Practical Assay for Nitrite and Nitrosothiol as an Alternative to the Griess Assay or the 2,3-Diaminonaphthalene Assay

Nitrite is a heavily assayed substrate in the fields of food safety, water quality control, disease diagnosis, and forensic investigation and more recently in basic biological studies on nitric oxide physiology and pathology. The colorimetric Griess assay and the fluorimetric 2,3-diaminonaphthalene (DAN) assay are the current gold standards for nitrite quantification. They are not without limitations, yet have amazingly survived 156 and 44 years, respectively, due to the lack of a practical alternative. Both assays exhibit slow detection kinetics due to inactivation of nucleophiles under strongly acidic media, require an extensive incubation time for reaction to go completion, and hence offer a limited detection throughput. By converting an intermolecular reaction of the Griess assay intramolecularly, we designed a novel probe (NT555) for nitrite detection, which displays superior detection kinetics and sensitivity. NT555 was constructed following our “covalent-assembly” probe design principle. Upon detection, it affords a gigantic bathochromic shift of the absorption spectrum and a sensitive turn-on fluorescence signal from a zero background, both of which are typical of an “assembly” type probe. Overall, NT555 has addressed various difficulties associated with the Griess and the DAN assays and represents an attractive alternative for practical applications.