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

Analytical Chemistry
Khaled A Mahmoud, John H T Luong

Abstract

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 observ...Continue Reading

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