A New Method For The Synthesis Of Silver Nanoparticles Stabilized By Mercapto DNA And The Detection Of Endonuclease Activity

Metal nanoclusters (MNCs) are a class of materials considered as intermediates between metal atoms and nanoparticles, they usually composed of a few to roughly a hundred atoms and behave like a molecule with discrete energy levels due to their limited electron transition. The optical/electronic characteristics of MNCs are tremendously influenced by their sizes. Lately, small silver clusters stabilized by DNA (DNA-AgNCs) have become a new class of fluorophores for wide applications, such as biolabeling and biosensing/chemical sensing. However, the development of fluorescent DNA-AgNCs is still in their infancy, as the stability of fluorescent DNA-AgNCs is not better than that of fluorescent nanoparticals, such as semiconductor quantum dots.Therefore, it is a high desirable on the synthesis of DNA-AgNCs with enhanced stability. In this thesis, a new method for the synthesis of stable DNA-SH-AgNCs templated by thiolated DNA (DNA-SH) has been developed and the characteristics of the DNA-SH-AgNCs have been also investigated.The exploration of restriction enzymes activity and inhibition has important significance to help diagnose pathogenic infections and drug development. Suffering from the problem that the existing detection methods (e.g., low sensitivity and poor accuracy), we developed a method for detecting restriction endonuclease EcoRI activity, based on that the restriction enzyme could cleavage DNA in specific sites and the fluorescence intensity of DNA-AgNCs changed with structural transformation. The assay had high sensitivity to detect low level EcoRI. The thesis composed by three parts as follows:In chapter one, the properties of DNA-AgNCs, synthetic methods and research progress on their applications were described, then, the detection methods of restriction endonuclease, as well as the advantages and disadvantages of each method, were summarized.In chapter two, the synthetic methods of stable DNA-SH-AgNCs were explored. First, the thiolated DNA was used as a scaffold for synthesis of DNA-SH-AgNCs. In comparision to the fluorescent intensity and stability of DNA-AgNCs, the results showed that the fluorescence intensity and stability of DNA-SH-AgNCs were better than that of DNA-AgNCs. Then, the composition, the possible structure, anti-oxidative capacity and Ag-S bond binding mode of DNA-SH-AgNCs were studied by means of mass spectrum, circular dichroism spectroscopy, cyclic voltammetry and X-ray Photoelectron Spectroscopy, respectively. The enhanced fluorescence and the stability were possible due to the synergistic protective effect of the mercapto group and DNA.In chapter three, a method based on a label-free DNA-templated silver nanocluster probe for fluorescence on-off detection of endonuclease activity and inhibition has been developed. This method for nuclease activity assay by combining the high specificity of DNA cleavage reactions with ultrahigh fluorescence turn-on abilities of guanine-rich (G-rich) DNA sequences in proximity to silver nanoclusters (AgNCs). The probe consisted of two single strand DNAs, one of the DNA strand(DNA) containing EcoRI recognition sites and a nucleation sequences at the5’-end can form AgNCs with weak fluorescence, the other(cDNA) with a G-rich sequences at the3’-end is partly complementary with DNA. Thus, the fluorescence of DNA-AgNCs probes was activated because of DNA hybridization. When these DNA-AgNCs probes were exposed to the targeted endonucleases, specific DNA cleavages occurred, and pieces of G-rich DNA fragments separated from AgNCs, resulting in fluorescence turn-off. The endonuclease activity was quantified by monitoring the change in the fluorescence intensity. The fluorescence reduction efficiency was linear with the EcoRI concentration in the range of5.0×10-4U· μl-1to3.0×10-3U· μL-1, with a detection limit of3.5×10-4U· μl-1, which is much better than or at least comparable with that in previous reports. The potential application of the proposed method for screening endonuclease inhibitors was also demonstrated. The inhibitors we finally selected was5-fluorouracil, it played a well inhibition in this system, the IC50value (i.e., the inhibitor concentration required to reduce enzyme activity by50%) of5-fluorouracil was found to be0.10mM. The presented assay protocol proved to be convenient, effective, sensitive, and easy in preparing the fluorescent probes.