Measurement of acetylcholinesterase activity by electrochemical analysis method utilizing organocatalytic reactions

Abstract

An electrochemical method for measuring acetylcholinesterase (AChE) activity was developed using nortropine-N-oxyl (NNO), an organocatalyst. The increase in catalytic current as NNO oxidizes choline allowed real-time monitoring of the AChE hydrolysis reaction. Compared to conventional H₂O₂-based sensors, this method eliminates one reaction step, enabling more direct and real-time monitoring of enzymatic activity. Amperometric measurements enable AChE activity determination over a range of 50-2000 U L^-1 and the limit of detection and limit of quantification in the low concentration range were calculated to be 14.1 U L^-1 and 46.9 U L^-1, respectively, with a correlation coefficient (R ²) of 0.9989. These results demonstrate that serum cholinesterase measurement using this method can be utilized for various diagnoses, such as liver and heart diseases. Furthermore, given the relevance of AChE in neurotoxicity evaluation, diagnosis of neurological disorders such as Alzheimer's disease, and environmental toxicity monitoring, this method has diverse potential applications. Moreover, this approach can be extended to other enzymatic reactions, indicating its promise for various analytical and diagnostic applications.

Fig. 1. Principle of AChE activity measurement.

Fig. 2. (A) Amperometric responses obtained using various concentrations of NNO (1 mM: black; 5 mM: blue; 10 mM: red) toward sequential additions of choline chloride at +0.6 V vs. Ag/AgCl in 100 mM phosphate buffer (pH 7.4). (B) Plots of current increase (ΔI) versus choline concentration (n = 3, mean ± SD).

Fig. 3. Amperometric responses obtained upon the addition of various analytes at 300 s into a 100 mM phosphate buffer (pH 7.4) containing 1 mM NNO. The applied potential was +0.6 V vs. Ag/AgCl. (a) Choline chloride (10 mM), black; (b) acetylcholine chloride (10 mM), blue; (c) AChE (500 U L⁻¹), green; (d) acetylcholine chloride (10 mM) and AChE (500 U L⁻¹), red.

Fig. 4. Amperometric responses obtained upon addition (at 300 s) of varying concentrations of acetylcholine chloride (0.1–10 mM) to a phosphate buffer (100 mM, pH 7.4) containing 1 mM NNO and 1000 U L⁻¹ AChE. The applied potential was +0.6 V vs. Ag/AgCl.

Fig. 5. Amperometric responses obtained after the addition of acetylcholine chloride (10 mM) at 300 s to electrolyte solutions containing 1 mM NNO and varying concentrations of AChE (30, 50, 100, 300, 500, 1000, and 2000 U L⁻¹). Measurements were performed at +0.6 V vs. Ag/AgCl in 100 mM phosphate buffer (pH 7.4).

Fig. 6. Amperometric responses obtained after the addition of acetylcholine chloride (10 mM) at 300 s to electrolyte solutions containing 0.1 mM NNO and various concentrations of AChE (50, 100, 200, 300, 500, 1000, 1500, and 2000 U L⁻¹). Measurements were performed at +0.6 V vs. Ag/AgCl in 100 mM phosphate buffer (pH 7.4).

Fig. 7. Calibration curves plotting the current increase (ΔI) versus AChE activity at (A) 10 min and (B) 60 min after the addition of 10 mM acetylcholine chloride, other conditions as in Fig. 6 (n = 3, mean ± SD).

Fig. 8. Calibration curves plotting the initial slope (for 1 min after acetylcholine chloride addition) of the current versus time curve in Fig. 6versus AChE activity, other conditions as in Fig. 6 (n = 3, mean ± SD).