The Next Generation of IR Spectroscopy: EC-QCL-Based Mid-IR Transmission Spectroscopy of Proteins with Balanced Detection

• Published in: Analytical chemistry (Washington) 2020-07, Vol.92 (14), p.9901-9907
• Main Authors: Akhgar, Christopher K, Ramer, Georg, Żbik, Mateusz, Trajnerowicz, Artur, Pawluczyk, Jarosław, Schwaighofer, Andreas, Lendl, Bernhard
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Animals; Aqueous solutions; Cadmium; Cadmium tellurides; Cattle; Chemistry; Concanavalin A - chemistry; Fabaceae - chemistry; gamma-Globulins - chemistry; Hemoglobins - chemistry; High power lasers; Infrared spectroscopy; Laser beams; Laser cooling; Lasers, Semiconductor; Light sources; Luminous intensity; Mercury; Mercury (metal); Mercury cadmium telluride; Mercury cadmium tellurides; Monitoring instruments; Noise levels; Protein structure; Protein Structure, Secondary; Proteins; Qualitative analysis; Quantum cascade lasers; Room temperature; Sensitivity; Spectrophotometry, Infrared - methods; Spectrum analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.0c01406

We report a mid-IR transmission setup for the analysis of the protein amide I and amide II band in aqueous solutions that achieves a limit of detection as low as 0.0025 mg mL–1 (outperforming our previous results and other state-of-the-art mid-IR-based techniques by almost an order of magnitude). This large improvement is made possible by combining the latest-generation external cavity-quantum cascade laser (EC-QCL) operated at room temperature with an optimized double-beam optical setup that adjusts the path length (26 μm) to ensure robust sample handling. For minimizing the noise introduced by the high-intensity laser light source, a thermoelectrically cooled mercury cadmium telluride balanced detection module was employed. In this way, noise levels better by a factor of up to 20 were achieved compared with single-channel measurements. Characteristic spectral features of proteins with different secondary structures were successfully identified at concentrations as low as 0.1 mg mL–1. Furthermore, a highly linear response was demonstrated for concentrations between 0.05 and 10 mg mL–1. The total acquisition time of the setup can be adapted to fulfill the required sensitivity of the protein measurements and to ensure maximum flexibility for future applications. The presented setup combines high sensitivity, large optical path lengths, and short measurement times and thus outperforms previous research type EC-QCL setups as well as commercially available instruments. This opens a wide range of future applications including protein–ligand interaction studies as well as qualitative and quantitative analyses of proteins in complex matrices such as those found in up- and downstream bioprocess monitoring and similar challenging applications which can not be readily met by conventional FT-IR spectroscopy.