An rolling circle amplification-assisted CRISPR/Cas12a-based biosensor for protein detection

Glycated hemoglobin (HbA1c) has been extensively studied as a pivotal marker for monitoring and diagnosing diabetes. In this study, we introduce a highly sensitive and rapid enzyme-catalyzed rolling circle amplification (RCA)-assisted nicked-PAM CRISPR/Cas12a assay (termed "RANPC"). The RANPC system initially recognizes and captures the target using immunomagnetic beads and antibodies, forming a sandwich structure. The antibodies, upon ligating the primers, trigger the RCA reaction, resulting in the generation of single-stranded DNA with numerous repetitive sequences. The product strand of RCA then initiates the trans-cutting activity of nicked-PAM CRISPR/Cas12a, ultimately reporting the target concentration through fluorescent signaling. This study combines dual antibody capture, signal amplification through RCA, and signal output by Cas12a, resulting in high sensitivity, high specificity, and high signal-to-noise ratio. Moreover, the strategic incorporation of an incomplete PAM design in the CRISPR/Cas12a system substantially reduces background interference and enhances assay specificity relative to previous conventional Cas12a detection methods. RANPC possesses advantages such as ultra-sensitivity (with the lowest detection limit up to 0.129 pg/mL and a linear range of 12.8 pg/mL to 1 µg/mL), high specificity, rapid reaction time (approximately 50 min), and straightforward operation. Under optimized experimental conditions, we successfully detected HbA1c in eight real-world blood samples, and the results were in good agreement with the theoretical values. This suggests that this method holds promise as a new approach for the analysis of HbA1c levels in the clinical setting.