Rapid detection of hydrogen peroxide and nitrite in adulterated cow milk using enzymatic and nonenzymatic methods on a reusable platform

Abstract

Cow milk is readily adulterated due to its complex properties that can emulsify many adulterants. Among the commonly used adulterants in cow milk are hydrogen peroxide (HP) and nitrite. Commercially available HP is added to extend cow milk's shelf life, while nitrite enters through the tap or pond water added to increase cow milk's volume. HP disrupts human free radical balance, while nitrite can cause methemoglobinemia. This study aims to rapidly detect these adulterants on-site by developing a point-of-care kit. A modified streptavidin–horseradish peroxidase (Strep–HRP)–tetramethylbenzidine (TMB) assay was used for designing the biosensor for HP detection. The Strep–HRP complex was immobilized on 8-well polystyrene strips with glutaraldehyde crosslinking. TMB was used as the substrate to detect HP at concentrations of 0.04% (v/v) and higher. Nitrite was detected using a modified Griess assay, wherein for the biosensor, the Griess reagent was coated on polystyrene strips with polyethylene glycol (PEG) used as the stabilizer to identify nitrite concentrations of 32 μg mL⁻¹ and above. The Strep–HRP and Griess assay strips prepared in this investigation were stable for 25 and 10 days, with three times reusability for HP and twice for nitrite detection. Both strips were accurate, up to 95%, for detecting HP and nitrite in cow milk samples.