Colorimetric detection of alkaline phosphatase based on the off-on effect of light-responsive oxidase mimicking activity of covalent organic framework (Cu-TpBpy-COF) under near-neutral condition

Abstract

A colorimetric strategy has been developed for the detection of alkaline phosphatase (ALP) activity based on the off-on effect of the catalytic activity of light-responsive oxidase mimics covalent organic framework (Cu-TpBpy-COF) in near-neutral condition. Cu-TpBpy-COF can effectively catalyze the oxidation of the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to form a blue oxidized product (oxTMB) with an absorption peak at 652 nm. Cu²⁺ is the active center of Cu-TpBpy-COF and pyrophosphate (PPi) can form a complex with Cu²⁺ to weaken the catalytic activity of Cu-TpBpy-COF. In the presence of ALP, PPi is hydrolyzed into orthophosphates (Pi) with low affinity to Cu²⁺, thus resulting in absorbance restoration. The absorbance at 652 nm is related to ALP activity in the linear range 10-150 U·L^-1 with a detection limit of 7.17 U·L^-1. The recoveries of ALP in serum samples are in the range 94.7~107.0% with relative standard deviations (RSD) lower than 5%. The decisive role of Cu²⁺ on the enhancing catalytic activities of Cu-TpBpy-COF in neutral condition was verified by TpBpy-COF and TpBD-COF as controls, in which the main difference between them is that TpBpy-COF contains pyridine nitrogen. Upon Cu²⁺ modification, Cu-TpBpy-COF has better catalytic activity than TpBpy-COF in a broader pH range because of the in situ generation of Cu⁺ under irradiation.

Keywords: Alkaline phosphatase; Colorimetry; Light-responsive oxidase mimics.