A colorimetric strategy for assay of protease activity based on gold nanoparticle growth controlled by ascorbic acid and Cu(II)-coordinated peptide

Abstract

We developed a colorimetric method for assay of protease activity through the growth of gold nanoparticles (AuNPs) with ascorbic acid (AA) as the reducing agent. The method is based on the difference in the catalytic activity of various Cu2+ species toward AA oxidation. A mutational peptide is employed as the protease substrate, in which only one amino acid residue is replaced by the histidine residue. Specifically, HAuCl4 can be reduced into AuNPs by AA; Cu2+ ion promotes the oxidation of AA under oxygen atmosphere by a redox cycling, thus preventing the formation of AuNPs. Cleavage of the substrate peptide results in the exposure of an amino terminal Cu2+ and Ni2+-binding (ATCUN) motif in the NH2-terminus of one fragment. The resultant ATCUN peptide can sequestrate Cu2+ and thus depress the catalytic oxidation of AA. The consumption of AA is monitored by UV–vis spectra and differential pulse voltammetry. The high extinction coefficient of the generated AuNPs enables the quantitative and sensitive colorimetric analysis of protease. To demonstrate the analytical performances, β-secretase was tested as a model protease. By employing peptide-functionalized magnetic beads, β-secretase in serum was determined with a satisfactory result. This work provides valuable information for designing of novel protease biosensors.