Design, development and application of protein-templated gold nanoclusters in biosensing of glutathione, sodium and potassium ions

Protein-templated gold nanoclusters (AuNCs) are emerging nanomaterials with diverse applications in biomedical fields. AuNCs display discrete energy levels, large Stokes shift, good water solubility, distinctive fluorescence properties, high photostability and biocompatibility. These features make AuNCs a perfect candidate for imaging, biosensing and theranostics in the cellular and molecular level. This project aims to develop AuNCs-based platform tools for biosensing applications. First, a facile and optimised protocol for the fabrication of BSA/AuNCs is developed. The protocol requires shorter synthesis time (only 6 h) and lower protein amount (only 20 mg mL-1 ), while giving higher QY (10.62 %). Functionalisation of reduced graphene oxide (rGO) with folic acid (FA) is performed using EDC/NHS cross-linking agents and their interaction after loading with BSA/AuNCs is demonstrated. Finally, a fluorescence “turnoff” sensing strategy is developed using the as-synthesised FA-rGO-BSA/AuNCs for sensitive and selective detection of glutathione. The strategy requires incubation time as short as 2 min only to observe a linear range from 0 to 1.75 µM and LOD of 0.1 µM towards GSH under pH 7.4. Following this, RNase A/AuNCs is synthesised using the optimised protocol. An RNase A/AuNCs-based platform is used to detect the presence of metal ions in aqueous solution. Besides, the activity of RNase A protein after the formation of RNase A/AuNCs is studied. It is found that 50.8 % of RNase A remain active in RNase A/AuNCs. The addition of FArGO onto RNase A/AuNCs serve as a “turn-on” and “turn-off” fluorescence sensor for biosensing of sodium and potassium ions. Lastly, since electroanalytical methods are fast, simple, sensitive and cost-effective, electrochemical approaches have been used, as a proof-of-concept, to investigate the interaction between FA and folate receptor, on a graphene platform. With this, laser scribed graphene oxide (GO) and GO-FA electrodes have been prepared using different approaches for the studies. Ru(NH3)6Cl3 is found to be more suitable to be used as the redox probe in the present electrochemical measurements than Fe2+/Fe3+. Meanwhile, different GO platforms have been studied using various substrates. Overall, these facile fabricated AuNCs-based biosensors pave the way and highlight the possibilities to detect important biomarkers in cancer cells, presenting potential nanotheranostic applications in biological detection and clinical diagnosis