A novel upconversion nanoparticle-based fluorescent ratiometric probe for NADH detection

Background Conventional biochemical methods for detecting nicotinamide adenine dinucleotide (NADH) struggle with issues such as low sensitivity, limited cellular permeability, and challenges in longitudinal studies within intact organs or deep tissue signal detection. Upconversion nanoparticle-based fluorescence sensing technologies emerge as a promising alternative, offering benefits like low cytotoxicity and high sensitivity. This study introduces a novel ratiometric approach to overcome these limitations and improve NADH detection in cellular studies. Results We have developed highly sensitive upconversion nanoparticle-based probes that enhance NADH detection. These probes consist of core-shell upconversion nanoparticles (UCNPs) surface-modified with mesoporous silica, which immobilizes a NADH-specific recognition molecule, Probe A. This setup facilitates a spironolactone ring-opening reaction, enhancing fluorescence resonance energy transfer (FRET) between Probe A and NADH. Our approach demonstrates a linear response over a 0–70 nM concentration range with a low detection limit of 0.053 nM. Application in PC12 and ρ0 cells confirms the method's efficacy and potential for broader biomedical research applications. Significance This innovative ratiometric sensing approach significantly enhances the accuracy and sensitivity of NADH detection. By providing a robust and precise measurement tool, it offers a substantial improvement over traditional biochemical methods, facilitating deeper insights into cellular metabolism and pathophysiological processes. Its adaptability and effectiveness herald potential transformative applications in biomedical research, particularly in studies involving metabolic and degenerative diseases.