Synthesis Of Functional Nanomaterials And Its Applications In Biosensing

With the development of research on the essence of life and life course, the sensitive, rapid and accurate detection of biomolecules is very important for obtaining important information of life, understanding the relationship between structure and function of biological molecules, exploring the life activities, early screening of diseases and treatment. This thesis focuses on the exploring new methods to analyze and detect biomolecules based on the fabrication of functional nanomaterials. Detail are described as below:1. Sensitive detection of human procalcitonin based on nanobody and nanoprobe signal amplified systemA nanoparticle label capable of amplifying the electrochemiluminescence (ECL) signal of human procalcitonin (PCT) is fabricated by reverse microemulsion method. The as-prepared CdTe@SiO2 nanoparticles have a narrow size distribution. The CdTe@SiO2 nanoparticles were attached to anti-PCT nanobody to construct an ECL biosensor of PCT. To improve their chemical and photochemical stabilities, QDs are coated with SiO2,which is not only impedes the diffusion of charge carriers generated upon photoexcitation as well as the diffusion of oxygen from the environment, but also prevents the coated QDs from coagulating in aqueous dispersions. Enhanced detection sensitivity was achieved where the SiO2 nanoparticle carriers increased the amount of signal tags bound per sandwiched immunoreaction. The linear relation range of PCT concentration was 0.01 to 20 ng mL-1 with a detection limit of 3.4 pg mL-1. The preparation method of CdTe@SiO2 nanoparticles could be extended to other QDs coated with SiO2 materials. This proposed amplification performance opens new opportunities for ultrasensitive detection of other biomarkers.2. Construction of oriented photoelectrochemical immune interface for label-free detection of neutrophil gelatinase-associated lipocalinA tetraaminophthalocyanine (CoPc) sensitized TiO2 electrode was employed for immobilization of streptavidin. The C-terminus of nanobodies is situated at the opposite site of the antigen binding region, as this terminus is rarely involved in binding, and provides an optimal target for modification. The nanobody with the highest affinity was biotinylated at the C-terminus, which is situated at the opposite site of the antigen binding region. Therefore, an oriented and sensitive PEC immune interface for NGAL by utilizing biotinylated anti-NGAL nanobody orientedly immobilized to streptavidin-coated CoPc-sensitized TiO2 electrode by streptavidin-biotin binding. Oriented immobilization of nanobody to streptavidin-coated electrode surfaces provides higher binding capacity; thus, it can lead to a high sensitivity. This ideal stepwise band-edge structure achieves the ultimate purposes of broad absorption of solar light, reduced recombination of photogenerated electron-hole pairs, and high-speed transfer of the excited electrons to the electrode. Ascorbic acid (AA), a nontoxic and efficient electron donor, was further added to the electrolytes in order to suppress the electron-hole recombination and therefore enhance the photocurrent intensity. The limit of detection (LOD) of the proposed immunosensor has been significantly lowered to 0.6 pg mL-1, which is more sensitive than nanobody and full-sized antibody randomly coated sensors. Nanobodies exhibit increased resilience to heat that full-sized antibody. The proposed immunosensor with simple operation and rapid response from PEC would provide a promising technique for disease diagnosis with great clinical application potential.3. Construction of electrochemical impedance immunosensor based on nanobody for label-free detection of apolipoprotein B-100A label-free immunosensor for the detection of apolipoprotein B-100 was developed based on biotinylated nanobody and polyethyleneimine functionalized graphene nanosheets (PEI-RGO) modified electrode by using the electrochemical impedance spectroscopy (EIS) technique. The PEI-RGO nanocomposites were obtained by reducing the as-prepared graphene oxide with PEI. Positively charged PEI molecules were used to serve as reducing agent and surface modifier. The unique properties of the graphene make the functionalized graphene nanosheets an effective candidate as a functionalized electrode modified materials for immobilization of protein. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5 ng mL-1 with a detection limit of 0.03 ng mL-1. This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis.4. Synthesis of Fluorescent Dye-doped Silica Nanoparticles for Target-Cell-Specific Delivery and Intracellular MicroRNA ImagingA multifunctional fluorescent nanoprobe (FSiNPs-AS/MB) with AS1411 aptamer and molecular beacon (MB) co-immobilized on the surface of the fluorescent dye-doped silica nanoparticles (FSiNPs) for target-cell-specific delivery and intracellular miRNA imaging, respectively. The FSiNPs were prepared by a facile reverse microemulsion method from tetraethoxysilane and silane derivatized coumarin that was previously synthesized by click chemistry. The as-prepared FSiNPs possess uniform size distribution, good optical stability and biocompatibility. In addition, there is remarkable affinity interaction between AS 1411 aptamer and nucleolin protein on cancer cell surface. Thus, a target-cell-specific delivery system by the FSiNPs-AS/MB is proposed for effectively transferring MB into the cancer cells to recognize the target miRNA. Using miRNA-21 in MCF-7 cells (a human breast cancer cell line) as model, the proposed multifunctional nanosystems not only allow target-cell-specific delivery with the binding affinity of AS 1411, but also can track the simultaneously the transfected cells and detect intracellular miRNA in situ. The proposed multifunctional nanosystems are a promising platform for highly sensitive luminescent nonviral vector in biomedical and clinical research.5. Target-Cell-Specific Fluorescence Silica Nanoprobes for Imaging and Theranostics of Cancer CellsA target-cell-specific theranostics nanoprobe for target-cell-specific delivery, cancer cells and intracellular miRNA-21 imaging, and cancer cell growth inhibition was proposed. The nanoprobe (FS-AS/MB) was prepared by simultaneously coupling of AS1411 aptamer and miRNA-21 molecular beacon (anti-miR-21-MB) onto the surface of Ru(bpy)32+-encapsulated silica (FS) nanoparticles. The FS nanoparticles synthesized by a facile reverse microemulsion method showed nearly monodisperse spherical shape with a smooth surface, good colloidal stability, a fluorescence quantum yield of ～21%, and low cytotoxicity. The antibiofouling polymer PEG grafted onto silica shell reduced nonspecific uptake of cells. The ability of FS-AS/MB for target-specific cells delivery, simultaneous cancer cells and intracellular miRNA-21 imaging, inhibition of miRNA-21 function and suppression of cell growth in vitro were also demonstrated. The results of the present study suggested that the proposed nanoprobes would be a promising theranostics for different cancers by imaging and inhibiting other intracellular genes.