Concentric-Type Immunoblotting for Simultaneous Separation and Detection of Exosomal Proteins

The rapid, high-throughput, and sensitive detection of exosomal protein is the key to achieving accurate qualitative and quantitative analyses. In this study, a concentric-type immunoblotting (CIB) for the simultaneous separation, concentration, and detection of exosomal proteins was first proposed by integrating a circular nonuniform gradient electric field with Western blot (WB). By utilizing the characteristic of varying circular nonuniform electric field density with radius, differential electromigration of marker proteins based on different molecular weights can be realized through a gradient-driven electric field. Through coupling with targeted spiral injection design and direct immunoassay, antigen–antibody specific recognition can be directly achieved in their respective separation regions, hence facilitating high-throughput, high-sensitivity, and rapid detection of multiple biomarkers. This model demonstrated respective improvements in separation resolutions 2.41-fold, 1.89-fold, and 3.54-fold for high- ( M > 70 kDa), medium- (25 < M ≤ 70 kDa), and low-molecular-weight ( M ≤ 25 kDa) universal proteins, respectively, with a 3.33-fold shortened separation time compared to conventional WB. Due to the combined shrinking effect in the concentration zone of the circular nonuniform electric field and immunoblotting in CIB, the limits of detection (LODs) for exosomal proteins Alix, TSG101, and CD9 were 2.0, 0.25, and 0.6 μg, respectively, which represent a sensitivity enhancement of 2.11- to 3.88-fold in comparison to WB, with RSD < 10%. This technique overcomes the low resolution and poor sensitivity problems of traditional WB; thus, it holds promise as a versatile and universal tool for protein separation and detection.