Split Aptamer-Driven CRISPR-Cas12a biosensor with signal amplification for quantitative detection of adenosine deaminase

Abstract

We report the development of a novel split aptamer-driven electrochemical biosensor integrating entropy-driven Signal amplification (EDSA) and CRISPR-Cas12a technology for the highly sensitive and specific detection of adenosine deaminase (ADA). ADA is an important biomarker involved in immune regulation and purine metabolism, and its accurate quantification is critical for diagnosing various diseases. In our system, ADA catalyzes the deamination of adenosine to inosine, resulting in the release of Aptamer1 from a split aptamer structure. The liberated Aptamer1 initiates a strand displacement reaction within a Blocker/Bandage/Scaffold complex, triggering a cascade of entropy-driven amplification cycles that generate a DNA duplex capable of activating CRISPR-Cas12a. Activated Cas12a exhibits trans-cleavage activity, specifically cleaving a ferrocene-labeled single-stranded DNA (ssDNA) probe immobilized on a gold electrode, leading to a measurable decrease in electrochemical signal. The biosensor operates under isothermal and enzyme-free conditions, providing simplicity, rapid detection, and low cost. It achieves a low detection limit of 0.688 U·L⁻¹ with a wide linear range from 1 to 20,000 U·L⁻¹. The system demonstrates excellent specificity against ADA analogues and common interfering proteins and performs reliably in diluted human serum samples. This strategy represents a versatile and modular platform for point-of-care diagnostics. By altering the aptamer sequence, the biosensor can be adapted for the detection of other clinically relevant proteins and enzymes, contributing to advances in personalized medicine.