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 rea ctor with a flow-injection (FI) chemiluminescence (CL) detector was de veloped and applied to the determination of >10 nM adenosine-5'-tripho sphate (ATP). Alkaline phosphatase from Escherichia coli was immobiliz ed on a nitrocellulose membrane by adsorption. When a sample of ATP wa s circulated in this enzyme reactor, orthophosphate was produced from alkaline phosphatase-catalyzed hydrolysis of ATP. In the FI system, th e enzymatically generated phosphate in the sample solution was led int o the loop of an injection valve followed by its conversion into molyb dovanadophosphoric acid by mixing with a reagent mixture containing am monium molybdate, ammonium metavanadate and sulfuric acid. The heterop oly acid subsequently reacts with luminol in a basic medium to produce CL, proportional to ATP concentration. A detection limit (DL) of 10 n M and a dynamic range extending from the DL to 10 mu M were obtained f or ATP. The relative standard deviation for five replicate measurement s of 1.0 mu M ATP was 6.6%.