Sensitively Detecting Recombinant Hirudin Variant-2 with Capacitive Immunoassay Based on Self-Assembled Monolayers

Abstract

Recombinant hirudins have a definite role in patients with heparin-induced thrombocytopenia, and prevent coronary thrombosis and recurrences. Here, capacitive immunosensors based on self-assembled monolayers (SAMs) were developed to immobilize polyclonal recombinant hirudin antibodies on gold electrodes for detecting recombinant hirudin variant-2 (r-HV2), a low-weight protein, directly and sensitively. A fast electrochemical measurement, potentiostatic step method, was applied to measure the capacitance changes caused by the interaction between the antigens and antibodies. The insulating properties of the immunosensors were investigated by cyclic voltammetry in a buffer solution containing (Fe(CN)6)3−/4−. The detection capability of the immunosensors modified by short-chain alkanethiol, thioctic acid (TA), or mercaptoacetic acid (MAA), were compared. Based on TA SAMs, the immunosensor could detect r-HV2 in the range from 100 pg/mL to 5 ng/mL, with a detection limit of 50 pg/mL. For the MAA modified immunosensor, a linear detection range from 5 pg/mL to 1 ng/mL was obtained, with a detection limit of 2.5 pg/mL. This indicated capacitive immunosensors with thinner SAMs could detect the antigens with higher sensitivity. The success of detecting r-HV2 showed that the capacitive immunoassay might be employed in the practical application with characteristics of simplicity and sensitivity.