The Application Research Of The Homogeneous DNA Electrochemical Biosensor

DNA electrochemical biosensor because of the advantages of simple,easy and high sensitivity,has received the widespread attention.But the traditional DNA electrochemical biosensor generally need electrode modification process which is complex and has poor reproducibility.In this thesis,the homogeneous DNA electrochemical biosensor based on the discrimination of the aggregation of long and short DNA on a negatively charged indium tin oxide electrode were designed,constructed and characterized.The proposed biosensors were applied to ultra-sensitive detection of DNA species and food contaminants.This thesis is divided into four chapters to elaborate:The first chapter introduces the features and the advantages of DNA electrochemical biosensor,traditional DNA electrochemical biosensors and the progress of the homogeneous DNA electrochemical biosensors.Meanwhile,the research ideas were summarized.In second chapter,a simple but ultraselective electrochemical DNA biosensor had been designed.The complementary substrate strand of target DNA species contains a simple asymmetric sequence(eMB)had been modified with a methylene blue at the 3'terminal firstly,which can not diffuse easily to the negative charged ITO electrode surface.The reason is that it carry the abundant negative charges due to a large number of electronegative phosphoric acid residues itself.The present of the target DNA(Tl)would trigger the formation of ds-DNA.Then the nicking endonuclease can recognize the simple asymmetric sequence in the ds-DNA and cleave the substrate strand of ds-DNA,producing a short ssDNA(eT)linked with methylene blue.The eT can diffuse easily to the negative charged ITO electrode surface and results in the enhanced electrochemical response detected.At the same time,the target DNA can dissociate from the dsDNA and trigger the next round of hybridization,cleavage and releasing,which results in the signal amplification.The dynamic concentration range spanned from 1 pM to 10 pM and the detection limit 0.35 pM(S/N=3).The assay not only owns the merits of simple and high efficiency since performed in a homogeneous solution,but also exhibits high stability when it is used in the simulation of saliva samples.In third chapter,a simple,sensitive and selective immobilization free electrochemical aptasensor had been developed.Methylene blue(MB)labeled probe DNA had been hybridized with the OTA aptamer first.At present of target(OTA),the aptamer prefers to form OTA-aptamer complex in lieu of aptamer-DNA duplex,which results in the dissociation of probe DNA from the probe DNA/aptamer complex.The released probe DNA could be digested into mononucleotides including a MB-labeled electroactive mononucleotide(eT)due to the employment of the RecJf exonuclease.The eT can diffuse easily to the negative charged ITO electrode surface and results in the enhanced electrochemical response detected.At the same time;,the aptamer in the OTA-aptamer complex can be digested by RecJf exonuclease also to liberate the target,which can participate in the next reaction cycling and realize the electrochemical signal amplification.The dynamic concentration range spanned from 0.01 ng mL-1 to 1.0 ng mL-1 and the detection limit 0.004 ng mL-1(S/N=3).The fabricated biosensor has been applied to detect OTA in real samples with satisfied results.In fourth chapter,a simple but sensitive immobilization free DNAzyme based electrochemical biosensor had been proposed.Lead dependent DNAzyme had been hybridized with its substrate(which had been modified with a methylene blue at 3'terminal)beforehand.The prescence of lead would trigger the cleavage of DNAzyme/substrate complex and cause the releasing of methylene blue-labeled short-oligonucleotide into the solution.The short-oligonucleotide can diffuse easily to the surface of the negative charged ITO electrode and results in the enhanced electrochemical response detected.Each lead can cleave a lot of DNAzyme/substrate complex to realize signal amplification.The dynamic concentration range spanned from 0.05 ?M to 1.0 ?M and the detection limit 0.018 ?M(S/N=3).The proposed biosensor had been applied to detect Pb2+ in water samples with satisfied results.