The Applications Of DNA In The Gene Diagnosis And Heavy Metal Ions Analysis

With the implement of Human Genome Plan, gene diagnose has become a focus ofmolecular biology, biomedical and genetic research. Various DNA detection techniquesbased on the principle of complementary base pairing have attracted much attention ofbioanalysts. Amoung them, DNA electrochemical biosensor is a powerful tool in genedetection, due to its easy of operation, sensitivity, and compatibility with the DNAbiochips, which is of great significance in DNA research. The public events of“Shuiyubing” and “Tongtong disease” have initiated world-wide concern on thecontamination of heavy metal. Therefore, the accurate, sensitive and on-site tracking ofheavy metal ions seems to be an urgent task in environmental and food monitoring, aswell as clinical toxicology. With the development of the in-depth investigation into theinteractions of DNA with metal ions, DNA was found to have specific interactionswith metal ions, which opens new avenues for heavy metal ions detection.This paper combines the principle of complementary base airing, the specificbinding affinity of G-quadruplex to certain metal ions, and the electrochemical sensingtechnique to design two novel electrochemical DNA biosensors, investigate theactivity of an endonuclease towards metal ion-mediated duplex-like DNA, as well astwo lead biosensors based on G-quadruplex. This paper not only provides usefulexperimental information for gene screening and disease diagnosis, but also broadensthe research field of DNA in the heavy metal ions sensing.Chapter l The review of electrochemical biosensorsIn the beginning of this dissertation，the basic components of an electrochemicalbiosensor, detection principle, its classification and applications are systematicallyreviewed. Then the design of a DNA electrochemical biosensor, the applications ofgold nanoparticles, diazotization-coupling reaction and electrochemical reduction ofdiazonium salts in construction of electrochemical sensing platforms are reviewed indetail. Finally, the current research of interactions between metal ions and DNA, andits application in metal ions sensing are introduced． Chapter2Gold nanoparticles modified electrode via amercapto-diazoaminobenzene monolayer and its development in DNAelectrochemical biosensorA novel protocol for the gold nanoparticles （AuNPs） modification on theelectrode surface was proposed, which was based on the self-assembly of AuNPs onthe mercapto-diazoaminobenzene monolayer modified electrode. Themercapto-diazoaminobenzene monolayer was obtained by covalent immobilization of4-aminothiophenol （4-ATP） molecules onto another4-ATP monolayer functionalizedgold electrode by diazotization-coupling reaction. The DNA immobilization andhybridization on the AuNPs modified electrode was further investigated. The preparedAuNPs–ATP–diazo-ATP film demonstrated efficient electron transfer ability for theelectroactive species toward the electrode surface due to a large conjugated structure ofthe mercapto-diazoaminobenzene monolayer. The recognition of fabricatedelectrochemical DNA biosensor toward complementary single-stranded DNA wasdetermined by differential pulse voltammetry with the use of [Co（phen）₃]³⁺as anelectrochemical indicator. A linear detection range for the complementary target DNAwas obtained from3.01×10^–10to1.32×10^–8M with a detection limit of9.10×10^–11M.The fabricated biosensor also possessed good selectivity and could be regeneratedeasily.Chapter3Gold nanoparticles modified electrode via simple electrografting of insitu generated mercaptophenyl diazonium cations for development of DNAelectrochemical biosensorA novel protocol for development of DNA electrochemical biosensor based ongold nanoparticles （AuNPs） modified glassy carbon electrode （GCE） was proposed,which was carried out by the self-assembly of AuNPs on the mercaptophenyl film（MPF） via simple electrografting of in situ generated mercaptophenyl diazoniumcations. The resulting MPF was covalently immobilized on GCE surface via C–C bondwith high stability, which was desirable in fabrication of excellent performancebiosensors. Probe DNA was self-assembled on AuNPs through the well-knownAu–thiol binding. The recognition of fabricated DNA electrochemical biosensortoward complementary single-stranded DNA was determined by differential pulsevoltammetry with the use of [Co（phen）₃]³⁺as the electrochemical indicator. Takingadvantage of amplification effects of AuNPs and stability of MPF, the developedbiosensor could detect target DNA with the detection limit of7.2×1011M, whichalso exhibits good selectivity, stability and regeneration ability for DNA detection.Chapter4Triggered activity of a nicking endonuclease for mercuric（II）ion-mediated duplex-like DNA cleavageThe cleavage activity of a nicking endonuclease towards metal-ion mediatedduplex-like DNA can be triggered by the corresponding metal ions, which wasdemonstrated with mercuric（II） ion as a model via a simple electrochemical protocol. The nicking endonuclease can recognize the Hg²⁺-mediated dsDNA and cleave at thedesignated site. This regulation mechanism was speculated to be also applied toC–Ag+–C system.Chapter5Crystal violet as a G-quadruplex-selective probe for sensitiveamperometric sensing of leadA sensitive and selective amperometric sensing platform for lead (Pb²⁺) wasdeveloped based on a Pb²⁺-induced G-rich DNA conformational switch from arandom-coil to G-quadruplex with crystal violet （CV） as the G-quadruplex-bindingindicator. The electrochemical property of CV in the G-quadruplex–CV complex wasfully investigated by various electrochemical techniques and the structural change wasconfirmed by circular dichroism measurement. The efficient intercalation of CV intothe Pb²⁺-stabilized G-quadruplex resulted in a readily measurable “turn–on”electrochemical signal, and the signal gain was linearly dependent on the logarithm ofPb²⁺concentration over the range from1.0×10–9M to1.0×10–6M with the detectionlimit of4.0×10^–11M.Chapter6Sensitive amperometric sensing platform for lead based ontarget-induced strand releaseA novel strategy for selective and sensitive amperometric detection of lead ion(Pb²⁺) was proposed based on target-induced strand release. The underlying goldelectrode was pre-modified with dendric gold nanoparticles （DenAu） by directelectrodeposition of the electrode in2.8mM HAuCl4and0.1M H2SO4solution undera negative potential of1.5V. This was expected to afford enhanced active electrodesurface for immobilization of thiol group-containing capture DNA molecules. Afterself-assembly on the DenAu modified electrode, the capture DNA moleculeshybridized with Pb²⁺-specific aptamer molecules to form DNA duplex into whichmethylene blue was intercalated, giving measurable electrochemical signal. Uponaddition of Pb²⁺, Pb²⁺bound to its aptamer to form Pb²⁺-stablized G-quadruplex whichreleased from the electrode surface into solution. The release of intercalated MBoccurred as well, which is responsible for significant signal reduction. Experimentalresults revealed that the biosensor showed sensitive response to the logarithm of Pb²⁺concentration over the range from1.0×10^–10M to1.0×10–7M with the detectionlimit of7.5×10^–11M. In addition, this strategy afforded exquisite selectivity for Pb²⁺against other metal ions. The excellent sensitivity and selectivity signified the potentialof the biosensor for Pb²⁺detection in real environmental samples.