Determination of minute amounts of ATP by flow injection analysis using enzyme amplification reactions and...

A flow-injection system for assay of trace levels of ATP is described that incorporates a small column reactor containing co-immobilized hexokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase. In the presence of appropriate cofactors, ATP is by the synergistic operation of the enzymes repeatedly recycled, resulting in substrate amplification. The ultimately generated NADH is measured fluorometrically. By this approach, where the enzymatic degradation step and the detection step are completely separated, it is possible to operate them individually under optimal conditions. The amplification factor is directly proportional to the residence time of the sample zone within the enzyme reactor, which time might be manipulated by altering the flow-rate and in the extreme by performing stopped-flow experiments. Amplification factors between 15 and 1000 were obtained, but it was found that increased amplifications did not lead to significantly lower detection limits; thus, it appears that a practical lower limit of detection is of the order of 1-5 nM. An investigation of this paradoxical feature, and a possible explanation for it, is given.