| Antibiotics as a class of emerging pollutants have been detected in the food.These pollutants have been widely concerned to researchers.Because the antibiotics are the persistence of the "false positive" and it can cause bacterium with drug resistan.In addition,in real samples such as fish,the antibiotic content is very low,generally exists in the trace level(ng mL-1).And there are a lot of structural analogues.This makes it necessary to development a sensitive and specific strategy for antibiotics in seafood.The fluorescent and colorimetric aptasensor just can meet the above requirements.In this thesis,we use the enzyme linked polymer,vesicle,exonuclease-assisted target recycling to amplify the signal,and construct the probe.The detail contents are as follows:1.A sensitive and facile colorimetric aptasensor for chloramphenicol(CAP)detection based on enzyme-linked polymer nanotracers labeled by double-stranded DNA(ds-DNA)antibody.In this work,the ds-DNA containing thiolated aptamer for CAP(Apt)and its complementary strand(cDNA)was immobilized on Fe3O4@Au magnetic nanoparticles(AuMNPs)as capture probe.An enzyme-linked polymer nanotracer was fabricated by co-immobilizing the ds-DNA antibody and horseradish peroxidase(HRP)labeled nanogold on Envision reagents(ds-DNA Ab/ EV-AuNPsHRP)as signal tag.EnVision reagent(EV)contains about 100 HRP and some anti-IgG antibody molecules.In the presence of CAP,the aptamer preferentially bound with CAP to release ss-DNA that can't be recognized by ds-DNA Ab in the nanotracers and then the signal tag was substituted into supernatant after magnetic separation.The supernatant containing numerous HRP could efficiently catalyze 3,3',5,5'-tetramethylbenzidine(TMB)-H2O2 solution for the color development quantified by ultraviolet-visible spectroscopy.Experimental results showed the CAP detection owning a linear range of 0.05-100 ng mL-1 and sensitively respond down to 0.015 ng mL-1.Besides,the results of this method for CAP detection in the fish samples agreed well with the ELISA results,vertified its accuracy and reliability.The developed method can be used for in-situ CAP detection in food safety.2.Herein,an ultrasensitive and selective colorimetric assay for antibiotics,using chloraphenicol(CAP)as model analyte,was developed based on a magnetic aptamer-HRPplatinum composite probes and exonuclease-assisted target recycling.The composite probes were prepared through the immunoreactions between the double strand DNA antibody(anti DNA)labeled on core-shell Fe3O4@Au nanoparticles(AuMNPs-anti DNA)as capture probe,and the double strand aptamer(aptamer hybrid with its complementary oligonucleotides)labeled on Pt@HRP nanoparticles as nanotracer(ds-Apt-HRP-PtNPs).When the CAP samples were incubated with the probes for 30 min at room temperature,it can be captured by the aptamer to form the nanotracer-CAP complex,which was then released into the supernatant after magnetic separation.This is because anti DNA on capture probes can't 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 aptamer and the CAP in aptamer-CAP complex can be released again,which can further participate in new cycling to react with the probes.Pt and HRP in the nanotracer could both catalyze and dual amplify the absorbance at 650 nm ascribed to 3,3',5,5'-tetramethylbenzidine(TMB)-H2O2 system.Moreover,the 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.Experimental results showed that the CAP detection owned a linear range of 0.001-10 ng mL-1 and detection limit of 0.0003 ng m L-1(S/N=3).The assay was successfully employed to detect CAP in milk,which is much facile,time saving,sensitive than the commercial ELISA kits.3.A selective and facile fluorescence “switch-on” scheme is developed to detect antibiotics residues in food,using chloramphenicol(CAP)as model,based on a novel magnetic aptamer probe(aptamer-Pt-luminol nanocomposite labeled with hemin/G-quadruplex).Firstly,the composite probe is prepared through the immuno-reactions between the capture beads(antidsDNA antibody labeled on magnetic Dynabeads)and the nanotracer(nano Pt-luminol labeled with double-strand aptamer,as ds-Apt,and hemin/G-quadruplex).When the composite probe is mixed with CAP,the aptamer preferentially reacted with CAP to decompose the double-strand aptamer to ssDNA,which can not be recognized by the anti-dsDNA antibody on the capture probes.Thus,after magnetic separation,the nanotracer can be released into the supernatant.Because the hemin/G-quadruplex and PtNPs in nanotracer can catalyze luminol-H2O2 system to emit fluorescence.Thus a dual-amplified "switch-on" signal appeared,of which intensity is proportional to the concentration of CAP between 0.001 and 100 ng mL-1 with detection limit of 0.0005 ng mL-1(S/N=3).Besides,our method has good selectivity and was employed for CAP detection in real milk samples.The results agree well with those from conventional Gas chromatograph-Mass spectrometer(GC-MS).The switch-on signal is produced by one-step substitution reaction between aptamer in nanotracer and target.When the analyte is changed,the probe can be refabricated only by changing the corresponding aptamer.Thus,all features above prove our strategy to be a facile,feasible and selective method in antibiotics screening for food safety.4.A novel fluorescence aptasensor was successfully developed to detect chloramphenicol(CAP)in food based on magnetic aptamer-liposome vesicle probe.In order to fabricate it,aptamer labeled on functionalized magnetic beads(MB)was firstly employed as capture adsorbent(MBApt),and then SSB(single-stranded DNA binding protein)and DIL(1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate)coimmobilized by liposomes(SSB/DIL-Lip)was employed as vesicle signal tracer.The composite vesicle probe was formed between SSB/DIL-Lip and MB-Apt based on SSB's specific recognition towards aptamer on vesicle signal tracer.Upon the vesicle probe solution reacting with CAP,the aptamer on the magnetic beads preferentially bounded with CAP,and then SSB/DIL-Lip vesicle signal tracer was released into the supernatant after magnetic separation.The released tracer can emit fluorescence,the intensity of which was in correspondence with the concentration of the analyte.At the optimum conditions,the aptasensor exhibited a good linear response for CAP in the range of 0.003-10 nM with a detection limit of 1 pM.Importantly,the methodology was further validated by the comparison between the results of analyzing CAP in fish samples with the corresponding results obtained by ELISA kit,providing a promising approach for quantitative monitoring of CAP and significant anti-interference ability in food safety.5.In this work,a novel homogeneous and signal “off–on” aptamer based fluorescence assay was successfully developed to detect chloramphenicol(CAP)residues in food based on the fluorescence resonance energy transfer(FRET).The vesicle nanotracer was prepared through labeling single stranded DNA binding protein(SSB)on limposome-CdSe/ZnS quantum dot(SSB/L-QD)complexes.It was worth mentioning that the signal tracer(SSB/L-QD)with vesicle shape,which was fabricated being encapsulated with a number of quantum dots and SSB.The nanotracer has excellent signal amplification effects.The vesicle composite probe was formed by combining aptamer labeled nano-gold(Au-Apt)and SSB/L-QD.Which based on SSB's specific affinity towards aptamer.This probe can't emit fluoresce which is in “off” state because the signal from SSB/L-QD as donor can be quenched by the Au-aptas acceptor.When CAP was added in the composite probe solution,the aptamer on the Au-Apt can be preferentially bounded with CAP then release from the composite probe,which can turn the “off” signal of SSB/L-QD tracer into “on” state.The assay indicates excellent linear response to CAP from 0.001 nM to 10 nM and detection limit down to 0.3 pM.The vesicle probes with size of 88 nm have strong signal amplification.Because a larger number of QDs can be labeled inside the double phosphorus lipid membrane.Besides,it was employed to detect CAP residues in the milk samples with results being agreed well with those from ELISA,verifying its accuracy and reliability. |
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