Monitoring cellular glycolysis pathway kinetics in the extracellular medium using label-free, Raman spectroscopy

This study explored the potential of Raman spectroscopy to holistically monitor the glycolysis pathway kinetics as a function of time through the extracellular medium. Initially, the collinearity of individual metabolites of interest- glucose and lactic acid as a function of their concentration was tested followed by the sensitivity analysis of the approach by elucidating the limits of detection (0.85 mM and 2.8 mM) and quantification (2.5 mM and 9.5 mM) for glucose and lactic acid respectively in the biological range. In the process several datamining approaches were also explored. Finally, the A549 cell culture was used for kinetic spectral acquisition of the extracellular medium mimicking the kinetic glycolysis assay as a function of time under different pathway modulations. The spectra were resolved and fitted with a kinetically constrained-model (A → B → C) using the multivariate curve resolution- alternating least squares tool for all the modulated conditions to elucidate the pathway kinetics and the rate of change. The rate of change of the resolved components for the stimulated condition (k1: 0.005 min⁻¹, k2: 0.011 min⁻¹) was approximately twice as that of the control (k1: 0,045 min⁻¹; k2: 0.049 min⁻¹) while the inhibited condition (k1: 0.025 min⁻¹, k2: 0.017 min⁻¹) was substantially slower. The technique is superior to the targeted current gold standard kinetic assay approach, in that it is holistic in nature and has potential applications in drug discovery, bioprocessing, disease diagnostics, etc. Furthermore, this approach overcomes the limitations of the omics/multiomics approaches, limited to a snapshot of cellular metabolism. This study serves as a guideline for future, more complex subcellular kinetic spectroscopy experiments.