Overcoming acidic limitations: an Os-Ru bimetallic polymeric nanozyme at neutral pH for dual-mode detection of acetylcholinesterase activity and its inhibitors.

Accurate detection of acetylcholinesterase (AChE) activity and its inhibitors is indispensable for the early diagnosis and drug discovery of neurodegenerative diseases. In nanozyme-based assays, the sensing mechanism relies on thiocholine (TCh), the product of AChE-catalyzed acetylthiocholine hydrolysis, which specifically inhibits the peroxidase (POD)-like activity of the nanozyme, thereby reducing the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) to the fluorescent and chromogenic product resorufin, enabling indirect quantification of AChE activity. However, conventional nanozyme-based AChE assays are fundamentally limited by a long-standing paradigm conflict: highly active nanozymes typically require acidic conditions for optimal catalysis, whereas AChE and biological systems function exclusively at near-neutral physiological pH. This incompatibility forces tedious pH-switching protocols, which inevitably cause AChE inactivation, signal distortion, and poor reproducibility. Here, we address this incompatibility by engineering an amphiphilic osmium (Os)-ruthenium (Ru) bimetallic polymeric nanozyme (polymer@OsRu) that exhibits robust POD-like activity at neutral pH, achieving high catalytic efficiency without compromising biocompatibility. This work delivers two key advances: (i) materials innovation-the polymer and the Os-Ru bimetallic synergy enable robust catalytic activity under physiological conditions, overcoming the limitations of conventional nanozymes; (ii) platform innovation-the integration of dual-mode (colorimetric and fluorescent) readouts provides intrinsic cross-validation, significantly enhancing detection reliability. The platform achieves ultrasensitive acetylcholinesterase (AChE) detection (LOD = 0.0297 mU mL-1 for colorimetry and 0.0856 mU mL-1 for fluorometry) and reliable inhibitor analysis (IC50 = 0.716 µM and 0.92 µM for the clinical drug rivastigmine tartrate), outperforming most reported neutral-pH AChE assays. Beyond analytical performance, this study establishes a generalizable design principle for decoupling nanozyme catalytic efficiency from pH constraints, establishing a versatile framework for physiologically adaptive, multimodal biosensing with broad implications in neurodegenerative diagnostics and drug discovery.