The Construction Of Electrochemical Sensor Based On G-quadruplex DNAzyme And Its Application In The Detection Of Trace Antibiotics

Antibiotics are one of the most widely used drugs in the world,the principle of killing bacteria is to inhibit the synthesis of cell walls,change the permeability of cell membranes,inhibit nucleic acid replication and transcription,and interfere with protein synthesis.In addition to clinical treatment,antibiotics are also widely used in agriculture and breeding industry,making a great contribution to the development of human society.However,the overuse of antibiotics causes a large number of undegraded antibiotics to enter the water body.Residual antibiotics in the environment can enter the human body through the enrichment of the food chain,causing allergic reactions in the human body and inducing the generation of resistance genes,which poses a great threat to human health.At present,the traditional detection methods are restricted by various conditions,making the residual detection of antibiotics cumbersome and inefficient.Therefore,it is urgent to develop an efficient and sensitive method to detect trace antibiotics in the environment.Here,we constructed an electrochemical biosensor based on G-quadruplex/DNAzyme and combined with a variety of signal amplification methods to detect antibiotics in the environment.Our electrochemical biosensors show high specificity and sensitivity to target antibiotics and can be applied to the detection of real samples.The main research contents are as follows:Firstly,herein,a split G-quadruplex DNAzyme as signal reporter has been integrated into electrochemical sensing for detecting kanamycin with specificity and sensitivity.To improve the signal-to-noise,the two G-rich oligonucleotide sequences?G1 and G2?are blocked into two different hairpin probes,respectively,preventing the two segments from assembling into the spilt G-quadruplex structure.Besides,we design a double-arch probe consisting of aptamer as the recognition element and two steps enzymatic signal amplification.Concretely,the first is the Nt.BbvCI-assisted nicking cyclic reaction activated by target-aptamer binding,and the second is an Exonuclease III?Exo III?-aided cyclic amplification for generating abundant G1 and G2.The modified capture probe on the electrode is used to combine the G1and G2 to form the spilt G-quadruplex/hemin when the K⁺and hemin are present.The complex plays a role of DNAzyme with superior horseradish peroxidase activity in catalyzing H₂O₂ decomposition.Under the optimal conditions,this biosensor shows excellent performance for kanamycin with detection limits of 83 fM ranging from 100 fM to 1 nM.Hence,the proposed strategy has the potential to develop an efficient and actual platform of small molecular analysis.Secondly,an Exo ?-powered DNA walking machine has been integrated into electrochemical sensing for label-free and highly sensitive detection of ampicillin.The electrochemical sensing interface is prepared by co-assembly of hemin aptamer-containing DNA track?DT?and DNA walker?DW?-locking probe?LP?duplex on gold electrode surface.The specific target and aptamer interaction triggers Exo III-assisted cyclic amplification,releasing the unlocking probe?UP?that is used for the deblocking of DW.Then the autonomous moving of DW on electrode surface is propelled via Exo III digestion of hybridized DT.The consumption of DT induces the formation of G-quadruplex/hemin DNAzyme that can catalyze the reduction of H2O2,thus a significantly strong current signal is released.Using ampicillin as a model analyte,the proposed biosensor achieves a detection limit of 0.76 pM within a linear detection range from 1 pM to 10 nM.Additionally,this biosensing strategy can be readily extended for the detection of wide variety of analytes by using corresponding target recognition probes.Therefore,the proposed strategy indeed creates a highly sensitive and label-free electrochemical sensing platform for molecular diagnosis applications.Thirdly,we have integrated an entropy-driven DNA walking machine into electrochemical platform for label-free sensing of ampicillin.As far as we know,this work is the first time that entropy-driven DNA machine has been used for antibiotics detection.The designed DNA machine is activated by a target-triggered catalytic hairpin assembly to form a spliced DNA walker long-arm.Then the autonomous and progressive spliced walk long-arm on electrode surface via the toehold-mediated strand displacement reaction induces continuous release of signal probe?SP?from three-stranded DNA complex.The freedom of SP leads to the generation of abundant G-quadruplex DNAzyme which can catalyze decomposition of hydrogen peroxide,so an extremely intense electrochemical response is observed.Our spliced DNA walking machine precedes the previous DNA machine in terms of economy and stability due to the strategy of entropy-driven and label-free,and possesses a detection limit to 0.96 pM for detection of ampicillin.