The Research On Electrochemical Biosensor Based On DNA Walker Amplification Strategy For The Detection Of Antibiotics

As one of the greatest discoveries of the 20th century,antibiotics have saved countless lives in clinical medicine because of their antibacterial function.However,in order to improve the output of the animal husbandry products,antibiotics have used widely in animal husbandry and aquaculture as antimicrobial agents and animal growth promoters.This inevitably caused their residues in animal husbandry and aquatic products.At the same time,due to the difficult degradation nature of antibiotics,high concentration of antibiotics will be gathered in the water.And antibiotics in water will eventually accumulate through the food chain to human body,posing a serious threat to human health.Therefore,the methods such as high performance liquid chromatography（HPLC）,capillary electrophoresis（CE）,enzyme-linked immunosorbent assay（ELISA）and liquid chromatography-mass spectrometry（LC-MS）have been developed for the detection of antibiotics.However,due to the disadvantages of expensive equipment,long analysis time and tedious sample preparation,it is very necessary to develop a sensitive and efficient new detection methods to detection antibiotics.Here,we developed three electrochemical aptasensors to detect antibiotics residues.The aptamer was used as the biometric element of the sensors,and the electrochemical workstation was used as the transducer.DNA walker amplification strategy combined with strand displacement amplification（SDA）,catalytic hairpin assembly（CHA）and other isothermal nucleic acid amplification technologies were used as signal amplification strategy.The strategies have the advantages of high specificity,high sensitivity,convenience.And we applied the constructed sensors to the antibiotics detection of drinking water and milk and achieved good results.The main research contents are as follows:First of all,based on dual-SDA technology combined with DNA walker driven by Mn²⁺-DNAzyme,an ultrasensitive kanamycin（KANA）electrochemical aptasensor was developed for kanamycin detection was constructed.Firstly,kanamycin specifically binds to its aptamer with high affinity and the arch probe（AP,T1-T2）is unwinding.After unwinding,the dual SDA is initiated by two single DNA strands,T1 and T2.Since the hairpin of Primer 2 contains palindromic sequences,the SDA triggered by T2 and P2 is bidirectional,which further increases the amplification efficiency.Subsequently,Mn²⁺-DNAzyme walker（DW）was unsealed by a dual SDA output product（OP）.When DNAzyme walker walked on the gold electrode surface,the adenine ribosome nucleotide（r A）on the hairpin（HP）modified on the gold electrode surface by Au-S bond was cut through DNAzyme,exposing the G-quadruplex（G4）sequence.Due to the horseradish peroxidase-like properties of G-quadruplex,H₂O₂ is catalyzed to produce electrochemical signals.Under the optimal experimental conditions,the electrochemical aptasensor showed satisfactory performance,with a detection range of 100 f M-5 n M and a minimum detection limit of 87.1 f M.Then,a label free electrochemical aptasensor based on entropy-driven DNA walker technology was developed for the detection of ampicillin in milk.Firstly,we designed an arched probe（AP）,which is composed of aptamer of ampicillin（APT）and trigger chain（T）.When ampicillin is present,ampicillin binds to its aptamer with high affinity,so that the APT-T was unwound and the T chain was released.Then T chain initiated a series of toehold mediated CHA reactions,resulting in the opening of H2 and the binding of H4 on the electrode surface.At the same time,the G-quadruplex sequence is exposed on the electrode surface.In the presence of K⁺and hemin,the G-quadruplex/hemin complex is formed,which can catalyze H₂O₂ to generate electrochemical signals.The aptsensor can effectively detect ampicillin in the range of 1 p M-10 n M,and the limit of detection is 0.467 p M.Finally,an electrochemical aptasensor based on nonfixed DNAzyme walker amplification technology was constructed to detect antibiotics in drinking water.In this system,aptamer was used as a biological recognition element,G-quadruplex was used as a signal probe,Mg²⁺-DNAzyme walker was used as a signal amplification system,and electrochemical workstation was used as a transducer.Firstly,we designed an arched probe（AP,APT-L）.When ampicillin is present,ampicillin specifically binds with its aptamer,so that the APT-L was unwound and the L chain was released.The L chain can connect the two parts of the linked Mg²⁺-DNAzyme,DA and DB.In the presence of Mg²⁺,the linked Mg²⁺-DNAzyme can cleave r A on H1.After cleavage,a large number of Mg²⁺-DNAzyme walkers are formed.After the reaction droplets were added to the electrode surface,the DNAzyme walker also cleave r A on H2 on the electrode surface,exposing the G-quadruplex sequence on H2,and formed G-quadruplex/hemin complex in the presence of K⁺and hemin.The complex catalyzed H₂O₂ to produce obvious current signal.Thanks to the advantages of the aptamer and electrochemical workstation,the sensor has excellent specificity and sensitivity,and the use of DNAzyme walker further improves its sensitivity,with the detection range of 100 f M-1 n M and the limit of detection of 25.7 fM.