Nanomolar Level Amperometric Determination of ATP Through Substrate Recycling in an Enzyme Reactor in a FIA System

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Abstract
ATP, adenosine-5′-triphosphate, was determined by recycling in an enzyme reactor with co-immobilized pyruvate kinase and hexokinase in the presence of glucose, NAD+, and phosphoenolpyruvate, PEP. Recycling produces glucose-6-phosphate which is converted to an equivalent amount of NADH by glucose-6-phosphate dehydrogenase. The NADH is detected at a graphite flow-through electrode modified with an adsorbed 3,3′-bis(benzo[a]phenoxazin-7-ium, 5-amino-9-(diethylamino))1,4,N,N′-diamidobenzene, BPT. Oxidation of NADH takes place at 0 mM vs Ag/AgCl due to the adsorbed phenoxazine. The amplification factor is directly proportional to the residence time in the reactor and it is increased as the flow rate decreases; it becomes350 at a flow rate of 0.07 ml/min. The amplification factor can be increased further by a controlled stop-time recycling; it became 1200 at a stop-time of 12 min. A theoretical expression for the amplification factor was derived and it shows that the amplification depends o n the residence time and the pseudo-first order rate coefficients of the recycling enzyme systems. The response was linear over more than three decades, from 1 nM to 5 μM ATP. The detection limit, 1 nM ATP was set by cofactor impurities in the reagent rather than by system sensitivity or noise.