Abstract This study presents two alternative fluorescent nanoparticle-based oxytetracycline (OTC) detection methods in milk samples. Rhodamine 6G-coated fluorescent hybrid silica nanoparticles and fluorescent magnetic/silica nanoparticles functionalized with anti-OTC antibodies were used in this test. The sandwich test format was utilized to compare anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles with OTC/OTC antibody-conjugated fluorescent hybrid silica nanoparticles in an Eppendorf tube with magnetic separators. The magnetic separator helps to quickly retain all of the OTC captured by fluorescent magnetic core–shell nanoparticles in the milk sample. As a result, the assay time was dramatically shortened. The obtained linear range was 1.34 × 10−6 to 2.10 × 10−8 (M) (R 2 = 0.9954), the detection limit was 4.76 ng/mL, and the total assay time was 90 min. This approach was used to determine the OTC concentration in milk samples, and the maximum percentage (%) of interference was less than 3.0%, with a recovery rate of greater than 97.0%. This approach offers a high potential for residue detection in milk samples. With a total analysis period of less than 90 min, this approach provided the best way to determine the capture and detector nanoparticles’ response.
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