Stabilization of SERS nanotags by BSA: insights into protein conformational dynamics across pH environments

The colloidal stability of surface-enhanced Raman scattering (SERS) nanotags is vital for designing SERS-based biosensors with high accuracy and reproducibility. Bovine serum albumin (BSA) is commonly used as a blocking agent to improve the stability of SERS nanotags and prevent non-specific interactions in biological media. However, limited understanding of its pH-dependent behaviour in the conjugated form may partly hamper the clinical application of SERS nanotags. In this study, we investigated the role of pH-dependent conformational changes in BSA in the colloidal and optical stability of BSA-functionalised SERS nanotags by combining experimental spectroscopic techniques (<i>i.e.</i>, UV-vis, circular dichroism, and Raman spectroscopy) with finite element method simulations. Our results show that coating with BSA effectively stabilizes the SERS nanotags under most pH conditions, with notable aggregation and hotspot formation only at extremely acidic and basic pH values. CD spectroscopy revealed that BSA undergoes significant secondary structure transitions with pH changes, but when conjugated to AuNPs, it maintains greater structural stability. Finite element simulations further highlighted the role of BSA in modulating the enhanced electric field distribution and preventing nanoparticle aggregation. These findings provide crucial insights into the design of robust, pH-resistant SERS-based biosensors and underscore the importance of protein secondary structure stability in nanobiotechnology applications.