Biochemical Molecules Detection Based On Nucleic Acid-templated Fluorescent Copper Nanoparticles

Fluorescent metal nanoclusters (nanoparticals), a kind of novel fluorescence nanomaterials, are discovered in recent years. Due to theirs quantum size effect, surface effect, small size effect and macroscopic quantum tunnel effect, fluorescent metal clusters (nanoparticals) show a series of unique properties, such as chemical properties and biological compatibility and and so on, which have received widespread concern by scientists. So far, Fluorescent metal nanoclusters are usually prepared by a biomineralization manner, in which biomolecules (e.g. small thiol-molecule, polymer, protein and nucleic acid) are used as templates to induce the formation of fluorescent metal nanoparticles (nanoparticals). Among these templates, nucleic acid has become a hot research topic because of its flexible, easy synthesis, modification and low biological toxicity. Nucleic acid-templated formation fluorescent metal nanoclusters (nanoparticals) have many advantages, such as easy preparation, low cost, low toxicity, large Stokes shift and high fluorescence quantum yield. Owing to these excellences, fluorescent metal nanoparticles can be widely used in the field of biological analysis detection, biological marker and fluorescence imaging. In this thesis, we choose the nucleic acid-templated fluorescent copper nanoparticals as fluorescence reporters to design the fluorescence porbe for hydrogen peroxide, glucose and Dam methyltransferase detection. The primary research is as follows:1. Studies on hydrogen peroxide and oxidase-based biosensing based on poly(thymine)-templated fluorescent copper nanoparticles.In recent years, DNA-templated formation of fluorescent metal nano materials as a new type of fluorescence signal element has been widely studied and applied in analytical sensing field. In this paper, poly(thymine)-templated fluorescent copper nanoparticles(T-CuNPs), for the first time, has been exploited as an effective signal indicator for constructing a new fluorescent strategy for H2O2 sensing. In the absence of corresponding targets H2O2, the probe poly(thymine) can effectively template the formation of T-CuNPs by a fast in situ manner, and obvious fluorescent signal can be recorded. However, when in the presence of the target(H2O2), hydroxyl radical(OH) can be formed from the Fe2+-catalyzed Fenton reaction. The probe will then be cleaved into mono-and/or short-oligonucleotide fragments which lose the ability for templating the fluorescent T-CuNPs. Because of the declining degree of fluorescence intensity is proportional to the concentration of target (H2O2), which can be successfully detected through the fluorescence change of T-CuNPs. The fluorescence probe exhibits low background fluorescence signal, high sensitivity and good selectivity to H2O2 in aqueous solutions. By taking advantage of H2O2 as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and been detected; and its practical applicability has been investigated by the assay of the real clinical blood sample. Results demonstrate that the proposed strategy not only has good detection capability, but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications.2. Studies on DNA methyltransferase detection based on poly(AT/TA)-template fluorescent copper nanoparticles.DNA methyltransferases (MTases) are a family of enzymes that could recognize particular short palindromic sequences and transfer a methyl group from S-adenosyl-L-meth-ionine to target adenine or cytosine residues during the biological DNA methylation process. An abnormal level of DNA MTase leads to the aberrant level of DNA methylation which has been regarded as biomarkers of early cancers. Accordingly, DNA MTase is regarded as a potential target for anticancer therapy and drug screening. Therefore, monitoring the DNA MTase activity and screening inhibitors are of significance in fundamental biochemical research, clinical diagnostics, drug discovery, and disease therapy. In this work, we hope to construct a label-free and sensitive fluorescent method for MTase activity detection based on poly(AT/TA)-template CuNPs. A Dam methyl enzyme recognition site is contained in template sequence of CuNPs. When there is no target of Dam MTase, poly(AT/TA) cannot be cuted off by Dpn I (methylation-sensitive restriction endonuclease), which will template the formation of CuNPs as long as added to the copper ion and sodium ascorbate. Conversely, in the presence of Dam, The poly (AT/TA) will change to short chain that cannot template the formation of CuNPs, The resulting fluorescence will be very weak. Importantly, this method exhibit sensitive responses toward Dam MTase with detection limit was 0.5 U/mL, Furthermore, this probe our is a promising strategy to screen inhibitors or drugs for Dam MTase.