Northern Kentucky University

Simple and Inexpensive Electrochemical Detection of Proteolytic Enzymes and Their Inhibitors

Institution

Northern Kentucky University

Abstract

Proteolytic enzymes function by cleaving proteins at specifically and involve in all cellular activities. The detection of these enzymes and their inhibitors are critical for disease diagnosis and therapeutic processes. A number of techniques have been used for the assay of the activities of these enzymes and their inhibitors. The most common methods are spectroscopy with synthetic chromogenic and fluorogenic substrates. However, although these methods are sensitive, they cannot be reliably used in highly colored and turbid samples such as whole blood. Potentiometric ion-responsive electrodes have also been used for this purpose. But due to the irreversible nature of their response, these are limited to single use and are not capable of continuous monitoring. We report here a reversible electrochemical method, pulsed chronopotentiometry for detection of enzyme activity. This method works on the fact that the sensor gives very sensitive response to the bigger molecules (proteins or synthetic oligopeptides), while it is much less sensitive to smaller fragments. In this method, the response to the peptide (voltage/potential) was first measured. Then, the enzyme was added to the peptide solution. The concentration of the original peptide decreased as it was broken into smaller fragments by the enzymes and the potential response of the sensor, which is a function concentration of the peptide decreases proportionally. This change in potential was monitored as a function of enzymatic reaction time and used as the analytical signal for the assay of enzyme activity. Here we detected the activity of the enzyme thrombin using synthetic peptides as substrates.

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Simple and Inexpensive Electrochemical Detection of Proteolytic Enzymes and Their Inhibitors

Proteolytic enzymes function by cleaving proteins at specifically and involve in all cellular activities. The detection of these enzymes and their inhibitors are critical for disease diagnosis and therapeutic processes. A number of techniques have been used for the assay of the activities of these enzymes and their inhibitors. The most common methods are spectroscopy with synthetic chromogenic and fluorogenic substrates. However, although these methods are sensitive, they cannot be reliably used in highly colored and turbid samples such as whole blood. Potentiometric ion-responsive electrodes have also been used for this purpose. But due to the irreversible nature of their response, these are limited to single use and are not capable of continuous monitoring. We report here a reversible electrochemical method, pulsed chronopotentiometry for detection of enzyme activity. This method works on the fact that the sensor gives very sensitive response to the bigger molecules (proteins or synthetic oligopeptides), while it is much less sensitive to smaller fragments. In this method, the response to the peptide (voltage/potential) was first measured. Then, the enzyme was added to the peptide solution. The concentration of the original peptide decreased as it was broken into smaller fragments by the enzymes and the potential response of the sensor, which is a function concentration of the peptide decreases proportionally. This change in potential was monitored as a function of enzymatic reaction time and used as the analytical signal for the assay of enzyme activity. Here we detected the activity of the enzyme thrombin using synthetic peptides as substrates.