A three-dimensional paper-based isoelectric focusing device for direct analysis of proteins in physiological samples

On-site protein analysis is crucial for disease diagnosis in community and family medicine in which microfluidic paper-based analytical devices (μPADs) have attracted growing attention. However, the practical applications of μPADs in protein analysis for physiological samples with high complexity is still limited. Herein, we developed a three-dimensional (3D) paper-based isoelectric focusing (IEF) platform, which is composed of power supply, reservoirs, and separation channel and made by the origami and stacking method, to simultaneously separate and enrich proteins in both low-salt and high-salt samples. Under the optimized experimental conditions, standard proteins (bovine hemoglobin (BHb) and phycocyanin (Phy)) were separated within 18 min under a 36 V power supply and obtained a 10-fold enrichment using the 3D paper-based IEF platform. Then, the capability of the 3D paper-based IEF platform for direct pretreatment of high-salt samples using a 12 V battery as power supply was measured 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 pretreat and quantitatively analyze microalbuminuria and C-reactive proteins in clinical urine and serum samples with analytical results with relative deviations of <8.4% and < 13.1%, respectively, to the clinical test results. This work proposes a new strategy to minimize the difficulty of directly processing high-salt samples with the traditional IEF system and provides a versatile, miniaturized, and low voltage demand analytical platform for on-site analysis of proteins in physiological samples.