Study On The Nucleic Acid Signal Amplification-based Electrochemical Homogeneous Biosensing

The highly sensitive and accurate detection of the analytes like protein biomarkers and antibiotics in complex matrices is of great importance in the fields such as medical diagnosis and food safety.Compared with the analytial methods such as chemiluminescence,fluorescence and colorimetry,electrochemical biosensors have better practical application values because of their unique advantages such as simple instruments,high sensitivity and rapid signal response.In order to achieve accurate and sensitive detection of low-abundance target analytes,researches have rencently done considerable work on the signal amplification strategy studies of electrochemical biosensors.However,these biosensing methods are usually constructed on the basis of the mode of heterogeneous analysis.So the assembly or modification of the electrode interface often requires very professional operation skills,and the static incubation and multi-step operations involved of them often require a long time for analysis.In contrast,the electrochemical biosensing methods developed on the basis of the homogeneous analysis mode do not need to separate target analytes and recognition molecules.So they possess the advantages such as simple operation,time saving,high automatization and good repeatability.Herein we combine it with several biological signal amplification techniques to carry out two researches on the study of highly sensitive biosensing methods of protein biomarkers and antibiotics in this thesis,which is listed as follows:1. Hybridization chain reaction and enzyme-nanotag enhanced synthesis of molybdophosphate for protein electrochemical biosensingBy combination of the magnetic bead?MB?-based sandwich biorecognition reactions with a gold nanoprobe-induced homogenous synthesis of molybdophosphate,this work successfully develops a novel biosensing method for the sensitive electrochemical detection of the tumor biomarker of carcinoembryonic antigen?CEA?.The nanoprobe is prepared through the specific loading of numerous alkaline phosphatase?ALP?-functionalized gold nanoparticles?Au NPs?on a double-stranded DNA?ds DNA?produced by the CEA aptamer-triggered hybridization chain reaction?HCR?.Both the large amounts of PO₄^3-produced by the ALP-catalytic hydrolysis of pyrophosphate and the phosphate backbone of ds DNA can react with the added Mo O₄^2-to generate electroactive molybdophosphate.So the gold nanoprobe was used for signal tracing of the sandwich bioassay of CEA at a constructed antibody-functionalized MB platform.The sensitive electrochemical measurement of molybdophosphate produced from the quantitatively captured nanoprobes at a carbon nanotube-modified electrode enabled the convenient signal transduction of the method.Due to the dually enhanced synthesis of molybdophosphate by the HCR and multi-enzyme Au NP nanotag,this method shows a wide linear range over four orders of magnitude and a low detection limit of0.027 pg m L^-1.2. Endonuclease-assisted DNA walking machine signal amplification for electrochemical biosensing of KanamycinBy combination of the target biorecognition-based DNA walking machine signal amplification and the sensitive electrochemical signal transduction at the vertically aligned single-walled carbon nanotubes?SWCNTs?-modified electrode,this work successfully develops a novel electrochemical homogeneous biosensing method for the convenient detection of kanamycin?Kana?.The DNA walking machine is prepared through the dual surface functionalization of Au NPs with the walking DNA?Walker?and auxiliary DNA?S1?.In the absence of the target analyte of Kana,Walker can be protected by its hybridization with the Kana aptamer?S2?,leading to the stable existence of the DNA walking machine in the solution.Upon its specific biorecognition reaction with Kana,the Walker chain will be released from the DNA walking machine.It can further hybridize with S1 and thus trigger the specific catalytic cleavage reaction of the Nt.Bsm AI nuclease to release the single-stranded oligonucleotide labeled with ferrocene?Fc-DNA?from S1.Through the non-covalent adsorption and sensitive electrochemical detection of Fc-DNA at the as-prepared vertically aligned SWCNTs-modified electrode,the convenient electrochemical signal transduction can be successfully realized for this method.As both the nuclease-assisted walking of the DNA walking machine and the excellent electroconductivity of the carbon nanotube-modified electrode can significantly enhance the sensing signal,this method shows an ultrahigh analytical sensitivity.Under the optimal conditions,this method can be used for the convenient and accurate detection of Kana in a wide linear range from 0.01 p M to 500 p M with a detection limit of 4.3 fM.