Analyte-induced hindrance in the RCA-assisted CRISPR/Cas12a system for homogeneous protein assays

Heterogeneous assays, such as enzyme-linked immunosorbent assays, have become indispensable for in vitro diagnostics. However, the simple, sensitive, and accurate detection is limited by their multiple washing and incubation steps, and limited amplification methods. In this study, we design a novel approach utilizing analyte-induced hindrance within the rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for simple and highly sensitive homogenous protein detection. Streptavidin (SA) and digoxin antibody (anti-Dig) are employed as representative detection models. The specific recognition of target proteins using primers modified with small molecules hinders the RCA process, preventing the activation of Cas12a's trans-cleavage activity, thereby leading to a reduction in fluorescence intensity. Our developed platform exhibites exceptional detection performance characterized by high sensitivity, robust specificity, and significant potential for application in complex samples. By expanding the recognition elements, this platform can evolve into a versatile clinical diagnostic tool with universal applicability. In addition, this platform provides a novel direction for quantifying ultralow-concentration disease biomarkers in clinical practice.