The nanoscale structures and dynamical processes of proteins have been extensively studied by various imaging techniques such as electron microscopy and atomic force microscopy (AFM) in liquid. However, these imaging techniques can damage or perturbate the samples and do not provide chemical identification. This prevents a direct monitoring of structural and chemical evolution under physiological conditions. Herein, we demonstrate a new nondestructive platform that enables nanoscale Infrared (IR) spectroscopy for protein at graphene-aqueous solution interface by combining graphene liquid cell and Fourier Transform Infrared Nanospectroscopy (nano-FTIR). In brief, an ultra-thin film (single layer graphene, ALD oxide or lipid layer) is used to separate the assembly buffer solution and the AFM tip so that the destructive factors such as sample dragging, tip contamination and absorption of IR in liquid are minimized. In one typical example, the dynamic evolution of the bacterial S-layer protein was recorded by following the amide I and II vibration bands, and a good agreement with the dynamics measured by AFM in liquid was observed. Amide band line profile of protein assemblies revels the structural inhomogeneity, which is unexpected from liquid AFM images previously reported. These observations have never been reported and our results revealed unprecedented detail of S-layer protein assembly dynamics. Our platform opens up broad opportunities for <i>operando</i> study of soft materials or nanostructures (enzyme, other membrane protein, virus, and plastic material) in their realistic condition and under external stimuli.

Graphene-based platform for investigation of protein assembly by infrared nanospectroscopy

ACS Spring 2022

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