The Study Of Aptamer Coding Probe Combining Microchip Electrophoresis Methods For Detection Of Veterinary Drug Residues In Food And Biological Sample

At present,many residues of veterinary drugs such as antibiotics,hormones and so on have been found in the environment.They have great side effects on humans through the food chain.However,the concentration of them in food or biological fluids is very low?<?g/kg?,and their matrix interferences are usually severe.Furthermore,there are always multiple drugs coexisting in food or biological sample.It is urgent to develop an analytical method with highly sensitivity,excellent specificity and high-throughput.In this paper,a series of novel aptamer-based probes were developed,combined with microchip electrophoresis platform,to detect veterinary drugs residues?including:chloramphenicol,kanamycin,penicillin,estradiol,etc.?,and their analytical performance and principles were studied.Compared with the electrochemical and optical aptasensors,the above sensors were simple and portable.And had the advantages of high throughput,rapid,good matrix interference resistance as well as wider application prospect in field detection.This paper is divided into three parts to carry out research work:1.A label-free and universal platform for antibiotics detection based on microchip electrophoresis using aptamer probesA novel label-free,universal,and high throughput aptasensor was developed based on a microchip electrophoresis?MCE?platform for automatic detection of antibiotic residues in food.Firstly,chloramphenicol?CAP?was employed as a model to be captured by its aptamer probe?Apt?.Then,the partial complementary oligonucleotide of CAP's aptamer?c-DNA?was introduced into the reaction system.Because the Apt-CAP complex can't further hybrid with free c-DNA,the amount of hybrid Apt-c-DNA double strand DNA?dsDNA?was less than that without adding the target.Finally,the above mixture was introduced into the microchip electrophoresis?MCE?platform for detection,both dsDNA and Apt-CAP can be separated and produce different fluorescence signals in the MCE.In a certain concentration range,the ratio of signal between dsDNA and Apt-CAP(I_dsDNA/I _Apt-CAP)was proportional to the concentration of targets.Under the optimum conditions,the ratio showed a satisfactory linearity range from 0.008 to 1.000 ng/mL of CAP with a detection limit of 0.003 ng/mL.Thus,a universal MCE-based assay was developed for quantifying CAP automatically.The method was also successfully applied in the different food samples for CAP detection,which showed a good recovery?Milk:91.1-108%,Fish:86.1-114%?and the results were consistent with that of ELISA.Above all,it's a high-throughput and prospective method which can be applied in high throughput screening of antibiotics in food safety.This method owned many merits as follows:firstly,MCE was a high throughput screening platform and the detection time is limited to 3 minutes for each sample.Secondly,the aptamer probes can be directly used for detection without labeling any signal tag which can facilitate the preparation procedures of probes.Thirdly,the operation was simple.Moreover,the assay with MCE platform can be used to detect other targets just by changing the corresponding aptamer probe;it can even realize simultaneous detection when the targets have aptamers with different number of base pairs.Above all,it's a high-throughput and prospective method which can be applied in high throughput screening of antibiotics in food safety.2.Microchip electrophoresis array-based aptasensor for multiplex antibiotic detection using functionalized magnetic beads and polymerase chain reaction amplificationA microchip electrophoresis?MCE?array-based aptasensor for multiplex detection of antibiotics was developed using multi-capture DNA functionalized magnetic beads?AuMPs@captureDNA@assistantDNA?and polymerase chain reaction?PCR?for signal amplification.In this study,kanamycin?KANA?and chloramphenicol?CAP?were employed as model analytes.In the presence of a target,specific binding between the target and corresponding capture DNA?C-DNA?would induce the unwinding of double-strand structures,leading to the release of assistant DNA?A-DNA?into the supernatant after magnetic separation.The released A-DNA was co-amplified by PCR with an internal standard strand?I-DNA?.After 20 cycles of PCR,the ratio(I_A-DNA/I_I-DNA)between the above PCR products was proportional to the concentrations of the target with a detection limit of 0.0025 nmol/L and 0.0060 nmol/L for KANA and CAP,respectively.Under optimal conditions,the method exhibited high sensitivity and selectivity for the targets,and the average detection time was about 1 min.Moreover,this assay was successfully employed to detect KANA and CAP in milk and fish samples with consistent results to that of enzyme-linked immunosorbent assay,suggesting that it is a promising platform for detecting small molecules in food by changing the corresponding aptamer.3.Multiplex detection of quality indicator molecules targets in urine using programmable hairpin probes based on a microchip electrophoresis platform and isothermal polymerase-catalyzed target recycleRecently,it was crucial to detect and quantify small-molecular targets simultaneously in biological samples.Herein,a simple and conventional double-T type microchip electrophoresis?MCE?based platform for multiplex detection of quality indicator molecules targets in urine,with ampicillin?AMP?,adenosine triphosphate?ATP?and estradiol?E2?as models,was developed.Several programmable hairpin probes?PHPs?were designed for detecting different target and triggering isothermal polymerase-catalyzed target recycle?IPCTR?for signal amplification.Based on the target-responsive aptamer structure of PHP?Domain I?,the target recognition can induce PHP conformational transition and produce extension duplex DNA?dsDNA?assisted by primer&Bst polymerase.After then,the target can be displaced to react with another PHP and initiate next cycle.After several rounds of reactions,the dsDNA can be greatly produced by IPCTR.Three targets can be simultaneously converted to dsDNA fragments with different length which can be separated and detected by MCE.Thus,a simple double-T type MCE based platform was successfully built for homogeneous detection of multiplex targets in one channel.Under the optimal conditions,the assay exhibited high-throughput?48 samples per hour at most?and sensitivity to three targets in urine with a detection limit of 1.00 nmol/L?ATP?,0.05 nmol/L?AMP?and 0.10 nmol/L?E2?respectively.The multiplex assay was successfully employed for above three targets in several urine samples,it combined the advantages of high specificity of programmable hairpin probes,excellent signal amplification of IPCTR,and high through-put of MCE which can be employed to screen in biochemical analysis.