Novel Electrochemical Biosensors Based On Aptamers And DNAzyme

Thrombin is a multifunctional serine protease with procoagulant functions.X-ray structure determination of human?-thrombin and related structures reveals a protein with several functional regions.Quantitative detection of thrombin?TB?is very important in clinical diagnosis,owing to the crucial role of TB in the cardiovascular diseases,regulation of inflammation processes,anti-clotting therapeutics,and so onPrions?PrP?consist mainly of a protease resistant protein designated PrP^Sc?PrP for prion protein and Sc for scrapie?,a posttranslationally modified form of the proteinase K-sensitive host-encoded PrP^C?C for cellular?.Both isoforms share the same amino acid sequence but differ in their secondary structures.Transmissible spongiform encephalopathies,such as Kuru,Creutzfeldt-Jakob disease in humans,bovine spongiform encephalopathy in cattle,and scrapie in sheep or goats,are thought to be transmitted by prions?proteinaceous infectious part icles?which consist mainly or totally of the scrapie prion protein PrP^Sc.Neurodegenerative diseases are a huge public health problem affecting tens of millions of patients.Therefore,targeted therapies for these aggregates and their detection as diagnos tic indicators are still highly desirable.Lead is a major environmental pollutant.It is quite harmful to human health,especially to children,and a very small amount of lead ion could cause serious damage to the brain and central nervous system.It can cause memory loss,irritability,anemia,muscle paralysis and mental retardation in children.As lead is nondegradable,it is persistent in the environment and can produce toxic effects in plants andanimals.Therefore,it is essential to find a technique for routine and effective monitoring of lead.The electrochemical biosensor is a sensor based on the ion conduction and electrochemical properties of the analyte,which converts the chemical quantity of the analyte into an electrical quantity for sensing detection.Conventional electrochemical DNA sensors are mainly used to detect nucleotide targets,such as DNA,RNA,etc.The introduction of aptamers enables the detection of non-nucleic acid targets?such as proteins,drugs,organic or inorganic molecules,etc.?and broadens the application range of electrochemical DNA sensors.The electrochemical aptamer sensor has the advantages of simple structure,low cost,simple operation,stable,and easy to miniaturize.In this thesis,the sensitivity detection of thrombin,prion protein and Pb²⁺was achieved by electrochemical sensing method:?1?A new electrochemical aptamerbased biosensor has been developed for sensitive assay of the thrombin based on the DNA-based dual-signaling electrochemical ratiometric method and the DNA walker strategy.A double-stranded DNA was prepared by hybridizing the DNA walkers?DWs?and the thrombin aptamer?TBA?,and then the DWs/TBA double-stranded DNA and methylene blue-labeled DNA?MB-DNA?were immobilized on the surfaces of the gold nanoparticle?AuNPs?modified glassy carbon electrode?GCE?.When ferrocene-labeled DNA?Fc-DNA?was added,the MB-DNA hybridized with the Fc-DNA to form a MB-DNA/Fc-DNA duplex.In the presence of TB,DWs could be released due to the specific interactio n between TB and its aptamer?TBA?.The released DWs hybridized wit h part of Fc-DNA in the MB-DNA/Fc-DNA duplex and a duplex with a blunt end at the3?-termini of Fc-DNA was formed,which triggered the Exo III cleavage process accompanied by the throwing away of Fc from the electrode and release of DWs.And the DWs continued to hybridize another Fc-DNA to perform the DNA walker recycling process.The remaining single-stranded MB-DNA could form a hairpin structure in the presence of magnesium ions(Mg²⁺)due to its tailor-made complementary bases at both ends.This resulted in an enhancement of the MB signal(I_MB)and a suppression of the Fc signal(I_Fc).Based on the I_MB/I_Fc value,the proposed electrochemical aptamer-based biosensor showed superior analytical performance for sensitive assay of TB with a linear range from 0.1 pM to 10 pM and a low detection limit of 56 fM.?2?A simple and novel electrochemical aptamer sensor has been developed for prion protein assay based on?-CD and 3D DNA walking machine.Since Au@Fe₃O₄?AuMNPs?with the advantages of relatively large surface area and easy magnetic separation,it was used to immobilize both aptamer 1?Apt1?of PrP^C and substrate probes?support-MB?to form the 3D DNA walking machine,which could achieve the conversion and amplification of prion protein?PrP^C?.In the presence of PrP^C,aptamer1?Apt1?specific interact with PrP^C.After the incubation with aptamer 2?Apt2-DWs?,it forms an Apt1/PrP^C/Apt2-DWs sandwich structure.Then,Apt2-DWs is paired with support-MB and a duplex with a blunt end at the 3?-termini of support-MB was formed,which triggered the Exo III cleavage process accompanied by the throwing away of MB from near to far along the DNA-AuMNPs track and release of DWs.And the Apt2-DWs continued to hybridize another support-MB to perform the DNA walker recycling process.Due to the wide 3D walking space and high local concentration of DNA components,the 3D DNA walking machine resulted in an amazing amplification effect for PrP^C by generating a large number of intermediate DNAs?rich in shear ed DNA fragments and methylene blue signaling factors?.The sensing platform is constructed by immobilizing HS-?-CD??-cyclodextrin labeled with sulfhydryl groups?on AuNPs/GCE.When the above intermediate DNA is dropped onto the electrode,The MB was introduced to the electrode surface based on the host-guest interaction between?-CD and MB.This resulted in an enhancement of the MB signal.The response peak current was linear with the prion protein in the range of 0.5 pg/mL to10 pg/mL,and the detection limit is 0.4 pg/mL?3??,which has good reproducibility,selectivity and stability.?3?A novel electrochemical DNA sensor based on DNAzyme and dual signal amplification strategy for the detection of Pb²⁺.First,the substrate chain S1 and the catalytic chain S2?methylene blue-labeled?are hybridized to form a double-stranded DNA?ie,DNAzyme?with a convex ring portion,which is immobilized on the surfaces of the gold nanoparticle?AuNPs?modified glassy carbon electrode?GCE?.In the presence of Pb²⁺,Pb²⁺-dependent DNAzyme was specifically identified and cleaved by Pb²⁺,and the remained fragment in the electrode substrate captured the bioconjugates of 6-FcHT@AuMNPs/SH-S3 by hybridization reaction.And the signal factor MB was away from the electrode surface.Since Au@Fe₃O₄ has the advantages of relatively large surface area and easy magnetic separation.It is to the benefit of immobilization of 6-FcHT and amplification of Fc signal.This resulted in an enhancement of the Fc signal(I_Fc)and a suppression of the MB signal(I_MB).Based on the I_Fc/I_MB value,the proposed electrochemical biosensor showed superio r analytical performance for sensitive assay of Pb²⁺with a linear range from 0.05 pM to 1 pM and a low detection limit of 0.033 pM.