Novel Fluorescent Biosensor Method Based On Silver And Copper Nanomaterials And Nucleic Acid Dye

Nanomaterials have been the subject of intense research in the past decades with their unique properties in physical, electrical, chemical and optical areas, due to their small size effect, surface effect, quantum size effect and macroscopic quantum tunneling effect. Nucleic acid dye, like SYBR Gold, SYBR Green?have been widely studied and applied. Recently, a vaviety of novel fluorescent biosensors have been constructed based on nanomaterials and nucleic acid dye, and the biosensors have shown great importance in the fields of clinical diagnostics, drug discovery,environmental monitoring and tumor imaging diagnosis and therapy. In this paper, we take advantages of silver nanoclusters, copper nanoparticles and nucleic acid dye to construct novel fluorescent biosensors for the detection of nuclease and biothiols.(1) In chapter 2, A facile and sensitive method was proposed for endonuclease activity assay by making use of the fluorescence enhancement effect when DNA–silver nanoclusters(DNA–Ag NCs) are in proximity to guanine-rich DNA sequences. The system mainly consists of block DNA(B-DNA), G-DNA and Ag-DNA. B-DNA serves as the substrate of the endonuclease(S1 nuclease as the model enzyme). G-DNA,which is predesigned entirely complementary to B strand, contains a guanine-rich overhang sequence and hybridization part at the 5'-end. Ag–DNA involves a sequence for Ag NCs synthesis and a sequence complementary to the hybridization part of the G-DNA. In the“off”state, B-DNA plays the role as a blocker that inhibit the proximity between Ag NCs and guanine-rich DNA sequences, resulting in a low fluorescence readout. However, if S1 nuclease is introduced into the system, B-DNA was cleaved into mono- or short-oligonucleotides fragments, which could not hybridize with G-DNA. As a result, the subsequent addition of DNA–Ag NCs could bring guanine-rich DNA sequences close to the Ag NCs, accompanied by a significant fluorescence enhancement. Therefore, endonuclease activity could be successfully quantified by monitoring the variation of fluorescence intensity. In addition, this approach can also be applied for inhibitor screening of endonuclease. This label-free and turn-on fluorescent assays employing the mechanism proposed here for the detection of nuclease and inhibitors turn out to be sensitive, selective, and convenient.(2) In chapter 3, Taking advantage of a universal fluorescent dye, SYBR Gold as the signal reporter, we developed herein a novel label-free and sensitive fluorescentplatform for sensing the activity and inhibition of S1 nuclease. SYBR Gold is one of the most commonly used commercial fluorescent dyes, and exhibits great fluorescence enhancement via bounding to nucleic acids other than mononucleotides. In the absence of S1 nuclease, nucleic acids keep the original length. The dye will emit a high fluorescent readout when bound to the nucleic acid. When S1 nuclease is introduced into the system, the nucleic acids are digested into mono- or short-oligonucleotides fragments, resulting in a low fluorescence signal. The biosensor has become a useful platform for the detection of S1 nuclease and its inhibitor screening because of its high sensitivity, easy operation, and cost efficiency.(3) In chapter 4, On the basis of that poly(T)-templated fluorescent copper nanoparticles might serve as a sensing signal probe, we construct a simple, rapid,sensitive method to detect biothiols. In the presence of biothiols including Cys, Hcy,GSH, the fluorescence intensity of copper nanoparticles is found to be quenched effectively with the increase of biothiols. The proposed strategy integrates simple in opration, fast signal response, and low cost. Furthermore, the capabilities for biothiols detection from complex sample is verified.