Alkaline phosphatase determination via regulation of enzymatically generated poly(thymine) as a template for fluorescent copper nanoparticle formation

We propose a new fluorometric method for alkaline phosphatase (ALP) determination. This method is based on the regulation of enzymatically generated poly(thymine) for the preparation of copper nanoparticles (CuNPs). 2′-Deoxythymidine 5′-triphosphate (dTTP) serves as the source for polymerization mediated by terminal deoxynucleotidyl transferase (TdT). This process generates poly(thymine), which acts as the template for synthesis of fluorescent CuNPs. However, if ALP catalyzes the hydrolysis of dTTP, the TdT-mediated polymerization will be disabled. This prevents the formation of CuNPs and causes a drop in fluorescence. The findings were used to design a sensitive and selective fluorometric method for ALP determination. A linear response in the activity range from 0.1 to 20 U L-1 and a limit of quantification of 0.3 U L-1 were obtained. The results indicate that the proposed method can be successfully applied to ALP assay in spiked diluted serum. This demonstrates the method’s reliability and practicability.