Detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode

Abstract

A novel electrochemical method, termed flash chronopotentiometry (FCP), is used to develop a rapid and sensitive method to detect protease activities. In this method, an appropriate current pulse is applied across a polycation-selective polymer membrane to induce a strong flux of the polycationic peptides from the sample phase into the organic membrane of the electrode. During this current pulse, the cell potential (EMF) is monitored continuously, and is a function of the polypeptide concentration. The imposed current causes a local depletion of the polypeptide at the sample/membrane interface, which yields a drastic potential change in the observed chronopotentiogram at a characteristic time, called the transition time (τ). For a given magnitude of current, the square root of τ is directly proportional to the concentration of the polypeptide. Proteases cleave polypeptides into smaller fragments that are not favorably extracted into the membrane of the sensor. Therefore, a decrease in the transition time is observed during the proteolysis process. The degree of change in the transition time can be correlated to protease activity. To demonstrate this approach, the activities of trypsin and α-chymotrypsin are detected using protamine and synthetic polycationic oligopeptides that possess specific cleavage sites that are recognized by these proteases.

Selectivity Enhancement for Chloride Ion by In(III)‐Porphyrin‐Based Polymeric Membrane Electrode Operated in Pulsed Chronopotentiometric Mode

“…It is important to note that this approach can also be utilized in a different measurement mode, termed flash chronopotentiometry, where dilute primary ions are locally depleted at the membrane surface to yield a dramatic break point in the resulting chronopotentiograms [21–24]. However, in the present example, the goal is to use the current pulse to deplete the lower concentration of a strongly interferent anion, not the target analyte.…”

In vitro methods for testing antiviral drugs

“…Some recently described arrangements employ nanoparticles (Feltrup and Singh, 2012;Khalilzadeh et al, 2016;Udukala et al, 2016;Wang et al, 2014;Zeng et al, 2015) or quantum dot bioconjugates (Lee and Kim, 2015;Li et al, 2014;Medintz et al, 2006) with immobilized fluorescently or luminescently labeled peptide substrates. Alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (Hu et al, 2015;Joshi et al, 2017;Lathia et al, 2011;Rumlová et al, 2003), analytical HPLC (Teruya et al, 2016), or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (Gemene and Meyerhoff, 2011;Han et al, 1996). To study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, X-ray or NMR structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of HIV-1 [reviewed in (Ghosh et al, 2016)], HCV (Yilmaz et al, 2016), and MERS .…”

Detection of Sub-Nanomolar Concentration of Trypsin by Thickness-Shear Mode Acoustic Biosensor and Spectrophotometry

“…Peptidases, more frequently referred to as proteases, are a group of enzymes that irreversibly hydrolyze a peptide bond in an amino acid sequence through the nucleophilic attack and subsequent hydrolysis of a tetrahedral intermediate. They play critical roles in biological and physiological processes such as blood clotting, digestion, and a variety of cellular activities [ 1 , 2 ]. Proteases are highly involved in the dairy industry as well, where their activity is directly linked to the shelf life of dairy products [ 3 ].…”