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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Spectroscopy

Background:

• 3,3',5,5'-tetramethylbenzidine (TMB) is used in colorimetric nitrite assays.
• Existing TMB-based nitrite assays and their mechanisms are controversial.
• Classical Griess assay is a standard for nitrite detection.

Purpose of the Study:

• To elucidate the reaction mechanism between TMB and nitrite.
• To identify the final product of the TMB-nitrite reaction.
• To develop an improved TMB-based colorimetric nitrite assay.

Main Methods:

• Electrospray ionization mass spectrometry (ESI-MS) for reaction product identification.
• Spectrophotometry for colorimetric detection.
• Blue LED irradiation to enhance reaction kinetics.

Main Results:

• Nitrite reacts with TMB via diazotization, forming yellow diazo-TMB, not TMB2+.
• Visible light promotes diazo-TMB to an excited state, leading to TMB+• formation via photoreduction or photosensitized oxidation.
• A light-driven assay achieved a 0.1 μM limit of detection for nitrite.

Conclusions:

• The reaction mechanism of TMB with nitrite is clarified, involving diazotization and light-induced oxidation.
• A novel, sensitive, light-enhanced colorimetric assay for nitrite detection was developed.
• The assay shows potential for detecting nitrite in urine samples and diagnosing urinary tract infections.