| Hematologic maligancy is one of the most critical threats to the health of human beings. Dveloping an efficient method for cancer diagnosis in an early stage can greatly improve the cure rate of cancer patients. Therefore it has become an important topic in molecular biology and biomedical research. Biosensor is playing an important role in disease diagnosis, detection of food pollution, forensic analysis and environmental monitoring due to its high-sensitivity and high-selectivity. The trend for analytical chemistry is to develop a miniaturized, smart, and integrated analytical devices. Recently, the rapid development of biotechonolgy, materials and micro-machining has dramatically boosted the application of biosensors. For example, aptamers, selected by SELEX, remain not only the high-specificity and high-affinity as traditional antibodies, but also have unique characters, such as easy to synthetize, good stability, various kinds of targets, non-toxicity and easy modification. Taking aptamers as a novel bio-recognition molecule has opened new pathways for wide application of biosensors. On the other hand, nanomaterials have excellent chemical and physical properties, such as large surface area, sufficient surface reaction sites, high catalytic efficiency, high adsorption ability and excellent stability, which offer a new approach in bioassay and promote the rapid development of chemical and biological sensors. Moreover, microfluidic chips play an important role in bioanalysis owing to its small volume, short analytical time, high sensitivity and easy coupling with other technologies.In this dissertation, in order to tackle the problems in the diagnosis of the early-stage cancer by employing optical biosensors, such as the low concentration of tumor markers and membrane proteins that were excessively secreted by cancer cells, high background signal, instability and low specificity, we tried to construct a novel fluorescent biosensing microfluidic chips platform with a low background, high stability and specificity by combining with the advantages of nanomaterials, aptamers and microfluidic chips. The principle of the ’signal-on’ fluorescence aptasensor is based on Fluorescence Resonance Energy Transfer (FRET) between a fluorophore-labeled aptamers (as a donor) and graphene Oxide (GO) nanomaterials (as an acceptor and ananoquencher). The main contents are shown as follows: Chapter One:IntroductionIn the beginning of this dissertation, research background and traditional diagnostic methods of hematologic malignant cells were firstly introduced. Secondly, the basic composition, classification and research advance of aptasensors were systematically reviewd. Among these parts, we emphasized on the application of FRET aptasenors based on nanomaterials. Thirdly, we reviewed the applications of microfluidic chips in the field of bioanalysis, especially the combination of microfluidic chips with FRET. At last, the purpose and the significance of the dissertation were demonstrated.Chapter Two:A Novel Graphene Oxide-based Biosensor for Rapid and Sensitive Fluorescence Detection of Mixed Nucleic AcidsIn this chapter, we develop a rapid, sensitive, selective and multi-color fluorescent analysis of DNA in homogenous solution by using graphene oxide (GO) due to its large nanosheet surface and excellent quencher ability. First, the single-layered and well-dispersed GO was prepared by Hummers method and then with sonication. Then, the biocompatibility of GO was tested by CCK-8experiment. Here, GO served as a new nanoprobe, and a "postmixing" strategy was employed for the fluorescent detection of multiplex DNA. And the procedure of the detection is that targets firstly mixed and reacted with aptamers, and then GO was added to the mixture, and finally the quantitative results of targets were obtained through measuring the changes of fluorescence. The linear range of the assay for DNA1and DNA2is15-100nM and30-150nM, respecitively, and the detection limited is10nM and25nM, respecitively. Because GO had high quenching efficience, and specific recognition for ssDNA, the GO biosensor by using a "postmixing" strategy could quickly quench fluorescent dye-labeled DNA in2min, which is faster than those by using the "premixing" strategy. And, the specific hybridization of DNA assured the specific detection of targets in a mixed solution. Therefore, this GO biosensor by using a "Post-mixing" strategy could meet the rapid and highly-specific demand of detecting for multiplex targets in a complex sample.Chapter Three:A Graphene Oxide-based FRET Aptasensing Microfliudic Platform for Visual and High-throughput Detection of Cancer CellsIn this chapter, a miniature multiplex aptasensing platform based on FRET was designed to detect cancer cells by combing the high-specificity of aptamers, the high quenching efficiency of GO, and small volume of a multichannel microfluidic chip. Firstly, the single-layered and well-solubility GO was prepared by Hummers method and then with sonication. Secondly, the FAM-modified aptamer (Sgc8c) was chosen for its specific binding with CCRF-CEM cells, and the GO/FAM-Sgc8c complex was formed through π-π stacking and its fluorescence signal dramatically decreased due to FRET. Once added the target cells, Sgc8c could bind specifically with target cell and form a stable hairpin structure, which resulted the release of Sgc8c from GO, and the fluorescence was recovered. Based on the above biosensing principle, we designed a33-channel PDMS-microfluidic chip for visual and high-throughput detection of cancer cells. Fluorescence intensity measurement and image analysis demonstrated the linear response for target CCRF-CEM cells was2.5×101to2.5×104cells/mL, with a detection limit about25cells/mL, which is ten times lower than those of normal biosensors. The novel fluorescence biosensing microfluidic platform fulfill a rapid, visible and high-throughput detection of cancer cells, which will be of great importance in developing new methods for early cancer diagnosis.
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