Study Of Novel Method For Dna Detection And Intracellular Micrornaanalysis

The accomplishment of the Human Genome Project and the progress in research of the functional genomics make gene diagnoses a hot pot in the areas of molecular biology and biomedicine. Based on the hybridization of bases, DNA biosensors which could continuously, fast, sensitively detect the specific gene sequence, have developed quickly in recent years. MicroRNAs(miRNAs)are short endogenous noncoding small RNAs that exist in genes of humans, plants, and animals. miRNAs play critical roles in multiple cell biological processes including cell proliferation, differentiation, apoptosis, metabolism and tumorigenesis. miRNA expression detection and analysis is a basic and preliminary procedure in most miRNA studies such as understanding its biological functions, diagnosis of diseases, as well as discovering new drug targets. This thesis focuses on exploring new methods to analyze and detect nucleic acid.1. Ultra-sensitive electrochemical dna biosensors based on multiplex enzyme functional carbon sphere and graphen/gold nanoparticle hybrid material dual signal amplified systemAn ultra-sensitive electrochemical DNA biosensor was constructed based on a dual signal amplified system that consisted of multiplex enzyme functionalized carbon sphere and graphene/gold nanoparticle hybrid materials. The electrochemical reduced graphene (ERGO) and the thiol groups tagged DNA strands (d(GT)29SH) were firstly assembled via π-π stacking, and the resulting DNA-carbon bioconjugates were then employed to scaffold the gold nanoparticles with DNA probe (AuNPs-ssDNA) into metal-carbon hybrid nanostructures (ERGO-Au). Either ERGO or AuNPs maintains their structures and properties due to the noncovalent features, and the heteronanostructure materials exhibited high conductivity. The synthesized carbon nanospheres (CNSs) yielded a homogeneous and narrow size distribution (120 nm), which were further labeled with horseradish peroxidase-streptavidin (HRP-SA) as signal amplification platform. The dual signal amplification strategy of ERGO-DNA-Au and the multienzyme labeling enhanced the sensitivity of the DNA biosensor greatly. The proposed method could respond to 0.8×10-17M DNA and work within a wide linear calibration range (10-13-10-17M). Meanwhile it maintained high specificity and repetition. The dual signal amplification strategy provides a simple and reliable method of DNA detection with high sensitivity and specificity, indicating promising application in the biomedicine.2. Quantum-dot-functionalized poly(styrene-co-acrylic acid) microbeads:step-wise self-assembly, characterization, and applications for sub-femtomolar electrochemical detection of dna hybridizationA novel nanoparticle label capable of amplifying the electrochemical signal of DNA hybridization is fabricated by functionalizing poly(styrene-co-acrylic acid) microbeads with CdTe quantum dots. CdTe-tagged polybeads are prepared by a layer-by-layer self-assembly of the CdTe quantum dots (diameter=3.07 nm) and polyelectrolyte on the polybeads (diameter=323 nm). The self-assembly procedure is characterized using scanning and transmission electron microcopy, and X-ray photoelectron, infrared and photoluminescence spectroscopy. The mean quantum-dot coverage is (9.54±1.2)×103 per polybead. The enormous coverage and the unique properties of the quantum dots make the polybeads an effective candidate as a functionalized amplification platform for labelling of DNA or protein. Herein, as an example, the CdTe-tagged polybeads are attached to DNA probes specific to breast cancer by streptavidin-biotin binding to construct a DNA biosensor. The detection of the DNA hybridization process is achieved by the square-wave voltammetry of Cd2+ after the dissolution of the CdTe tags with HNO3. The efficient carrier-bead amplification platform, coupled with the highly sensitive stripping voltammetric measurement, gives rise to a detection limit of 0.52 fM and a dynamic range spanning 5 orders of magnitude. This proposed nanoparticle label is promising, exhibits an efficient amplification performance, and opens new opportunities for ultrasensitive detection of other biorecognition events.3. Fluorescence resonance energy transfer between quantum dots and graphene oxide for sensing biomoleculesThis work designed a novel platform for effective sensing of biomolecules by fluorescence resonance energy transfer (FRET) from quantum dots (QDs) to graphene oxide (GO). The QDs were first modified with a molecular beacon (MB) as a probe to recognize the target analyte. The strong interaction between MB and GO led to the fluorescent quenching of QDs. Upon the recognition of the target, the distance between the QDs and GO increased, and the interaction between target-bound MB and GO became weaker, which significantly hindered the FRET and, thus, increased the fluorescence of QDs. The change in fluorescent intensity produced a novel method for detection of the target. The GO-quenching approach could be used for detection of DNA sequences, with advantages such as less labor for synthesis of the MB-based fluorescent probe, high quenching efficiency and sensitivity, and good specificity. By substituting the MB with aptamer, this strategy could be conveniently extended for detection of other biomolecules, which had been demonstrated by the interaction between aptamer and protein. To the best of our knowledge, this is the first application of the FRET between QDs and GO and opens new opportunities for sensitive detection of biorecognition events.4. The use of polyethylenimine-grafted graphene nanoribbon for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNAA simple nanocarrier of polyethylenimine-grafted graphene nanoribbon (PEI-g-GNR) was proposed as an effective gene vector. The GNR was formed by longitudinally unzipping multiwalled carbon nanotubes (MWCNTs), and treated with strong acids and sonication to obtain surface carboxylic acid groups for graft of PEI via electrostatic assembly. The PEI-g-GNR appeared to protect locked nucleic acid modified molecular beacon (LNA-m-MB) probes from nuclease digestion or single-strand binding protein interaction, thus could be used as a nanocarrier of the probes for more efficient transfection of cells than PEI or PEI-g-MWCNTs due to the large surface area of the GNR and high charge density of PEI. The cytotoxicity and apoptosis induced by the PEI-g-GNR were negligible under optimal transfection conditions. Combining with the remarkable affinity and specificity of LNA to microRNA (miRNA), a delivery system by the LNA-m-MB/PEI-g-GNR was proposed for effectively transferring LNA-m-MB into the cells to recognize the target miRNA. Using HeLa cells as model, a method for detection of miRNA in single cell was developed. These results suggested that PEI-g-GNR would be a promising nonviral vector for in situ detection of gene in cytoplasm and gene therapy in clinical application.5. Target-cell-specific, intracellular imaging and detection of microRNA with functionalized Sno2 nanoparticleA multifunctional fluorescent nanoprobe of SnO2 (f-SnO2) was designed to target cell-specific, intracellular imaging and to detect microRNA (miRNA). For the fabrication of multifunctional fluorescent nanoparticle, the SnO2 was firstly modified with 2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[amino (polyethylene glycol) 2000] (DL-PEG2000-NH2), to obtain SnO2 high degree of size monodispersity. The primary amine groups on the surface were then either converted into pyridyldisulfide groups by treatment with sulfosuccinimidy 6-(3’(2-pyridyldithio) propionate (Sulfo-LC-SPDP) or functionalized with folic acid (FA). The resulting nanoparticles were treated with thiolated gene probe by disulfide groups. The f-SnO2 could target specific cell with folate-receptor-mediated type and be imaged in the cell by autogenous florescence. The disulfide linkages between gene probe and the nanoparticles could be cleaved readily in an intracellular environment, which facilitate to inhibit or detect miRNA. Meanwhile, it served to protect gene probes from nuclease digestion or single-strand binding protein (SSB) interaction, negligible cytotoxicity and apoptosis. The proposed multifunctional fluorescent nanoprobe could perform simultaneous specific delivery, cell imaging and detection of miRNA, which have potential advantages in vivo experiments and clinical applications.