Microchip Electrophoresis Based Simultaneous Detection Method For Multiclass Chemical Contaminants

At present,Organic contaminants and inorganic pollutants in food pose a risk to the health of human throughout the world,Some of the more common chemical contaminants are antibiotics,hormone,natural toxins,and heavy metal ions.Accordingly,it needs to develop a rapid and simultaneous method for measurement of multiclass chemical contaminants in in one sample.However,determination of the chemical contaminants was often very difficult in complex matrix samples,for the presences of various interferents and the low concentration?<?g/kg?of them in food or environment samples.In this paper,a series of novel aptamer-based endcoded probes as transducer and DNA signal amplification strategywere developed,combined with Microfluidic chip platform,to detect multiclass chemical contaminants residues?such as kanamycin,estradiol,heavy mental ions,etc.?.Compared with the traditional instrumental analysis method,the method has the characteristics of low cost and simple operation.Compared with the electrochemical fluorescence sensor method,it has high throughput,strong resistance to matrix interference,and can simultaneously detect various types of chemical pollutants.This thesis is divided into three parts to research.1.A microchip electrophoresis-based assay for ratiometric detection ofkanamycin by R-shape probe and exonuclease-assisted signal amplification.Excessive intake of kanamycin?KANA?can cause some serious drug-resistant diseases,so it is urgent to develop some accurate and rapid analytical methods for monitor KANA residues in foodstuffs with complex matrix.Recently,many ratiometric assays were reported to be capable of overcoming matrix interference.Herein,a ratiometric and homogeneous assay for KANA detection based on microchip electrophoresis?MCE?was developed.First,by one single strand DNA?S-DNA?and one hairpin DNA?H-DNA?,a novel R shape DNA probe?RDNA?was prepared.After the probe was incubated with KANA,the S-DNA-KANA complex was formed,and HDNA was released.Moreover,in the presence of exonuclease I?Exo-I?,S-DNA-KANA complex would be digested to release the captured KANA for triggering target recycling and signal amplification.With the reaction going on,the fluorescence intensity of H-DNA?IH?increased and that of R-DNA?IR?decreased.They can be separated at different voltage intensities and converted to fluorescent signals for signal readout by MCE.The signal ratio of IH/IR was found to be linear toward target from 0.5 pg mL^-1 to 10 ng mL^-1,and the limit of detection was 150 fg mL^-1.The R-DNA probe can quantitatively convert the amount of target to the intensity of DNA without label by MCE,and achieved exonuclease assisted signal amplification in homogenous solution.It was valuable to detect antibiotics residues in foodstuff with complex matrix.This approach broadened the application field of MCE to detect antibiotics without derivatization,which provided a promising platform for rapid screening of antibiotic residues in food.2.Microchip electrophoresis based multiplexed assay for silver and mercury ions simultaneous detection in complex samples using a stirring bar modified with encoded hairpin probes for specific extraction.It is crucially important to rapidly,simultaneously,and sensitively determine trace amounts of heavy metal ions in complex samples.Herein,a stirring bar modified with two kinds of encoded hairpin DNA probes?H0 and H0??was used in a multiplexed strategy allowing for specific extraction of Hg?II?and Ag?I?coupled to microchip electrophoresis?MCE?separation and LED induced fluorescence?LIF?detection.The extraction step utilizes stir bars,which are functionalized with designed hairpin DNA probes?H₀ with T-T and H₀?with C-C mismatches in stems?.This allows the specific capture of Hg?II?and Ag?I?through C-Ag-C and T-Hg-T interactions.These complexes are then enzymatically degraded by the action of exonuclease III?Exo III?.The ions released during this enzymatic reaction can initiate a new cycle of interactions with hairpin structures and enzymatic reactions and so on.This cyclic step is specific to the presence of Hg?II?and Ag⁺and represents the first round of amplification of the presence of the selected ions.The resulting single strand DNAs on the stirring bars after enzymatic degradation were used in the second step as primers to trigger the catalytic hairpin assembly?CHA?in the presence of a couple of hairpin structures in solution.Such a reaction allows producing duplexes that can be monitored by MCE-LIF.The fluorescence intensity of CHA products?I_P?increased and that of hairpin DNAs?I_R?decreased with the increase of target concentrations.The signal ratios?I_P/I_R and I_P?/I_R??consisted of targets.The assay was employed for Hg?II?and Ag?I?detection in several mediums including water,milk,and fish samples with complex matrices.The results showed that the assay could avoid matrix interference to increase the sensitivity.Therefore,the multiplexed assay was ideal to simultaneously and quickly detect metal ions in complex samples.3.Combination of magnetic encoded aptamer probes and microfluidic chip for simultaneous detection of multiclass chemical contaminants in food with multi-branched DNA nanostructures as signal tagsIt has aroused increasing attention to develop some multiplex assays to simultaneously monitor multiclass chemical contaminants which commonly co-exist in foods,such as heavy metal ions(Pb²⁺,Cd²⁺),antibiotics?Kanamycin,chloramphenicol?and estrogen?estradiol,diethylstilbestrol?residues.In the study,a microfluidic chip?MC?-based multianalysis method coupled with magnetic encoded aptamer probes for simultaneous detection of some organic contaminants?Kanamycin and estradiol?and inorganic one(Pb²⁺),was firstly developed.In detail,the magnetic-beads?MBs?-based encoded probes labeling with triple aptamer hybrid chains were firstly used to selectively capture the targets,and then generate single-stranded primers.Then,the primers triggered hybridization chain reactions?HCR?to form triple types of multi-branched DNA nanostructures.Finally,three kinds of complementary strands?C-DNAs?with different length which can generate different MCE signals simultaneously were added to hybrid with the branches in the nanostructures,respectively.The decrement signals from cDNAs were employed for qualification of targets.The DNA nanostructures corresponding to different targets as signal tags,can realize“one target to massive C-DNA's decrease”to improve the sensitivity.Under the optimum conditions,the method can be employed to simultaneously detect0.176 pM?0.118 pM and 0.129 pM for kanamycin,estradiol and lead ions in foods?fish and milks?,respectively.Furthermore,the MCE platform can be reusable to detect more than 3000 samples.The recoveries were from 98%to 105%with relative standard deviation?RSD,n=3?less than 5%.The assay combining MCE,magnetic encoded probes with multi-branched DNA signal tags,can not only realize high-throughput,-sensitive and-selective detection,but also show excellent capacity of resisting interference from complicated food matrix.It offers a universal,resuable and high-throughput detection platform for screening multiclass chemical contaminants in food samples with complex matrix.