Spot test of urinary protein using Erythrosin B and a membrane film

Abstract

Point-of-care testing is currently one of the subjects of growing interest in analytical chemistry. Elevated levels of urinary protein imply renal failure, which is one of the world's biggest public health problems. In spite of the urgent necessity for a screening test of protein in urine, there are no reports of a simple yet sensitive method for its detection. In this study, we developed a new visual method, using Erythrosin B and a cellulose acetate membrane film as the substrate for a new spot test of urinary protein in the presence of poly(ethylene glycol) (PEG). The noteworthy point of our work is that when a drop of dye–protein solution containing PEG is set on a membrane film, a red ring-shaped stain of the dye-bound protein is formed on the film surface. PEG plays a significant role in eliminating the reagent blank, thus providing a clear contrast. Measurements taken using a dynamic light scattering particle size analyzer indicated that the underlying mechanism of this contrast is brought about by the different sizes of the excess dye and dye–protein particles. The visual detection limit is 0.5 mg dm⁻³ for human serum albumin (HSA), the main protein in urine. Our visual method is sufficiently sensitive to detect urinary protein even for healthy subjects, providing a higher sensitivity than test strips by a factor of 60–200. When 0.15 cm³ of urine is used to prepare 10 cm³ of sample solution, the practical detection threshold is 30 mg dm⁻³ in urine using a 67× dilution factor. The proposed method will be useful as a simple, rapid, and cost-effective screening test for the diagnosis of renal failure at an early stage.