Study On The Simultaneous Detection Of Multiple Antibiotics Based On Aptamers

Antibiotics have been widely used in the treatment of various bacterial infections of human and animal diseases. However, it is easy to cause a variety of antibiotic residues in animal-derived foods, which can be hazard for health. Therefore, to establish efficient methods for the detection of multiple antibiotic residues in food is of great importance in food safety control. We report a simultaneous electrochemical detection of streptomycin(STR), chloramphenicol(CHL) and tetracycline(TET) based on aptamers and quantum dots(QDs).Aptamers are synthetic oligonucleotides specific for a wide range of target molecules with high affinity. We used STR, CHL and TET binding aptamer DNAs(Ap-DNAs) for our research. Three complementary DNA1 sequences(cDNA1s) were designed according to corresponding Ap-DNAs. Capture DNAs(Cap-DNAs) and complementary DNA2 sequences(cDNA2s) were designed according to 3’-terminal and 5’-terminal of corresponding cDNA1 s, respectively. PbS, CdS and ZnS QDs modified with mercaptoacetic acid were synthesized by water phase method. Then the amino group of streptavidin(SA) was covalently linked to the carboxyl group of mercaptoacetic acid on QDs. After that, 3’-terminal biotinylated cDNA2 s were attached to the corresponding SA-modified QDs respectively via the strong biotin-streptavidin affinity.Then an electrochemical sensor was constructed based on aptamers and quantum dots. Three cDNA1 s are complementary to part of the corresponding Ap-DNAs, and allowed to form duplex DNAs initially. When STR, CHL and TET are present, the three antibiotics bind with the corresponding Ap-DNAs specifically with higher affinity, which dehybridizes duplex DNAs and releases those cDNA1 s. Then the 3’-terminal of the liberated cDNA1 s hybridize with the corresponding thiol-modified Cap-DNAs which have been immobilized on the surface of gold electrode by self-assembly. After that, the prepared QDs-cDNA2 s conjugates are further introduced to the electrode surface due to the hybridization between the 5’-terminal of cDNA1 s and cDNA2 s. After formation of the sandwich complexes, the surface-captured QD tags are dissolved with HNO3, and then the released Pb2+, Cd2+ and Zn2+ are monitored electrochemically. The distinct SWASV stripping signals for Pb2+, Cd2+ and Zn2+ reflect the identities(potentials of Pb2+, Cd2+ and Zn2+ at-0.6 V,-0.8 V and-1.1 V correspond to STR, CHL and TET, respectively) and concentrations of the targets.The feasibility and performance of the assay for single antibiotic were firstly evaluated. This method can detect STR, CHL, TET in the range of 10~5000 nmol·L-1, 5~3000 nmol·L-1 and 20~8000 nmol·L-1, respectively. Compared with the HPLC method, this method also showed high accuracy through the detection of different concentrations of each antibiotic.Moving to the simultaneous detection of three antibiotics, the experiments were carried out as above. CV and EIS were employed to characterize the surface of gold electrode. The validity of the assay in the detection of antibiotic residues in real food samples was also evaluated by employing artificially contaminated milk containing STR, CHL and TET. The results showed the proposed method can be suitably applied for the simultaneous detection of multiple antibiotics in milk samples and achieved satisfactory reproducibility.