Dual-Mode Sensing Platform Based on Laccase-like Sm-L-CDs for Highly Sensitive Detection of NADH

Nicotinamide adenine dinucleotide (NADH) is a key coenzyme in biological metabolism, and its abnormal levels are closely associated with metabolic diseases. Therefore, accurate and sensitive detection of NADH is of great significance for the diagnosis and treatment of related diseases. However, most existing dual-mode sensors rely on the same recognition process to generate two types of signals, which leads to signal crosstalk and insufficient cross-validation capability. To address this limitation, we constructed a Sm-L-CDs-based fluorescence/colorimetric dual-mode sensing system for the sensitive detection of NADH. The fluorescence signal of the CDs is effectively quenched by NADH through the inner filter effect and static quenching mechanism. Meanwhile, the CDs exhibit excellent laccase-like activity, enabling them to catalyze the oxidative color development of 4-AP to produce a pink product, which could be subsequently reduced and decolorized by NADH for colorimetric detection. These two completely independent detection mechanisms enable dual-readout cross-validation and offer superior anti-interference performance and application flexibility compared with conventional signal-coupled dual-mode sensors, achieving detection limits of 0.49 μM for fluorometry and 0.67 μM for colorimetry. Furthermore, to improve the practicality of the method, we integrated this sensing system with a smartphone to construct a portable detection platform. Collectively, this study provides a novel technical route for the accurate and convenient detection of NADH based on two independent mechanisms, representing the first example of fluorescence/colorimetric dual-mode NADH sensing with signal crosstalk-free cross-validation, and offers new insights for research on enzyme activity assays and related metabolic diseases.