Highly selective and sensitive detection of NADP coenzymes using co-immobilized glucose-6-phosphate dehydrogenase/diaphorase reactors as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system

Two enzyme reactors prepared by the co-immobilization of two different glucose-6-phosphate dehydrogenases (G6PDH; from Leuconstoc mescenteroides (LM) and yeast (Y) and diaphorase are employed to enhance the sensitivity of NAD(P) coenzymes as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system. The NAD(P) coenzymes are recycled enzymatically during passage through the reactor in the presence of sufficient glucose-6-phosphate and oxygen in the carrier solution to produce a large amount of hydrogen peroxide, which is detected chemiluminometrically in the subsequent flow line. The G6PDH(LM)/diaphorase co-immobilized reactor is not specific between the NAD and NADP coenzymes, but shows a six fold selectivity towards NADP coenzymes compared to NAD coenzymes; the amplification factors for NAD and NADP coenzymes are 60 and 380, respectively, at a flow rate of 0.3 ml min(-1). In contrast, the G6PDH(Y)/diaphorase co-immobilized reactor is specific for NADP coenzymes with an amplification factor of about 600 (at a flow rate of 0.3 ml min(-1)). The detection limit is 6 fmol for both NADP(+) and NADPH.