Rapid pre-analysis derivatization-enabled high-throughput immunoassay for nitrite detection in foods

Nitrite is a well-known food additive and water pollutant that has garnered extensive attention due to both of its adverse and beneficial effects on human health, calling for rapid and reliable analytical methods. However, the simple and diminutive structure of nitrite has impeded the development of screening immunoassay for this substance. In this work, based on the rapid and selective derivatization of nitrite into benzotriazole (BTA) by o-phenylenediamine, two haptens targeting the structure of BTA were designed and synthesized, and a specific monoclonal antibody against BTA was generated. Using this antibody, an indirect competitive enzyme-linked immunosorbent assay (ELISA), involving a rapid pre-analysis derivatization, was developed for the detection of nitrite in foods. The limits of detection in drinking water, pickle, and ham sausage were 2.0 μg/L, 0.05 mg/kg, and 0.05 mg/kg, respectively. The recoveries ranged from 82.7% to 114.6%, and the results of this ELISA correlated well with those from the standard spectrophotometry method. This pioneering immunoassay for nitrite holds great promise for routine monitoring, ensuring food safety and consumer health. • Haptens against benzotriazole (BTA) was firstly synthesized. • A highly specific anti-BTA monoclonal antibody was generated. • A rapid nitrite derivatization strategy suitable for ELISA was designed. • An ic-ELISA for high-throughput detection of nitrite was firstly developed.