Coupling of membrane-immobilized enzyme reaction and heteropoly acid luminol chemiluminescence reaction for the determination of adenosine-5'-triphosphate

A method based on the combination of a membrane-immobilized enzyme reactor with a flow-injection (FI) chemiluminescence (CL) detector was developed and applied to the determination of >10 nM adenosine-5'-triphosphate (ATP). Alkaline phosphatase from Escherichia coli was immobilized on a nitrocellulose membrane by adsorption. When a sample of ATP was circulated in this enzyme reactor, orthophosphate was produced from alkaline phosphatase-catalyzed hydrolysis of ATP. In the FI system, the enzymatically generated phosphate in the sample solution was led into the loop of an injection valve followed by its conversion into molybdovanadophosphoric acid by mixing with a reagent mixture containing ammonium molybdate, ammonium metavanadate and sulfuric acid. The heteropoly acid subsequently reacts with luminol in a basic medium to produce CL, proportional to ATP concentration. A detection limit (DL) of 10 nM and a dynamic range extending from the DL to 10 mu M were obtained for ATP. The relative standard deviation for five replicate measurements of 1.0 mu M ATP was 6.6%.