A Peptide Microarray-Based Fluorescent and Resonance Light Scattering Assay for Screening Thrombin Inhibitor

Abstract

A peptide microarray-based fluorescent and resonance light scattering (RLS) two readout assay has been developed for screening thrombin inhibitors in blood samples. In this assay, the biotinylated peptide microarray was used as the reaction platform. The peptide C-terminal fragments carrying biotin sites departed from the slide when the biotinylated peptides were digested by thrombin hydrolysis reaction. The hydrolysis progress was labeled by fluorescent and 30-nm peptide-stabilized gold nanoparticles (GNPs) through the biotin-avidin interaction. In the presence of thrombin inhibitors, the hydrolysis reactions were blocked, and the inhibition capability of inhibitors could be detected by detecting fluorescent and RLS signal changes. The half maximal inhibitory concentration (IC50) of thrombin inhibitors in pure thrombin solution and spiked human serum was in order of argatroban < human antithrombin-III (HAT-III) < AEBSF. The absolute differences of IC50 between pure thrombin solution and spiked human serum increased with the decreasing inhibition specificity. The order of the inhibition activities of five compounds (7.5 μM) in human plasma was: argatroban > HAT-III > trypsin inhibitor > E-64 > AEBSF. The reversible or irreversible characters of argatroban and HAT-III have been estimated in human plasma. Compared with experimental data of fluorescent and RLS assay in blood sample, the RLS assay labeled by 30-nm GNPs is proven to be more suitable for the inhibitor detection in complex blood sample.