Visible Light-Driven Direct Colorimetric Detection of Nitrite with 3,3′,5,5′-Tetramethylbenzidine

Due to the unique features of 3,3',5,5'-tetramethylbenzidine (TMB) in analytical chemistry, a series of TMB-based colorimetric nitrite assays (particularly nanozyme-involved) were developed to supplement the classical Griess assay. However, the exact reaction mechanisms and the final product between TMB and NO₂^- are controversial. Herein, we found that the widely adopted mechanism, namely the reaction between NO₂^- and the one-electron oxidation product of TMB (TMB^+•), may be not valid. Alternatively, we found the reaction between NO₂^- and TMB primarily proceeded via diazotization (confirmed with ESI-MS) and yielded yellow diazo-TMB (not TMB²⁺, the two-electron oxidation product of TMB). In the presence of an easily ignored condition of visible light, diazo-TMB was promoted to its excited state, followed by two photoreactions to yield blue TMB^+•, namely photoreduction ([diazo-TMB]* + TMB → TMB^+•) and photosensitized oxidation ([diazo-TMB]* + O₂ → ¹O₂, ¹O₂ + TMB → TMB^+•). To facilitate the colorimetric process, a blue LED with a lighting wavelength matching with the maximum absorption of diazo-TMB, was employed to accelerate the generation of TMB^+•. The light-driven colorimetric nitrite assay offered a limit of detection (LOD) of 0.1 μM, which was further explored to nitrite detection in urine samples and diagnosis of urinary tract infections caused by Escherichia coli.