Design Of Label-Free Fluorescent Nucleic Acids Probes Using DNA/Carbon Nanomaterials Assemblies

Fluorescence probe is mainly consisted of two functional parts, the recognition moiety and signal moiety. When the recognition moiety selectively binds with the target molecule, signal moiety is responsible for outputting the fluorescence signals. With predesigned and selected structures, functional nucleic acids can selectively bind and recognize different kinds of analytes, including cations, small biomolecules, proteins, DNA and even cancer cells. For most nucleic acids probes, signal moiety linked with recognition moiety through covalent interaction, which makes it laborious, time-consuming and costly. Even more modification of nucleic acid may alter its recognition ability. On the other hand, due to their unique optical and electronic properties, carbon nanomaterials, such as carbon nanotubes and graphenes, when combining with nucleic acids can thus enhance the molecular recognition interactions and amplify the detection signals. They have been well-developed in nanotechnology, biosensing and drug delivery.In this thesis, with the purpose of designing label-free nucleic acid fluorescent probes with lower background signal, higher selectivity and sensitivity, and being capable of applying in complex enviroments, DNA is combined with various carbon nanomaterials, including single walled carbon nanotubes (SWNTs) and carbon nanoparticles (CNPs), to fabricate high performance chem/biosensing platforms. The contents of this thesis include as following:1. Design of time-resolved luminescence sensing platform of proteins using Eu3+ complex and aptamer-wrapped SWNTs. Using SWNTs as universal quencher, Eu3+ complex could absorb on the surface of SWNTs and the Eu3+ fluorescence is quenched. Due to the lower affinity of aptamer/protein to SWNTs than that of single stranded DNA and SWNTs are more stable in solutions with high concentration of Na+, this platform is used to sense lysozyme. Compared with traditional methods, the proposed sensor presents lower background signal in complicated biological samples.2. Fluorescent single-nucleotide polymorphisms(SNPs) analysis based on carbon nanoparticles and DNA ligase. A new method for SNPs analysis was developed based on the fluorescence quenching effect of carbon nanoparticles and nucleic acids ligation reaction. In the presence of DNA ligase, the two half primer DNA probes are linked by the perfectly complementary DNA target to form a duplex, but the duplex could not formed by the single-base mismatched DNA. The fluorescence of SYBR Green I (SG)-single-stranded DNA (ssDNA) is efficiently quenched when they are mixed with CNPs unless they hybridize with their perfectly matched DNA target.3. Fluorescent assay of DNA adenine methylation methyltransferase (Dam Mtase) activity. SG shows different binding abilities to double-stranded DNA (dsDNA) over ssDNA by intercalating within the internally stacked bases of dsDNA, which result in an increase of their fluorescence quantum yields. Dam MTase could methylate the N6-adenine in the symmetric tetranucleotide 5′-G-A-T-C-3′, it has also been found that Dpn I endonuclease can only cut the sequence of 5′-G-Am-T-C-3′when the internal adenine is methylated. Thus, in the presence of Dam MTase, the dsDNA containing the sequences of 5′-G-A-T-C-3′can be methylated and then be cut into four pieces of ssDNA, emitting weak fluorescence. While in the absence of Dam MTase, or the enzyme is inactive, the dsDNA is remained in the solution, having strong intensity of fluorescence. Coupling SG with CNPs not only significantly reduces the background signal when SG binds to ssDNA, but also improves the specificity.