A visual detection of protein content based on titration of moving reaction boundary electrophoresis

A visual electrophoretic titration method was firstly developed from the concept of moving reaction boundary (MRB) for protein content analysis. In the developed method, when the voltage was applied, the hydroxide ions in the cathodic vessel moved towards the anode, and neutralized the carboxyl groups of protein immobilized via highly cross-linked polyacrylamide gel (PAG), generating a MRB between the alkali and the immobilized protein. The boundary moving velocity (V_MRB) was as a function of protein content, and an acid–base indicator was used to denote the boundary displacement. As a proof of concept, standard model proteins and biological samples were chosen for the experiments to study the feasibility of the developed method. The experiments revealed that good linear calibration functions between V_MRB and protein content (correlation coefficients R > 0.98). The experiments further demonstrated the following merits of developed method: (1) weak influence of non-protein nitrogen additives (e.g., melamine) adulterated in protein samples, (2) good agreement with the classic Kjeldahl method (R = 0.9945), (3) fast measuring speed in total protein analysis of large samples from the same source, and (4) low limit of detection (0.02–0.15 mg mL⁻¹ for protein content), good precision (R.S.D. of intra-day less than 1.7% and inter-day less than 2.7%), and high recoveries (105–107%).