Biomimetic point-of-care testing of trace free bilirubin in serum by using glucose selective capture and surface-enhanced Raman spectroscopy

The concentration of bilirubin in serum is a key indicator for liver function, because excess bilirubin accumulation may lead to serious health problems like Jaundice. It is known that in comparison with total bilirubin, the concentration of hydrophobic free bilirubin is more closely associated with Jaundice because of its capability to cross the capillary walls and even blood-brain barrier. However, it is not easy to detect the low concentration of free bilirubin (∼ nM) in the presence of large amount of protein-bound bilirubin (> 150 μM), and no point-of-care detection tool is available. In the present work, direct detection of free bilirubin was realized by using surface enhanced Raman spectroscopy (SERS). By mimicking the in vivo chemical interaction between glucuronic acid and bilirubin, glucose functionalized Au nanoparticles were synthesized for selective capture and detection of free bilirubin. By developing a convenient detection procedure of using robust Au array chip and internal standard calibration method, the sensitive detection of free bilirubin with detection limit down to 0.1 nM was achieved on a handhold Raman spectrometer by monitoring its fingerprint spectra. The clinical applicability was demonstrated with analyzing the spiked and real blood samples. As a proof of concept, the binding constant between bilirubin and human serum albumin (HSA) was determined with the present method. The influence of the binding capacity of the HSA for bilirubin in the presence of competing molecule salicylate sodium was also quantitatively measured, which demonstrated the importance of detection of free bilirubin instead of total bilirubin for warning the risk of bilirubin encephalopathy.