Studies On The Detection Of DNA And Hg²⁺ Based On Novel Molecular Beacon And Thymine-mercury(â…¡) Coordination

With the completion of the human genome project, there has been a rapid shift in focus from simply collecting and archiving genomic data to utilizing genetic analysis for prediction and discovery. The development and utilization of new quantitative tools for research across disciplinary interfaces will prove vital in achieving these objectives. Molecular beacons (MBs) are ideally suited for and hold great promise in genomics and proteomics. More research is expected for MB applications in mutation detection for a variety of disease diagnostics and disease mechanism studies. Gene expression monitoring in living cells and tissues under different conditions with precise quantitation also provides an avenue for further investigation. The extraordinary target-specific capability along with the availability of different fluorophore-quencher pairs makes MB probes extremely useful for multiple analyte applications as well. These properties also make MBs suited for use in the identification of genetic alleles or particular strains of infectious agents. All of these developments will open the possibility of using easily obtainable and designer DNA molecules for genomics and proteomics studies, for molecular diagnosis of diseases, and for new drug development.Because of continuing concern over mercury in the environment and its deleterious effects on human health, obtaining new mercury detection methods that are cost-effective, rapid, facile and applicable to the environmental and biological milieus is an important goal. This objective has recently emerged as a focal point in the chemistry and, more broadly, sensing communities. DNA's structure and its functioning in the organism are known to be very sensitive to the influence of heavy metals. So, it is very attractive to use DNA-containing systems, e.g., DNA-based biosensors, to perform heavy metal assays. Recently, it has been reported that mercury ions can selectively bind thymine-thymine pairs in DNA sequences to form T-Hg2+-T complexes. For the moment, thymine-Hg2+-thymine chemistry has been highlighted in the development of Hg2+ sensors because thymine-thymine mismatch shows high selectivity to Hg2+ against many other metal ions.The goal of the present study is to design and research novel molecular beacon and sensors with high sensitivity and selectivity for Hg2+. This paper combines the excellent characteristics of nanoparticles, the specific recognition of molecular recognition elements and the electrochemical technique. The details are given as follows:Chapter One:PrefaceIn this chapter, we review the principles, characteristics of molecular beacon and its application in analytical chemistry field. Several assays based on T-Hg2+-T coordination were introduced. Finally, expounded the aim and the significance, pointed out the research content and the innovation in this paper.Chapter Two:Electrochemically active-inactive switching molecular beacon for direct detection of DNA in homogenous solutionA new kind of electrochemical molecular beacon was reported, termed "electrochemically active-inactive switching molecular beacon", for direct detection of DNA in homogenous solution. The electrochemical molecular beacon consists of a stable stem-loop oligonucleotide carrying two carminic acid moieties (acting as electrochemical reporter) attached at its termini. In a close form, the electrochemical signal is quenched because two carminic acid moieties are close enough to associate into dimer. In the presence of the complementary DNA target, the electrochemical molecular beacon undergoes a conformational transformation from closed (hairpin) to open (linear) structure, which is associated with an increase in electrochemical signal. We found that the electrochemical molecular beacon is as effective as conventional molecular beacon in signaling the presence of complementary target and discriminating targets that differ by a single nucleotide. The proposed electrochemical molecular beacon has a great potential for investigating the interactions of DNA-protein and developing electrochemical real-time polymerase chain reaction.Chapter Three:colorimetric detection of SNPs based on label-free molecular beacon modified gold nanoparticles through non-crosslinking aggregationA new colorimetric approach has been developed for the detection of single nucleotide polymorphisms (SNPs). One label-free molecular beacon was immobilized on the surface of gold nanoparticles through Au-S bonds. The sensitive detection of SNPs was achieved by non-crosslinking aggregation of MB-functionalized gold nanoparticles induced by hybridization of target DNA. The mutant target can be determined in a mixed solution containing 19 times higher concentration of wild target. The allele frequency of 5% was accurately determined in a homogeneous and cost-effective manner.Chapter Four:Polythymine oligonucleotide-modified gold electrode for voltammetric determination of mercury (Ⅱ) in aqueous solutionPolythymine oligonucleotide (PTO)-modified gold electrode (PTO/Au) was developed for selective and sensitive Hg2+ detection in aqueous solutions. This modified electrode was prepared by self-assembly of thiolated polythymine oligonucleotide (5'-SH-T15-3') on the gold electrode via Au-S bonds, and then the surface was passivated with 1-mercaptohexanol solution. The proposed electrode utilizes the specific binding interactions between Hg2+ and thymine to selectively capture Hg2+, thereby reducing the interference from coexistent ions. After exchanging the medium, electrochemical reduction at-0.2V for 60s, voltammetric determination was performed by differential pulse voltammetry using 10mM HEPES (pH 7.2,1M NaClO4) as supporting electrolyte. This electrode showed increasing voltammetric response in the range of 0.2-1nM Hg2+, with a relative standard deviation of 5.32% and a practical detection limit of 60pM. Compared with the conventional stripping approach, the modified electrode exhibits good sensitivity and selectivity, and is expected to be a new type of green electrode. Chapter Five:Simple and rapid colorimetric mercury sensing assay using mercury-specific DNA-functionalized gold nanoparticlesA new colorimetric assay for Hg2+ based on mercury-specific DNA (MSD)-functionalized gold nanoparticles (AuNPs) is described. The sensing mechanism of the assay is based on thymine-Hg2+-thymine (T-Hg2+-T) chemistry and a unique colloidal stabilization effect associated with conformational change of MSD attached on AuNP surfaces. Upon binding of Hg2+, the surface-tethered MSD undergoes a structure switch and forms hairpin-like structure of T-Hg2+-T complexes. As a result, the Hg2+-induced conformational change significantly enhances the colloidal stability of AuNPs toward salt-induced aggregation. Moreover, the colloidal stability of AuNPs increases as the concentration of Hg2+ increases, accompanied by the color changes of solution from purple to red. The proposed sensor enables the colorimetric detection of Hg2+in the concentration range of 0-10μM Hg2+ ions with a detection limit of 60nM (S/N=3), and allows for the selective discrimination Hg2+ ions from 8 other environmentally and physiologically relevant metal ions.Chapter Six:Simple and rapid colorimetric assay for mercuric ions based on TCA-capped gold NanoparticlesA simple, rapid, colorimetric assay for sensing Hg2+ in aqueous solution based on trithiocyanuric acid-capped gold nano-particles (TCA-AuNPs). Trithiocyanuric acid (TCA) is a star-shape trithiol molecule that can spontaneously attach to citrate-capped AuNPs through the displacement of the weakly bound citrate ions and the formation of the Au-S covalent bonds. In the absence of Hg2+, the resulting TCA-AuNPs are dispersed in aqueous solution owing to the high negative charge density of TCA on each AuNP surface. In the presence of Hg2+, the coordination between the mercapto groups of TCA and Hg2+ induced the significant aggregation of TCA-AuNPs via the bridging of neighboring nanoparticles, thereby resulting in a color change that can be observed by the naked eye. Under the optimal conditions, the ration of absorbance (A620/A520) was linearly related to the concentration of Hg2+in the range of 0μM to 1.7μM and the assay allowed detection at levels as low as 50 nM Hg2+. The method is relatively simple and easily operated. The response time upon exposure to Hg2+ is instantaneous. The sensor exhibits highly selective detection of Hg2+ in aqueous solution.