Matrix Effects on the Quantification of NanoLuc Bioluminescence in Ex Vivo Organ Homogenates

Bioluminescent reporters and biosensors are widely used to quantify gene expression, evaluate delivery systems and assess biodistribution because they provide low-background readouts over a broad dynamic range. The NanoLuc–furimazine system is particularly attractive owing to its high brightness and stability. Ex vivo analysis of organ homogenates offers a controlled approach for measuring tissue-associated reporter activity, yet matrix-dependent effects are often overlooked in quantitative interpretation. Here, we systematically evaluated how mouse organ homogenates affect NanoLuc–furimazine measurements by spiking defined amounts of purified NanoLuc into different organ matrices. Across matrices, optical profiles, blank signals, calibration slopes, and estimated limits of detection and quantification varied, resulting in substantial differences in the measured NanoLuc signal. Further analysis showed that NanoLuc-independent furimazine luminescence arises from lysis-buffer components and protein-containing environments. Matrix-matched blank subtraction reduced background-related bias, whereas matrix-specific calibration demonstrated differences in analytical response, particularly for weak signals and optically dense samples. These findings highlight the extent of matrix-dependent variability in ex vivo NanoLuc measurements and support more accurate interpretation of reporter signals in delivery, biodistribution, biosensing, and gene-expression studies.