Screening Of Antibiotics-specific Aptamer And Fabrication Of Antibiotics Aptasensor Based On Graphene

The overcommitment of antibiotics as veterinary drugs will lead to antibiotic residues in animal-derived food, which can cause the damage of human health, and therefore become an important issue in food safety control. In order to improve the safety of animal-derived food, it is very important to establish efficient, accurate, fast, and economical methods for detection of antibiotics. Aptamer-based assay exhibits high specificity and sensitivity, and has great prospect in the detection of antibiotics. Ofloxacin and kanamycin are both commonly-used clinical antibiotics and veterinary drugs. This thesis focuses on the screening of antibiotics-specific aptamer and the fabrication of aptasensor for antibiotics.Functionalized graphene, graphene oxide was introduced in the process of systematic evolution of ligands by exponential enrichment(SELEX). Graphene oxide SELEX(GO-SELEX) was used to screen the aptamers that bind to ofloxacin with high specificity and affinity from a 79-nt initial library containing 35-nt random sequences. The initial library was incubated with ofloxacin after pretreatment, then graphene oxide was added to adsorb ss DNA. which cannot binding with ofloxacin and separate them from ofloxacin-aptamer complexes. After centrifugation and purification, ofloxacin-binding ss DNA in the supernatant was amplified through PCR. Then the single strand was prepared by using streptavidin-coated magnetics beads. The recovery of ss DNA was evaluated after each SELEX round. The recovery became unchanged after 6 rounds of SELEX. Then one round of negative selection was carried out to remove ss DNAs which bind with other antibiotics, followed by the last round of SELEX. The PCR amplification products after the last SELEX round were cloned and sequenced. Twelve ofloxacin-specific aptamers were obtained. Among them, four sequences with higher homology, aptamers ap1, ap3, ap4 and ap5 were picked out. Their secondary structure were simulated, and the dissociation constants(Kd) were measured, which were 251.3, 130.1, 159.1, 304.4 nmol·L-1 respectively. The ap3 and ap4 were proven to be highly-specific to ofloxacin.Aptamer-based detection of kanamycin was established by using a graphene modified glassy carbon electrode. Kanamycin-specific aptamer can be adsorbed onto graphene modified electrode, which prevents the electron transfer between the probe [Fe(CN)6]3-/4- and the electrode. However, after incubation with samples containing kanamycin, kanamycin can bind with aptamer and desorb from the electrode, which in turn decline the resistance of the electron transfer and recover the electrochemical signal. The procedure was firstly characterized by cyclic voltametry(CV) and atomic force microscopy(AFM). Then this replacement effect was utilized to electrochemically determine kanamycin. Under the optimized condition, kanamycin can be determined by using differential pulse voltammetry(DPV) in a linear range of 1×10-6~1×10-5 mol·L-1, with the detection limit of 5×10-7 mol·L-1. The method was successfully applied to detect kanamycin in milk samples. A similar linear detection range was derived in milk sample as in standard kanamycin samples. The value of relative standard deviation(RSD) indicates satisfactory reproducibility of the method.