跳到主要內容

臺灣博碩士論文加值系統

(18.97.9.172) 您好!臺灣時間:2025/02/18 04:06
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳昱佑
研究生(外文):CHEN, Yu-Yu
論文名稱:耦合抗體之磁性奈米粒子及耦合抗體與葡萄糖氧化酶之膠珠的製備 暨電化學反應分析
論文名稱(外文):Preparation and Electrochemical Analysis of Magnetic Nanoparticles Conjugated with Antibody and Silica Nanoparticles Conjugated with Antibody and Glucose Oxidase
指導教授:龍鳳娣
指導教授(外文):LUNG, FENG-DI
口試委員:林宗欣楊雅倩
口試委員(外文):LIM, TSONG-SHINYANG, YA-CHIEN
口試日期:2019-07-04
學位類別:碩士
校院名稱:東海大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:97
中文關鍵詞:奈米粒子抗體葡萄糖氧化酶免疫反應電化學
外文關鍵詞:NanoparticlesAntibodyGlucose OxidaseImmunoreactionElectrochemistry
相關次數:
  • 被引用被引用:0
  • 點閱點閱:286
  • 評分評分:
  • 下載下載:3
  • 收藏至我的研究室書目清單書目收藏:0
材料之表面經過修飾後可與蛋白質、胜肽或小分子藥物以共價鍵結合,形成之耦合物可廣泛應用於生醫檢測方法和治療藥物之開發。本研究推算奈米粒子表面之羧基量,以及蛋白質耦合量,其目的為優化合成方法,降低活化試劑與蛋白質使用量,並應用於耦合抗體之磁性奈米粒子及耦合抗體與葡萄糖氧化酶之膠珠的製備暨電化學反應分析以期建立偵測免疫反應之電化學分析法。實驗流程首先分析奈米粒子與蛋白質耦合之優化條件,接著應用耦合反應之最佳條件製備抗體-奈米粒子或抗體-奈米粒子-蛋白質之耦合物。實驗方法包括: (1) 應用Ninhydrin 方法推算奈米粒子之羧基含量,以降低活化劑與蛋白質之用量;(2) 應用 Bradford 定量未反應之蛋白質,推算耦合反應所需蛋白質之用量,建立耦合反應優化的條件;(3) 應用耦合反應之優化條件,製備葡萄糖氧化酶-奈米粒子之耦合物,並應用電化學分析法之電流訊號,判別耦合物的製備是否成功; (4) 應用優化之條件,將捕捉抗體耦合至奈米粒子,專一性地辨識其抗原;(5) 應用優化之條件,將偵測抗體與葡萄糖氧化酶耦合至奈米粒子;(6) 應用電化學分析其完整免疫反應。本研究電化學方法之建立,應用於偵測特定抗體與抗原之間的專一性反應,研究成果有助於疾病檢測方法之開發。
The surface of the material can be modified to form covalent bonds with proteins, peptides or small molecular drugs, and the coupling products can be widely used in the development of biomedical testing methods and therapeutic drugs. This study estimated the amount of carboxyl groups and protein coupling on the surface of nanoparticles. The purposes are to optimize the synthesis method and reduce the amount of activation reagents and protein. The preparation and electrochemical analysis of magnetic nanoparticles conjugated with antibody and silica nanoparticles conjugated with antibody and glucose oxidase, are expected to establish an electrochemical analysis method for detecting immunoreaction. The first of experimental procedure analyzed the optimal conditions for the protein-conjugated nanoparticles, and prepared antibody-nanoparticles or antibody-nanoparticle-protein coupling products with the optimal conditions of the coupling reaction. The experimental methods include: (1) Apply Ninhydrin method to estimate the carboxyl group of the nanoparticles to reduce the amount of activation reagent and protein; (2) Apply Bradford assay to quantify the unreacted protein, estimating the amount of protein required for the coupling reaction, and establishing the optimal condition; (3) Glucose oxidase was conjugated with nanoparticles by the optimal conditions and detected whether the conjugation was successful by the electrochemical analysis; (4) The capture antibody was conjugated with nanoparticles by the optimal conditions, and the antibody-conjugated nanoparticles specifically identified the antigen; (5) Antibody and glucose oxidase were conjugated with nanoparticles by the optimal conditions; (6) Apply electrochemical method to analyze immunoreaction. In this study, the established electrochemical analysis method can be applied to detect the specific reaction between antibody and antigen, and the results contribute to the development of disease detection.
謝誌 i
摘要 ii
Abstract iii
圖目錄 vii
表目錄 ix
專有名詞縮寫列表 xi
第一章、前言 1
第二章、文獻探討與研究動機 2
2-1 奈米粒子 2
2-1-1 奈米粒子之特性與應用 2
2-2 磁性奈米粒子 3
2-2-1 磁性奈米粒子之特性 3
2-2-2 磁性奈米粒子之應用 3
2-3 二氧化矽奈米粒子 4
2-3-1 二氧化矽奈米粒子之特性 4
2-3-2 二氧化矽奈米粒子之應用 4
2-4 葡萄糖氧化酶 5
2-4-1 葡萄糖氧化酶之特性 5
2-4-2 葡萄糖氧化酶之應用 5
2-5 奈米粒子應用於生醫檢測 6
2-5-1 抗體耦合奈米粒子之製備與應用 6
2-5-2 奈米粒子應用於生醫檢測之研究動機與目的 6
第三章、材料與方法 7
3-1 實驗設計與流程: 7
3-2 Ninhydrin 反應之方法推算磁性奈米粒子與二氧化矽奈米粒子之羧基量 8
3-3 Bradford 法定量之方法推算磁性奈米粒子與二氧化矽奈米粒子之 BSA 耦合量 10
3-4 葡萄糖氧化酶耦合磁性奈米粒子與電化學分析 GOx-MNP 11
3-5捕捉抗體耦合磁性奈米粒子之步驟 12
3-6 應用優化條件於捕捉抗體耦合磁性奈米粒子 14
3-7 葡萄糖氧化酶耦合二氧化矽奈米粒子與電化學分析 GOx-NSP 16
3-8 不同條件葡萄糖氧化酶耦合二氧化矽奈米粒子並呈色分析耦合物 17
3-9 不同比例葡萄糖氧化酶與偵測抗體耦合二氧化矽奈米粒子並呈色分析其免疫反應 18
3-10 偵測抗體與葡萄糖氧化酶耦合二氧化矽奈米粒子並呈色分析其完整免疫反應 20
3-11 偵測抗體與葡萄糖氧化酶耦合二氧化矽奈米粒子並電化學分析其完整免疫反應 22
第四章、結果與討論 25
4-1 應用與探討不同優化條件於奈米粒子之目的 25
4-2 應用 Ninhydrin 反應定量磁性奈米粒子與二氧化矽奈米粒子之羧基量結果 27
4-3 Bradford 法推算磁性奈米粒子與二氧化矽奈米粒子之 BSA 耦合量結果 28
4-4 應用優化條件於葡萄糖氧化酶耦合磁性奈米粒子並電化學分析結果 29
4-5 應用優化條件於抗體耦合磁性奈米粒子並電化學分析免疫反應結果 30
4-6 電化學分析葡萄糖氧化酶耦合二氧化矽奈米粒子之結果 31
4-7 不同條件之葡萄糖氧化酶耦合二氧化矽奈米粒子並呈色分析耦合物結果 32
4-8 偵測抗體與葡萄糖氧化酶耦合二氧化矽奈米粒子並呈色分析免疫反應結果 33
4-9 偵測抗體與葡萄糖氧化酶耦合二氧化矽奈米粒子並電化學分析免疫反應結果 34
第五章、結論 36
未來展望 37
圖附錄 38
表附錄 54
參考文獻 78


1.Ibrahim Khan, K.S., Idrees Khan, Nanoparticles: Properties, applications and toxicities. Arabian Journal of Chemistry, 2017.
2.Rehana, D., A.K. Haleel, and A.K. Rahiman, Hydroxy, carboxylic and amino acid functionalized superparamagnetic iron oxide nanoparticles: Synthesis, characterization and in vitro anti-cancer studies. Journal of Chemical Sciences, 2015. 127(7): p. 1155-1166.
3.Sperling, R.A. and W.J. Parak, Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles. Philosophical Transactions of the Royal Society a-Mathematical Physical and Engineering Sciences, 2010. 368(1915): p. 1333-1383.
4.Yu, M.K., J. Park, and S. Jon, Targeting Strategies for Multifunctional Nanoparticles in Cancer Imaging and Therapy. Theranostics, 2012. 2(1): p. 3-44.
5.Bartczak, D. and A.G. Kanaras, Preparation of Peptide-Functionalized Gold Nanoparticles Using One Pot EDC/Sulfo-NHS Coupling. Langmuir, 2011. 27(16): p. 10119-10123.
6.Avvakumova, S., et al., Biotechnological approaches toward nanoparticle biofunctionalization. Trends in Biotechnology, 2014. 32(1): p. 11-20.
7.Conde, J., et al., Revisiting 30 years of biofunctionalization and surface chemistry of inorganic nanoparticles for nanomedicine. Frontiers in Chemistry, 2014. 2.
8.Kurdekar, A.D., et al., Fluorescent silver nanoparticle based highly sensitive immunoassay for early detection of HIV infection. Rsc Advances, 2017. 7(32): p. 19863-19877.
9.Chailyan, A., P. Marcatili, and A. Tramontano, The association of heavy and light chain variable domains in antibodies: implications for antigen specificity. Febs Journal, 2011. 278(16): p. 2858-2866.
10.Mahan, A.E., et al., A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis. Journal of Immunological Methods, 2015. 417: p. 34-44.
11.Sela-Culang, I., V. Kunik, and Y. Ofran, The structural basis of antibody-antigen recognition. Frontiers in Immunology, 2013. 4.
12.Sakamoto, S., et al., Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. J Nat Med, 2018. 72(1): p. 32-42.
13.Abuknesha, R.A., et al., Labeling of biotin antibodies with horseradish peroxidase using cyanuric chloride. Nat Protoc, 2009. 4(4): p. 452-60.
14.Abuknesha, R.A., et al., Efficient labelling of antibodies with horseradish peroxidase using cyanuric chloride. Journal of Immunological Methods, 2005. 306(1-2): p. 211-217.
15.Nayak, S., et al., Point-of-Care Diagnostics: Recent Developments in a Connected Age. Analytical Chemistry, 2017. 89(1): p. 102-123.
16.Xu, J., et al., Family-Based Big Medical-Level Data Acquisition System. Ieee Transactions on Industrial Informatics, 2019. 15(4): p. 2321-2329.
17.Xiaohua Huang, M.A.E.-S., Gold nanoparticles: Optical properties and implementations in cancer diagnosis and photothermal therapy. Journal of Advanced Research, 2010. 1(1): p. 13-28.
18.Akbarzadeh, A., M. Samiei, and S. Davaran, Magnetic nanoparticles: preparation, physical properties, and applications in biomedicine. Nanoscale Research Letters, 2012. 7: p. 1-13.
19.Banerjee, R. and A. Jaiswal, Recent advances in nanoparticle-based lateral flow immunoassay as a point-of-care diagnostic tool for infectious agents and diseases. Analyst, 2018. 143(9): p. 1970-1996.
20.Zhao, L.J., et al., Sensitive detection of protein biomarkers using silver nanoparticles enhanced immunofluorescence assay. Theranostics, 2017. 7(4): p. 876-883.
21.Draz, M.S. and H. Shafiee, Applications of gold nanoparticles in virus detection. Theranostics, 2018. 8(7): p. 1985-2017.
22.Wahajuddin and S. Arora, Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers. International Journal of Nanomedicine, 2012. 7: p. 3445-3471.
23.Singamaneni, S., et al., Magnetic nanoparticles: recent advances in synthesis, self-assembly and applications. Journal of Materials Chemistry, 2011. 21(42): p. 16819-16845.
24.Schladt, T.D., et al., Synthesis and bio-functionalization of magnetic nanoparticles for medical diagnosis and treatment. Dalton Transactions, 2011. 40(24): p. 6315-6343.
25.Du, P.F., et al., A Competitive Bio-Barcode Amplification Immunoassay for Small Molecules Based on Nanoparticles. Scientific Reports, 2016. 6.
26.Golberg, A., et al., Cloud-Enabled Microscopy and Droplet Microfluidic Platform for Specific Detection of Escherichia coli in Water. Plos One, 2014. 9(1).
27.Wang, S.F., et al., Electrochemical immunoassay of carcinoembryonic antigen based on a lead sulfide nanoparticle label. Nanotechnology, 2008. 19(43).
28.Shukla, S., et al., Detection of Cronobacter sakazakii in powdered infant formula using an immunoliposome-based immunomagnetic concentration and separation assay. Scientific Reports, 2016. 6.
29.Kim, E.Y., et al., Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method. Nanomedicine, 2008. 3(3): p. 293-303.
30.Almstatter, I., et al., Characterization of Magnetic Viral Complexes for Targeted Delivery in Oncology. Theranostics, 2015. 5(7): p. 667-685.
31.Koudelka, K.J., et al., Virus-Based Nanoparticles as Versatile Nanomachines. Annual Review of Virology, Vol 2, 2015. 2: p. 379-401.
32.Lee, A.H.F., et al., Preparation of iron oxide silica particles for Zika viral RNA extraction. Heliyon, 2018. 4(3): p. e00572.
33.Tang, L. and J.J. Cheng, Nonporous silica nanoparticles for nanomedicine application. Nano Today, 2013. 8(3): p. 290-312.
34.Bitar, A., et al., Silica-based nanoparticles for biomedical applications. Drug Discovery Today, 2012. 17(19-20): p. 1147-1154.
35.Ab Rahman, I. and V. Padavettan, Synthesis of Silica Nanoparticles by Sol-Gel: Size-Dependent Properties, Surface Modification, and Applications in Silica-Polymer Nanocomposites-A Review. Journal of Nanomaterials, 2012.
36.Kim, J.W., L.U. Kim, and C.K. Kim, Size control of silica nanoparticles and their surface treatment for fabrication of dental nanocomposites. Biomacromolecules, 2007. 8(1): p. 215-222.
37.Bouchoucha, M., et al., Antibody-conjugated mesoporous silica nanoparticles for brain microvessel endothelial cell targeting. Journal of Materials Chemistry B, 2017. 5(37): p. 7721-7735.
38.Narayan, R., et al., Mesoporous Silica Nanoparticles: A Comprehensive Review on Synthesis and Recent Advances. Pharmaceutics, 2018. 10(3).
39.Wu, Y.Y., et al., A novel ratiometric fluorescent immunoassay for human alpha-fetoprotein based on carbon nanodot-doped silica nanoparticles and FITC. Analytical Methods, 2016. 8(27): p. 5398-5406.
40.Qu, W., et al., Folic acid-conjugated mesoporous silica nanoparticles for enhanced therapeutic efficacy of topotecan in retina cancers. International Journal of Nanomedicine, 2018. 13: p. 4379-4389.
41.Wohlfahrt, G., et al., The chemical mechanism of action of glucose oxidase from Aspergillus niger. Molecular and Cellular Biochemistry, 2004. 260(1-2): p. 69-83.
42.Leskovac, V., et al., Glucose oxidase from Aspergillus niger: the mechanism of action with molecular oxygen, quinones, and one-electron acceptors. International Journal of Biochemistry & Cell Biology, 2005. 37(4): p. 731-750.
43.Meyer, M., et al., Aspects of the mechanism of catalysis of glucose oxidase: A docking, molecular mechanics and quantum chemical study. Journal of Computer-Aided Molecular Design, 1998. 12(5): p. 425-440.
44.Bankar, S.B., et al., Glucose oxidase - An overview. Biotechnology Advances, 2009. 27(4): p. 489-501.
45.Hecht, H.J., et al., Crystal-Structure of Glucose-Oxidase from Aspergillus-Niger Refined at 2 .3 Angstrom Resolution. Journal of Molecular Biology, 1993. 229(1): p. 153-172.
46.Wohlfahrt, G., et al., 1.8 and 1.9 angstrom resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes. Acta Crystallographica Section D-Biological Crystallography, 1999. 55: p. 969-977.
47.Zhai, H., et al., Colorimetric and Ratiometric Fluorescence Dual-Mode Sensing of Glucose Based on Carbon Quantum Dots and Potential UV/Fluorescence of o-Diaminobenzene. Sensors, 2019. 19(3).
48.Bohm, A., et al., Covalent Attachment of Enzymes to Paper Fibers for Paper-Based Analytical Devices. Frontiers in Chemistry, 2018. 6.
49.Lee, S.R., et al., Development of a disposable glucose biosensor using electroless-plated Au/Ni/copper low electrical resistance electrodes. Biosensors & Bioelectronics, 2008. 24(3): p. 410-414.
50.La Belle, J.T., et al., Self-monitoring of tear glucose: the development of a tear based glucose sensor as an alternative to self-monitoring of blood glucose. Chemical Communications, 2016. 52(59): p. 9197-9204.
51.Su, D., et al., Magnetic bead-based mimic enzyme-chromogenic substrate and silica nanoparticles signal amplification system for avian influenza A (H7N9) optical immunoassay. Rsc Advances, 2017. 7(67): p. 41989-41999.
52.Nam, J.M., K.J. Jang, and J.T. Groves, Detection of proteins using a colorimetric bio-barcode assay. Nature Protocols, 2007. 2(6): p. 1438-1444.
53.Luo, Y.H., W.C. Dou, and G.Y. Zhao, Rapid electrochemical quantification of Salmonella Pullorum and Salmonella Gallinarum based on glucose oxidase and antibody-modified silica nanoparticles. Analytical and Bioanalytical Chemistry, 2017. 409(17): p. 4139-4147.
54.Liu, L., et al., Nanomaterials-Based Colorimetric Immunoassays. Nanomaterials, 2019. 9(3).
55.Gao, Z.Q., et al., Magnetic Bead-Based Reverse Colorimetric Immunoassay Strategy for Sensing Biomolecules. Analytical Chemistry, 2013. 85(14): p. 6945-6952.
56.Jia, C.P., et al., Nano-ELISA for highly sensitive protein detection. Biosensors & Bioelectronics, 2009. 24(9): p. 2836-2841.
57.Frampton, J.P., et al., Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA. Scientific Reports, 2014. 4.
58.Lahiri, J., et al., A strategy for the generation of surfaces presenting ligands for studies of binding based on an active ester as a common reactive intermediate: A surface plasmon resonance study. Analytical Chemistry, 1999. 71(4): p. 777-790.
59.Medda, L., M. Monduzzi, and A. Salis, The molecular motion of bovine serum albumin under physiological conditions is ion specific. Chemical Communications, 2015. 51(30): p. 6663-6666.
60.Tonigold, M., et al., Pre-adsorption of antibodies enables targeting of nanocarriers despite a biomolecular corona. Nature Nanotechnology, 2018. 13(9): p. 862-+.
61.Puertas, S., et al., Designing novel nano-immunoassays: antibody orientation versus sensitivity. Journal of Physics D-Applied Physics, 2010. 43(47).

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊