GTP as a peroxidase-mimic to mediate enzymatic cascade reaction for alkaline phosphatase detection and alkaline phosphatase-linked immunoassay

Guanosine triphosphate (GTP), a substrate for RNA synthesis, plays a pivotal role in life-form metabolism at the cellular level. Herein, we first showed that GTP exhibited an intrinsic peroxidase-mimic activity, accelerating H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to produce a color reaction. Compared to GTP, weaker catalytic activity was observed from guanosine diphosphate (GDP) and virtually no catalytic activity was observed from guanosine monophosphate (GMP) and guanosine. Alkaline phosphatase (ALP) can catalyze the dephosphorylation of GTP with in situ generation of GDP, GMP and guanosine, which followed by receding the oxidation of TMB. Inspired by this, a novel colorimetric sensor for ALP detection was designed based on the enzymatic cascade reaction by coupling ALP-catalyzed dephosphorylation of GTP with GTP-catalyzed oxidation of TMB. This method allowed ALP sensing in a range from 0.01 to 100 U/L with a detection limit of 0.009 U/L, which was sensitive enough for ALP activity assay in biological samples (the normal ALP level range of adult serum is 20-140 U/L). Moreover, ALP is widely used in enzyme-linked immunosorbent assays (ELISA) as a signal reporter, the proof-of-concept ELISA for alpha-fetoprotein (AFP) detection was established. This unexpected discovery not only extends the new biological function of GTP, but also holds great promise for development of versatile biosensors that initiate the concentration changes of GTP.