A Colorimetric/Fluorescence Dual Detection for Acid Phosphatase Based on Copper Nanoclusters

A simple and effective fluorescent/colorimetric dual detection was established toward acid phosphatase (ACP). Photoluminescent copper nanoclusters with blue emission were synthesized. These nanoclusters were also found with peroxidase-like activity and capable of accelerating the conversion of 3,3',5,5'-tetramethylbenzidine (TMB) into a colored oxidized form (oxTMB). As a strong ligand toward copper, pyrophosphate (PPi) inhibited this catalysis. When ACP was present, it hydrolyzed PPi into non-binding phosphate, and thus the inhibited catalysis was recovered. This brought in an increase in absorbance from oxTMB for a colorimetric signaling. Meanwhile, due to a good overlap of the emission of the CuNCs and absorption of oxTMB, the fluorescence signal was attenuated through inner filter effect (IFE). Both the increase of the absorption and decrease in fluorescence were applied for the quantification of ACP activity. The colorimetric signals provided a linear response toward ACP in the range 0.1-10 U/L, with a detection limit of 0.01 U/L, while the fluorimetry responded within 0.005-10 U/L, with a detection limit of 0.001 U/L. Because the same reagents and steps were applied, the method allowed a facile switching between these two modes, depending the performances required and instrumentations available. The applicability of the detection was validated through analysis of human serum samples.