Optical Spectroscopic Detection of Mitochondrial Biomarkers (FMN and NADH) for Hypothermic Oxygenated Machine Perfusion: A Comparative Study in Different Perfusion Media.

Ex situ machine perfusion has emerged as a pivotal technique for organ preservation and pre-transplant viability assessment, where the real-time monitoring of mitochondrial biomarkers-flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH)-could significantly mitigate ischemia-reperfusion injury risks. This study develops a non-invasive optical method combining fluorescence and UV-visible spectrophotometry to quantify FMN and NADH in hypothermic oxygenated perfusion media. Calibration curves revealed linear responses for both biomarkers in absorption and fluorescence (FMN: λ = 445 nm, λ = 530-540 nm; NADH: λ = 340 nm, λ = 465 nm) at concentrations < 100 μg mL. However, NADH exhibited nonlinear fluorescence above 100 μg mL, requiring shifted excitation to 365 nm for reliable detection. Spectroscopic analysis further demonstrated how perfusion solution composition alters FMN/NADH fluorescence properties, with consistent reproducibility across media. The method's robustness was validated through comparative studies in clinically relevant solutions, proposing a strategy for precise biomarker quantification without invasive sampling. These findings establish a foundation for real-time, optical biosensor development to enhance organ perfusion monitoring. By bridging spectroscopic principles with clinical needs, this work advances translational sensor technologies for transplant medicine, offering a template for future device integration.