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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 …