A Novel Multiplex Mycotoxin Surface-Enhanced Raman Spectroscopy Immunoassay Using Functional Gold Nanotags on a...

A novel multiplex mycotoxin surface-enhanced Raman spectroscopy (SERS) immunoassay was established for the first time on different artificial antigen-modified silica photonic crystal microspheres (SPCMs), which can be integrated into a biochip array to achieve multiplex detection using corresponding antibody-functionalized gold nanoparticles (AuNPs) as the SERS nanotag. The unique optical structure of SPCMs is helpful to find the detection spots easily, accommodate a large amount of probe molecules, and enhance the Raman signal intensity. Such enhancement was confirmed by the simulation result, showing the electric field enhancing effect in SPCMs with AuNPs being 7 times. A competitive SERS immunoassay was established using antigen-modified SPCMs and mycotoxins to compete for binding antibody-functionalized SERS nanotags, displaying broad linear detection ranges of 0.001-0.1 ng/mL for aflatoxin B₁ (AFB₁), 0.01-10 ng/mL for ochratoxin A (OTA), and 0.001-0.1 ng/mL for zearalenone (ZEN) and low detection limits of 0.82 pg/mL for AFB₁, 1.43 pg/mL for OTA, and 1.00 pg/mL for ZEN. In the spiked cereal samples, recovery rates of the method were measured in the range of 70.35-118.04% for the three mycotoxins, which was in agreement with that of the traditional enzyme-linked immunosorbent assay method. The SERS immunoassay for mycotoxin detection also showed high specificity and good repeatability and reproducibility. The new microsphere-based SERS immunoassay biochip only requires a one-step reaction and overcomes the disadvantages of fluorescence and chemiluminescence background signals. The work paves the way for further developing SERS-based microsphere suspension arrays for new targets.