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DNA Biosensor and Its Application Based on G Guadruplex and Three-arm Junction Probe

Author: ZhangJing
Tutor: FuFengFu
School: Fuzhou University
Course: Analytical Chemistry
Keywords: G-quadruplex Juction probe DNA biosensors SNPs DNAzyme2’-deoxyinosine
CLC: O657
Type: PhD thesis
Year: 2011
Downloads: 36
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Abstract

In this thesis, several novel DNA biosensors, which based on G Guadruplex andJunction probe, were developed. The prepared DNA biosensors were used to thesensitive determination of metal ion, short DNA species related to some diseases andvirus or proteins. There are six chapters in this thesis and the main contents are listedas follows:Chapter1: In this chapter, the DNA structure, diversity, characteristics andresearch progress are overviewed. Especially, knowledge on the biosensing designbased on the conformational changes and the polymorphism of DNA including thestructure of different types of DNA probes （especially the G quadruplex structure andthe juction probe） was introduced in detail. Meanwhile, the biosensor for the detectionof metal ion, small molecule drugs, protein, hybridization and gene diagnosistechnology studies were summarized. Finally, the research purpose and main contentof this thesis was also summarized.In chapter2, a new electrochemical biosensor for the monitoring of ultratraceterbium based on the conformational change of DNA containing a single guanine（G）-rich stretch was described. The biosensor was fabricated by immobilizing athiolated DNA containing a single G-rich stretch on the gold surface as probe surface.The G-rich DNA probewas found to be capable of changing its configurationfromflexible single-stranded structures to rigid tetramolecular G-quadruplex in thepresence of terbium III, which provided a switchable charge transport path for theoxidation of [Fe（CN）6]4. The switchable surface provided a sensing platformfor thesingle-step and reagentless detection of Tb³⁺. Using this reusable electrochemicalsensing platform, a simple, rapid, and selective biosensor for the determination ofultratrace terbiumions has been developed. The success in the present biosensorservednas a significant step toward the development of monitoring ultratrace Tb³⁺inriver water or seawater.In chapter3, a Tb³⁺-promoted G-quadruplex-hemin DNAzyme was firstlyreported. We demonstrated that trace Tb³⁺is able to induce guanine-rich DNAfolding into a compact antiparallel G-quadruplex structure and thus allows the formation of G-quadruplex-hemin DNAzyme. The proposed DNAzyme caneffectively catalyze the H2O2-mediated oxidation of TMB （3,3’,5,5’-tetramethylbenzidine sulfate） and leads to a change from colorless to blue in solutioncolor, which provides a sensing platform for the label-free visual detection of Tb³⁺.Using above sensing platform, a selective and sensitive label-free visual method forthe detection of trace Tb³⁺was developed. The proposed method can be used to detectas low as1.13×10^-7M of Tb³⁺by the naked eye observation and9.0×109M of Tb³⁺by UV-vis spectrophotometry with a better stability and reproducibility. Comparedwith K+-promoted G-quadruplex-hemin DNAzyme reported in previous study, thenovel Tb³⁺-promoted G-quadruplex-hemin DNAzyme has much higher peroxidaseactivity and better specificity, which lead to a great potential in the field of optical,electrochemical and chemiluminescence DNAzyme-based biosensors.In chapter4, a novel junction-probe electrochemical biosensor for thesequence-specific detection of DNA with higher sensitivity and higher discriminationability was described. This DNA biosensor is based on junction-probe detectionstrategy, which operates via a concept called template-enhanced hybridizationprocesses （TeHyP）. TeHyP encompasses a design strategy whereby two probes that donot hybridize to each other at a specific temperature can be made to anneal to eachother in the presence of a template （target） via the formation of a ternary complex（“Y” junction structure）. The resulting structure that forms after thetemplate-enhanced hybridization then was detected by electrochemical method with[Ru（NH₃）₆]³⁺as signal molecule. We demonstrated that the formation of “Y” junctionstructure brings more [Ru（NH₃）₆]³⁺to the electrode surface via electrostaticinteraction and results in an increasing electrochemical signal. By employing abovestrategy, this DNA biosensor could detect target DNA and exhibited highdiscrimination ability even against single-base mismatch.In chapter5, a novel electrochemical biosensor for single nucleotidepolymorphism （SNP） detection within an individual saliva DNA, which was based onjunction probes containing2’-deoxyinosine （dI） residues substituted two basesadjacent to the SNP site, was described. The sensor operates via a concept calledtemplate enhanced hybridization processes （TeHyP）. TeHyP encompasses a designstrategy whereby two probes that do not hybridize to each other at a specifictemperature can be made to anneal to each other in the presence of a template （acomplementary target DNA） via the formation of a ternary complex （“Y” junction structure）. The formation of “Y” junction structure electrostatically bonded more[Ru（NH₃）₆]³⁺（RuHex）, and lead to a bigger redox signal in electrochemicalmeasurement. However, in the presence of a mismatch DNA, the “Y” junctionstructure cannot be formed completely, and less RuHex electrostatically binding to“Y” and results in a lower redox signal. The biosensor, which takes advantage ofjunction probes, dI residues, and electrochemical sensor，provides a new SNP typingmethod with high discrimination ability and high sensitibity.In chapter6, a novel signal-on junction-probe electrogeneratedchemiluminescence （ECL） aptamer biosensor has been developed for the detection ofultratrace thrombin based on a structure-switching ECL quenching mechanism. TheECL aptamer biosensor comprises two main parts: an ECL substrate and an ECLintensity switch. The ECL substrate was made by modifying the complex of Aunanoparticle and ruthenium （II） tris-bipyridine (Ru（bpy）₃²⁺–AuNPs) on the surface ofgold electrode （GE）, and the ECL intensity switch contains three probes designedaccording to the “junction-probe” strategy. The first probe is capture probe （CP）which was functionalized with a thiol group at one end and covalently attached to GE.The second probe is aptamer probe （AP）. The third one is ferrocene-labeled probe（FP）, which was functionalized with ferrocene tag at one end. We demonstrated that,in the absence of thrombin, CP, AP and FP will hybridize to form a ternary “Y”junction structure and resulted in a quenching of ECL of Ru（bpy）₃²⁺. Whereas, in thepresence of thrombin, the Ap prefers to form the G-quadruplex aptamer–thrombincomplex and lead to an obvious recovery of ECL of Ru（bpy）₃²⁺, which provided asensing platform for the detection of thrombin. Using this reusable sensing platform, asimple, rapid and selective signal-on ECL aptamer biosensor for the detection ofthrombin.

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