Near-infrared fluorometric determination of protein by shifting the ion-association equilibrium between cationic heptamethylene cyanine and poly-glutamate

Abstract

A new method based on near-infrared (near-IR) fluorescence recovery, employing a two-reagent system, which is composed of a cationic heptamethylene cyanine and poly-glutamate, is presented for the determination of protein. The fluorescence of cationic heptamethylene cyanine, with the maximum excitation and emission wavelengths at 800 and 815 urn, respectively, was quenched by poly-glutamate with a proper concentration, but recovered by adding proteins at pH 3.5. Under optimum conditions, the recovered fluorescence was in proportional to the concentration of proteins. The linear ranges of the calibration curves were 50-1000, 100-1500 and 100-1000ng/mL with the detection limit of 37, 40, 43 ng/mL for BSA, HSA, and gamma-IgG, respectively. The relative standard deviation (n = 8) was 1.7% for 400 ng/mL bovine serum albumin (BSA). The proposed method was applied to the determination of proteins in real serum samples with satisfactory results.