Selection Of Aptamers Of Neomycin And Streptomycin And Detection Of Their Multi-residues Labeled By Quantum Dots

Neomycin and streptomycin are aminoglycoside antibiotics which are widely used in animal husbandry.The unreasonable use can cause their residues in animal-derived foods,which affect human health after being consumed by humans.Therefore,a fast method useable for the on-site detection of streptomycin and neomycin is urgently needed to strengthen market supervision.Aptamer is a kind of single-stranded oligonucleotide with high affinity for a specific target.Quantum dot(QD)is a kind of fluorescent nanomaterial that can be excited at mono wavelength and emitted at multi-wavelengths.Their combination will be helpful to highly specifically recognize neomycin and streptomycin,and fast detect their multi-residues by fluorescence method.The selection of aptamers of neomycin and streptomycin,the fabrication of aptamer-QD complex fluorescent probes,and the detection method of multi-residues of neomycin and streptomycin by use of the fluorescent probes are studied in this thesis.The results are as follows:(1)The methods for screening highly specific aptamers of neomycin and streptomycin were developed.The main methodology parameters are as follows:the maximum mass ratio of carboxylated magnetic beads to neomycin and streptomycin is 34:1and 26:1;the PCR annealing temperature is 60�C;the ratio of forward and reverse primers is 30:1.(2)Highly specific aptamer N1 for neomycin and aptamer S11 for streptomycin are obtained first time by the methods,which there is not interactive recognition between N1and S11.N1 and S11 are highly affinitive to neomycin and streptomycin,and their dissociation constant K_d values are 40.96�11.56 nmol/L and 44.94�7.37 nmol/L,respectively.(3)A non-SELEX method for screening streptomycin aptamers was developed first time.The time window of which the target-ssDNA complex was collected was within 0-17min.Affinity-stabilized aptamer candidates were obtained after only 3 rounds of screening.The aptamer NS2 obtained by this method has high affinity and high specificity for streptomycin,and the dissociation constant K_d value is 26.56�8.57 nmol/L.(4)The conditions of fabricating two composite fluorescent probes used for the highly specific and affinitive recognition to neomycin and streptomycin were optimized.The emission wavelengths of QD1 and QD2 are 548 and 580 nm,and their active time are4 and 15 min.The molar ratios of QD1:N1 and QD2:NS2 are 1:1.95 and 1:1.41,and the concentrations of N1-QD1 and NS2-QD2 are 123 and 81 nmol/L.These two conjugations react with graphene oxide(GO)for 30 min to form composite fluorescent probes.The concentration of GO in N1-QD1/GO?NS2-QD2/GO is 300?g/mL.(5)The two composite fluorescent probes-based method was developed first time for rapid detection of neomycin and streptomycin multi-residues.The reaction time of sample and fluorescent probes is 30 min.The detection wavelengths of neomycin and streptomycin were set as 550 and 586 nm,and their correction wavelengths 618 and 518 nm,respectively.The linear ranges of neomycin and streptomycin were 50-1000?g/mL and 10-1000?g/mL,and their detection limits were 10.02 and 6.65?g/L.The spiked recoveries of neomycin and streptomycin in milk samples were 96.12-104.65%and 95.86-106.69%,respectively.The method had good specificity for both antibiotics.