Vibrational spectroscopic fingerprinting and DFT-assisted band assignment of the resazurin–resorufin redox transformation in Escherichia coli

Resazurin is a widely used redox-active dye whose reduction to resorufin forms the basis of numerous biological viability and metabolic assays. Despite its extensive use, a detailed vibrational spectroscopic characterization of the resazurin–resorufin redox transformation, supported by rigorous molecular-level band assignments, particularly in biologically relevant environments, remains lacking. Here, we investigate vibrational spectroscopic monitoring of the resazurin–resorufin redox transformation during bacterial metabolic activity in an Escherichia coli strain, using a correlative approach that integrates Raman spectroscopy, infrared (IR) spectroscopy, polarization-dependent Raman measurements, and density functional theory (DFT) simulations. Raman and IR spectra collected during the redox process show clear and reproducible changes in both band positions and relative intensities. In particular, a consistent Raman band shift from 1643 to 1652 cm⁻¹ is observed, along with pronounced intensity redistribution in low-frequency modes associated with ring deformation and aromatic skeletal vibrations. Quantitative analysis of selected Raman band ratios in the 480–450, 1179–1149, and 1450–1397 cm⁻¹ regions provides a simple and robust way to follow the progression of the redox transformation. Polarization-dependent Raman measurements further reveal changes in vibrational symmetry and molecular polarizability as reduction proceeds. DFT simulations reproduce the key experimental features and enable assignment of the observed Raman bands to specific molecular motions. UV–Visible and fluorescence measurements provide independent confirmation of the redox state of the dye. Together, this work establishes a DFT-supported vibrational framework for monitoring the resazurin–resorufin redox transformation in biological systems, providing a molecular-level basis for future spectroscopic studies employing resazurin as a redox reporter.