Selective and sensitive detection of free bilirubin in blood serum using human serum albumin stabilized gold nanoclusters as fluorometric and colorimetric probe

Abstract

We report here a fluorescence quenching based non-enzymatic method for sensitive and reliable detection of free bilirubin in blood serum samples using human serum albumin (HSA) stabilized gold nanoclusters (HSA-AuNCs) as fluorescent probe. The fluorescence of the nanoclusters was strongly quenched by bilirubin in a concentration dependent manner by virtue of the inherent specific interaction between bilirubin and HSA. A strong binding constant of 0.55×106Lmole−1 between the HSA-AuNC and bilirubin was discerned. The nano clusters each with size ~1.0nm (in diameter) and a core of Au18 were homogeneously distributed in HSA molecules as revealed from the respective high resolution transmission electron microscopic and mass spectroscopic studies. The fluorescence quenching phenomena which obeyed a simple static quenching mechanism, was utilized for interference free detection of bilirubin with minimum detection limit (DL) of 248±12nM (S/N=3). The fluorescence response of HSA-AuNCs against bilirubin was practically unaltered over a wide pH (6–9) and temperature (25–50°C) range. Additionally, peroxidase-like catalytic activity of these nanoclusters was exploited for colorimetric detection of bilirubin in serum sample with a DL of 200±19nM by following the decrease in absorbance (at λ440nm) of the reaction and its rate constant (Kp) of 2.57±0.63mLμg−1min−1. Both these fluorometric and colorimetric methods have been successfully used for detection of free bilirubin in blood serum samples.