Novel Proximity-Based CRISPR-Cas12a Assay for Protein Detection and its Application in CRP Diagnosis

Accurate and sensitive detection of protein biomarkers is critical for early disease diagnosis and therapeutic monitoring. This study introduces a novel isothermal proximity CRISPR-Cas12a assay for the detection of C-reactive protein (CRP), a key biomarker for inflammation and disease progression. By employing a DNA-antibody bioconjugate strategy, this method converts protein recognition into DNA signals, enabling multiple signal amplification cascades. The proximity hybridization of antibody probes triggers the Cas12a trans-cleavage activity, releasing detectable signals in fluorescence and commercial test strips. Dual-mode detection was realized, with detection limits of 0.57 ng/mL for fluorescence and 2.63 ng/mL for test strips. Linearity detection ranges were found to be 0.1–100 µg/mL. Furthermore, it exhibited high specificity against interfering substances in blood. Clinical serum samples were analyzed, demonstrating the assay’s robustness and practicality under physiological conditions. This approach enhances the detection of CRP and provides a versatile framework for the quantitative analysis of various protein biomarkers, offering an alternative solution for clinical protein diagnosis for point-of-care tests.