The Study On Fluorescence And Microfluidic Methods Based On Aptamer Probe For Detection Of Antibiotics Residues In Food

At present,many types of antibiotic residues have been found in the environment.They slow degradation in the environment,easily deposited in the soil sediment,and through the food chain into the human body,causing harm to the human body.In addition,the concentration of antibiotics in the environment and food is very low,and their matrix interferences are usually severe.Therefore,it is urgently needed to develop an analytical method for the determination of antibiotic residues in complex matrices such as food.In this paper,a series of novel aptamer sensors were developed for the detection of antibiotic residues(including chloramphenicol,kanamycin and oxytetracycline)based on nucleic acid aptamer probes combined with fluorescence and microfluidic methods.And its performance and principle are studied and explored.Compared with some aptamer sensors,the above-mentioned analytical method reduces the experimental cost,simplifies the experimental steps,enhances the detection sensitivity and the accuracy,and has wider application prospect.The study of the thesis is performed as the following fields: 1.A facile signal-on and homogeneous fluorescence assay for specific detection of antibiotics residues based on aptamer-dsDNA antibody-quatumn dots probesIn this work,a facile,signal-on and homogenous fluorescence assay using novel aptamer-dsDNA antibody-quatumn dots probes was designed for detecting antibiotics residues.In this assay,the fluorescent probes were fabricated by using double-stranded DNA antibody(dsDNA Ab)covalent bonded on the surface of CdSe quantum dots.The antibody can bind double-stranded DNA(dsDNA)specifically.When dsDNA was added,the fluorescence could be efficiently quenched because the probes contacted each other with dsDNA as bridge.The dsDNA was formed between an aptamer towards chloromycetin(CAP)and its complementary strand DNA(cDNA).When CAP was added into the reaction system,the aptamer reacted with CAP preferentially and the dsDNA was unwinded to single strand DNA(ssDNA)which can't be recognized by the dsDNA antibody.Thus the probes were separated and fluorescence was recovered.Based on the scheme,a signal-on fluorescent assay for detecting antibiotics in homogenous aqueous phase was developed.Under the optimized conditions,the assay shows a good liner range from 0.05 ng mL-1 to 100 ng mL-1 and detection of limit(LOD)is 0.002 ng mL-1.If the analyte is changed,the assay can also be employed only by changing the corresponding aptamer and its cDNA.What's more,the probes can be reusable at least 10 times with the recovery above 90 %.The fluorescence assay is facile and has a promising application for detecting antibiotic contaminants in biological and environmental samples.2.Single-stranded DNA binding protein-assisted fluorescence aptamer switch based on FRET for homogeneous detection of antibioticsIn this work,a smart single-stranded DNA binding protein(SSB)-assisted fluorescence aptamer switch based on fluorescence resonance energy transfer(FRET)was designed.The FRET switch was synthesized by connecting SSB labeled quantum dots(QDs@SSB)as donor with aptamer(apt)labeled gold nanoparticles(AuNPs@apt)as acceptor,and it was employed for detecting chloramphenicol(CAP)in a homogenous solution.In the assay,the interaction between core-shell QDs@SSB and AuNPs@apt leads to a dramatic quenching(turning off).After adding CAP in the detection system,AuNPs@apt can bind the target specifically then separate QDs@SSB with AuNPs@apt-target,resulting in restoring the fluorescence intensity of QDs(turning on).Consequently,the fluorescence intensity recovers and the recovery extent can be used for detection of CAP in homogenous phase via optical responses.Under optimal conditions,the fluorescence intensity increased linearly with increasing concentrations of CAP from 0.01 to 100 ng mL-1.The limit of this fluorescence aptamer switch was around 3 pg mL-1 for CAP detection.When the analyte is changed,the assay can be applied to detect other targets only by changing relative aptamer in AuNPs@apt probe.Furthermore,it has potential to be served as a simple,sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.3.Novel label-free and high-throughput microchip capillary electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplificationIn this work,a novel label-free microchip electrophoresis(MCE)platform has been developed here for simultaneous detection of two antibiotics which are kanamycin(Kana)and oxytetracycline(OTC),based on two aptamer probes and target catalyzed hairpin assembly(CHA)for signal amplification.These probes contained six DNA strands termed as Kana aptamer-catalysis strand(Kana apt-C),Kana inhibit strand(Kana inh),OTC aptamer-catalysis strand(OTC apt-C),OTC inhibit strand(OTC inh),hairpin structures H1 and H2 which were partially complementary.Upon the addition of Kana or OTC,the binding event of aptamer and target triggered the self-assembly between H1 and H2,resulting in the formation of many H1-H2 complexes.They could show amplified signals in the MCE system and represent the corresponding concentration of Kana or OTC respectively.With the help of the well-designed and high-quality CHA amplification,the assay could yield 300-fold signal amplification.Under optimal conditions,this assay exhibited a linear correlation in the ranges from 0.001 ng mL-1 to 10 ng mL-1,with the detection limits of 0.7 pg mL-1 and 0.9 pg mL-1(S/N=3)toward Kana and OTC,respectively.The platform has the following advantages: firstly,the aptamer probes can be fabricated easily without labeling signal tags for MCE detection;Secondly,the targets can just react with probes and produce the amplified signal in one-pot.Finally,the targets can be simultaneously detected within 10 mins in different channels,thus high-throughput measurement can be achieved.Based on this work,it is anticipated that this detection platform will be universally served as a simple,sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.