Glass Fiber Interfaced Special Rolling Circle Amplification for Multiplexed Protein Detection with Paper Strip Digital Readouts

Point-of-care testing (POCT) for proteins faces challenges, including limited sensitivity and multiplexing capabilities. The recently developed minimum secondary structured rolling circle amplification (MSS-RCA) promised POCT with desirable sensitivity in nucleic acid detection under isothermal conditions, but with a long assay time. This study integrates the 5' specific enzymatic activity of T7 exonuclease with the high-speed template elongation ability of MSS-RCA in a one-pot system to establish the dual amplification T7-MSSRCA detection platform. T7-MSSRCA employed a specialized reporter modified with human chorionic gonadotrophin (hCG) molecules for signal output on hCG test strips, which could be measured by a lateral flow assay (LFA) analyzer. The integration of T7-MSSRCA with glass fiber interfaced immunoassays enabled multiplexed detection of low-abundance protein targets (α-synuclein, IL-6, TNF-α, and IFN-γ) at subpicogram levels in serum samples of Parkinson's disease with high specificity and accuracy, promising its application in biomarker identification of chronic diseases.