Monitoring Enzymatic Reactions through Probing Chemical Bond Changes by the CD₃ Group Using Multiplex Coherent Anti-Stokes Raman Scattering

We propose a method to measure enzymatic reactions using the C-D stretching vibrational spectra of CD₃ groups that exist next to the site where a chemical bond changes. In the proposed method, temporal changes in the concentrations of the substrate and product are measured from changes in the C-D stretching spectra of the CD₃ groups. Vibrational spectra are obtained using multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy, which can obtain vibrational spectra much faster than spontaneous Raman spectroscopy. We tested the proposed method by examining the dehydrogenation reaction of isopropyl alcohol-1,1,1,3,3,3-D6 (IPA-D6) catalyzed by alcohol dehydrogenase and found that the change in concentrations of the substrate and product, IPA-D6 and acetone-D6, respectively, was successfully measured from the C-D stretching spectra.