Sensitive detection of acid phosphatase enzyme and screening of inhibitors using an anionic polyfluorene derivative

Abstract

An assay method, that includes a ferric iron bound to a novel anionic water soluble polymer (P1), is developed, for the continuous and real time fluorescent amplification detection of acid phosphatase (ACP) enzyme activity under acidic conditions in nanomolar quantities. The ferric iron bound P1 offers a sensitive and rapid turn on assay for inorganic phosphate (Pi) selectively. The most frequently used phosphatase substrate p-nitro phenyl phosphate (p-NPP), which was inactive towards the dequenching of P1–Fe³⁺ fluorescence, has been utilized in the study as a model compound for the enzymatic hydrolysis and a very small concentration of enzyme in nanomolar regime was sufficient to generate a significant fluorometric change during enzymatic hydrolysis. Kinetic parameters were derived by observing the fluorometric changes of P1 during enzymatic hydrolysis providing vital information on the changes observed in the enzyme kinetics in a competitive environment. Additionally, this assay also offers high throughput screening of ACP inhibitors that may render the usefulness of this method in drug discovery.