Tunable Interband Cascade Laser-Based Mid-Infrared Spectroscopy for Protein Sensing: Wavenumber-Resolved Performance Evaluation within the Amide I Region

Abstract Compact mid-infrared (MIR) spectrometers based on tunable interband cascade lasers (tICLs), which are integrated with thin-film waveguides, represent a promising platform for discrete-emission spectroscopic measurements in aqueous systems, operating across the amide I region (1692–1640 cm − 1 ). This study presents a wavenumber-resolved performance evaluation of a prototype tICL-based spectrometer using bovine serum albumin (BSA) standards at different concentrations to assess the limit of detection (LOD), limit of quantification (LOQ), and signal-to-noise ratio (SNR), while benchmarking its performance against a compact Fourier-transform infrared attenuated total reflectance (FTIR-ATR) system to contextualize analytical behavior. Although the prototype showed acceptable initial performance, system-induced variability affected the analytical sensitivity and reproducibility of the measurements. To address these limitations, a wavenumber-resolved analytical framework was applied to quantify sample-induced spectral variations. Absorbance variation was analyzed at individual emission points, focusing on concentration dependence and replicate consistency. Using the strictly standardized mean difference (SSMD) metric and z -score standardization, wavenumbers were ranked by concentration-level discriminability, with those having positive z -scores selected for further analysis. When applied to an independent dataset, the SSMD-based selection yielded lower LOD (1.24% to 0.71%) and LOQ (3.75% to 2.17%), reflecting improved analytical sensitivity through channel selection. Partial least-squares regression (PLSR) score-space clustering further demonstrated enhanced separation of BSA concentrations with SSMD-filtered subsets. Overall, the proposed wavenumber-resolved performance framework enabled the selection of high-quality emission points, supporting the optimization of compact tICL-based MIR spectrometers for protein-relevant analytical measurements and industrial process-monitoring contexts.