A triple-amplification colorimetric assay for antibiotics based on magnetic aptamer–enzyme co-immobilized platinum nanoprobes and exonuclease-assisted target recycling

Abstract

Herein, an ultrasensitive and selective colorimetric assay for antibiotics, using chloramphenicol (CAP) as the model analyte, was developed based on magnetic aptamer–HRP–platinum composite probes and exonuclease-assisted target recycling. The composite probes were prepared through immunoreactions between the double stranded DNA antibody (anti-DNA) labeled on core–shell Fe₃O₄@Au nanoparticles (AuMNP-anti-DNA) as the capture probe, and the double stranded aptamer (aptamer hybrid with its complementary oligonucleotides) labeled on Pt@HRP nanoparticles as the nanotracer (ds-Apt–HRP–PtNPs). When the CAP samples were incubated with the probes for 30 min at room temperature, they could be captured by the aptamer to form a nanotracer–CAP complex, which was then released into the supernatant after magnetic separation. This is because the anti-DNA on the capture probes cannot recognize the single strand aptamer–CAP complex. The exonuclease I (Exo I) added into the supernatant can further digest the aptamer–CAP from the 3′-end of the aptamer and the CAP in the aptamer–CAP complex can be released again, which can further participate in a new cycling process to react with the probes. Pt and HRP in the nanotracer could both catalyze and dual amplify the absorbance at 650 nm ascribed to the 3,3′,5,5′-tetramethylbenzidine (TMB)–H₂O₂ system. Moreover, Exo I can assist the target recycling, which can further amplify the signal. Thus, the triple amplified signal can be quantified by ultraviolet-visible spectroscopy. The experimental results showed that the CAP detection possessed a linear range of 0.001–10 ng mL⁻¹ and a detection limit of 0.0003 ng mL⁻¹ (S/N = 3). The assay was successfully employed to detect CAP in milk, which is much more facile, time saving, and sensitive than the commercial ELISA kits.