Multimodal optical system for quantitative discrimination of bilirubin species

Bilirubin occurs in human serum in multiple molecular forms: free unconjugated, albumin-bound, and conjugated species, and distinguishing between them is clinically relevant for assessing liver function and bilirubin metabolism. However, current routine assays do not resolve these subtypes individually. Here, we present a compact, multimodal optical platform that combines absorbance spectroscopy, fluorescence intensity, fluorescence anisotropy, and photobleaching analysis to investigate the optical signatures of bilirubin species in solution. Using well-defined synthetic standards representing free, bound, and conjugated bilirubin, we characterize each modality's discriminative capability. Absorbance measurements enable total bilirubin determination, fluorescence provides high sensitivity to albumin-bound bilirubin, and anisotropy measurements, done with a plate reader, reveal different responses to each species through their differing rotational mobilities. Photobleaching kinetics further highlight species-dependent photostability under controlled irradiance conditions. Taken together, these complementary, label-free optical readouts enable improved resolution of bilirubin subtypes in synthetic samples and establish a foundation for future translational studies in biologically complex matrices.