成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	包志豪 Pau, Chi-Ho
論文名稱：	透過金屬螯合表面快速生產功能性蛋白質微陣列 Rapid Fabrication of functional Protein Microarrays via Metal Chelating surfaces
指導教授：	許觀達 Syu, Guan-Da
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技與產業科學系 Department of Biotechnology and Bioindustry Sciences
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	59
中文關鍵詞：	功能性蛋白質微陣列、蛋白質純化鍍層、微陣列性能、親和性捕獲
外文關鍵詞：	Protein Microarray, Rapid fabrication, IMAC, Coating
相關次數：	點閱：59 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

在功能性蛋白質微陣列上，會使用純化的蛋白質印於微陣列上並用於了解生化功能。在這裡，我們的目標是開發一種塗層，無需額外純化步驟即可固定細胞裂解物中的 his-tagged 蛋白質。我們通過 1,4-丁二醇二縮水甘油醚 (BDGE) 將亞氨基二乙酸 (IDA) 固定在載玻片表面，然後在載玻片中加入飽和量的 CuSO4, CaCl2,MgCl2,NiCl2以獲得離子，並存儲在-80。C 以供進一步使用。在這項研究中，開發了一種具有銅離子表面的微陣列。該平台允許對 His Tag 重組蛋白進行片上純化，為 His Tag 探針高通量實驗提供了一種策略。由於這些陣列具有捕獲 his-tagged 蛋白質的潛力，我們打印了純化的 his-tagged 蛋白質、純化的 non-his-tagged 蛋白質、未純化的 his-tagged裂解物以測試陣列性能。最後，為了展示親和捕獲陣列的應用，我們將製作一些Aska library overexpression strain細胞裂解物陣列來演示它。在此研究，我們發現在各種離子的鍍層下，銅離子擁有最優異的性能，其對Hist-tagged protein的固定量為49453a.u，非專一結合為0.4%，而且擁有最好的疏水性，液滴在其表面的大小僅為1140pixel，將蛋白質純化集成到芯片上不僅有助於高效可靠地製造 His 標籤蛋白微陣列，而且還簡化了工藝和通量形式。這種勞動力和成本效益高的方法可以促進蛋白質微陣列在診斷、藥理學、蛋白質組學和其他實驗室項目中的使用。

On the functional protein microarray, it poses purified proteins on the array for interrogating biochemical functions. Here, we aim to develop an in-house coating to capture the his-tagged proteins in the cell lysates without additional purifications.we use Piranha Solution to functionalize the slide surface and fix Iminodiacetic acid (IDA) on the surface of the slide through 1,4-Butanediol diglycidyl ether (BDGE), and then add a saturated amount of CuSO4, CaCl2,MgCl2,NiCl2 to the slide to obtain ions, and stored in -80。C for further usage. in this study, a microarray with a ion surface was developed. This system allows on-chip purification of His Tag recombinant proteins, which provides a strategy for His Tag probe high-throughput experiments. In order to skip the traditional rate-limiting steps, we developed an on-chip purification process and fixed His Tag protein with recombinant lysates. A streamlined method for rapid manufacturing and purification of His tag protein microarrays was studies.. Since these arrays had potential to capture the his-tagged proteins, we printed purified his-tagged proteins, purified non-his-tagged proteins, unpurified his-tagged lysates to test the array performance. Finally, to show the application of the affinity capture array, we will fabricate a Aska library overexpression strain cell lysate array to demonstrate it. The integration of protein purification on a chip not only contributes to the efficient and reliable manufacturing of His tag protein microarrays, but also simplifies the process and throughput formats. This labor and cost-effective approach can promote the use of protein microarrays in diagnostics, pharmacology, proteomics, and other laboratory programs.

Chinese Abstract	 II

Acknowledgement	V

Table of Contents	VI

Contents of Table	IX

Contents of Figures	X

Abbreviation List	XII

1.	Research Background	1

1-1	Introduction to protein and DNA microarray	1

1-2	Protein purification	2

1-3	Immobilized Metal ion Affinity Chromatography(IMAC)	3

1-4	Metal chelator	4

1-5	Mechanism of IMAC Desorption	4

1-6	Chemical modification of substrates	5

1-7	Research aims	6

2.	Materials and Methods	8

2-1	Materials	8

2-2	Methods	10

3.	Results	15

3-1   A surface coating with specific selectivity to His-tagged protein was established, and the coating conditions were optimized.	15

3-2   EDTA wash proves that BDGE-IDA-ion chip immobilizes proteins by metal affinity.	16

3-3   To verify whether there is a competitive relationship between the Anti-His antibody and the immobilization strategy of the chip.	17

3-4   Compared with the Aldehyde chip, the BDGE-IDA-ion chip can complete the fixation of the sample in a short time, and the short fixation time can reduce the non-specific binding	18

3-5   Compare the binding capacity and nonspecific binding of different ions,and Copper ions have the highest specificity and binding capacity for His-tagged proteins	19

3-6   The surface features of BDGE-IDA-ion and Aldehyde chips were observed under SEM, and their hydrophobicity and surface roughness were detected, and it was found that the roughness was proportional to the non-specific binding	19

3-7   Compare BDGE-IDA-ion and Aldehyde chip whether adding or not adding blocking buffer will increase the background intensity	21

3-8   Use Aska library E.coli to make cell lysate to demonstrate the ability of BDGE chip to immobilize His-tagged proteins	21

3-9   Validation of protein activity by interaction between ACE2 and SARS-CoV-2 Spike protein	22

4.	Discussion	24

References	34

Table	38

Figures	39

Avijit K. Adak, Ben-Yuan Li, Li-De Huang, Ting-Wei Lin, Tsung-Che Chang, Kuo Chu Hwang, and Chun-Cheng Lin,Fabrication of Antibody Microarrays by Light-Induced Covalent and Oriented Immobilization,ACS Applied Materials & Interfaces, 6 (13), 10452-10460, 2014.

A-Jun WAN, Kun WANG, Hong-Cai ZHANG, Hui-Li LI, De-Nong WANG, Modern Carbohydrate Microarray Biochip Technologies,Chinese Journal of Analytical Chemistry, Volume 40, Issue 11,1780-1788, 2012.

Alessandro Gori, Laura Sola, Paola Gagni, Giulia Bruni, Marta Liprino, Claudio Peri, Giorgio Colombo, Marina Cretich, and Marcella Chiari. Screening Complex Biological Samples with Peptide Microarrays: The Favorable Impact of Probe Orientation via Chemoselective Immobilization Strategies on Clickable Polymeric Coatings,Bioconjugate Chemistry, 27 (11), 2669-2677, 2016.

Bo-Wen Li, Yi Zhang, Yin-Chun Wang, Yang Xue, and Xin-Yi Nie.Rapid Fabrication of Protein Microarrays via Autogeneration and on-Chip Purification of Biotinylated Probes.ACS Synthetic Biology, 9 (9), 2267-2273, 2020.

Chandradoss SD, Haagsma AC, Lee YK, Hwang JH, Nam JM, Joo C. Surface passivation for single-molecule protein studies. J Vis Exp.(86),50549. 2014

Carmona P, Röding M, Särkkä A, von Corswant C, Olsson E, Lorén N. Structure evolution during phase separation in spin-coated ethylcellulose/hydroxypropylcellulose films. Soft Matter. ;17(14):3913-3922. 2021.

Feng Wang, Lu Yu, Chao Li, Xiaofeng Xia, Fuming Zhang, Robert J. Linhardt, Site-specific immobilization of papain on DDI-modified polystyrene beads for the oligo(γ-ethyl-L-glutamate) synthesis, Applied Catalysis A: General, 10.1016/j.apcata.2021.118472, 630, (118472), 2022.

Guoting Qin, Catherine Santos, Wen Zhang, Yan Li, Amit Kumar, Uriel J. Erasquin, Kai Liu, Pavel Muradov, Barbara Wells Trautner, Chengzhi Cai. Biofunctionalization on Alkylated Silicon Substrate Surfaces via “Click” Chemistry, Journal of the American Chemical Society, 10.1021/ja1025497, 132, 46, 16432-16441, 2010.

Hao Jia ORCID logo, Ao Zhang, Yuquan Yang, Yaqi Cui, Jianrong Xu, Hewei Jiang, Shengce Tao, Dawei Zhang, Heping Zeng, Zhaoyuan Hou, Jijun Feng.A graphene oxide coated tapered microfiber acting as a super-sensor for rapid detection of SARS-CoV-2.Lab on chip,21,2398-2406,2021.

Hsiao, H.-Y.; Chen, M.-L.; Wu, H.-T.; Huang, L.-D.; Chien, W.-T.; Yu, C.-C.; Jan, F.-D.; Sahabuddin, S.; Chang, T.-C.; Lin, C.-C. Fabrication of Carbohydrate Microarrays through Boronate Formation Chem. Commun, 47, 1187– 1189, 2011.

Holstein, C.A., Chevalier, A., Bennett, S. et al. Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers. Anal Bioanal Chem 408, 1335–1346, 2016.

Harbers GM, Emoto K, Greef C, et al. A functionalized poly(ethylene glycol)-based bioassay surface chemistry that facilitates bio-immobilization and inhibits non-specific protein, bacterial, and mammalian cell adhesion. Chem Mater.;19(18):4405-4414, 2007.

Hyoung Sub Kim, Ji Young Hyun, Seong-Hyun Park and Injae Shin. Analysis of binding properties of pathogens and toxins using multivalent glycan microarrays,RSC Adv, 8, 14898-14905, 2018.

H. Zhu, M. Bilgin, R. Bangham, D. Hall, A. Casamayor, P. Bertone, N. Lan, R. Jansen, S. Bidlingmaier, T. Houfek, T. Mitchell, P. Miller, R. A. Dean, M. Gerstein and M. Snyder.Global Analysis of Protein Activities Using Proteome Chips, Science, 293, 2101-2105,2001.

Joana Moreira, A, Catarina Vale ORCID ,Natália M. Alves. Spin-coated freestanding films for biomedical applications,Journal of Materials Chemistry B ,9 (18) , 3778-3799, 2021.

Jian Yan, Changwen Zhao, Yuhong Ma and Wantai Yang. Covalently Attaching Hollow Silica Nanoparticles on a COC Surface for the Fabrication of a Three-Dimensional Protein Microarray, Biomacromolecules, 23, 2614, 2022.

Ji Young Hyun,Cheol Wan Park,Yanna Liu,Daeun Kwon,Seong-Hyun Park,Sungjin Park,Jaeyoung Pai,Prof. Dr. Injae Shin. Carbohydrate Analogue Microarrays for Identification of Lectin-Selective Ligands,Chembiochem,Volume18 Issue12,1077-1082, 2017.

Jaeyoung Pai, Ji Young Hyun, Jieun Jeong, Sohee Loh , Eun-Hee Cho, Young-Sun Kang, Injae Shin. Carbohydrate microarrays for screening functional glycans,Chem. Sci, 7, 2084-2093, 2016.

Kwon, K., Grose, C., Pieper, R. et al. High quality protein microarray using in situprotein purification. BMC Biotechnol 9, 72 ,2009.

Li-Te Yin, Chih-Yu Hu, Cheng-Hao Chang, A single layer nitrocellulose substrate for fabricating protein chips, Sensors and Actuators B: Chemical, Volume 130 Issue 1,374-378, 2008.

Laura Sola, Francesco Damin, Marcella Chiari, Array of multifunctional polymers for localized immobilization of biomolecules on microarray substrates, Analytica Chimica Acta, Volume 1047,188-196, 2019.

Mazin A. Zamzami, Gulam Rabbani, Abrar Ahmad, Ahmad A. Basalah, Wesam H. Al-Sabban, Saeyoung Nate Ahn, Hani Choudhry, Carbon nanotube field-effect transistor (CNT-FET)-based biosensor for rapid detection of SARS-CoV-2 (COVID-19) surface spike protein S1, Bioelectrochemistry, Volume 143,1567-5394,2022.

María Gallardo-Gómez, Loretta De Chiara, Paula Álvarez-Chaver, Joaquin Cubiella. Colorectal cancer screening and diagnosis: omics-based technologies for development of a non-invasive blood-based method. Expert Review of Anticancer Therapy, 21:7, 723-738, 2021.

Marina Cretich, Valentina Sedini, Francesco Damin, Maria Pelliccia, Laura Sola, Marcella Chiari, Coating of nitrocellulose for colorimetric DNA microarrays, Analytical Biochemistry,Volume 397 Issue 1,84-88, 2010.

Jerker Porath,Immobilized metal ion affinity chromatography,Protein Expression and Purification,Volume 3 Issue 4, 263-281, 1992.

Samuel D Conzone, Carlo G Pantano,Glass slides to DNA microarrays, Materials Today,Volume 7, 20-26, 2004.

Sunkara V., Park D.K., Hwang H., Chantiwas R., Soper S.A., and Cho Y.K.. Simple room temperature bonding of thermoplastics and poly(dimethylsiloxane). Lab on a Chip, 11(5), 962-965, 2011

Sheng-Ce Tao & Heng Zhu. Protein chip fabrication by capture of nascent polypeptides,Nature Biotechnology ,24, 1253–1254 , 2006.

Sungjin Park, Jeffrey C. Gildersleeve, Ola Blixt,Injae Shin. Carbohydrate microarrays,Chem. Soc. Rev ,42, 4310-4326, 2013.

X.-Y. Zhu, Y. Jun, D. R. Staarup, R. C. Major, S. Danielson, V. Boiadjiev, W. L. Gladfelter, B. C. Bunker, and A. Guo.Grafting of High-Density Poly(Ethylene Glycol) Monolayers on Si(111).Langmuir, 17 (25), 7798-7803, 2001.

Ying-Wei Lu, Chih-Wei Chien, Po-Chiao Lin, Li-De Huang, Chang-Yang Chen, Sz-Wei Wu, Chia-Li Han, Kay-Hooi Khoo, Chun-Cheng Lin, and Yu-Ju Chen. BAD-Lectins: Boronic Acid-Decorated Lectins with Enhanced Binding Affinity for the Selective Enrichment of Glycoproteins,Analytical Chemistry, 85 (17), 8268-8276, 2013.

Yongseok Jun, Taewoon Cha, Athena Guo, X.-Y. Zhu,Patterning protein molecules on poly(ethylene glycol) coated Si(111),Biomaterials,Volume 25, 3503-3509, 2004.

校內：2027-06-30公開
校外：2027-07-31公開電子論文尚未授權公開，紙本請查館藏目錄

簡易檢索 / 詳目顯示

相關論文