CRISPR-Cas12a Amplified RNase Activity Sensor Powered by Gold Nanoparticle-Barcode DNA Multipliers

Abstract

Ribonuclease A (RNase A) is a clinically relevant biomarker whose aberrant activity compromises RNA stability and interferes with RNA-based therapeutics, highlighting the need for rapid and ultrasensitive detection tools. In this work, we developed a CRISPR/Cas12a-assisted biosensing platform integrated with a substrate-bridged magnetic bead-gold nanoparticle assembly (SB-MAC) for highly sensitive and selective RNase A detection. By optimizing AuNP loading density, RNA substrate/barcode DNA molar ratio, and enzymatic incubation conditions, the prepared dual-functionalized SB-MAC architecture enabled efficient substrate/RNase A cleavage interaction and significant signal amplification, yielding a limit of detection (LOD) of 0.16 pg mL⁻¹ for RNase A.The sensor exhibited excellent specificity against structurally and functionally related biomolecules and demonstrated strong analytical performance when tested on serum and water samples with recoveries obtained ranging from 104 to 110%. Owing to its modular substrate design and robust signal amplification, this DNA-assisted platform offers a versatile and clinically relevant tool for monitoring RNase A activity and can be readily adapted for detecting other nuclease-based biomarkers.