CRISPR/Cas12a-Based Aptasensor Enhanced by Functionalized AuNPs for Sensitive Full-Range C-Reactive Protein Detection

Abstract

C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections, autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng/mL and a quantifiable range of 0.1 -150 µg/mL. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical application.