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

AuNP-mediated oriented immobilization enhances AChE loading capacity by 8.6-fold. • Rotating-splicing design improves operational convenience and enzyme reusability. • AuNPs@enzyme OIMERs show superior performance: K m = 0.051 ± 0.001 mM and RSD < 1.0%. • AuNPs@enzyme OIMERs retain 87.0% after 80 runs and 93.3% after 40 days storage. • System enables bioassay-guided fractionation via visual/quantitative evaluation. Background 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. Results 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 (IC 50 ) values of seven compounds from natural products were determined, correlating well with the reference UV method, confirming the reliability for practical inhibitor screening. Significance 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. Download: Download high-res image (291KB) Download: Download full-size image