The authors used the photothermal-based AFM-IR technique to achieve the measurements of infrared absorption spectra and chemical maps at the single-molecule level for the first time. The high sensitivity in their measurement enabled the accurate determination of the secondary structure of a single protein molecule that is consistent with the structure of the bulk protein material. The results demonstrated the high sensitivity and high spatial resolution of the AFM-IR technique and paved the way for direct probing of individual biomolecules in a broader context.
To achieve AFM-IR characterization of a single protein molecule, an innovative approach was adopted in the experimental setup. This approach, namely ORS-nanoIR, used an off-resonance, low-power, and short pulse of the infrared beam to excite the single protein molecule. The off-resonance excitation, i.e., laser pulse rate detuned to a frequency 1-2 kHz less than the peak of contact resonance frequency, gave the maximum contrast in laser-induced cantilever deflection signal between the protein sample and the gold substrate. The low power and short pulse operation scheme established a linear response regime of the cantilever to the thermal expansion of the sample induced by infrared absorption. Such conditions also avoided damage of the soft protein sample during the measurement. This innovative experimental approach enabled the acquisition of AFM-IR spectra and maps from a single protein on a time scale of 1 s, with a ~10-20 signal-to-noise ratio. As examples to demonstrate the capabilities of this technique, single molecules of two different protein species, apoferritin and thyroglobulin, were successfully measured, and their secondary structures were accurately determined from the high-quality AFM-IR spectra.