| Cancer is one of the most serious threats to human beings. In recent years, the incidence of cancer is increasing. Rapid and efficient detection of cancer cells at their earliest stages is one of the central challenges in cancer diagnostics. Nanotechnology, born in the late 1980s, has caused a revolution in many areas including chemistry, biology, medicine and other fields. Because nanomaterials have some unique properties, such as large specific surface area, a lot of reactive sites, high catalytic efficiency, strong adsorption capacity, and good stability, many new nanomaterial-based technologies and methods were developed for the research of biomolecular interaction, human disease, early diagnosis and treatment of cancer. Among them, the detecttion of cancer cells by biosensors based on nanomaterials is one of the current hotspots for the researchers.For example, gold nanoparticles are biocompatible, which make them suitable for covalent binding with biorecognization molecules, such as the oligonucleotides, antibodies, and proteins. Functionalized gold nanoparticles have not only better dispersion and stability but also the cability to recignize targets. In addition, their surface plasmon resonance absorption in the UV-visible area will change with the shape of the gold nanoparticles, the distance between the particles, the solution dielectric constant, the temperature, and other factors. And the change will leads to different color of gold nanoparticles solution. At present, gold nanoparticle-based colorimetric method has been widely used to detect inorganic metal ions, DNA, enzymes, proteins,tumor cell and so on. But the limited sensitivity and selectivity affects their use in early cancer diagnosis.Graphene oxide(GO) is a two-dimensional honeycomb crystal packed by flat mono layer of carbon atoms densely and each carbon atom is sp2 hybridized. There are a large number of oxygen functional groups on the surface of GO, which causes that GO can disperse preferably in aqueous solution. Also the biocompatibility of GO is very good, and GO is available to be a biosensor element. Meanwhile due to its unique electronic properties, GO is an efficient and universal fluorescence quenching material. Currently, the GO-based fluorescence resonance energy transfer (FRET) biosensors have been widely applied to detect inorganic metal ions, DNA, enzymes, proteins, cancer cells and so on. However, exploration of GO for living cells analysis and in situ monitoring still remains at a very early stage.Based on this, in the thesis we designed a simple, sensitive, selective, and cost-effective colorimetric aptasensor for the detection of cancer cells based on cell-triggered cyclic enzymatic signal amplification, and a GO-based FRET biosensor to detect Cyclin A2 in living cancer cells. Cyclin A2 is a prognostic indicator in early-stage cancers and has been reported overexpressed in many types of cancers including breast cancer, liver cancer, lung cancer, soft tissue sarcoma, leukemia, and lymphoma. The main contents are as follows: Chapter One:IntroductionFirstly, the research background and traditional diagnostic methods of cancer were introduced. Secondly, we described the detection of cancer cells by biosensors based on nanomaterials systematically, and focused on the preparation, the biological function, the properties, and the application of gold nanoparticles and GO. Finally, significance and purpose of this research were demonstrated. Chapter Two:Visual and highly sensitive detection of cancer cells by a colorimetric aptasensor based on cell-triggered cyclic enzymatic signal amplificationWe developed a simple, cost-effective, and highly sensitive colorimetric method for visually detecting rare cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and linker DNAs stably coexist in solution, and the linker DNA assemble DNA-AuNPs, producing a purple solution. In the presence of target cells, the specific binding of HAPs to the target cells triggers a conformational switch that results in linker DNA hybridization and cleavage by nicking endonuclease-strand scission cycles. Consequently, the cleaved fragments of linker DNA can no longer assemble into DNA-AuNPs, resulting in a red color. UV-vis spectrometry and photograph analyses demonstrated that this CTCESA-based method exhibited selective and sensitive colorimetric responses to the presence of target CCRF-CEM cells, which could be detected by the naked eye. The linear response for CCRF-CEM cells in a concentration range from 100 to 10000 cells was obtained with a detection limit of 40 cells, which is approximately 20 times lower than the detection limit of normal AuNP-based methods without amplification. Given the high specificity and sensitivity of CTCESA, this colorimetric method provides a sensitive label-free and cost-effective approach for early cancer diagnosis and point-to-care applications. Chapter Three:Visual and sensitive detection of Cyclin A2 in living cells using a graphene oxide-based FRET biosensorIn this chapter, we achieved sensitive and selective detection of Cyclin A2 in living cancer cells using a graphene oxide-based FRET biosensor. A peptide probe (p1) which could recognize Cyclin A2 specifically was modified by FAM. FAM-p1 can interact to nano-GO by π-π and electrostatic reaction to form a stable p1 peptide-nano GO probe. Since the fluorescence resonance energy transfer occurs between FAM-p1 and nano-GO, p1 peptide-nano GO probe has weak fluorescence background signal. In homogeneous solution, once added the target protein, p1 could bind with it specifically and be released from nano-GO, and the fluorescence was recovered. The quantitative results of target protein were obtained through measuring the changes of fluorescence. The linear range of the assay for Cyclin A2 is 1-6 nM and the detection limit is 0.16 nM,3750-fold better than the reported value of 0.6 μM using the Tb3+ chelating macrocycle modified p21(WAF-1) peptide. Most importantly, when p1 peptide-nano GO probe was incubated with the cancer cells, it could enter the cells and the specific binding between target protein and peptide can make p1 away from the surface of nano-GO, then fluorescence recovery also occurs. Using confocal laser technology, the visual detection of Cyclin A2 in living cells can be achieved. |
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