Abstract
The extensive use of cefapirin (CP) in milk has caused various health issues, resulting in an urgent need for an efficient assay for its detection. This study aimed to perform a colorimetric/fluorescence dual-mode assay based on graphene oxide–assisted systematic evolution of ligands by exponential enrichment (GO-SELEX) in vitro screening method for detecting CP aptamers. The aptamer (Apt22) had a high affinity (Kd = 13.818 ± 1.405 nM), with only 33 bases. Hence, we designed the G-quadruplex (G4) into a hairpin structure complemented by the shear effect of Exo III, and constructed a colorimetric/fluorescent dual-mode sensor. A colorimetric signal was generated using 3,3′,5,5′-tetramethylbenzidine discoloration catalyzed by a DNA-mimicking enzyme constructed from G4 and hemin. The formation of G4 implied that the hairpin structure was disrupted and the FAM and BHQ at the ends of it were separated, thus generating a fluorescent signal. Under optimal experimental conditions, the detection limits were 0.0168 and 0.0136 μM when the linear ranges of the colorimetric and fluorescent aptasensors were 1–8 μM and 0.9–2.3 μM. The sensor had high specificity and repeatability. It combined the advantages of visual colorimetric and fluorescence precision detection, providing a new approach to the practical monitoring of CP in milk.
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