The Research And Application Of Nucleic Acid And Nucleic Acid Mimics-based Electrochemical Biosensing

As a promising handy detection technique, electrochemical biosensors have received increasing attention due to their intrinsic advantages of simple instrumentation, high sensitivity, low cost, promising response speed and trace analysis, etc. Combined electrochemical sensor with the bioassay, monitoring of life system or related process is ascribed to sensitive biological materials（including enzymes, nucleic acids and other biologically active substances）. A number of novel nanotechnologies are introduced gradually in electrochemical analysis and molecular biology, presenting a broad prospect. Nano-materials with large specific surface area and high catalytic activity are used as sens ing materials which possess high selectivity, high sensitivity, fast and convenient detection. With the development of DNA nanotechnology, a number of two- and three-dimensional structure of DNA nanomaterials are assembled through base pairing complementary principle of DNA, and used gradually in bio-sensing and nanotechnology equipment field. The development of these DNA nanomaterials provides a powerful tool for the evolution of analytical chemistry.Based on the above considerations and relevant reports in the literature previously, we developed a series of new electrochemical biosen sing methods. Specific work of this paper includes the following aspects:（1） We describe a novel label-free amplified multifunctional strategy of dendritic electrochemical DNA sensor based on terminal deoxynucleo tidyl transferase（Td T）. We have found that the sequence composition of Td T-yielded DNA is largely dependent on the constitution of substrate deoxynucleotides（d NTPs） pool. After rational design of d NTPs pool and controllable Td T polymerization, dendritic protocol has been developed involving two-type amplification strategies; one is the formation of “trunk” and “branch” of the dendritic electrochemical sensor by Td T amplification; the other is the introduction of nucleic acid functionalized Au nanoparticle s（DNA-Au NPs） for multiple branching. The results indicate that the G-rich ss DNA, which is synthesized under the condition of 40% deoxyadenosine triphosphate（d ATP） and 60% deoxyguanosine triphosphate（d GTP）, can be induced to form a long signal strand to G-quadruples（G4） in the presence of Pb2 +. The electrochemical sensing platform is employed for sequence-specific DNA detection and the detection limit is as low as 1 f M. Our multifunctional strategy is further extended to Pb²⁺ detection and thrombin aptas ensor. This proposed sensor displays excellent sensitivity and selectivity, and is applied for detection in complicated samples successfully.（2） Since two ends of coenzyme A（CoA） are free thiol group and adenine respectively, the potential coordination p olymer formed by CoA and Ag（I） was supposed as the chain-like structure with（-CoA-Ag（I）-） repeated units and multiple adenine bases as the side groups along the polymer backbone, which is similar to the structure of poly A single-stranded nucleic acid（ss NA）, for example RNA. We investigated the interaction of CoA-Ag（I） coordination polymer（CoA-Ag（I） CP） and GO and discovered that CoA-Ag（I） CP, similar to ss NA, can easily and effectively bind with GO through its adenine side groups-caused π-π stacking interaction. Moreover, CoA-Ag（I） CP adsorbed on GO-modified electrode possesses high electro-catalytic activity to H2O2 reduction. In this chapter, fluorescence spectroscopy, ultraviolet spectroscopy, infrared spectroscopy, dynamic light scattering, atomic force microscopy, and electrochemical method were employed to study properties and electro-catalytic mechanism of CoA-Ag（I） CP. Then, our work gave the first example of electrochemical sensor for detection of HAT or protein acetyltransferase activity. HAT p300 catalyzes the transfer of an acetyl group from Ac-CoA to lysine residue of substrate peptide, producing ε-N-acetyl lysine residue and CoA. In the presence of p300 HAT, CoA was produced and interacted with Ag（I） to form CoA-Ag（I） CP. The activity of p300 HAT was quantitatively measured by monitoring CoA generation during the acetylation. It is highly prom ising for development of miniaturized and cost-efficient biosensing platform for epigenesist-related clinical diagnosis and anti-carcinogenic drug discovery.（3） In this work, we focused on designing DNA prism nanostructures probe and its application in el ectrochemical biosensors. The DNA prism nanostructure was hierarchically assembled from one thiolated DNA fragment, one probe-containing DNA fragment, and one triangular facet DNA fragment. The prism contains three thiol groups at three vertices and is exp ected to readily and strongly anchor at Au surfaces by the three thiols. By incorporating DNA prism nanostructures into macroscopic gold interfaces, not only the number of probes is large, but also the lateral space between the DNA probes could be finely controlled. We designed a DNA prism structure with pendant probe D NA at the other three vertices, which has a much thicker layer, and places the probes in a solution-phase-like environment with enhance d capture-target probe binding affinity. Target DNA was appended to the rigid DNA prism nanostructure on electrode surface, then signal DNA was modified with a biotin tag to introduce signals. The biotin tag can bind specially to horseradish peroxidase-conjugated avidin（avidin-HRP）, which catalyze the reductio n of hydrogen peroxide to generate quantitative electrochemical current signals in the presence of 3,3’,5,5’-Tetramethylbenzidine（TMB）. By comparison of prism DNA nanostructure electrode and single-stranded DNA substrate electrode, we found that DNA prism nanostructure substrate electrode can be stably upright on the electrode surface without mercaptoethanol（MCH） passivation. The relatively high protein-repelling ability of the DNA prism nanostructure surface permits the use of prism-based sensors directl y in real samples.（4） It is a challenge to design programmable engineering of a biosensing interface in the field of biomolecule detection. Micro RNAs become a promising biomarker because of their functions in regulationg many cellular processes, however, the same micro RNA with the short size and low abundance displays different expression level in the serum of breast cancer, it is challenging to develop fast, inexpensive, and simple biosensor to detect them. To solve this problem, based on the third chapte r, DNA prism nanostructure has been improved to design seven different DNA strands. The new DNA prism nanostructures were assembled by control of 1: 1: 1 ratio to build three different pendant probe DNA, and used to simultaneously detect three different ta rgets without limitations. The electrochemical signals are expected by different metal ions(Zn2 +, Cd2 + and Pb²⁺) modified metallothionein coupled to signal DNA, which are produced by electrochemical differential pulse stripping voltammetry, thus capable of specifically detecting the expression of three kinds of micro RNAs. Nevertheless, to improve the sensitivity, supersandwich method is used by introducing an auxiliary DNA which can hybridize with signal DNA to form a long chain DNA, where these metallothi onein can release metal ions in an acidic solution, thus achieving the detection of micro RNAs. Finally, we also demonstrated its application by analyzing micro RNA expression levels in clinical serum samples from breast cancer patients, yielding a promising method in medical diagnosis of cancer.