A derivative of 3,3′,5,5′-tetramethylbenzidine for highly robust colorimetric sensing of nitrite in food samples

As an approved additive, nitrite has been widely used in food processing, but excessive intake can lead to serious health issues. Griess assay is currently the worldwide standard method for nitrite detection in food samples. However, the required highly acidic media (pH = 1) makes the use of corrosive strong acids unavoidable. 3,3 ',5,5 '-tetramethylbenzidine (TMB), the most widely used chromogenic substrate in ELISA, was proved to be nitrite-responsive in a buffered media (pH 2-3). However, the TMB-based nitrite assay suffered from signal instability that may be problematic for quantitative detection. Herein, we found that a TMB derivative, 3,3 '-dimethoxy-5,5 '-dimethylbenzidine (<bold>2OCH</bold>(<bold>3</bold>)<bold>2CH</bold>(<bold>3</bold>)), allowed colorimetric sensing of nitrite with much improved stability over TMB. Detailed mechanistic analysis indicated both TMB and <bold>2OCH</bold>(<bold>3</bold>)<bold>2CH</bold>(<bold>3</bold>) could be diazotized with nitrite. However, the product of <bold>2OCH</bold>(<bold>3</bold>)<bold>2CH</bold>(<bold>3</bold>) is more stable due to the higher electron-donating ability of the methoxyl group that can stabilize the cationic diazotization product. Compared with the Griess assay (2 reagents, LOD 0.1 mu M), the proposed <bold>2OCH</bold>(<bold>3</bold>)<bold>2CH</bold>(<bold>3</bold>)-based nitrite assay exhibited similar sensitivity (only one reagent, LOD 0.2 mu M), but much lower acidity (pH = 3, buffered media). The practical application of the <bold>2OCH</bold>(<bold>3</bold>)<bold>2CH</bold>(<bold>3</bold>)-based assay was validated through detection of nitrite in various food samples. Moreover, to facilitate on-site monitoring, a self-assembled nitrite test kit was constructed and confirmed for nitrite testing in food samples.