New insight at determining G6PD activity: Selective enzyme biosensor

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that catalyzes the first step of the pentose phosphate pathway (PPP). The only source for NADPH formation in erythrocytes is the PPP, and NADPH production is significantly reduced in G6PD deficiency. Current G6PD activity measurement methods can determine the enzyme level as zero. However, the absolute zero activity of the "housekeeping" enzyme, G6PD, is incompatible with life. In order to clarify this situation, we designed a selective enzyme biosensor system that can precisely determine the enzyme level. The experiment group consisted of cases whose G6PD activity was measured as zero or low. In our study, a triple electrode system was used. Gold was used as the working electrode, the platinum wire was used as the counter electrode, and Ag/AgCl was used as the reference electrode. The bioactive layer was formed with both BSA/Gelatin and HEMA-MAC polymers. SEM image of the bioactive layer was taken, and FTIR analysis was performed. The glutathione reductase enzyme was immobilized on the gold electrode to use the NADPHs, formed after the G6PD enzyme activity, as a substrate. Crosslinking was performed with glutaraldehyde. In biosensor, the optimization of the bioactive layer, the immobilization conditions and the working conditions of the enzyme were done. The potential difference that emerged when NADPH was oxidized to form NADP was measured with a Differential Pulse Voltammetry (DPV). Precise measurement of G6PD level was made using the standard chart. In our study, by using the selective enzyme biosensor system, the actual values of the G6PD activity levels of the cases, which were measured as zero by traditional methods, were determined. It was revealed that the G6PD activity was not absolute zero due to the DPV measurements obtained. Thus, the activity of G6PD, the most common enzymopathy in the world, was measured more sensitively using a biosensor system. Predictably, this selective enzyme biosensor design will contribute significantly to the diagnosis and follow-up of patients with erythrocyte G6PD enzyme deficiency.