Neutral-pH Cu-Lcys nanozymes for colorimetric detection of ascorbic acid under physiological conditions

Most nanozyme-based colorimetric platforms require acidic conditions (pH 3.0-5.0) that are incompatible with biological fluids, necessitating sample acidification that risks protein denaturation and analyte degradation. Herein, we report Cu-Lcys nanozymes, synthesized via a simple one-step coordination of Cu(II) with L-cysteine, that exhibit robust peroxidase-like (POD-like) activity optimized at physiological condition (pH 7.5). The Cu-Lcys nanozymes catalyze the decomposition of H₂O₂ to generate reactive oxygen species (ROS) that oxidize o-phenylenediamine (OPD) to produce a colorimetric signal at 420 nm. Ascorbic acid (AA) competitively scavenges these ROS, producing a concentration-dependent decrease in absorbance. Under optimized conditions, the platform exhibits a linear response to AA over 0.25-8.0 µg mL^-1 (R² = 0.993) with a detection limit of 0.211 µg mL^-1. The method demonstrates high selectivity for AA in the serum and achieves recoveries of 96.5-103.8% in spiked goat serum samples without pH adjustment. Additionally, the intrinsic antibacterial properties of the copper core (>96% inhibition of S. aureus and E. coli at 20 ng mL^-1) provide built-in biosafety for handling biological specimens. This work offers a practical neutral-pH colorimetric strategy for AA analysis in complex biological matrices.