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

Highlights: What are the main findings? First systematic optical characterization of FMN/NADH in clinically relevant perfusion media, revealing medium-specific fluorescence quenching effects. Identification of critical sensor design parameters: optimal excitation wavelengths (FMN: 455 nm; NADH: 360 nm), concentration-dependent NADH nonlinearity (>100 µg mL−1), and FMN-NADH spectral interference. What is the implication of the main finding? Providing standardized calibration protocols enabling direct translation to portable optical sensors for real-time perfusion monitoring. Providing the theoretical framework to develop portable optical sensors for clinical perfusion monitoring, with specific guidelines for excitation wavelengths, interference mitigation, and medium-specific calibration. 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: λex = 445 nm, λem = 530–540 nm; NADH: λex = 340 nm, λem = 465 nm) at concentrations < 100 μg mL−1. However, NADH exhibited nonlinear fluorescence above 100 μg mL−1, 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.