The Study On Colorimetric Aptsensor For Rapid Screen Antibiotics Residue In Foods

Antibiotics have been widely used in the treatment of various non-viral infections in veterinary clinics.However,excessive and inappropriate use of antibiotics leads to its residue in lactating animal milk.As we all know,antibiotics will accumulate in the human body through the food chain,which will make the body resistant to antibiotics and endanger people’s health.Therefore,the detection of antibiotic residues in foods is not to be delayed.However,the content of antibiotics in foods is often very low,usually in the trace level(ng mL^-1),and there are serious matrix interferences in foods,so it is extremely difficult to achieve on-site testing.Colorimetric aptasensor is a kind of spectral analysis method with high sensitivity,good selectivity,easy operation and visual inspection results.It has constructed rapidly developed into one of the main methods of food safety field detection in recent years.This article has constructed a series of highly sensitive colorimetric probes based on the specific recognition of aptamers to antibiotics,using Au nanoparticles modified SiO2,enzyme-linked polymers,Exo I,catalytic chain self-assembly（CHA）,and cyclic chain displacement polymerization（CSDP）-assisted targets cycling for signal amplification.Compared with some current aptasensors,the above-mentioned methods have reduced the experimental cost,shortened the detection time,improved the detection sensitivity,and provided broad application prospects for on-site detection of antibiotic residues in food.The detail contents are as follows:1.A facile colorimetric aptamer assay for chloramphenicol detection in food based on a magnetic single-stranded DNA binding protein-linked composite probeHerein,a promising colorimetric strategy for small molecule detection was developed based on one novel magnetic single-stranded DNA binding protein（SSB）-linked composite probe.Firstly,a magnetic probe was synthesized by labeling SSB on gold magnetic particles（Au MNPs-SSB）and detection probe by co-immobilizing aptamer（Apt）and horseradish peroxidase（HRP）on the core-shell SiO₂@Au nanoparticles（Apt-SiO₂@Au-HRP）;furthermore,Au MNPs-SSB was bound with Apt-SiO₂@Au-HRP to construct a composite probe relied on the specific affinity between SSB and free aptamer.When the target,with chloramphenicol（CAP）as model,was introduced into the reaction system,the formation of the target-Apt-SiO₂@Au-HRP complex can release from its parent probe to supernatant after magnetic separation because SSB can’t bind target-aptamer complex.The target-Apt-SiO₂@Au-HRP complex in supernatant could efficiently catalyze3,3’,5,5’-tetramethylbenzidine（TMB）-H₂O₂ system for the color development.Under the optimized reaction conditions,the linear range was from 0.05 to100 ng mL^-1 with a low detection limit of 0.02 ng mL^-1 for CAP.Moreover,the aptamer assay was employed to detect CAP in milk and the results were consistence with those from ELISA.2.A POCT colorimetric aptasensor for streptomycin detection using porous silica beads-enzyme linked polymer aptamer probes and exonuclease-assisted target recycling for signal amplificationIn this work,a point of care testing（POCT）colorimetric aptasensor was developed to detect streptomycin（STR）using porous SiO2 micro beads（pore size of 200 nm）-enzyme linked polymer(PowerVision^TM,PV)probes and exonuclease-assisted target recycling for signal amplification.The probe was fabricated by single-stranded DNA binding protein labeled SiO₂ micro particles（P-SiO2-SSB）as capture probes and Au NPs&aptamer（Apt）co-immobilized on PV（Apt-Au-PV）as nanotracer.In the presence of STR and exonuclease I（Exo I）,the nanotracer would combine with STR to form the complex（STR/Apt-Au-PV）.Hereafter,Exo I further digest the aptamer on STR/Apt-Au-PV,then STR was released again to participate new cycling,the approach lead to generate more nanotracers into supernatant to improve the sensitivity.Moreover,the dendrimer PV which contains large quantities of HRPs can efficiently catalyzed H₂O₂-TMB（3,3’,5,5’-tetramethylbenzidine）system for color development and the results can observed by the naked eye.Due to the excellent multiple signal amplification effects for porous SiO2 micro particles to label more nanotracer,PV and exonuclease-assisted target recycling,the aptasensor exhibit a low detection limit with 1 pg mL^-1（S/N=3）.In addition,the developed method might provide a prospect assay for food tests at point of care.3.Mimicking enzyme-based colorimetric aptasensor for antibiotic residue detection in milk combining magnetic Loop-DNA probes and CHA-assisted target recycling amplificationIn this study,A mimicking enzyme based colorimetric aptasensor was developed for kanamycin（KANA）detection in milk,using magnetic loop-DNA-NMOF-Pt（m-L-DNA）probes and catalytic hairpin assembly（CHA）-assisted target recycling for signal amplification.The m-L-DNA probes were constructed based on hybridization between two components:hairpin DNA H1（containing aptamer sequence）immobilized magnetic beads（m-H1）,signal DNA（sDNA,partial hybridization with H1）labeled nano Fe-MIL-88NH₂-Pt（NMOF-Pt-sDNA）as nanotracer.In the presence of KANA and another hairpin DNA H2 which was fully complementary to H1 under external magnetic field,m-L-DNA probes decomposed and formed m-H1/KANA intermediate which triggered CHA reaction to form stable duplex strand（m-H1-H2）while releasing KANA again for recycling.Consequently,numerous NMOF-Pt-sDNA as mimicking enzyme can synergistically catalyze 3,3’,5,5’-tetramethylbenzidine for color development.The aptasensor exhibited high selectivity and sensitivity for KANA in milk with the detection limit of 0.2 pg mL^-1within 30 mins.The assay can conveniently extended to on-site screening other antibiotic in food while changing the base sequence of probes.4.A multiplex signal amplified colorimetric aptasensor for antibiotics measurement using DNAzyme labeled Fe-MIL-88-Pt as novel peroxidase mimic tags and CSDP target-triggered cyclesIn this report,An ultrasensitive colorimetric aptasensor was developed for detection of antibiotics,with chloramphenicol（CAP）as model target,using labeled Fe-MIL-88-Pt as novel peroxidase mimic signal tags and target-triggered circular strand-displacement polymerization（CSDP）for signal amplification.The system consists of two parts which can partially hybridize with each other:one is capture probe which was formed through immobilizing hairpin DNA containing aptamer sequence on magnetic beads（MB-cDNA）,another is signal tag which was constructed through labeling single strand DNAzyme（G-quadruplex/Hemin）which can partially hybrid with cDNA on platinum nanoparticles functionalized Fe-MIL-88（MIL-88-Pt-DNAzyme）.All components of MIL-88,Pt and DNAzyme in the tag can act as peroxidase mimic to triply catalyze the oxidation of 3,3’,5,5’-tetramethylbenzidine（TMB）by H₂O₂ into a blue-colored oxidized TMB（oxTMB）for the colorimetric readout.Thus distinctive signal can be observed by naked eye even in presence of very few tags.In the presence of target and primer,cDNA loop can open to form cDNA/CAP intermediates,enabling primer to hybridize with the exposed sequences of the cDNA,which initiated target assisted CSDP recycles.Then numerous signal tags were released into supernatant to catalyze TMB for color development.The detection limit of the aptasensor was 0.03 pM.Furthermore,this method can produce signals being observed by naked eye to facilitate in-situ detection.