Novel development of cell-based bioassays for biomedical applications.

• Other Authors: Cheung, Ka Lun.
• Format: Others
• Language: English; Chinese
• Published: 2012
• Subjects: Biological assay > Technique; Biological Assay > methods
• Online Access: http://library.cuhk.edu.hk/record=b5549158; http://repository.lib.cuhk.edu.hk/en/item/cuhk-328697

細胞為本生物檢測法（CBB）是指任何一種以細胞系統及其身上發生的生物反應為基礎原之直接檢測方法。在這篇文中，有關CBB 的工作大致分為個主要章節，描述如下： === 第一章節探討以可調控式納米孔為基礎的電阻脈衝感應檢測法（RPS）進人血紅細胞的毒學研究。我們成功檢測到血紅細胞在低滲環境下的體型大小變化，並以式細胞儀及激光共焦掃描顯微鏡驗證上述實驗結果。此外，我們亦使用重皂甙（PD）及四溴雙酚A（TBBPA）種化合物以進毒試驗。在此章節，我們展示以RPS 在人體細胞系統的首次應用。這種RPS 無疑成為一種創新及低成本的技術，有助快速研究與血液有關的疾病。 === 第二章節探討以細胞為本的納米毒學研究。由於納米子已被廣泛應用於生物醫學上，它的應用同時帶出有關其毒性及其帶出的健康問題。在這章節，我們主要研究種同塗層的納米條（Au-NRs），其塗層分別為十烷基三甲基溴化氨（CTAB）及聚乙二醇（PEG）。透過一在人嗜鹼性細胞KU812 的毒實驗，我們展示Au-NRs 的生物相容性取決於它的塗層。CTAB 塗層的Au-NRs 會引發KU812 細胞死亡及其過敏性反應，而PEG 塗層的Au-NRs 則會引發以上反應。 === Cell-based Bioassay (CBB) refers to any kind of assay which detection principle is based on direct monitoring of biological events in living cell systems.The work in this thesis is divided to two main chapters described as follows. === The first focus was the characterization of human erythrocytes toxicology using tunable nanopore-based resistive pulse sensing (RPS). We successfully monitored the size changes of the erythrocytes in hypotonic environment. Results were confirmed with flow cytometry and confocal laser scanning microscopy (CLSM). Furthermore, drug assays were performed, in which the erythrocytes were treated with polyphyllin D (PD) or tetrabromobisphenol A (TBBPA). Here we reported the first application of the tunable nanopore-based RPS on human live cell system. This RPS system served as a novel and low-cost technique for rapid analysis of bloodrelated diseases at point-of-care. === Another focus was cell-based bioassays in nanotoxicology. As nanoparticles have been widely applied in biomedical and biological aspects, health issues have been raised and the toxicity of nanoparticles is much concerned. In this study, we investigated the cytotoxicity of CTAB- and PEG-coated gold nanorods (Au-NRs). Through a series of toxicological studies and using human basophils cell line KU812, we demonstrated the biocompatibility of coating of Au-NRs was critical to the cytotoxicity to cells. CTAB-coated Au-NRs, rather than PEG-coated Au-NRs, were able to induce cell death and allergic response in KU812 cells. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Cheung, Ka Lun. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. === Includes bibliographical references. === Abstracts also in Chinese. === Members of Examination Committee --- p.i === Declaration --- p.ii === Publications --- p.iii === Abstract --- p.iv === Acknowledgements --- p.viii === List of Figures --- p.xi === Table of Content --- p.xii === General Introduction --- p.1 === Chapter Chapter 1 --- Cell-based Bioassays using Resistive Pulse Sensing --- p.13 === Chapter 1.1 --- Introduction --- p.14 === Chapter 1.2 --- Materials and Methods --- p.26 === Chapter 1.2.1 --- Preparation of human erythrocytes --- p.26 === Chapter 1.2.2 --- Resistive pulse sensing (RPS) --- p.26 === Chapter 1.2.3 --- Hemolysis assay --- p.27 === Chapter 1.2.4 --- Osmolarity assay --- p.28 === Chapter 1.2.5 --- Confocal laser scanning microscopy --- p.28 === Chapter 1.2.6 --- Flow cytometry --- p.29 === Chapter 1.2.7 --- Polyphyllin D (PD) assay --- p.29 === Chapter 1.2.8 --- Tetrabromobisphenol A (TBBPA) assay --- p.30 === Chapter 1.2.9 --- Statistical analysis --- p.30 === Chapter 1.3 --- Results --- p.31 === Chapter 1.3.1 --- Measurement of human erythrocytes using RPS --- p.31 === Chapter 1.3.2 --- RPS measurement of human erythrocytes in hypotonic enviornment --- p.36 === Chapter 1.3.3 --- RPS measurement of PD-treated human erythrocytes --- p.48 === Chapter 1.3.4 --- RPS measurement of TBBPA-treated human erythrocytes --- p.55 === Chapter 1.4 --- Discussions and Conclusion --- p.64 === Chapter 1.5 --- References --- p.68 === Chapter Chapter 2 --- Cell-based Bioassays on Human Basophils --- p.71 === Chapter 2.1 --- Introduction --- p.72 === Chapter 2.2 --- Materials and Methods --- p.74 === Chapter 2.2.1 --- Reagents --- p.74 === Chapter 2.2.2 --- Preparation of the CTAB-coated and PEG-coated nanorods --- p.74 === Chapter 2.2.3 --- Cell culture and preparation --- p.75 === Chapter 2.2.4 --- Alamar blue assay --- p.76 === Chapter 2.2.5 --- Calcein leakage assay and propidium iodide staining assays --- p.77 === Chapter 2.2.6 --- Histamine release assay --- p.78 === Chapter 2.2.7 --- Beta-hexosaminidase release assay --- p.79 === Chapter 2.2.8 --- Statistical analysis --- p.80 === Chapter 2.3 --- Results --- p.81 === Chapter 2.3.1 --- Physical and optical properties of the gold nanorods --- p.81 === Chapter 2.3.2 --- CTAB-, but not PEG-coated Au-NRs elicits allergic degranulation --- p.83 === Chapter 2.3.3 --- CTAB-coated Au-NRs are more cytotoxic than PEGcoated Au-NRs --- p.88 === Chapter 2.3.4 --- CTAB-, but not PEG-coated, Au-NRs cause plasma membrane permeabilization --- p.96 === Chapter 2.3.5 --- CTAB-, but not PEG-coated, Au-NRs disrupt plasma membrane asymmetry --- p.108 === Chapter 2.4 --- Discussions and Conclusion --- p.113 === Chapter 2.5 --- References --- p.116 === General Discussions and Conclusion --- p.118 === References --- p.124