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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 牟中原(Chung-Yuan Mou) | |
dc.contributor.author | Hsin-Yi Chen | en |
dc.contributor.author | 陳欣宜 | zh_TW |
dc.date.accessioned | 2021-06-16T05:41:47Z | - |
dc.date.available | 2019-09-04 | |
dc.date.copyright | 2014-09-04 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-11 | |
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L., A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: Evaluation in human polymorphonuclear leukocytes. Free Radic. Biol. Med. 2001, 31 (7), 894-901. 39. Dickinson, B. C.; Huynh, C.; Chang, C. J., A Palette of Fluorescent Probes with Varying Emission Colors for Imaging Hydrogen Peroxide Signaling in Living Cells. J. Am. Chem. Soc. 2010, 132 (16), 5906-5915. 40. Luo, Y. P.; Liu, H. Q.; Rui, Q.; Tian, Y., Detection of Extracellular H2O2 Released from Human Liver Cancer Cells Based on TiO2 Nanoneedles with Enhanced Electron Transfer of Cytochrome c. Anal. Chem. 2009, 81 (8), 3035-3041. 41. Belousov, V. V.; Fradkov, A. F.; Lukyanov, K. A.; Staroverov, D. B.; Shakhbazov, K. S.; Terskikh, A. V.; Lukyanov, S., Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 2006, 3 (4), 281-286. 42. Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari, M.; Sun, C.; Petros, J.; Taylor, W. R.; Murthy, N., In vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles. Nat. Mater. 2007, 6 (10), 765-769. 43. Jv, Y.; Li, B. X.; Cao, R., Positively-charged gold nanoparticles as peroxidiase mimic and their application in hydrogen peroxide and glucose detection. Chem. Commun. 2010, 46 (42), 8017-8019. 44. Oh, W. K.; Jeong, Y. S.; Kim, S.; Jang, J., Fluorescent Polymer Nanoparticle for Selective Sensing of Intracellular Hydrogen Peroxide. ACS Nano 2012, 6 (10), 8516-8524. 45. Crow, J. P., Dichlorodihydrofluorescein and Dihydrorhodamine 123 Are Sensitive Indicators of Peroxynitritein Vitro:Implications for Intracellular Measurement of Reactive Nitrogen and Oxygen Species. Nitric Oxide 1997, 1 (2), 145-157. 46. Pryor, W. A., Oxy Radicals And Related Species Their Formation Lifetimes And Reactions. In Berne, R. M., 1986, 657-668. 47. Miller, A. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56680 | - |
dc.description.abstract | 活性氧物質(ROS) 在細胞生理反應中扮演著細胞內訊息傳遞者的角色。在正常情況下,細胞內有足夠的抗氧化機制與活性氧物質達成平衡,但當細胞受到特定刺激時,細胞內過量的活性氧物質造成細胞產生氧化壓力,導致細胞內蛋白質、脂質、遺傳物質不可逆的破壞,造成細胞凋亡,甚至老化、糖尿病、癌症等疾病的發生。因此,若能夠在早期即有效的監控細胞內活性氧物質的水平,能夠及早預防疾病的產生。
可穿透細胞的二氧化矽空心球酵素載體搭配活性氧物質螢光試劑的組合非常適合應用在細胞內氧化壓力的偵測。在這份研究中,我們將辣根過氧化酶(HRP)以溫和的製程包覆在奈米空心球內部,稱之為HRP@HSN。此酵素載體具有好的催化活性、高穩定性、細胞膜穿透性、生物相容性、能夠保護酵素避免受到蛋白酶的分解,並且具有核內體 (endosome) 脫離的機制。當HRP@HSN 被細胞吞噬後,這些酵素載體內的HRP 能夠與活性氧物質反應,進一步與能通透細胞膜的DHR123 試劑作用,在波長530 nm 偵測其螢光值。在我們的系統中,我們以濃度為每毫升 100 微克的酵素載體在1 小時內即可進行細胞內活性氧物質的偵測。並且,利用此具有高靈敏度的系統,我們能夠在細胞發炎反應發生時進行偵測以及定量細胞所產生的活性氧物質。 | zh_TW |
dc.description.abstract | Reactive oxygen species (ROS) which served as intracellular messengers are associated with cellular functions through affecting cellular signaling. Under certain stimulations, ROS would be overproduced and further caused oxidative stress resulting in the cell irreversible damage or death in many diseases. Therefore, it is useful to monitor the cellular level of ROS for prevention of cell damage at the early stage as well as indication of the staging of disease. ROS sensitive chromosphere agent combining with transmembrane nanoparticles was considered to apply for intracellular reporter signal.
In this study, we developed a mild synthesis protocol to load horseradish peroxidase (HRP) into the interior cavity of hollow silica nanospheres (HSN) to form the HRP encapsulated HSNs, named HRP@HSN. This particle shares several advantages such as effectual catalytic activity, high stability, membrane permeability, cell biocompatibility, the ability to escape from endosome, and enzyme protection from protease. When HRP@HSNs were introduced to HeLa cells and RAW264.7 macrophage cell line, the particles were able to convert ROS into hydroxyl radical which reacting with dihydrorhodamine 123, a cell-permeable dye. In our system, we could detect the cellular ROS within 1 hour at a dose limitation as low as 100 μg/mL, and illuminate at emission wavelength 530 nm. We exploited this system with high sensitivity and also demonstrated the cellular example at ROS-associated inflammatory status. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T05:41:47Z (GMT). No. of bitstreams: 1 ntu-103-R01223208-1.pdf: 2946175 bytes, checksum: b0a86a4a25a2e30851f0b9269f1fb64d (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 謝誌 ................................................................................................................................... I
中文摘要 ..........................................................................................................................II Abstract ......................................................................................................................... III Table of Contents .......................................................................................................... IV List of Figures .............................................................................................................. VII List of Tables .............................................................................................................. XIV Chapter 1 Introduction .............................................................................................. 1 1.1 Background of Hollow Silica Nanostructures (HSNs)............................... 1 1.1.1 Hard Template ..................................................................................... 2 1.1.2 Soft Template ....................................................................................... 3 1.1.3 Self-Template/Template free Method ................................................ 6 1.1.4 Literature review in application of silica nanoparticles in enzyme delivery ................................................................................................. 9 1.2 Reactive Oxygen Species (ROS) ................................................................ 15 1.2.1 Superoxide Anion (O2•-) .................................................................... 16 1.2.2 Hydroxyl Radical (•OH) .................................................................... 16 1.2.3 Peroxynitrite (ONOO-) ..................................................................... 17 1.2.4 Hydrogen peroxide (H2O2) ............................................................... 17 1.3 Literature review of nanoparticles applications on ROS detection ....... 19 Chapter 2 Materials and Methods .......................................................................... 23 2.1 Materials ...................................................................................................... 23 2.2 Synthesis of hollow silica nanospheres (HSNs) ........................................ 23 IV 2.3 Encapsulation of horseradish peroxidase in the hollow silica nanospheres (HRP@HSNs) ....................................................................... 24 2.4 Synthesis of FITC-HSNs and HRP@FITC-HSNs ................................... 24 2.5 Loading efficiency of horseradish peroxidase in HSNs ........................... 25 2.5.1 Conjugate Rhodamine B isothiocyanate (RITC) with HRP ......... 25 2.5.2 Synthesis of RITC-HRP@HSNs ...................................................... 25 2.5.3 Examine loading efficiency of HRP in HSNs .................................. 25 2.6 Activity assay of HRP@HSNs ................................................................... 25 2.7 Reactivities of HRP@HSN with various ROS ......................................... 26 2.8 Cell culture .................................................................................................. 26 2.8.1 HeLa cell ............................................................................................ 26 2.8.2 RAW264.7.......................................................................................... 27 2.9 Cell viability and proliferation .................................................................. 27 2.10 Cell uptake analysis .................................................................................... 28 2.11 Reactive oxygen species (ROS) detection ................................................. 28 2.11.1 Fluorescence images and data analysis ........................................... 28 2.12 Semi-quantitative analysis ......................................................................... 29 2.13 Material Characterization and Instrument.............................................. 29 2.13.1 Transmission Electron Microscopy (TEM) ..................................... 29 2.13.2 Dynamic Light Scattering and Zeta Potential ................................ 30 2.13.3 Fluorescence spectrophotometer...................................................... 30 Chapter 3 Result and Discussion ............................................................................ 31 3.1 Synthesis and Characterization of Enzyme Encapsulated Silica Nanospheres ................................................................................................ 31 3.1.1 The physical properties of HRP@HSNs ......................................... 31 V 3.1.2 Loading efficiency of HRP in HRP@HSN ...................................... 35 3.1.3 Catalytic activity of HRP in HRP@HSN ........................................ 37 3.1.4 Reactivities of HRP@HSN with various ROS ................................ 38 3.2 Biological Application of HRP@HSN ....................................................... 41 3.2.1 Cytotoxicity of HRP@HSNs and HSNs .......................................... 42 3.2.2 Cell uptake of HRP@HSN and HSN ............................................... 44 3.3 Intracellular ROS detection with HRP@HSN ......................................... 47 3.3.1 Reactive oxygen species detection for HeLa cell ............................ 47 3.3.2 Fluorescence intensity showed dose-dependent for HeLa cell ...... 52 3.3.3 Reactive oxygen species detection inRAW264.7 macrophages...... 54 3.3.4 Fluorescence intensity showed dose-dependent for RAW264.7 macrophages ...................................................................................... 59 3.3.5 Particle dosage effect for RAW264.7 ............................................... 60 3.3.6 Semi-quantitative analysis ................................................................ 61 Chapter 4 Conclusion .............................................................................................. 63 Reference ........................................................................................................................ 64 | |
dc.language.iso | en | |
dc.title | 利用二氧化矽奈米酵素載體進行細胞內活性氧物質偵測 | zh_TW |
dc.title | Intracellular imaging of reactive oxygen species with enzyme encapsulated hollow silica nanospheres | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳建志(Chien-Tsu Chen),簡汎清(Fan-Ching Chien) | |
dc.subject.keyword | 二氧化矽空心球,活性氧物質,蛋白質輸送,辣根過氧化?, | zh_TW |
dc.subject.keyword | Hollow silica nanoparticle,reactive oxygen species,protein delivery,horseradish peroxidase, | en |
dc.relation.page | 69 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-12 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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