Internally Calibrated Electrochemical Enzyme Assay

The majority of existing enzyme assays rely on changes in the optical properties of enzyme solution. However, such assays often require auxiliary enzymes and/or toxic chromogenic agents involve a large number of liquid-handling steps require a time-consuming incubation have a limited utility in turbid solutions Researchers at UT San Antonio have developed a simple, reliable and cost effective, electrochemical assay for fast quantification of enzyme activity. The assay requires only a small amount of enzyme to quickly determine its activity with no need for the enzyme-based external calibration or the reactivation of electrode surface. This assay is amenable to automation and miniaturization and is well suited for the fast analysis of commercial batches of enzymes, quantification of enzymes. The assay is performed in the constant-potential amperometric mode in a stirred solution of enzyme’s substrate. It requires three solutions only to quantify the enzyme activity: 1. A solution of enzyme’s substrate. 2. A solution of redox active species participating in enzymatic reaction. 3. A solution of assayed enzyme. The assay involves only three simple steps: 1. Record a baseline current in solution A. 2. Known aliquots of solution B are added to solution A to record current steps. 3. Add Solution C and trigger enzymatic reaction and record an angled current-time segment, which is calculate the enzyme activity. Typically, such “current steps-current slope” amperometric trace is recorded in a short 5-minute experiment. Robert Graham (210) 458-8139

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