"Apple tree"-like AuNPs@enzyme microreactor: A reusable microfluidic paper-based analytical device for improved acetylcholinesterase inhibitors screening

Acetylcholinesterase (AChE) inhibitors are key therapeutic agents for neurological disorders such as Alzheimer's disease, yet conventional screening of natural product inhibitors are often time-consuming, instrument-dependent, and limited in reusability. To address this, we developed a simple, colorimetric platform based on a reusable microfluidic paper-based analytical device integrated with Au nanoparticle (AuNPs)-mediated enzyme microreactors, coupled with smartphone-based RGB values (blue (B), green (G), and red (R) channel) analysis for rapid and on-site screening of AChE inhibitors from natural products. The microreactors feature an "apple tree"-like architecture, in which AuNPs serve as the "fruits" enabling high-density oriented enzyme immobilization, enhanced substrate accessibility, and excellent reusability.

AChE was oriented immobilized on concanavalin A-modified AuNPs surface via affinity self-assembly, achieving an 8.6-fold increase in enzyme loading compared to direct adsorption. The immobilized enzyme retained 87.0% of its initial activity after 80 consecutive assays, demonstrating excellent reusability and stability. The method showed superior repeatability with intraday, interday, and batch-to-batch relative standard deviations (RSD) of the colorimetric readouts all below 1.0%. The system enabled visual, quantitative evaluation during bioassay-guided fractionation, substantially reducing unnecessary separation steps. Moreover, half-maximal inhibitory concentration (IC₅₀) values of seven compounds from natural products were determined, correlating well with the reference UV method, confirming the reliability for practical inhibitor screening.

This platform offers a cost-effective, reusable, and user-friendly tool for early-stage screening of AChE inhibitors in natural extracts, with potential to accelerate the discovery of lead compounds for neurodegenerative disease therapy while minimizing resource consumption and analytical workload.