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.
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