A convenient label free colorimetric assay for pyrophosphatase activity based on a pyrophosphate-inhibited Cu2+–ABTS–H2O2 reaction

Abstract

The evaluation of pyrophosphatase (PPase) activity plays an important role in diagnosing diseases and understanding the function of PPase in the related biological events. In this work, an inhibition effect of pyrophosphate (PPi) on Cu²⁺-catalyzed H₂O₂-mediated oxidation of 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) was observed. We utilize this inhibition effect to develop a convenient label free visual method for PPase activity detection. A hydroxyl radical could be generated from a Cu²⁺-based Fenton-like reaction, and then reacted with ABTS to produce colored ABTS˙⁺. The strong complexation between PPi and Cu²⁺ disturbed this Cu²⁺-catalyzed ABTS–H₂O₂ reaction probably due to changing redox potentials of Cu²⁺ towards H₂O₂. The PPase-catalyzed hydrolysis of PPi into Pi prohibited the complexation, resulting in the recovery of catalytic capability of Cu²⁺. As a result, the solution color changed from colorless to green with a remarkable increase of absorbance. Compared with the traditional PPase assays, the developed visual assay is cost-effective and easy to implement. And a high sensitivity for PPase with a detection limit of 0.027 U mL⁻¹ was achieved. Moreover, the proposed colorimetric strategy was also applied to evaluate PPase inhibition and exhibited a good assay performance in complex biological samples.