Pulsed Chronopotentiometric Detection of Thrombin Activity Using Reversible Polyion Selective Electrodes

Abstract

AbstractA simple and rapid electrochemical method for the detection of thrombin activity is presented here for the first time using a synthetic polypeptide substrate and a pulsed chronopotentiometry transduction protocol with polyion selective electrodes. A cathodic current applied across a polyion selective membrane electrode causes the extraction of the polypeptides from the sample into the membrane and the membrane potential, which is a function of the concentration of these polypeptides in the sample, is measured at the same time. Since the polyion extraction is a diffusion‐controlled mass transfer process, depletion of the polypeptides at the membrane‐sample interface at a characteristic transition time occurs. The square root of the transition time is directly proportional to the concentration of the polypeptide substrate according to the Sand equation. Upon addition of thrombin, the polypeptide substrate undergoes proteolysis and yields smaller peptide fragments that exhibit smaller measured electromotive force (emf) responses since they are less preferred by the membrane, and the transition times become shorter. The rate of change of the square root of the transition time is directly proportional to the change in the polypeptide concentration, which in turn relates to the polypeptide hydrolysis time, and can be used for monitoring enzyme activity. Indeed, the square root of transition time was found to be linear with the proteolysis time with R2=0.98. The activity of thrombin was determined to be 3.6 μg substrate per μg thrombin per Min.