核酸適體篩選技術之自動化微流控系統研發及其於 SARS-CoV-2 S1 protein適體之篩選

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研究生：	吳紘賓 Wu, Hung-Bin
論文名稱：	核酸適體篩選技術之自動化微流控系統研發及其於 SARS-CoV-2 S1 protein適體之篩選 An automatic microfluidic system for screening SARS-CoV-2 S1 protein aptamer
指導教授：	李國賓 Lee, Gwo-Bin
口試委員:	沈延盛 Shan, Yan-Shen 張晃猷 Chang, Hwan-You
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	76
中文關鍵詞：	人工適體、嚴重特殊傳染性肺炎、微流體、新冠病毒、配體指數增強進化技術
外文關鍵詞：	Aptamers, COVID-19, Microfluidics, SARS-CoV-2, Systematic evolution of ligands by exponential enrichment
相關次數：	點閱：2 下載：0
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自嚴重特殊傳染性肺炎COVID-19（Coronavirus Disease-2019）變的更加嚴重以來，被感染之患者人數持續增加，死亡總人數也持續上升。引起傳染的主要為新型冠狀病毒SARS-CoV-2 （severe acute respiratory syndrome coronavirus 2）導致。然而，SARS-CoV-2 進入人體的方式是藉由刺突蛋白(S protein, spike protein) 與宿主細胞的受體血管張力素轉化酶2(ACE2, Angiotensin-Converting Enzyme 2)結合。 S蛋白由兩個亞基S1和S2組成。其中，S1亞基含有一個受體結合結構域。因此，S1蛋白為檢測COVID-19的重要生物標誌物之一。在此研究中使用配體指數增強進化技術(SELEX, systematic evolution of ligands by exponential enrichment)篩選出能與S1蛋白結合的適體，可用於早期檢測開發。為了提供穩定的篩選適體，本研究針對SARS-CoV-2 S1 protein開發了自動化適體篩選的集成微流控系統，該系統所篩選出的適體能應用於COVID-19早期檢測。本研究中，適體篩選包括 5輪正向選擇（S1 蛋白）、3輪負向選擇（非目標病毒、細胞和細菌）以及2輪藉由人類唾液進行的競爭選擇，透過10輪的篩選以增強特異性和親和力。最終，通過基於 FAM（羧基熒光素）適體的親和力測試，測得篩選出的適體的解離常數為 63.06 nM。此適體被證明可以捕獲SARS-CoV-2非活性病毒和SARS-CoV-2偽病毒，該適體將來能用於診斷 COVID-19。

Since the 2019 coronavirus disease (COVID-19) has become serious, the number of infected patients has continued to increase, and the total number of deaths has also continued to rise. COVID-19 was caused by the virus which was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is noted that SARS-CoV-2 infection is through the spike (S) protein binding to a host cell receptor, angiotensin converting enzyme-2 (ACE2). The S protein is composed of two subunits, namely S1 and S2. Among them, the S1 subunit contains the receptor binding domain. Therefore, S1 protein is one of the important biomarkers for detecting COVID-19. In this work, systematic evolution of ligands by exponential enrichment (SELEX) technology was used to screen aptamers that could bind to the S1 protein such that they could be used for diagnosis of SARS-CoV-2. An integrated microfluidic system for automating SELEX was developed in this study. Screening of aptamers involved 5 rounds of positive selection (S1 protein), 3 rounds of negative selection with (non-target viruses, cells and bacteria), and a newly developed approach of competitive selection using human saliva for 2 rounds of competitive selection to enhance specificity and affinity. Finally, the dissociation constant of the screened aptamers was measured to be 63.06 nM by using a carboxyfluorescein-based aptamer affinity test. Furthermore, the aptamer was shown to be capable of capturing SARS-CoV-2 inactive viruses and pseudo viruses. The aptamer may be used in diagnosis of COVID-19 in the near future.

誌謝    I
Abstract    II
中文摘要    III
Table of contents    IV
List of tables    VI
List of figures    VII
Abbreviations and nomenclature    XI
Chapter 1: Introduction    1
1-1 COVID-19 and SARS-CoV-2    1
1-2 Detection of SARS-CoV-2    3
1-3 Aptamers and antibodies    5
1-4 Systematic evolution of ligands by exponential enrichment (SELEX)    8
1-5 Microfluidic SELEX    10
1-6 Motivation and Objectives    13
Chapter 2 Materials and methods    15
2-1 ssDNA library    15
2-2 Protein and pre-loading reagent preparation    15
2-3 Chip design and chip microfabrication    18
2-4 Experimental setup    22
2-4-1 Experimental procedure    22
2-4-2 Pneumatic control and electromagnetic valves control module    27
2-4-3 Peltier devices for temperature control    30
2-4-4 Magnet control module    32
2-4-5 An automatic system for protein-SELEX    34
2-5 Determination of the dissociation constants (Kd)    37
2-6 Binding with SARS-CoV-2 inactive virus    40
2-6-1 Preparation of SARS-CoV-2 S1 aptamer conjugated beads    40
2-6-2 SARS-CoV-2 S1 aptamer binding with inactive virus    41
2-7 SARS-CoV-2 S1 aptamer binding with pseudoviruses    44
2-8 Specificity test of SARS-CoV-2 aptamer    46
Chapter 3 Results and discussion    49
3-1 Pumping volume of micropump    49
3-2 Mixing index of micromixer    51
3-3 Screening results of on-chip SELEX    53
3-4 Sequence and structure determination of aptamers    57
3-5 Dissociation constant determination (Kd)    59
3-6 Aptamer binding with inactive virus    62
3-7 Aptamer binding with pseudoviruses    64
3-8 Specificity tests    66
Chapter 4 Conclusions and future works    70
4-1 Conclusions    70
4-2 Future works    71
References    72
Publication list    76


                                

[1] M. Ciotti, M. Ciccozzi, A. Terrinoni, W.C. Jiang, C.B. Wang, S. Bernardini, The COVID-19 pandemic, Critical reviews in clinical laboratory sciences, 57, 2020, 365-388.
[2] A.S. Fauci, H.C. Lane, R.R. Redfield, Covid-19—navigating the uncharted, New England Journal of Medicine, 382, 2020, 1268-1269.
[3] D. Wu, T. Wu, Q. Liu, Z. Yang, The SARS-CoV-2 outbreak: What we know, International journal of infectious diseases, 94, 2020, 44-48.
[4] S.F. Pedersen, Y.C. Ho, SARS-CoV-2: a storm is raging, The Journal of clinical investigation, 130, 2020, 2202-2205.
[5] M. Hasoksuz, S. Kilic, F. Sarac, Coronaviruses and sars-cov-2, Turkish journal of medical sciences, 50, 2020, 549-556.
[6] A.R. Bourgonje, A.E. Abdulle, W. Timens, J.L. Hillebrands, G.J. Navis, S.J. Gordijn, M.C. Bolling, G. Dijkstra, A.A. Voors, A.D. Osterhaus, P.H. van der Voort, D.J. Mulder, H. van Goor, Angiotensin-converting enzyme 2 (ACE2), SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19), The Journal of pathology, 251, 2020, 228-248.
[7] L. Falzone, N. Musso, G. Gattuso, D. Bongiorno, C.I. Palermo, G. Scalia, M. Libra, S. Stefani, Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection, International journal of molecular medicine, 46, 2020, 957-964.
[8] V.M. Corman, O. Landt, M. Kaiser, R. Molenkamp, A. Meijer, D.K. Chu, T. Bleicker, S. Brunink, J. Schneider, M.L. Schmidt, D.G. Mulders, B.L. Haagmans, B. van der Veer, S. van den Brink, L. Wijsman, G. Goderski, J.L. Romette, J. Ellis, M. Zambon, M. Peiris, H. Goossens, C. Reusken, M.P. Koopmans, C. Drosten, Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR, Eurosurveillance, 25, 2020, 2000045.
[9] D. Stadlbauer, F. Amanat, V. Chromikova, K. Jiang, S. Strohmeier, G.A. Arunkumar, J. Tan, D. Bhavsar, C. Capuano, E. Kirkpatrick, P. Meade, R.N. Brito, C. Teo, M. McMahon, V. Simon, F. Krammer, SARS‐CoV‐2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup, Current protocols in microbiology, 57, 2020, 100-114.
[10] Q.X. Long, B.Z. Liu, H.J. Deng, G.C. Wu, K. Deng, Y.K. Chen, P. Liao, J.F. Qiu, Y. Lin, X.F. Cai, D.Q. Wang, Y. Hu, J.H. Ren, N. Tang, Y.Y. Xu, L.H. Yu, Z. Mo, F. Gong, X.L. Zhang, W.G. Tian, L. Hu, X.X. Zhang, J.L. Xiang, H.X. Du, H.W. Liu, C.H. Lang, X.H. Luo, S.B. Wu, X.P. Cui, Z. Zhou, M.M. Zhu, J. Wang, C.J. Xue, X.F. Li, L. Wang, Z.J. Li, K. Wang, C.C. Niu, Q.J. Yang, X.J. Tang, Y. Zhang, X.M. Liu, J.J. Li, D.C. Zhang, F. Zhang, P. Liu, J. Yuan, Q. Li, J.L. Hu, J. Chen, A.L. Huang, Antibody responses to SARS-CoV-2 in patients with COVID-19, Nature medicine, 26, 2020, 845-848.
[11] C. Tuerk, L. Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, science, 249, 1990, 505-510.
[12] A.D. Ellington, J.W. Szostak, In vitro selection of RNA molecules that bind specific ligands, nature, 346, 1990, 818-822.
[13] M.R. Dunn, R.M. Jimenez, J.C. Chaput, Analysis of aptamer discovery and technology, Nature Reviews Chemistry, 1, 2017, 1-16.
[14] R. Stoltenburg, C. Reinemann, B. Strehlitz, SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands, Biomolecular engineering, 24, 2007, 381-403.
[15] S.D. Mendonsa, M.T. Bowser, In vitro evolution of functional DNA using capillary electrophoresis, Journal of the American Chemical Society, 126, 2004, 20-21.
[16] J.G. Bruno, J.L. Kiel, Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods, Biotechniques, 32, 2002, 178-183.
[17] W.T. Liu, W.B. Lee, Y.C. Tsai, Y.J. Chuang, K.F. Hsu, G.B. Lee, An automated microfluidic system for selection of aptamer probes against ovarian cancer tissues, Biomicrofluidics, 13, 2019, 014114.
[18] P. Bayat, R. Nosrati, M. Alibolandi, H. Rafatpanah, K. Abnous, M. Khedri, M. Ramezani, SELEX methods on the road to protein targeting with nucleic acid aptamers, Biochimie, 154, 2018, 132-155.
[19] J.C. Cox, A.D. Ellington, Automated selection of anti-protein aptamers, Bioorganic & medicinal chemistry, 9, 2001, 2525-2531.
[20] G. Hybarger, J. Bynum, R.F. Williams, J.J. Valdes, J.P. Chambers, A microfluidic SELEX prototype, Analytical and bioanalytical chemistry, 384, 2006, 191-198.
[21] T.R. Olsen, C. Tapia-Alveal, K.A. Yang, X. Zhang, L.J. Pereira, N. Farmakidis, R. Pei, M.N. Stojanovic, Q. Lin, integrated microfluidic selex using free solution electrokinetics, Journal of the Electrochemical Society, 164, 2017, B3122.
[22] J. Chen, X. Liu, M. Xu, Z. Li, D. Xu, Accomplishment of one-step specific PCR and evaluated SELEX process by a dual-microfluidic amplified system, Biomicrofluidics, 15, 2021, 024107.
[23] W. Jia, Z. Wang, Z. Lu, B. Ding, Z. Li, D. Xu, The discovery of lactoferrin dual aptamers through surface plasmon resonance imaging combined with a bioinformation analysis, Analyst, 145, 2020, 6298-6306.
[24] Y. Liu, N. Wang, C.W. Chan, A. Lu, Y. Yu, G. Zhang, K. Ren, The Application of Microfluidic Technologies in Aptamer Selection, Frontiers in cell and developmental biology, 2021, 2548.
[25] H. JIANG, L. Xue-Fei, Z. Ke-Xin, Progress of aptamer screening techniques based on microfluidic chips, Chinese Journal of Analytical Chemistry, 48, 2020, 590-600.
[26] X. Liu, H. Li, W. Jia, Z. Chen, D. Xu, Selection of aptamers based on a protein microarray integrated with a microfluidic chip, Lab on a Chip, 17, 2017, 178-185.
[27] S.L. Hong, Y.T. Wan, M. Tang, D.W. Pang, Z.L. Zhang, Multifunctional screening platform for the highly efficient discovery of aptamers with high affinity and specificity, Analytical chemistry, 89, 2017, 6535-6542.
[28] H. Stoll, H. Kiessling, M. Stelzle, H.P. Wendel, J. Schütte, B. Hagmeyer, M. Avci-Adali, Microfluidic chip system for the selection and enrichment of cell binding aptamers, Biomicrofluidics, 9, 2015, 034111.
[29] H. Tsutsui, C.M. Ho, Cell separation by non-inertial force fields in microfluidic systems, Mechanics research communications, 36, 2009, 92-103.
[30] M. Evander, L. Johansson, T. Lilliehorn, J. Piskur, M. Lindvall, S. Johansson, M. Almqvist, T. Laurell, J. Nilsson, Noninvasive acoustic cell trapping in a microfluidic perfusion system for online bioassays, Analytical chemistry, 79, 2007, 2984-2991.
[31] A. Sinha, P. Gopinathan, Y.D. Chung, H.Y. Lin, K.H. Li, H.P. Ma, P.C. Huang, S.C. Shiesh, G.B. Lee, An integrated microfluidic platform to perform uninterrupted SELEX cycles to screen affinity reagents specific to cardiovascular biomarkers, Biosensors and Bioelectronics, 122, 2018, 104-112.
[32] C.S. Lin, Y.C. Tsai, K.F. Hsu, G.B. Lee, Optimization of aptamer selection on an automated microfluidic system with cancer tissues, Lab on a Chip, 21, 2021, 725-734.
[33] Y. Cao, A. Yisimayi, F. Jian, W. Song, T. Xiao, L. Wang, S. Du, J. Wang, Q. Li, X. Chen, Nature, 2022, 1-10.
[34] K. Khan, F. Karim, Y. Ganga, M. Bernstein, Z. Jule, K. Reedoy, S. Cele, G. Lustig, D. Amoako, N. Wolter, Omicron BA. 4/BA. 5 escape neutralizing immunity elicited by BA. 1 infection, Nature communications, 13, 2022, 1-7.

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