Infrared nanospectroscopy (NanoIR) is a new experimental technique to research the secondary structure of protein-based nanoarchitectures in recent years. Compared with the conventional IR, NanoIR reveals to be an exquisite, sensitive, and accurate tool to analyze and image the single molecule secondary structure, which can reach up to high spatial resolution (10 nm). Here we present a detailed protocol to introduce how to study single silk nanofibril (SNF) and process the results by this routine. This protocol provides a useful method to demonstrate the microstructure of nanomaterials by NanoIR, displaying the potential application in analytical chemistry, biomaterials, and nanotechnologies.
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