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

Khaled Mahmoud, John H T Luong

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71 Citations (Scopus)

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 (Ki) attested that lopinavir/ritonavir (Ki = 20 ± 3 pM) and saquinavir (Ki = 57 ± 8 pM) even at 10 pM competed strongly with pepstatin for effective binding to HIV-1 PR. Indinavir (Ki = 630 ± 22 pM) only competed well with pepstatin at a much higher concentration (1 nM). No significant inhibitory effect was observed for the fosamprenavir (Ki = 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.

Original languageEnglish
Pages (from-to)7056-7062
Number of pages7
JournalAnalytical Chemistry
Volume80
Issue number18
DOIs
Publication statusPublished - 15 Sep 2008
Externally publishedYes

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Carbon Nanotubes
Single-walled carbon nanotubes (SWCN)
Electric Impedance
Nanoparticles
Screening
Electrodes
Peptides
Assays
HIV Protease Inhibitors
Dielectric Spectroscopy
Protease Inhibitors
Electrochemical impedance spectroscopy
Gold
Saquinavir
Lopinavir
Indinavir
Ritonavir
Prodrugs
Sulfhydryl Compounds
Immobilization

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

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title = "Impedance method for detecting HIV-1 protease and screening for its inhibitors using ferrocene-peptide conjugate/Au nanoparticle/single-walled carbon nanotube modified electrode",
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 (Ki) attested that lopinavir/ritonavir (Ki = 20 ± 3 pM) and saquinavir (Ki = 57 ± 8 pM) even at 10 pM competed strongly with pepstatin for effective binding to HIV-1 PR. Indinavir (Ki = 630 ± 22 pM) only competed well with pepstatin at a much higher concentration (1 nM). No significant inhibitory effect was observed for the fosamprenavir (Ki = 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.",
author = "Khaled Mahmoud and Luong, {John H T}",
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