Conformational dynamics of proteins are critical for their biological functions but are challenging to study at the single-molecule level. Here, we present our ongoing research in using aperture-based plasmonic nanotweezers to investigate the conformational dynamics of single unmodified proteins in aqueous solutions (Figure 1). We utilise gold double-nanohole (DNH) structures to induce a localized surface plasmon resonance, creating a highly confined optical field capable of trapping individual proteins for extended periods (Fig. 1b). The transmitted optical signal through the DNH correlates to the local refractive index within the hotspot, which is related to the polarizability of the trapped protein [1-2]. In this work, we trap individual proteins in the DNH for hours and monitor their conformational dynamics of proteins in response to sequential environmental changes, such as pHs and substrate concentrations [3]. We demonstrate that plasmonic nanotweezers can resolve conformational states of single proteins and monitor transitions between these conformations (Fig. 1c). This presentation will showcase the capabilities of optical nanotweezers in various applications, including thermal folding and unfolding dynamics, enzyme kinetics and pathways [1], and protein disassembly kinetics [4] etc. The approach presented here can be widely adopted by other biophysical studies on conformational dynamics of single proteins without the requirement of site-specific modifications.

[1] Y. Pang & R. Gordon, Nano Lett 12, 402–406 (2012).
[2] C. Ying, et al., ArXiv Prepr. ArXiv210706407 (2021).
[3] A. Yousefi, et al., Nano Lett. 23, 3251–3258 (2023).
[4] A. Yousefi, et al., bioRxiv, 2023-09. 22.558948 (2023).