. The estimated inhibition constant (K_i) attested that lopinavir/ritonavir (K_i = 20 ± 3 pM) and saquinavir (K_i = 57 ± 8 pM) even at 10 pM competed strongly with pepstatin for effective binding to HIV-1 PR. Indinavir (K_i = 630 ± 22 pM) only competed well with pepstatin at a much higher concentration (1 nM). No significant inhibitory effect was observed for the fosamprenavir (K_i = 11 ± 0.5 nM) as expected from this pro-drug. Such results agreed well with the values reported in the literature. This assay format is a definite asset for the expedited development of effective HIV-1 PR inhibitors with low molecular weights.