Abstract: A sensitive and selective assay of proteins is proposed based on measuring the total internal-reflected resonance light scattering(TIR-RLS) signals produced on the water/tetrachloromethane(H₂O/CCl₄) interface. In an aqueous medium with pH value in the range of 3.29-3.78, electrostatic attraction occurs between the negatively charged Evans Blue(EB) and positively charged proteins, forming hydrophobic ion associates and resulting in EB-protein adsorption on H₂O/CCl₄ interface. The presence of cetyltrimethylammonium bromide prompts this adsorption, resulting in strongly enhanced TIR-RLS signals. The intensity of the enhanced TIR-RLS at 360-370 nm was found to be proportional to the concentration of proteins. For bovine serum albumin and human serum albumin, the linear range of detection is 0.07-1.2 μg/mL and the limits of detection are 6.68 and 6.30 ng/mL(3σ), respectively, while for lysozyme, the linear range of detection is 0.06-1.0 μg/mL and the limit of detection is 6.0 ng/mL(3σ). The content of the total albumin in a human urine sample could be directly determined by using the standard addition method with a percent recovery of 97.6%-104.1%, and the RSD ranging from 1.9% to 4.2%.

Key words: Total internal-reflected resonance light scattering(TIR-RLS), Protein, Evans Blue(EB), Cetyltrimethylammonium bromide(CTMAB)