Development of enzyme-based bar code-style lateral-flow assay for hydrogen peroxide determination

A unique approach of developing a bar code version of lateral-flow enzymatic-based assay for the semi-quantification of hydrogen peroxide is described. The proposed assay system is mainly composed of a goat anti-mouse IgG–horseradish peroxidase conjugate (Gt anti-M IgG–HRP)-coated nitrocellulose (NC) membrane and a peroxidase substrate pad. Unlike the bar code immunochromatographic assay which depends on the stepwise capture of analyte, the principle of enzyme-based bar code lateral-flow assay is based on the different reaction time on successive lines due to the delay in 3,3′,5,5′-tetramethylbenzidine (TMB) release. Hydrogen peroxide (H2O2) acts as a limiting factor which controls the rate of the enzymatic conversion of TMB to blue color complex. The system expresses the concentration of H2O2 in micromole range as three distinct ladder bars in 9 min therefore without the need of any reading device. The major advantages of this assay are its easily readable result, and also its simplicity and low-cost in production offers a cheaper alternative for testing those expensive biosensors might not be available to the third world countries. By incorporating with H2O2-generating oxidoreductases, the assay can be further extended to detect a variety of analytes with clinical and environmental importance. Glucose was chosen to be the model analyte where the proposed system gave signal response at between 5 μM and 100 μM.