Abstract:
The present investigation is focused on the utilization of human serum albumin-stabilized gold nanocluster (HSA-AuNC) as biorecognition element for detection of bilirubin in optical and electrochemical transducer platforms. The natural affinity of bilirubin to HSA and some unique optical and electrochemical features of gold nanoclusters (AuNCs) were exploited to develop the detection techniques. Following a simple microwave-based technique AuNCs were synthesized in the protein matrix of HSA. Using transmission electron microscopy (TEM) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), the size and the number of gold atom in these NCs were determined to be ~ 2.5 nm and 18, respectively. The formation of AuNCs in the protein matrix though caused some perturbation in the secondary structure of the protein, the binding of bilirubin to the HSA-AuNC assembly was not much altered as evident from the reasonably good binding constant of 0.55 x 106 L mole-1. Zeta potential studies revealed that the formation of nanoclusters caused to increase the surface charge density of the HSA protein. The HSA-stabilized AuNC, when excited at λ380 nm, produced an intense fluorescence at λ640 nm. Interestingly, the binding of bilirubin with HSA-AuNC quenched the fluorescence in a concentration dependent manner. 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 ± 12 nM at a signal-to-noise (S/N) ratio of 3. Additionally, a peroxidase-like catalytic activity of these nanoclusters was observed and exploited for the detection of bilirubin following a colorimetric approach. The detection involves a decrease in absorbance of bilirubin at λ440nm in the reaction solution upon addition of H2O2 due to peroxidative catalytic effect of the AuNCs. The minimum DL for bilirubin as discerned from the analysis was 200 ± 19 nM.
