紫膜生物光電晶片之類風溼性關節炎檢測應用與原子力顯微鏡分析

簡易檢索 / 詳目顯示

回結果列表

研究生：	王詠毅 Yung-Yi Wang
論文名稱：	紫膜生物光電晶片之類風溼性關節炎檢測應用與原子力顯微鏡分析 Rheumatoid arthritis detection application of purple membrane-based photoelectric chips and AFM analysis
指導教授：	陳秀美 Hsiu-Mei Chen
口試委員:	林景堉蔡伸隆
學位類別：	碩士 Master
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	106
中文關鍵詞：	紫膜、生物晶片、類風溼性關節炎檢測、原子力顯微鏡
外文關鍵詞：	purple membrane, photoelectric chips, Rheumatoid arthritis detection, atomic force microscopy
相關次數：	點閱：386 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

細菌視紫質 (bacteriorhodopsin)是一種存在於紫膜 (purple membrane, PM) 中的特殊光敏感蛋白，具有光質子驅動幫浦功能，受到光照後會使質子從膜內傳送到膜外，進而啟動ATP合成，可用以產生光電流；本實驗室過去已利用此特性製作PM光電感測晶片。本研究主要分為兩個部分，首先，就先前使用異型與同型雙官能基兩種不同架橋所分別製作的aptamer-PM與protein A-PM複合晶片，利用原子力顯微鏡分析其表面形貌，結果發現在PM晶片上以前者架橋所塗覆的aptamer覆蓋率比以後者架橋所塗覆的protein A高。其次，利用PM晶片檢測類風溼性關節炎。以具有特定序列之胜肽當作辨識分子以同型雙官能基架橋固定化在PM晶片上後可直接捕捉血清中的抗體，再以標記有奈米金之抗人體IgA抗體來偵測被捕捉的IgA。具有遮光性的奈米金可增加PM檢測晶片的光電流下降程度，即增加靈敏度。研究中探討胜肽固定化濃度、血清稀釋倍率、以及抗IgA抗體與奈米金複合物之稀釋倍率對晶片檢測成效的影響，以決定晶片製程與檢測的最適化條件。最後分別利用20位常人以及20位類風濕性關節炎患者之血清，以所製備的胜肽-PM晶片進行檢測，並就晶片的光電流下降比例做比較，結果發現以二組不同檢體檢測的晶片光電流下降平均值具有明顯差異 (p =1.4×10-12)，可訂出分界線，因此本研究證實PM晶片可被應用於類風溼性關節炎檢測。

Bacteriorhodopsin is a special light-sensitive protein in purple membranes (PM) with a light-driven proton pump function. Upon illumination, it transports a proton from the cytoplasmic to the extracellular side of PM and thus drives ATP synthesis. The generated proton gradient can power photocurrents, which has been used to develop PM-based photoelectric sensors in this laboratory. This thesis contains two parts. First, atomic force microscopy was used to examine the surface morphology of the aptamer-PM and protein A-PM composite chips whose preparations have been previously developed using heterobifunctional and homobifunctional crosslinkers, respectively, for conjugation on PM-coated chips. The results showed aptamers had a better coverage on PM chips than protein A did. Secondary, PM chips were applied to detect rheumatoid arthritis. A peptide with a specific sequence was first immobilized on PM chips via the homobifunctional crosslinker, and the resulting sensor chips were subsequently used to directly adsorb antibodies in serum, followed by the labeling of the captured IgA using anti-human IgA antibodies conjugated with nanogold particles (AuNPs). AuNPs were employed to enhance the detection sensitivity due to their light-shielding effect. The optimization of peptide immobilization concentrations, serum dilution rates, as well as the dilution rates of the antibodies-AuNPs complex was investigated to determine the best conditions for chip preparation and serum detection. Finally, the serum of 20 normal people and 20 rheumatoid-arthritis patients were analyzed with the as-prepared sensor chips and the reduction levels of the photocurrents generated by the chips were compared. A significant difference (p = 1.4×10-12) was observed between the averages of the photocurrent reduction levels obtained from the normal-control group and the patient group, enabling the determination of the cut-off value as well as demonstrating the application potential of the PM chips in rheumatoid-arthritis detection.

中文摘要             I
Abstract           II
致謝               III
目錄               IV
表目錄             VI
圖目錄             VII
第1章 緒論                                                                       11
第2章 文獻回顧                                                                    13
2-1 類風溼性關節炎 (Rheumatoid arthritis, RA)                                     13
2-1-1類風溼性關節炎之症狀及誘發因素                                                13
2-1-2 檢測類風溼性關節炎之項目                                                     14
2-1-2-1 類風濕因子 (RF)                                                          14
2-1-2-2 瓜氨酸化 (Citrullination)以及抗環瓜氨酸抗體 (Anti-CCP)                     15
2-1-3-3 C反應性蛋白及紅血球沉降速率                                                17
2-1-3 免疫檢測原理與方式                                                          17
2-1-3-1酵素結合免疫分析法                                                         18
2-1-3-2 放射免疫分析法                                                            19
2-1-3-3 免疫螢光分析法                                                            20
2-1-4美國風濕病學院（American Collegeof Rheumatology, ACR）所制定之分類標準。       22
2-2 細菌視紫質 (bacteriorhodopsin, BR)                                           23
2-2-1 Halobacterium salinarum與BR結構                                            23
2-2-2 BR的光循環與質子傳遞過程                                                    27
2-2-3 BR光電響應                                                                 29
2-2-4 PM之單層貼附及固定化                                                        32
2-2-5 PM晶片之應用                                                               34
2-3 原子力顯微鏡 ( atomic force microscopy, AFM)                                  36
2-3-1 AFM介紹                                                                    36
2-3-2 AFM之掃描種類                                                              38
2-3-2-1 接觸模式                                                                 38
2-3-2-2 非接觸模式                                                               38
2-3-2-3 輕敲模式                                                                 38
2-3-2-4 PeakForce QNM 模式                                                       39
2-3-3 PM在AFM下之形貌                                                            40
第3章 實驗                                                                       41
3-1 實驗目的                                                                     41
3-2 實驗流程                                                                     42
3-3 量測                                                                         48
3-3-1 微分光電流量測                                                              48
3-3-2 AFM分析與操作                                                              48
第4章 實驗結果                                                                    50
4-1 mica-PM晶片之AFM影像                                                         50
4-1-1 利用微流體沖洗改善PM堆疊情形                                                 51
4-1-2 利用NHS-PEG2-NHS作為架橋以protein A製程製作的mica晶片之層層AFM影像            53
4-1-3以Sulfo-SIAB當架橋以aptamer製程製作mica晶片之層層AFM影像                      57
4-1-4 兩種不同架橋在經過AFM掃描後之探討                                            58
4-2 自體免疫PM檢測晶片製作                                                        63
4-2-1 自體免疫PM檢測晶片初步製作                                                   63
4-2-2 在接附血清前加入glycine，試以glycine填補來消除各層之間的非特異性吸附現象        66
4-2-3 Native peptide與Citrullinated peptide在不同血清 (常人、病患)的情形對光電流密度與光電流比例所造成之影響。                                                           70
4-2-4 最適化citrullinated peptide濃度之探討                                       75
4-2-5 調整血清稀釋倍率對光電流比例之影響                                            81
4-2-6 抗體-奈米金之最佳稀釋倍率探討                                                86
4-2-7 以最適化條件製作自體免疫PM檢測晶片，並以常人、病患血清製作光電流比例分界線。     91
第5章 結論                                                                       99
第6章 參考文獻                                                                   101

                                

Balashov SP. Protonation reactions and their coupling in bacteriorhodopsin. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2000, 1460, 75-94.

Bedell SE and Bush BT. Erythrocyte sedimentation rate. The American Joumal of Medicine. 1985, 78, 1001-1009.

Binnig G, Quate CF, Gerber CH. Automatic Force Microscope. Physical Review Letter. 1986, 56.

Boster Biological Technology: Types of ELISA assay. 2017. https://www.bosterbio.com/newsletter-archive/20170728-which-elisa

Bruker: PeakForce Tapping & QNM. 2011. https://blog.brukerafmprobes.com/guide-to-spm-and-afm-modes/peakforce-tapping-onm/

Carlson CS, Aldred SF, Lee PK, Tracy RP, Schwartz SM, Rieder M, Liu K, Williams OD, Iribarren C, Lewis EC, Fornage M, Boerwinkle E, Gross M, Jaquish C, Nickerson DA, Myers RM, Siscovick DS, Reiner AP. Polymorphisms within the c-reactive protein (crp) promoter region are associated with plasma crp levels. American Journal of Human Genetics. 2005, 77, 64-77.

Chen HM, Lin CJ, Jheng KR, Kosasih A, Chang JY. Effect of graphene oxide on affinity-immobilization of purple membranes on solid supports. Colloids and Surfaces B: Biointerfaces. 2014, 116, 482-488.

Chen HM, Jheng KR, Yu AD. Direct, label-free, selective, and sensitive microbial detection using a bacteriorhodopsin-based photoelectric immunosensor. Biosensors and Bioelectronics. 2017, 91, 24-31.

Darwish I. A.Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances. International journal of Biomedical science. 2006

Greiner A , Plischke H , Kellner H , Gruber R. Association of anti-cyclic citrullinated peptide antibodies, anti-citrullin antibodies, and IgM and IgA rheumatoid factors with serological parameters of disease activity in rheumatoid arthritis. Annals New York Academy of Sciences . 2005, 1050, 295-303.

Hampp N. Bacteriorhodopsin as a Photochromic Retinal Protein for Optical Memories. Chemical Reviews. 2000,100, 1755-1776.

Jacobson DL, Gange SJ, Rose NR, Graham NMH. Epidemiology and Estimated Population Burden of Selected Autoimmune Diseases in the United States. Clinical Immunology and Immunopathology. 1997, 84, 223-243.

Jalili N & Laxminarayana K. A review of atomic force microscopy imaging systems: application to molecular metrology and biological sciences. Mechatronics. 2004, 14, 907-945

Jin Y, Honig T, Ron I, Friedman N, Sheves M, Cahen D. Bacteriorhodopsin as an electronic conduction medium for biomolecular electronics. Chemical Society Reviews. 2008, 37, 2422-2432.

Khorana HG. Vectorially oriented purple membrane: characterization by photocurrent measurement and polarized-Fourier transform infrared spectroscopy. 1988, 263, 7349-7442.

Kochi Y, Suzuki A, Yamada R, Yamamoto K. Ethnogenetic heterogeneity of rheumatoid arthritis-implications for pathogenesis. Nature Reviews Rheumatology. 2010, 6, 290-295.

Koivunen ME & Krogsrud RL. Principles of Immunochemical Techniques Used in Clinical Laboratories. Laboratory Medicine. 2006, 37, 490-497.

Kuo CF , Luo SF , See LC , Chou IJ , Chang HC , Yu KH. Rheumatoid arthritis prevalence, incidence, and mortality rates: a nationwide population study in Taiwan. Rheumatol Int. 2013, 33, 355-360.

Liao CC, Chou PL, Cheng CW, Chang YS, Chi WM, Tsai KL, Chen WJ, Kung TS, Tai CC, Lee KW, Chen YC, Lin CY. Comparative analysis of novel autoantibody isotypes against citrullinated-inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3)(542-556) peptide in serum from taiwanese females with rheumatoid arthritis, primary sjogren's syndrome and secondary sjogren's syndrome in rheumatoid arthritis. Journal of Proteomics. 2016, 141, 1-11.

Lu T, Li BF, Jiang L, Rotheb U, Bakowsky U. Temperature and pH-dependent inversion of photoelectric response in bacteriorhodopsin. Journal of the Chemical Society, Faraday Transactions. 1998, 94, 79-81.

Mayo Clinic: Rheumatoid arthritis overview. 2017. https://www.mayoclinic.org/diseases-conditions/rheumatoid-arthritis/symptoms-causes/syc-20353648

Pruijn Ger JM. Citrullination and carbamylation in the pathophysiology of rheumatoid arthritis. Frontiers in Immunology. 2015, 6.

Spudich JL , Yang CS , Jung KH , Spudich EN. Retinylidene Proteins: Structures and Functions from Archaea to Humans. Annual Review of Cell and Developmental Biology. 2000, 16, 365-392.

Trier N, Houen G. Epitope specificity of anti-citrullinated protein antibodies. Antibodies. 2017, 6.

Valesini G , Gerardi MC, Iannuccelli C, Pacucci VA .Citrullination and autoimmunity. Autoimmunity Reviews. 2015, 14, 490-497.

Van Venrooij WJ , Van Beers Joyce JBC, Pruijn Ger JM. Anti-CCP Antibody, a Marker for the Early Detection of Rheumatoid Arthritis. New York Academy of Sciences. 2008, 1143, 268-285.

Voïtchovsky K , Antoranz Contera S , Kamihira M , Watts A , Ryan JF. Differential stiffness and lipid mobility in the leaflets of purple membranes. Biophysical Journal. 2006, 90, 2075-2085.

Vossenaar ER & Van Venrooij WJ. Anti-CCP antibodies, a highly specific marker for (early) rheumatoid arthritis. Clinical and Applied Immunology Reviews. 2004, 4, 239-262.

Wang J. Vectorially oriented purple membrane: characterization by photocurrent measurement and polarized-fourier transform infrared spectroscopy. Thin Solid Films. 2000, 379,224-229.

Wang JP, Yoo SK, Song L, El-Sayed MA. Molecular mechanism of the differential photoelectric response of bacteriorhodopsin. The Journal of Physical Chemistry B. 1997, 101, 3420-3423.

Wickstrand C, Dods R, Royant A, Neutze R. Bacteriorhodopsin: Would the real structural intermediates please stand up? Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2015, 1850, 536-553.

中華民國類風濕性關節炎之友協會.2011. http://www.raag.org.tw/knowra_view.php?guid=933693f0-29e2-11e0-8f50-0016e6676417&page=1

林其融. AFM探討分別以結合氧化石墨烯之親和吸附與共價鍵結作用固定化之紫膜. 2012. 國立台灣科技大學化學工程研究所碩士論文.

林承德. 氧化石墨烯濃度對以親合吸附作用製備細菌視紫質光電晶片之影響. 2012. 國立台灣科技大學化學工程研究所碩士論文.

陳冠辰. 適體-細菌視紫質生物光電感測晶片之探討. 2015. 國立台灣科技大學化學工程研究所碩士論文.

吳欣穎. 細菌視紫質泛用型免疫光電晶片製備與原子力顯微鏡分析. 2015. 國立台灣科技大學化學工程研究所碩士論文.

廖信銓. 微流體於細菌視紫質光電晶片製備之應用. 2015. 國立台灣科技大學化學工程研究所碩士論文.

龔庭萱. 新穎性血清蛋白質之丙二醛修飾在類風溼關節炎致病角色之探討. 2016. 國立台北醫學大學醫學檢驗暨生物技術研究所碩士論文.

全文公開日期 2023/08/27 (校內網路)
全文公開日期本全文未授權公開 (校外網路)
全文公開日期本全文未授權公開 (國家圖書館：臺灣博碩士論文系統)

簡易檢索 / 詳目顯示

相關論文