Fabrication Of The Aptamer-functionlied Novel Probes For Rapid Detection Of Antibiotics

Antibiotic was developed in 1950s and widely used as a specific medicine for bacterial infections.Nowadays,it is due to this abused use,abtitiotics have become the"pollutants"in food products such as animal origin food and milk because these kinds of pollutants possess the durability and also can cause the resistance of environmental bacterias.The contents of amtibiotics in real samples such as fish and milk is very low,it’s usually a trace level(ng mL^-1).In addition,most of antibiotics have structural analogues and usually accompanied by complicated matrix interference in foodstuff.Hence,we developed a series of methods possess the advantages of sensitivity,specificity and simultaneity for the detection of antibiotics,the aptamer functionalied probes may fufill this purpose.In this paper,we construct a series of assays for the antibiotics determination based on the aptamer functionalized magnetic beads probe and hydrophilic nano metal-organic frameworks material probes coupled with chromatography and electrochemical methods.1.An ultrasensitive electrochemical aptasensor for multiplex antibiotics detection based on endonuclease and exonuclease assisted dual recycling amplification strategy was proposed.Kanamycin and chloramphenicol were selected as candidates.Firstly,aptamers of the antibiotics were immobilized on bar A and then binding with their endonuclease labeled complementary DNA strands to construct enzyme-cleavage probes.Secondly,The nano zirconium-metal organic framework（NMOF）particles with 1,4-benzene-dicarboxylate（BDC）as linker was defined as UiO-66.And its updated version,hierarchically porous UiO-66（HP-UIO-66）decorated with different electroactive materials as signal tags were synthesized.Then they were immobilized on bar B linked by two duplex DNA strands which can be specifically cleaved by corresponding enzyme-cleavage probes in bar A.Once targets were introduced into system,aptamers can capture them and then release enzyme-cleavage probes.In the presence of exonuclease-I,exonuclease assisted target recycling amplification was triggered and more enzyme-cleavage probes were released into solution.The probes can trigger endonuclease assisted recycles and repeatedly cleave their corresponding duplex DNA strands on bar B then released numerous signal tags into supernatant.Thus two recycling amplification was performed in the system.Finally,MB and Fc in the signal tags were detected by square wave voltammetry after removing bar A/B and the current intensities were correspondent with the concentration of KANA and CAP respectively.Under the optimum condition,the limits of detection for the KANA and CAP were 35 fM and 21 fM respectively with a wide linear range from 1×10^-4 to 50 nM.This dual recycling amplification detection system exhibited high sensitivities and specificity.It can be easily extended to detect other targets if changing the corresponding aptamers and has potential application values for screening of multiplex antibiotics residues in food safety.2.A novel aptamer functionalized magnetic adsorbent was developed and combined with magnetic dispersive solid phase extraction（MDSPE）for selective enrichment of several amphenicol antibiotic residues（Chloramphenicol（CAP）,thiamphenicol（TAP）and florphenicol（FF））in foodstuff,then determined by High Performance Liquid Chromatography（HPLC）-Diode array detector（DAD）.This adsorbent can specifically and simultaneously recognize and enrich CAP,TAP,and FF with high adsorption amount from some complicated food matrix,e.g.milk based on the high affinity of aptamer towards the analytes.The saturated extraction capacities for CAP,TAP and FF by the adsorbent were 2.82,2.56,2.72μg/g（mass of target/adsorbent）respectively and the enrichment folds were more than 100 times.Afterwards,the target analytes were washed away by pH 8.5 0.1 M Tris-HCl buffer solution and detected by HPLC-DAD.The parameters including extraction temperature,extraction capacity,extraction&desorption pH,extraction&desorption time were investigated.With the optimized conditions,the limits of detection（LOD）and limits of quantitation（LOQ）were 0.12-0.17 ng/mL and 0.40-0.55 ng/mL for the amphenicols in milk.The adsorbent also has good reproducibility for extraction which can be reused at least for 60 cycles with the recovery over 80%.The MDSPE combined with HPLC-DAD detection possessed the advantages of high selectivity,extraction capacity and very convenient for magnetic separation.In addition,this method is environment friendly and employed littler organic solution（microliter grade）in the period of pretreatment and extracting.It is a universal platform which can be extended to selective enrichment other organic pollutants residues if changing the modified aptamers.3.In this study,fluoride-selective electrode（FSE）,together with nano metal organic frameworks（NMOF）tags and double stir-bars assisted toehold-mediated target recycling signal amplification strategy was proposed to construct a FSE based aptasensor for kanamycin.Firstly,nano porous zirconium based MOF particles with 2-amino-1,4-benzenedicarboxylic acid as linker was synthesized which was defined as UIO-66-NH₂ for adsorption of fluoride to construct signal tags.Then they were labeled on strand 1（S1:consists aptamer and toehold sequence）and further immobilized on bar-A through DNA self-assembly.On the other hand,the predesigned strand 3（S3）was immobilized on bar-B,In the presence of target,aptamer in S1 capture kanamycin and the signal tags will disperse into the solution.Meanwhile,the dissociative target@aptamer@signal tags compound hybridize to the S3 on bar-B due to the toehold sequence on the S1 will trigger the complementation reaction towards S3,and the target will participate in the subsequent cycling.After that,bar-B was transferred in beaker-2 and the total ionic strength adjustment buffer（TISAB）was added.Finally,the fluoride was released by TISAB and the potentiometer can read the valve which corresponds to the amounts of kanamycin.Under the optimum condition,the limit of detection for kanamycin is 65 nM with a wide linear range from 0.2μM 10.0μM.This FSE based aptasensor detection system exhibited high sensitivities and specificity.And most importantly,the proposed assay possesses the comparable effect with that of ELISA method but more cost-effective,and successfully broaden the application area of the fluoride-selective electrode.