Colorimetric sol gel based biosensor platform for determination of reduced glutathione

Abstract

Reduced glutathione (GSH) is a biochemical and antioxidant marker in various examinations of pathological states. Though standard tests for determination GSH in biological samples are available, there are significant drawbacks. The method for determination of GSH based on enzymatic reaction applying enzyme glutathione-S-transferase (GST) was established and introduced to provide a simple but reliable platform for determination of GSH in biological samples. Sol-gel membrane was utilized for immobilization of the homogenate from pig liver containing high activity of GST. Enzymatic method was compared with currently used Ellman method. The results of experiments showed, that Ellman method as well as enzymatic sol-gel method are appropriate for the determination of GSH in tissues. Michaelis-Menten constant of the novel method was set to be 1.9mmol/l and limit of detection (LOD) of Ellman method was determined to be 30μmol/l while LOD of enzymatic method was set to be six times higher. Concerning to interferences, enzymatic sol-gel technique showed better results compared to the standard Ellman method. No interfering substances were revealed in the GST based assay. On the other hand, bovine serum albumin, ascorbic acid (AA) and N-acetylcysteine (N-AC) were confirmed as strong interferents in Ellman method. Spiked plasma samples were measured using the GST based method and their kinetic curve has very similar shape as kinetic curve of standard solutions.