A Clickase-Mediated Immunoassay Based on Nanopore and Bionic Signal Labels for Ultrasensitive, Portable, and On-Site Detection of Ricin

It is of particular importance to develop an effective method that possesses several merits simultaneously of rapid, ultrasensitive, portable, and on-site detection potential for food safety detection. Herein, we propose a clickase-mediated immunoassay based on nanopore and bionic signal labels for the detection of ricin. The introduction of Cu/Cys clickase and nanopore simultaneously effectively addressed the inherent limitations of natural enzymes and colorimetric signal output, respectively. Using this method, bionic signal labels can be easily formed through DNA and Gram-positive bacterial cell wall terminal peptide fragments (labeled by alkynyl and azide, respectively) and vancomycin. Translocation of the D‑P@​vancomycin through the nanopore generated highly specific oscillation current traces. This method showed a great on-site detection potential and superior analytical performance owing to the combination of the specificity of antibodies, high CuAAC click reaction catalytic efficiency of clickase, ultrasensitivity of the nanopore, and high signal resolution of D‑P@​vancomycin. Moreover, the practical applicability of the established method was also verified, achieving a limit of detection (LOD) down to 200.9 ag/mL with a wide linear relationship under the optimized conditions. In conclusion, this method is promising for rapid, portable, ultrasensitive, and on-site food safety detection.