Adenosine deaminase-regulated aptakiss circuit coupled with hairpin-tube amplification for ratiometric ECL/SERS...

Adenosine deaminase (ADA) is a key purine-metabolism enzyme that catalyzes the deamination of adenosine into inosine and is closely associated with immune regulation, inflammation, and disease-related metabolic disorders. Herein, an ADA-responsive ratiometric electrochemiluminescence/surface-enhanced Raman scattering (ECL/SERS) biosensor was developed by integrating an adenosine-regulated aptakiss recognition module, a hairpin-tube-mediated interfacial amplification reaction, and a CsPbBr₃@PDA@AuNPs/MXene (PAM)-modified glassy carbon electrode. In the absence of ADA, adenosine promotes formation of the DNA1/DNA2/adenosine aptakiss complex, leading to DNA3 release and activation of the hairpin-tube reaction on the PAM/GCE interface. The resulting cyclic displacement recruits DNA4-Fc to the electrode, producing a strong Fc-associated SERS signal while suppressing the CsPbBr₃-based ECL emission. In contrast, ADA converts adenosine into inosine, prevents aptakiss complex formation, inhibits DNA3 release, and suppresses DNA4-Fc accumulation, resulting in high ECL and low SERS signals. Owing to these anti-correlated responses, the ratiometric signal R = I_ECL/I_SERS increased with ADA activity. Under the optimized conditions, the normalized ECL and SERS responses displayed good concentration-dependent trends over 0.001-10 U L^-1 ADA, and the ratiometric calibration gave a low detection limit of 0.0022 U L^-1. The biosensor also showed favorable selectivity, repeatability, electrode-to-electrode reproducibility, operational stability, storage stability, and SERS spatial uniformity, demonstrating its potential for reliable ADA activity analysis.