Fluorescence dual “turn-on” detection of acid phosphatase via fluorescence resonance energy transfer and dual quenching strategy to improve sensitivity

A fluorescent dual “turn-on” method is utilized to detect acid phosphatase (ACP) via dual quenching and fluorescence resonance energy transfer (FRET) strategy to improve sensitivity for the first time. Bovine serum albumin protected gold nanoclusters (BSA-AuNCs) are utilized as fluorescence probe and peroxidase-mimicking catalyst. BSA-AuNCs oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to TMB (oxTMB) with the absorbance peak at 652 nm. Fluorescence spectrum of BSA-AuNCs with emission peak at 635 nm is largely overlapped with absorption spectrum of oxTMB, which brings FRET from BSA-AuNCs to oxTMB and fluorescence is quenched. When Ce3+ is added, BSA-AuNCs catalyze the oxidation of Ce3+ to Ce4+ by H2O2. Ce4+ has oxidizing properties, which can oxidize TMB into oxTMB and enhance FRET. It achieves the fluorescence is quenched further more. This dual fluorescence quenching effect brings a low background for ACP detection. ACP catalyzes ascorbic acid 2-phosphate (AA2P) hydrolysis into phosphate ion (PO43−) and ascorbic acid (AA). Owing to high coordination between Ce3+ and PO43−, TMB oxidation is prevented and fluorescent is turned-on. AA reduces oxTMB into TMB, which makes FRET be prevented and fluorescence is also increased. Therefore it is utilized in ACP detection from 0.01 to 2 U/L with high selectivity and accuracy.