Study On The Preparation Methods Of Aptasensor For Aminoglycoside Antibiotic Residues In Milk

The wide application of antibiotics in animal husbandry industry causes the diversity and complexity of antibiotic residues in animal derived food,such as milk.At present,the main detection method of antibiotics is highly sensitive instrumental analysis.However,the high cost and the complexity of pre-processing,it has been unable to meet the increasing demand for rapid,timely and efficient detection of large number samples.It is urgent to develop a detection technology for simultaneous detection of multiple antibiotic residues.Sensor analysis based on aptamers has attracted wide attention.In recognition,the aptamer has an advantage over the antibody.The problem of simultaneous detection of multiple antibiotics can be solved by using special element marking technology.Aptamer is a short factitious RNA or single-stranded DNA sequence screened by an in vitro selection technique,which specifically recognizes and binds to its pre-selected targets including small molecules,ions,proteins,and even whole cells.According to previous reports,a variety of aptamers with high specificity for antibiotics have been screened and used.The aptasensor was constructed to analyze the target using electrical signal transformation before and after the combination of high-specificity,high-affinity aptamer and target substance.This paper focuses on the construction of single antibiotic aptasensors and multiple aptasensors via RNA and DNA aptamers.The specific research contents are as follows:?1?Study on an electrochemical aptasensor for detecting a single antibiotic residue based on RNA aptamerAn aptasensor was developed for highly sensitive detection of antibiotic,neomycin?NEO? as model,which was based on the platform of ferrocene-multi-walled carbon nanotubes?Fc-MWCNTs?and silica hybridized mesoporous ferroferric oxide nanoparticles?SiO₂@Fe₃O₄?.The sensitivity of the electrochemical aptasensor was improved attributing to high conductivities of SiO₂@Fe₃O₄ and Fc-MWCNT.MWCNTs was used as an electron transfer mediator.The electrical signal was greatly improved in the presence of Fc due to its good electron-transfer ability.SiO₂@Fe₃O₄ with large specific pore volume and high specific surface area was successfully prepared by hydrothermal and chemical activation treatment.SiO₂@Fe₃O₄ was served as label to immobilize more NEO aptamer and promote the electron transfer.NEO and two different aptamers were used to recognize the different parts of NEO to form a sandwich structure.The electrochemical aptasensor exhibited a detection limit of 759 pM and excellent analytical performances.In addition,the as-prepared aptasensor was successfully utilized for the detection of NEO in spiked milk samples with the acceptable recovery percentage of 91.47%-106.35%.?2?Study on a multiplex electrochemical aptasensor for detecting multiple antibiotic residues based on RNA aptamerA label-free aptasensor based on gold nanoshells?AuNSs?was achieved for highly sensitive detection of two antibiotics,kanamycin?KAN?and tobramycin?TOB?as model.The AuNSs with a shell structure had good conductivity and can better combined to aptamer.Streptavidin?SA?was modified to cadmium sulfide composites?CdS?and conjugated with biotin-labeled KAN aptamer?Bio-KAP?.In the same way,SA was modified to lead sulfide composites?PbS?and conjugated with biotin-labeled TOB aptamer?Bio-TAP?.Complex of KAP-Bio-SA-CdS and TAP-Bio-SA-PbS was modified to AuNSs/GE?gold electrode?to construct an aptasensor for detection of KAN and TOB.The devised aptasensor exhibited detection limits?LODs?of 0.125 nM to KAN and 0.489 nM to TOB.The proposed aptasensor was examined with a real milk sample.?3?Study on an electrochemical aptasensor for detecting a single antibiotic residue based on DNA aptamerWe developed an aptasensor by double-stranded DNA?dsDNA?modified with cadmium sulfide?CdS?nanoparticles and gold nanoparticles?AuNPs?onto gold electrode?GE?for antibiotic detection,KAN as model.The CdS nanoparticles were employed to strongly adsorb on the surface of GE via Au-S interactions.AuNPs as the mediator improved electron relay during the entire electron transfer process and the aptasensor response speed.The proposed aptasensor exhibited a low detection limit of 2.85 nM.Aptasensor by ssDNA showed low limit of detection with 9.76 nM.The aptasensor displayed high specificity for KAN and was free from interference in common milk adulterants.The proposed aptasensor had excellent analytical performances.The aptasensor could selectively identify targets even in complexes such as only skimmed milk and that the proposed aptasensor could be used for KAN detection in milk.Next,an ultrasensitive electrochemical aptasensor for KAN detection was constructed based on a dual-signal amplification strategy with the ordered mesoporous carbon-chitosan?OMC-CS?/gold nanoparticles-streptavidin?AuNPs-SA?and DNA2 labelled with ferrocene?Fc-DNA2?.The AuNPs-SA was used to immobilize the DNA1 strand?biotin labelled?with the biotin-streptavidin system.The DNA2 strand containing the KAN aptamer was labelled with ferrocene to increase the current signal on the electrode surface when bound to KAN.The proposed aptasensor provided a detection limit as low as 47.2 pM for KAN.This aptasensor had satisfactory electrochemical performances.Additionally,it also displayed a good specificity for KAN without interference from competitive analogues.Furthermore,the constructed aptasensor was successfully used to detect KAN in a real milk sample.The proposed method for KAN detection has great potential for the detection of other antibiotics.?4?Study on a multiplex electrochemical aptasensor for detecting multiple antibiotic residues based on DNA aptamerA dual-target electrochemical aptasensor was developed for the simultaneous detection of multiple antibiotics based on metal ions as signal tracers and nanocomposites as signal amplification strategy,KAN and STR as models.The designed aptasensor had some characteristics of high electrochemical conductivity and high specific surface area because of rod-like mesoporous carbon-gold nanoparticles?OMC-AuNPs?and carbon nanofibers?CNFs?.Moreover,CNFs and OMC-AuNPs could be homogeneously and firmly adhered to the surface of SPCE,and complimentary strands of aptamers could also be well immobilized on the surface of the electrode modified with CNFs and OMC-AuNPs.Metal ions such as Cd²⁺and Pb²⁺could generate distinct differential pulse voltammetry?DPV?peaks.In the absence of KAN and STR,the aptamers bound to their complimentary strands.Upon addition of KAN and STR,the aptamers bound to their targets,which led to the complementary strands released from the aptamers and more changes of current peaks because of the aptamers labelled with CdS and PbS.Under the optimal conditions,this aptasensor showed a selectivity toward KAN and STR with limits of detection?LODs?as low as 87.3 and 45.0pM,respectively.The developed aptasensor was successfully assessed by detection of KAN and STR in a spiked milk sample.At the same time,a dual-target electrochemical aptasensor was developed for the simultaneous detection of multiple antibiotics based on metal ions as signal tracers and nanocomposites as signal amplification strategy.Complementary strand of KAP?cKAP?and STP?cSTP?were linked with the poly?A?structure?cSTP-PolyA-cKAP?to increase their conformational freedom.Carbon nanofibers-gold nanoparticles?CNFs-AuNPs?and graphitized multi-walled carbon nanotubes(MWCNT_Gr)as a sensor platform enhanced the surface area to capture a large amount of cSTP-PolyA-cKAP.The aptasensor could detect KAN and STR as low as 74.50 pM and 36.45 pM respectively.This aptasensor showed promising applications for the detection of other analytes by replacing aptamers and using more nanometal ion-labeled aptamers.