Developmental aspects of amperometric ATP biosensors based on entrapped enzymes

A novel concept for a dual-enzyme-based microbiosensor for the detection of adenosine-5′-triphosphate (ATP) was developed. The employed enzymes pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and hexokinase were entrapped, using pH-shift-induced precipitation of electrodeposition paint (EDP) at platinum microelectrodes (diameter of 25 µm). PQQ-GDH is known showing a superior activity for glucose conversion at the relevant conditions (low oxygen concentration) for ATP detection in targeted biomedical studies. For immobilizing the two enzymes PQQ-GDH and hexokinase, the deposition conditions of EDP Resydrol AY498w/35WA were adapted to ensure high immobilization rates. Prior to ATP sensing, the conversion of glucose, which is the co-substrate for both enzymatic reactions, was optimized. Optimization was targeted towards ATP measurements in biomedical environments by optimizing the PQQ-GDH sensor for glucose. Therefore, different mediators were tested regarding their electron transfer rate and their compatibility with the enzyme: free-diffusing N-methylphenazonium methyl sulfate (PMS) and ferrocenemethanol, and an immobilized chromium hexacyanoferrate layer at platinum electrode. Free-diffusing ferrocenemethanol reveals high sensitivity towards glucose of 1.5?±?0.4 nA/mM. In a next step, hexokinase was co-entrapped in the polymer film resulting in a sensitivity of up to 290 pA/µM.