Hydrogen-Bond-Engineered Photoactivated Au ₁₀ Clusterzymes for Amplex Red Specificity and Highly Efficient Fluorescence Sensing

Despite the promise of gold clusterzymes in biosensing, their applications are predominantly confined to peroxidase (POD)-mimicking systems reliant on unstable, cytotoxic hydrogen peroxide (H 2 O 2 ) under acidic conditions. Therefore, constructing ultrasensitive fluorescence platforms independent of POD-H 2 O 2 at neutral pH remains challenging. Herein, we propose a highly active, substrate-specific photoactivated oxidase-like Au 10 clusterzymes functionalized with a thymine derivative (ATT) for sensitive and efficient fluorescence sensing using amplex red (AR) as the substrate. Hydroxyl and pyrimidine nitrogen groups of ATT collectively construct the biomimetic substrate pocket on the Au 10 clusterzyme surface. This unique pocket selectively recruits and orients AR via hydrogen-bonding interaction, dramatically enhancing the catalytic efficiency and specificity at pH 7.4. Furthermore, the photoexcitation of Au 10 clusterzymes generates reactive oxygen species (ROS) and electron–hole pairs, driving H 2 O 2 -independent AR oxidation with rapid signal saturation of 5 min and exceptional kinetics ( K m = 0.3201 μM, K cat = 100.1 s –1, K cat / K m = 3.128 × 10 8 s –1 ·M –1 ). The platform thus delivered selective, ultrasensitive quantification of AChE and ALP (limit of detection: 0.0125 and 0.128 mU/mL, respectively) and inhibitor screening over wide dynamic ranges, demonstrating robust performance in real human serum (recoveries: 96.60–102.10%; RSDs < 4.33%). The hydrogen-bond microenvironment engineering optimizes clusterzymes–substrate interaction, advancing high-performance biosensing platforms.