Direct Analysis of Proteins in Physiological Samples with Three-Dimensional Paper-Based Isoelectric Focusing

Abstract

Point-of-care testing (POCT) of proteins is crucial for on-site and rapid diagnosis of diseases in community and family medicine. Microfluidic paper-based analytical devices (µPADs) have attracted growing attention in POCT of proteins, while the practical applications of µPADs in the analysis of proteins in physiological samples is still limited as physiological samples are usually high-salt and complex. Herein, we developed a three-dimensional (3D) paper-based isoelectric focusing (IEF) platform, which is composed of power supply, reservoirs and separation channel make by origami and stacking of cellulose acetate membrane, to simultaneously separate and enrich proteins in both low-salt samples and high-salt samples, especially for directly pretreating and analyzing proteins in physiological samples. Under the optimized experimental conditions, standard proteins (Phy and BHb) were separated in 18 min under a 36.0 V power supply and obtained 10-fold enrichment using the 3D paper-based IEF platform. Then, the capability of the 3D paper-based IEF platform with using a 12.0 V battery as power supply for direct pre-treatment of high-salt samples was proven through separating three standard proteins in saline (0.9 % NaCl) with separation resolution (SR) >1.29. Through further coupling with colorimetric and lateral flow strip measurements, the 3D paper-based IEF platform was applied to directly pre-treat and quantitatively analyze the two disease biomarkers, i.e., microalbuminuria and C-reactive proteins in clinical human serum. The analytical results had the relative deviations of <8.4% and <13.1% to clinical results, respectively. As the first report of the paper-based IEF platform for directly pretreating high-salt samples, this work proposes a new strategy to minimize the difficulty of directly processing high-salt samples with traditional IEF system and provides a versatile, miniaturized and low voltage demand analytical platform for POCT of proteins in physiological samples.