| Nanomaterials, due to their diverse sizes, shapes, compositions, and versatile optical, electronic, magnetic properties, have attracted tremendous research interests in the last decade or two, leading to a wide range of applications in different areas such as tracking, biodetection, optoelectronic and photovoltaic devices. The development of such nanoscience is critically dependent on the ability to synthesize nanomaterials with desired structure and properties. In vitro fabrication methods often involve strong reducing agents, harsh reaction conditions and extremely rapid process, which is difficult to exquisitely control. With the development of interdisciplinary subject among the materialogy, chemistry, and life science, live cells have been exploited to synthesize nanomaterials with exquisitely controlled size, shape, composition, and crystal structure of the desired inorganic nanomaterials due to the high degree of organization and sophisticated molecular controls in these biological systems. Thus, using biochemical reactions in living cells to synthesize materials offers a very attractive way to obtain the designer nanomaterials under easily feasible conditions.We have previously developed a novel "space-time coupling" strategy that allows the synthesis of inorganic nanomaterials in a highly tunable way in live cells. The key feature is to temporally and spatially couple intracellular unrelated biochemical reactions or metabolic pathways that do not encounter each other under normal condition to enable the synthesis of nanomaterials with desired properties through elaborate design and facile chemical handling. For example, by coupling of intracellular selenium reduction and Cd(11) detoxification, fluorescent CdSe quantum dots with tunable fluorescence spectra can be synthesized in live yeast cells just by'culturing'the live cells with chemical compounds. Furthermore, many efforts have been devoted by us to exploring the biosynthetic mechanism of CdSe QDs using molecular genetics methods, which have demonstrated the important role of expression of the glutathione metabolic gene in the intracellular CdSe QDs formation. Based on knowledge on the biosynthetic mechanisms underlying the intracellular nanomaterial formation, our laboratory has successfully created a quasi-biological system for the controllable synthesis of water-solution nanomaterials. Although such "space-time coupling" strategy has overcome the bottleneck on the intracellular synthesis of high-quality quantum dots, but it is a significant challenge to generate and use the intracellular fluorescence from nanomaterials with a simple and effient method. And, there is an ungent need for a convenient strategy to modify the cell surface. Based on these, the main research work in this doctoral thesis has been summarized as follows:(1) Uniform fluorescent nanobioprobes based on the live cells have been constructed for pathogen detection. Using "space-time coupling" strategy, we have successfully transformed Staphylococcus aureus (S. aureus) cells into fluorescent cellular beacons. The cellular beacons have high brightness, outstanding photostability, high luminance, good monodipersibility and perfect uniformity, which can be directly and easily conjugated to monoclonal antibodies (mAbs) based on specific interaction between the protein A self-expressed on the cell surface and the Fc region of mAbs to generate fluorescent-biotargeting biofunctional probes without any additional modification. Coupling with immunomagnetic nanosphere, the resulting nanobioprobes can be applied to detection H9N2 virus. With this method, H9N2 virus can be detected specifically and reproducibly with the virus concentration of 8.94 ng/mL. Just by changing the antibodies conjugated to the cells, such new bioprobes can be constructed to detect other pathogens, such as pseudorabies virus, baculovirus, Salmonella typhimurium bacteria, and SK-BR-3 cells. Such a simple, sensitive, but specific approach can become a general platform for detection of pathogens and other biotargets.(2) A novel cell labeling method based on wheat germ agglutinin (WGA)-modified cellular beacons has been developed for fast and efficiently labeling tumor cell. The stability of the cellular beacons with different batches was inverstigated, including their fluorescence properties, fluorescence productivity, dispersibility and sizes, which indicated the cellular beacons could be regarded as good photoluminescence reporters for cell labeling. Furthermore, N-acetylglucosamine endogenously expressed on the surface of S. aureus makes WGA molecular conjugation easy. Thus, just by mixing the cellular beacons with WGA molecular and simply centrifuging to move excess WGA, WGA-modified cellular beacons can be regarded as fluorescent-biotargeting bioprobes for fast and efficiently labeling tumor cells.(3) We report a novel approach that harnesses cellular pathways for in vitro synthesis of high-quality nanomaterials with tunable lengths and optical properties. We first demonstrated that in vivo biochemical pathways could be used to synthesize Te nanorods via the intracellular reduction of TeO2- in living S. aureus cells. We then utilized the pathways to set up a quasi-biological system for Te nanorod synthesis in vitro. This allowed us to successfully synthesize in vitro, under routine laboratory conditions, Te nanorods with uniform and tunable lengths, ranging about from 10 nm to 200 nm, and controllable optical properties with high molar extinction coefficients. Our approach should open new avenues for controllable, facile, and efficient synthesis of designer nanomaterials for diverse industrial and biomedical applications. More important to the ever expanding applications of nanomaterials, the in vitro systems such as the one reported here can be easily scaled up and manipulated to produce and purify large amounts of high quality and homogenous nanomaterials under conditions that are user friendly and reliable, which has long been a major road block for broad applications of nanomaterials.(4) A reliable and biofriendly strategy for fluorescent cell-drived microvesicles biolabeling based on MCF-7 cells self-synthesizing fluorescent quantum dots for in vivo imaging was developed. Just by facile chemical handling and rational utilization of intracellular biochemical processes, fluorescent CdSe quantum dots can be synthesized in live MCF-7 cells. Furthermore, cells can release into the extracellular environment diverse types of plasma membrane named microvesicles from the surface of cells upon stimulation. Based on that, the fluorescence microvesicles were formed by direct budding of the plasma membrane that coated intracellular fluorescent QDs. Then, using this convenient procedure, the microvesicles can be efficiently labeled by the intracellular fluorescent QDs with reliable reproducibility and excellent biocompatibility, which can further be applied for in vivo imaging. Moreover, the strategy to lable microvesicles via intracellular biochemical reactions presented herein provides a new insight to the use of live organisms for a wide spectrum of biomedical applications. |
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