Molecularly Tailored Pd@Pt Nanozymes for Real-Time Smartphone-Assisted Acetylcholinesterase Detection and Therapeutic Inhibitor Assessment

Abstract

The detection of acetylcholinesterase (AChE) activity is of paramount importance for diagnosing neurological disorders and screening potential therapeutic inhibitors. Herein, we present a novel colorimetric biosensor for AChE activity and inhibitor detection, enabled by L-isoleucine-modified Pd@Pt nanoparticles (IlePd@Pt NPs) with enhanced peroxidase-like (POD-like) activity. Utilizing twenty-two amino acids as surface ligands, L-isoleucine was identified as the optimal modifier, achieving remarkable catalytic performance through a unique interaction between its side-chain structure and the nanoparticle surface. Molecular dynamics simulations revealed the mechanistic basis of this enhancement, providing atomic-scale insights into the reduced binding energy with substrates (H₂O₂ and TMB) and the resulting catalytic optimization. The biosensor exhibits high sensitivity, with limits of detection (LODs) of 0.205 mU/mL and 0.799 mU/mL when using a microplate reader and a smartphone, respectively. Its integration with smartphone-based analysis enables portable, cost-effective, and user-friendly point-of-care testing (POCT). Furthermore, the biosensor demonstrated robust performance in real biological samples, including human and fetal bovine serum, with excellent recovery rates. The platform also facilitated the screening of AChE inhibitors, effectively evaluating tacrine with an IC₅₀ of 14.0 nM, underscoring its potential for drug discovery and clinical applications. This work provides a significant advancement in the field of nanozyme-based biosensors by combining high catalytic activity, computational insights, and practical applicability. The proposed system offers a simplified yet powerful tool for real-time diagnostics and pharmaceutical research, addressing critical needs in healthcare and biomedical science.