SERS-based error calibration of a TMB-H₂O₂ colorimetric system

3,3',5,5'-tetramethylbenzidine (TMB)-H₂O₂ is widely used as an effective colorimetric system, in which the color reaction is implemented with peroxidase-catalyzed TMB oxidation by H₂O₂ that usually measured UV-vis absorption spectra or Raman spectra. However, its low accuracy significantly limits its application. Blue charge transfer complex (CTC), which is the product of TMB and H₂O₂ reaction and is used as the basis for partial colorimetric methods, usually causes colorimetric error owing to changes in the UV-vis absorption and Raman spectra during TMB oxidation under various environmental conditions (catalyst type, temperature, H₂O₂ concentration). Herein, we propose a surface-enhanced Raman spectrum (SERS)-based error calibration method to improve the accuracy of the TMB-H₂O₂ colorimetric system. It is found that under 633 nm laser excitation, TMB has three Raman peaks at 1189, 1335 and 1609 cm^-1 in the single-electron oxidation phase, and these peaks disappear completely in the two-electron oxidation phase. By comparing these Raman peaks, we can conveniently obtain the actual process information during TMB oxidation. Using the proposed method, the accuracy of the TMB-H₂O₂ colorimetric system improved by more than 15%. Importantly, this SERS-based TMB-H₂O₂ error calibration method will open a new horizon for enzyme-linked immunosorbent assay (ELISA) and other biomedical applications.