High-Sensitivity Mid-IR Absorption Spectroscopy for Proteins in Aqueous Solution

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Although infrared (IR) absorption spectroscopy has the ability to quantify complex biomolecules and their structural conformations, analyzing proteins in aqueous solutions with the technique can be a challenge due to the fact that strong IR absorption of water will overwhelm the detection system’s limited dynamic range. As a result, the yield is only a very short path and a limited concentration sensitivity. Young Jong Lee and his colleagues at the Biosystems and Biomaterials Division of the National Institute of Standards and Technology in Gaithersburg, Maryland, have developed a solvent absorption compensation (SAC) approach which, by distinguishing the analyte signal over the full dynamic range at each wavelength, can improve the sensitivity of the concentration and extend the available pathlength. Lee spoke to Spectroscopy about this approach.

In a recent paper (1), you present a SAC approach to analyze proteins in an aqueous solution using infrared spectroscopy that can improve the concentration sensitivity and extend the available pathlength by distinguishing the analyte signal over the full dynamic range at each wavelength. Why is this important? In what analyses or research would this ability be especially beneficial?

The measurement of an absorption spectrum of a sample with a transparent solvent is typically not limited by the dynamic range of the detection system. However, when a solvent or a reference material used for an absorption measurement absorbs the incident light strongly at a narrow spectral range, the available dynamic range becomes reduced and cannot distinguish the analyte signal with the full dynamic resolution in the local spectral range. The strong IR absorption by water notoriously has limited its application to hydrated samples, such as protein solutions and live cells. In particular, the most prominent IR band of protein overlaps with the broad bending peak of water, suffering significantly reduced sensitivity. The SAC method can eliminate the substantial water absorption contribution and, thus, measure the protein spectrum with a > 100 times improved signal-to-noise ratio. This new approach will be especially beneficial to biosciences and biotechnologies that need to characterize biomolecules in intact hydrated conditions.

Please briefly explain how the adaptive SAC–IR technique is implemented. Are commercial instruments capable of this measurement available?

SAC requires an optical unit that can adjust the transmission efficiency sufficiently fast while the laser wavelength scans. We tested acousto-optic modulators and rotating polarizers for the SAC unit. There is no commercially available system using the SAC technique currently, but there have been discussions on licensing the method with multiple companies.

You utilized IR absorption spectroscopy in your efforts. What advantages does IR have over Raman or other techniques for biomolecule investigations?

IR and Raman have both pros and cons for biomolecule characterization. The advantages of IR include the high concentration sensitivity due to higher IR absorption cross section and the SI-traceability of the absorbance data.

How does this work differ from what has been previously done by yourself or others?


About a decade ago, quantum cascade lasers were introduced to IR spectroscopy of biological molecules. Thanks to the intense laser, quantum cascade laser (QCL)-based IR spectroscopy was able to measure proteins through a thick optical pathlength with greatly improved sensitivity, compared to traditional Fourier transform infrared (FT–IR) methods. However, due to the limited dynamic range imposed by the strong IR absorption by water, researchers were able to measure only a narrow spectral range (~100 cm-1). This new technique allows for a broader spectral range (Δω ~ 400 cm-1: shown in the published paper in 2021; and Δω ~ 900 cm-1: results are unpublished).

Please summarize your conclusions from this work.

We have demonstrated an adaptive SAC technique that can enhance the signal-to-noise ratio of QCL-IR absorption spectroscopy of analytes in hydrated solutions. We have shown that this simple optical technique can improve the sensitivity by two orders of magnitude without changing the detection system. The same SAC approach can be applied to other types of scanning absorption spectroscopy that investigate samples in strongly absorbing solvents to improve the sensitivity and the limit of detection.

Were there any particular challenges or limitations encountered in your work?

Technical challenges specific to the SAC technique include improving the optical performance of the acousto-optic modulators for a wider wavelength range.

Can this technique be applied to any other absorption spectroscopies of analytes? If so, which might find the technique to be especially useful?

This technique can be directly applied to other types of absorption spectroscopies characterizing highly absorbing solvents, such as near-infrared (NIR) and terahertz (THz) spectroscopy for hydrated samples. Similarly, taking advantage of the full dynamic range over a broad spectral range will help to improve the overall sensitivity when used for highly absorbing matrices.

Can you please summarize the feedback that you have received from others regarding this work?

Many pharmaceutical analytical scientists were interested in this technique and wanted to see this technique used in commercial measurement systems for the quantitative analysis of therapeutic drugs. Some instrument manufacturers asked if this technique could be applied to non-transmission techniques, such as fluorescence spectroscopy, Raman spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy.

What are the next steps in this research?

We are applying this technique for the characterization of various types of drug products by using improved sensitivity, spectral breadth, acquisition speed, and microfluidics. We are also developing SAC-based IR microscopy to image live cells and hydrated tissues.


(1) Chon, B.; Xu, S.; Lee, Y. J. Compensation of Strong Water Absorption in Infrared Spectroscopy Reveals the Secondary Structure of Proteins in Dilute Solutions. Anal. Chem. 2021, 93, 2215–2225. DOI: 10.1021/acs.analchem.0c04091

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