| Due to their versatility, sensitivity and quantitative capabilities, the development of sensitive and selective fluorescent probes has become a very active research field in recent years. Fluorescent probes find great potential applications in a wide variety of fields, including disease diagnosis, drug discovery, clinical toxicology, environmental monitoring and so on. With high affinity and specificity to their target molecules, aptamers which are selected in vitro from random pools of DNA or RNA molecules, are ideal recognition elements in the development of fluorescent probes. Compared with antibodies, aptamers offer another two main advantages. First, aptamers can be produced by chemical synthesis with extreme accuracy and reproducibility. Second, aptamers are highly compatible with a variety of signal amplification strategies for highly sensitive bioassay. With these desirable characteristics, aptamer-based fluorescent probes have been developed to detect various targets, including metal ions, small molecules, proteins, cells and so on. However, there are many problems in the area of aptamer fluorescent probes, such as sensitive bioanalysis in complex biological systems, design of fluorescent switchs compatible with biological systems, and fluorescent imaging of cells via an intelligent manner.Dynamic DNA nanotechnology has facilitated the development various DNA devices, which can be engineered to serve as programmable molecular machines, chemical amplifiers and logic gates. Instead of purely classical hybridization mechanisms, these dynamic DNA devices harness a process called toehold mediated DNA strands displacement. In this process, two partial or full complementary DNA strands hybridize to each other by initiating at toeholds domains and then displace another pre-hybridized DNA strands. As a result, such adaptive and reconfigurable DNA molecular devices can be developed as a novel signal transduction format for the design of aptamer-based fluorescent probes in an enzyme-free, isothermal manner. More importantly, this toehold mediated DNA strands displacement can be adapted to engineer several powerful amplification cascades with polynomial or exponential signals amplification ability. Without using protein enzymes, such signal amplification schemes show great potential to develop low-cost and point-of-care diagnostics. Specially, the hybridization chain reaction (HCR) and catalyzed hairpin assembly have been frequently employed in many aptamer-based probes for sensitive detection of numerous targets. Take advantage of the toehold mediated DNA strands displacement mentioned above, several novel aptamer-based fluorescent probes have been designed in this doctoral dissertation to obtain unique signal transduction formats and high sensitivity. In addition, gold nanoparticles and MoS2nanosheets have aslo been imployed in the design of probes for bioimaging or bioanalysis. The texts are summarized in details as follows:(1) Aptamer-based fluorescence anisotropy (FA) assays have attracted great interest in recent years. However, a key factor that determines FA value is molar mass, thus limiting the utility of this assay for the detection of small molecules. To solve this problem, streptavidin, as a molar mass amplifier, was used in a hybridization chain reaction (HCR) to construct a target-triggered cyclic assembly of DNA-protein hybrid nanowires for highly sensitive detection of small molecules by fluorescence anisotropy. In this assay, one blocking DNA strand could be released by target-aptamer recognition, and then it served as an initiator to trigger enzyme-free autonomous cross-opening of hairpin probes via HCR to form a DNA nanowire for further assembly of streptavidin. Using adenosine triphosphate (ATP) as the model small molecule, this novel dual-amplified, aptamer-based FA assay afforded high sensitivity with a detection limit of100nM, which is much lower than that of the disassembly approach without HCR amplification or the assembly strategy without streptavidin. In contrast to the previous turn-off disassembly approaches based on nonspecific interactions between the aptamer probe and amplification moieties, the proposed aptamer-based FA assay method exhibits a turn-on response to ATP, which can increase sensing reliability and reduce the risk of false hits. Moreover, because of its resistance to environmental interferences, this FA assay has been successfully applied for direct detection of0.5μM ATP in complex biological samples, including cell media, human urine, and human serum, demonstrating its practicality in real complex biological systems.(in chapter2)(2) DNA strand displacement cascades have been engineered to construct various fascinating DNA circuits. However, their further biological applications are limited by insufficient cellular internalization of naked DNA structures, as well as the separated multicomponent feature. In this work, these problems are addressed through the development of a novel DNA nanodevice, termed intelligent layered nanoflare, which integrates DNA computing at the nanoscale via self-assembly of DNA flares on a single gold nanoparticle. As a "lab-on-a-nanoparticle", the intelligent layered nanoflare could be engineered to perform a variety of Boolean logic gate operations, including three basic logic gates, one three-input AND gate, and two complex logic operations, in a digital, nonleaky way. In addition, the layered nanoflare can serve as a programmable strategy to sequentially tune the size of nanoparticles, as well as a new fingerprint spectrum technique for intelligent multiplex biosensing. More importantly, the nanoflare developed here can also act as a single entity for intracellular DNA logic gate delivery without the need for commercial transfection agents or other auxiliary carriers. By incorporating DNA circuits on nanoparticles, the presented layered nanoflare will broaden the applications of DNA circuits in biological systems and facilitate the development of DNA nanotechnology.(in chapter3)(3) Owning a series of advantages over antibodies, aptamers provide the basis for the design of excellent imaging probes with high specificity under complex biologic environments. However, most of the aptamers-based fluorescent probes were in an uncontrolled way, in which the fluorescence signal couldn’t be altered after the recognition of aptamer to its target. This uncontrolled characteristic is unfavorable for multiplex imaging, as well as temporal and spatial imaging with specific need, such as the research of probes’internalization. To overcome the limitations of previous aptamer probes, we adapted a new class of dynamic DNA complexes as programmable aptamer probes for imaging cell-surface marker. It allowed AND-logic controlled imaging and reiterative fluorescence labeling. Both confocal imaging and flow cytometry have been employed to confirm the response and features of each imaging probes. The two-label approach in these probes offers the opportunity to study a single binding event by different fluorescence signals, and provides a flexibility choice of fluorophores to overcome the shortcomings and utilize the advantages of individual ones. Specifically, one of the fluorescence labels could be used as an internal control for another one, thus improving imaging by accounting for cell-to-cell variations in probe binding amount and background,(in chapter4)(4) Similar to graphene’s morphology and properties, MoS2has received much attention from researchers, and it may find great potential applications in many areas including biomedicine. However, to our best knowledge, the possibility of using aptamer as targeting moiety for MoS2-based drug delivery platforms has not been demonstrated until now. In this study, a new MoS2-based nanosystem was developed to facilitate the application of MoS2in biomedical applications. In this nanosystem, the chemical exfoliated MoS2nanosheets were used as a drug carrier, while the aptamer served as a targeting moiety for cancer cells. Due to the defect sites in the surface of MoS2nanosheets, the Aptamer Sgc8, targeting leukemia T cells, could be easily conjugated via a thiol-based covalent bond and was also labeled with FITC as fluorescent signal for cell imaging. Form the results of flow cytometry and confocal imaging, the Aptamer Sgc8could greatly and specifically bind to its target cell due to a multivalent effect. In addition, the exceptional surface-area-to-mass ratio of MoS2nanosheets makes it a promising class of nano-carriers for molecule drugs, doxorubicin (DOX) and chlorin e6(Ce6), with high loading capacities. Further results from in vitro cytotoxicity experiments showed that MoS2nanosheets could be used for deliver therapeutic molecules into cell to effectively kill target cells, thus demonstrating the potential application of MoS2nanosheets in cancer therapeutics.(in chapter5)(5) Gold nanoparticles-based colorimetric assay possesses several unique advantages, and has been applied for a wide range of targets, varying from nucleic acids to different metal ions. However, due to the lack of proper coordinating ligand, gold nanoparticles-based colorimetric sensing system for Au3+has not been developed so far. It is well-known that Au3+could induce the oxidation transition of thiol compounds to disulfide compounds. In this article, we for the first time converted such thiol masking reaction into colorimetric sensing system for label-free detection of Au3+via a target-controlled aggregation of nanoparticles strategy. In the new proposed sensing system, fluorosurfactant-capped gold nanoparticles were chosen as signal reporter units, while an Au3+-triggered oxidation of cysteine (Cys), which inhibited the aggregation of gold nanoparticles, acted as the recognition unit. By varying the amount of Cys, a tunable response range accompanied with different windows of color change could be obtained for Au3+, illustrating the universality of the sensing system for Au3+samples with different sensitivity requirements. Under optimized condition, the proposed sensing system exhibits a high sensitivity towards Au3+with a detection limit of50nM, which is lower than previously reported spectroscopic methods. It has also been applied for detection of Au3+in practical water samples with satisfactory result,(in chapter6)... |
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