The Synthesis Of Novel DNA Functional Nanomaterials And Its Application In The Detection Of Nucleic Acids And Proteins

As a kind of smart macromolecule, DNA becomes the most popular subject in the science research area, due to its biological function, biocompatibility, molecular recognition ability, and nanoscale controllability. The functional nanomaterials, which are formed on the DNA template or functionalized with DNA as the ligand, possess the unique photoelectric properties and better tunability in nanoscale. With the application of nano-biotechnology widely used in analytical chemistry, DNA functional materials have become the powerful tool in the developing of novel analytical method. Nevertheless, the present DNA functional nanomaterials have many disadvantages, including tedious preparation process, low stability and bio-toxicity. Thus, it is highly desirable to develop new methods for DNA functional nanomaterials preparation and explore the application of these materials in bioanalytical research.The main line of this dissertation is developing new methods for the preparation of DNA functional nanomaterials. The systematical research of the preparation and properties of the DNA functional nanomaterials are carried out from different aspects of the nanomaterials, including the enzymatic synthesis, self-assembly, morphology control and heterogeneous formation. The obtained nanomaterials are applied in nuclear acid and protein detection through the following works:1. A new enzymatic synthesis method of Cu/Ag alloy nanocluster has been developed. The complexes, which formed through the coordination between the pyrophosphate and copper ions, are selected as the substrate of alkaline phosphatase. The copper ions are released when the pyrophosphate is broken down by the alkaline phosphatase. In the presence of DNA template, the DNA-templated Cu/Ag alloy nanoclusters are formed during the reduction of silver ions and copper ions by NaBH4. Alkaline phosphatase has been widely used in enzyme-linked immunosorbent assay due to its universality in enzyme-labeled antibody. We combine the enzymatic synthesis of alloy nanocluster with enzyme-linked immunosorbent assay for the detection of cancer biomarker. This approach improves the selectivity of protein detection based on the DNA-templated noble nanocluster and gets rid of the interference from the other proteins.2. Room temperature hybridization method to self-assemble DNA-templated Ag nanoclusters is presented. In this method, self-assembly of Ag nanoclusers is triggered in the presence of the DNA scaffolds which are the products of rolling circle amplification. Through DNA hybridization, the Ag nanocluster nanowires are obtained at room temperature resulted in the morphology change from spot to wire. In each assembly unit, the dark fluorescence of Ag nanocluster can be enhanced when placed in proximity to guanine-rich sequences. After the self-assembly of Ag nanoclusters, the red nanowires can be observed directly by the confocal microscopy. Taking advantage of rolling circle amplification and fluorescence enhancement of silver nanocluster, a novel method is developed for sensitivity detection of DNA based on the silver nanocluster wire.3. A new method for control synthesis of Cu nanoparticles is developed. The poly-thymine fragment is elongated at the 3’of primer DNA by the template-independent polymerization of terminal deoxynucleotidyl transferase (TdT). In the presence of ascorbic acid and copper ions, the red fluorescent copper nanoparticles are formed at the poly-thymine sequence. It is found that the concentration of TdT was more effective than incubation time in extending the primer DNA. The results in the characterization of the obtained Cu nanoparticles show the size distribution and fluorescence life time of Cu nanoparticles can be mediated by the concentration of TdT. Because of the specific interaction between polynucleotide kinase and 5’phosphate terminus of primer DNA, a sensitive method for the detection of polynucleotide kinase is developed.4. The DNA functional metal organic frameworks (MOFs) are synthesized. The DNA probes are absorbed on the surface of MOFs through the π-π stacking interaction between the ligand of MOFs and the nucleoside in DNA molecule. The fluorescent of the dyes are efficiently quenched by the Cu complex in the porous structure of MOFs. In the presence of the target, the immersion of nucleotide bases into formed duplex structure protected the DNA from being absorbed. With the relative distance between ds-DNA and MOFs increasing, the photo-induced electron transfer-based quenching is weakened which resulted in the fluorescence recovery. The simultaneous detection of two different targets is implemented by the synchronous scanning fluorescence spectrometry.