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Under the excitation of a 980 nm excitation light, the fluorescence signals of the synthesized core-shell NaYF4Yb@NaYF4Ho and monolayer NaYF4Yb,Tm upconversion nanoparticles (UCNPs) were simultaneously detected at 656 and 696 nm, respectively. The two upconversion materials were coupled with anti-clothianidin and anti-imidacloprid monoclonal antibodies by the glutaraldehyde cross-linking method as signal probes. Imidacloprid (IMI) and clothianidin (CLO) could compete with antigen-conjugated amino Fe3O4 magnetic nanomaterials for binding