Engineering a Lab-Free Microfluidic Chip Biosensor Integrating Immuno-Proximity Ligation and On-Cleavage RCA for Multiplex Meat Adulteration Monitoring

Abstract

Traditional analytical methods for food authentication heavily rely on laboratory-based instrumentation and inconsistent operational procedures, limiting their practical applicability for on-site identification in complex food markets. Herein, a lab-free centrifugal microfluidic chip-based biosensor was developed for the simultaneous identification of chicken, duck, pork, beef, mutton, and horseflesh adulteration through endogenous IgG recognition. By integrating an immuno-proximity ligation assay (PLA) with nicking enzyme-assisted rolling circle amplification (RCA), the proposed platform innovatively transformed protein detection into highly efficient nucleic acid signal amplification, enabling continuous “amplification−cleavage” cyclic generation of G-quadruplex DNAzymes with peroxidase-mimicking activity for colorimetric detection, alongside berberine-binding aptamer fragments for fluorescence enhancement. The biosensor achieved accurate quantification of IgGs from six meat species within 90 min, with detection limits as low as 0.056 ng/mL for the colorimetric mode and 0.013 ng/mL for the fluorescent mode, respectively, and was capable of detecting adulterants at levels as low as 0.5% (w/w). Commercially available meat products were successfully analyzed using the proposed platform, obtaining excellent recovery rates of 95.0%−108.9%. This work presents a portable, automation-enabled, and highly scalable biosensing platform for standardized multi-species meat authentication, providing a versatile strategy for on-site food adulteration monitoring and nucleic acid signal amplification-assisted protein diagnostics.