Physicochemical Understanding of Protein-Bound Quantum Dot-Based Sensitive Probing of Bilirubin: Validation with Real Samples and Implications of Protein Conformation in Sensing.

Precise and rapid determination of free bilirubin (BR), a key biomarker of pathological conditions of the liver, is important clinical issue. The present study demonstrates that the combination of the strong specific affinic properties of protein, bovine serum albumin (BSA), toward bilirubin and luminescence of well-characterized semiconductor quantum dots (QDs) can offer a simple, fast, and sensitive technique for the determination of free bilirubin through quenching analysis. Here, BSA molecule not only stabilizes the quantum dots in an aqueous environment but also helps bring BR closer to QDs during the interactions of CdSe-BSA QDs with BR. Further, it is revealed through photophysical investigation that the conformation of protein molecule may play an important role in biomolecular sensing considering bilirubin as a model target molecule. The luminescence of CdSe-BSA QDs was highly responsive toward bilirubin, where nearly 90% of emission intensity was quenched on adding only 40 μM bilirubin, suggesting strong interactions involved between synthesized QDs and bilirubin. Solvent polarity dependence on luminescence changes confirms strong electrostatic interaction between the QDs and BR. The applicability of the synthesized quantum dots in sensing bilirubin has been checked in the presence of different possible interfering agents and also with plasma isolated from real blood samples of both normal and hepatitis patients. It was observed that bilirubin as control sample as well as in human serum sample can be optimally measured at pH 7.5, 25 °C. Thus, the proposed strategy being able to measure free BR even at least two orders of magnitude lower than bilirubin level in normal blood may provide a reasonable protocol to determine BR in the pathophysiology of many critical human diseases, like hepatitis and Gilbert's syndrome in the near future.