Development of a creatinine enzyme-based bar-code-style lateral-flow assay

A lateral-flow, enzyme-based, bar-code assay for creatinine employing the concept of combination of diffusion and kinetics controlled has been developed. Unlike the traditional bar-code version of immunochromatographic assay, which depends on the stepwise capture of colorimetric tracer-labeled antibody-antigen complex by the immobilized antibody on each successive line, the principle of our proposed assay is based on the delay in TMB release and its diffusion in combination with horseradish peroxidase kinetics. Hydrogen peroxide (H2O2) produced from enzymatic reactions acts as a limiting factor, which controls the rate of conversion of TMB to blue color complex. The assay takes advantage of giving ladder bar result therefore without the need of any reading device. Depending on the amount of enzymes used, the assay can be one (9 min) or two steps (19 min). The strip assay semiquantitatively measures creatinine concentrations ranging from 0 to 400 mu M. Thirty urine samples and thirty serum samples were tested, and the assay showed 90.0% and 86.7% agreement compared with conventional Jaffe method, respectively. This assay provides a tool for quick identification of creatinine for patients without the requirement of any instrument.