Ratiometric fluorescence and colorimetric strategies for assessing activity of butyrylcholinesterase in human serum using g-C₃N₄ nanosheets, silver ion and o-phenylenediamine

Abstract: Ratiometric fluorescence and colorimetric strategies for detecting activity of butyrylcholinesterase (BChE) in human serum were developed by using g-C₃N₄ nanosheets, silver ion (Ag⁺) and o-phenylenediamine (OPD) as chromogenic agents. The oxidation-reduction reaction of OPD and Ag⁺ generates 2,3-diaminophenazine (oxOPD). Under exciation at 370 nm, g-C₃N₄ nanosheets and oxOPD emit fluorescence at 440 nm (F₄₄₀) and 560 nm (F₅₆₀), respectively. Additionally, oxOPD exhibits quenching ability towards g-C₃N₄ nanosheets via photoinduced electron transfer (PET) process. Thiocholine (TCh), as a product of BChE-catalyzed hydrolysis reaction of butylthiocholine iodide (BTCh), can coordinate with Ag⁺ intensively, and consequently diminish the amount of free Ag⁺ in the testing system. Less amount of free Ag⁺ leads to less production of oxOPD, resulting in less fluorescence quenching towards g-C₃N₄ nanosheets as well as less fluorescence emission of oxOPD. Therefore, by using g-C₃N₄ nanosheets and oxOPD as fluorescence indicators, the intensity ratio of their fluorescence (F₄₄₀/F₅₆₀) was calculated and employed to evaluate the activity of BChE. Similarly, the color variation of oxOPD indicated by the absorbance at 420 nm (ΔA₄₂₀) was monitored for the same purpose. These strategies were validated to be sensitive and selective for detecting BChE activity in human serum, with limits of detection (LODs) of 0.1 U L⁻¹ for ratiometric fluorescence mode and 0.7 U L⁻¹ for colorimetric mode. Graphical abstract: (Figure presented.).