表面增強拉曼散射探針及微流道系統 用於癌症細胞及外泌體表面生物標記物的多重檢測

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：20

、訪客IP：3.144.154.208

姓名

闕華恩(Hua-En Chueh) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

表面增強拉曼散射探針及微流道系統用於癌症細胞及外泌體表面生物標記物的多重檢測
(Immuno-functionalized Surface-Enhanced Raman Spectroscopy-Probes (SERS-Probes) and Microfluidic System for Multiplexed Detection of Cell and Exosome Surface Biomarkers in Cancer Diagnosis)

相關論文

★ 具生物沾粘性免疫奈米磁珠之電化學平台於急性冠心病標誌物檢測	★ 離子液體應用於脂溶性蛋白之快速萃取及檢測
★ 深度學習計算成像系統於生物醫學顯微影像之重建與分析	★ 磁電化學免疫分析系統於新型冠狀病毒感染檢測之研製
★ 計算照明高通量生物醫學成像顯微鏡系統	★ 菲涅耳數位全像顯微系統於全血細胞分析之研製
★ 遮罩區域卷積類神經網路於醫學影像物件偵測分析應用	★ 基於深度學習之高通量顯微影像系統於血液分析
★ 可功能化編碼之表面增強拉曼光譜標籤探針於高靈敏與多重生醫分子檢測	★ 高通量計算顯微影像系統之研製於生物醫學成像與分析
★ 功能性抗生物沾黏單層膜於冠狀動脈心血管疾病標誌物之檢測應用	★ 鼻咽癌外泌小體藉由EB病毒潛伏膜蛋白1促進巨噬細胞免疫抑制型分化

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

癌症的早期檢測有助於在疾病最前期識別出癌症或癌前病變，這可以提高存活率及減少發病率。過去十年中，使用體液進行癌症相關分析的液態檢體引起了廣泛的關注。體液中含有很多生物分子，並有可能代表疾病狀態，且檢體方便取得，所以是一個新興的疾病檢測方法。隨著研究的成熟，許多研究者發現，使用多個生物標記物而不僅是單一生物標記物是實現此目標更可行的方法。因此，在液態檢體中檢測細胞或外泌體上的不同膜蛋白標記物，在癌症診斷中具有重要意義。目前多重檢測最常用的儀器是流式細胞儀，其面臨的問題是螢光染劑的散射光帶寬重疊，無法有效區分不同檢測物，而拉曼光譜有著很窄的帶寬，可以透過光譜解析以獲得不同成分的組成。此外，微流道系統是處理少量液態檢體(如血液)的好工具，但傳統的製造方法(如曝光顯影技術)耗時耗財。
此研究合成出不同組免疫功能化的奈米等級表面增強拉曼探針（SERS-probes）來檢測多個目標分子，且使用雷射雕刻和熱壓黏合方法快速製作微流道試片，建立一套癌症生物標記物的檢測系統。另外，使用COMSOL模擬可以優化微流道系統的設計，而將最小平方法用在化學成分推論，則可以進行拉曼光譜解析以獲得不同成分的組成。再者，由於乳癌的廣泛流行且該領域已有相當深入的研究，我們先選擇其進行概念驗證。
本研究著眼於拉曼多重檢測的拉曼分子選擇方式、表面增強拉曼探針合成步驟的探討、及簡易的微流道製程方法，並且提出了一種結合免疫功能化的表面增強拉曼探針和微流道技術的方法，為細胞和外泌體表面生物標記物的多重檢測建立了有前景的平台。這項研究有助於開發透過液態檢體進行早期癌症檢測的新型診斷工具，並為未來的研究者提供適合更深入研究的方向。

摘要(英)

Liquid biopsies have the ability to reveal the presence or absence of illness. It is especially suitable for early cancer detection because of the easy availability of body fluids. Furthermore, many researchers concluded that using multiple biomarkers instead of one is more feasible. As a result, identifying different membrane proteins as biomarkers on cells or exosomes in liquid biopsy has gained importance in cancer diagnosis. Currently, the most common instrument for multiplexing is flow cytometer. One of its challenges is fluorescence overlapping, making it difficult to effectively distinguish different analytes. On the other hand, Raman spectroscopy features a narrow bandwidth, various components can be differentiate through spectral analysis. Moreover, microfluidics are good tools for handling small quantities of liquid samples, but traditional fabrication methods like soft lithography are time-consuming and costly.
This study employed laser cutting and thermal bonding method to quickly fabricate microfluidic chips and also successfully synthesized different immuno-functionalized surface-enhanced Raman scattering-probes (SERS-probes) to detect multiple target molecules at the same time, taking advantage of its narrow bandwidth compared to fluorescence. Also, COMSOL simulation is used to optimize microfluidics designs, while classical least square demultiplexs SERS spectra. Additionally, Breast cancer is chosen as the primary demonstration for this study due to its wide prevalence and the in-depth research conducted in this field.
Our results proposed an integrated method of immuno-functionalized SERS-probes combined with microfluidics to provide a promising platform for the multiplex detection of cell and exosome surface biomarkers. This research potentially contributes to developing new diagnostic tools for the early detection and also point a direction for future research.

關鍵字(中)

★ 表面增強拉曼探針
★ 表面生物標記物
★ 拉曼多重檢測
★ 最小平方法
★ 微流道
★ 熱壓黏合法
★ COMSOL模擬

關鍵字(英)

★ Immuno-functionalized SERS-probes
★ Surface Biomarkers
★ SERS Multiplexed Assay
★ Classical Least Square
★ Microfluidics
★ Thermal Bonding
★ COMSOL simulation

論文目次

中文摘要 i
ABSTRACT ii
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS iv
LIST OF FIGURES vii
LIST OF TABLES ix
ABBREVIATIONS ix
Chapter 1. Introduction 1
1-1 Preface 1
1-2 Motivation and Research Questions 2
Chapter 2. Literature Review 3
2-1 Diseases Diagnostics 3
2-1-1 ELISA (Enzyme-Linked Immunosorbent Assay) 5
2-1-2 Flow Cytometry 7
2-2 Surface-Enhanced Raman Spectroscopy-probe (SERS-probe) 9
2-2-1 Raman Spectroscopy 10
2-2-2 Surface-Enhanced Raman Spectroscopy 12
2-2-3 SERS-Substrates 14
2-3 Microfluidics 17
2-3-1 Spiral Microfluidics 19
2-3-2 Fluid Dynamics 20
2-4 Immuno-Magnetic Beads 23
2-5 Breast Cancer 24
2-5-1 Cell Surface Biomarkers 25
2-5-2 Exosome Biomarkers 27
2-6 Brief Summary of Literature Review 29
Chapter 3. Materials and Methods 30
3-1 Experimental Framework 30
3-2 Materials and Instruments 31
3-2-1 Chemical Compounds 31
3-2-2 Biological Sample 32
3-2-3 Microfluidics 32
3-2-4 Instruments 32
3-3 SERS-probe 33
3-3-1 Experimental Background and Principle 33
3-3-2 Gold Nanoparticle Synthesis 36
3-3-3 SERS-tags and Protection Layer Synthesis 37
3-3-4 Antibody Conjugation 38
3-4 SERS-probe Surface Antibody Coverage Quantification 39
3-4-1 Experimental Background and Principle 39
3-4-2 Bradford Assay Standard Curve with BSA 40
3-4-3 BSA Standard Curve and Data Point Plotting Steps in OriginPro 8 40
3-4-4 Lyophilization Sample Preparation and Calculation 41
3-5 Biological Sample Processing 41
3-5-1 Cell Culture 41
3-5-2 Exosome Preparation 42
3-6 Magnetic Beads for Exosome Capturing 43
3-6-1 Experimental Background and Principle 43
3-6-2 Conjugating Antibodies on Dynabead-Carboxylic Acid (COOH) 43
3-6-3 Capturing Exosomes (sEVs) with Antibody-conjugated Dynabead 44
3-6-4 Dynabead-captured sEV Staining and Imaging with PKH-26 44
3-7 SERS-probe Function Test 45
3-7-1 Dynabead-captured sEV Labeling with SERS-probe 45
3-7-2 Cell Surface Labeling with SERS-probe 45
3-8 Microfluidics Fabrication 46
3-8-1 Experimental Background and Principle 46
3-8-2 Laser Cutting 47
3-8-3 Thermal Bonding 47
3-8-4 Simulation with COMSOL Multiphysics 48
3-9 Detection System Setup (Data Collection) 50
3-9-1 Raman Microscopy 50
3-9-2 Microfluidics System 52
3-10 Raman Spectrum Data Analysis 52
3-10-1 Matlab Code for Quick Visualization of Raman Data 52
3-10-2 Demultiplexing with Classical Least Square 53
Chapter 4. Results and Discussions 54
4-1 Characterization of SERS-probes 54
4-1-1 Absorbance 54
4-1-2 Transmission Electron Microscope (TEM) Imaging 54
4-1-3 Size Distribution from TEM images 55
4-1-4 SERS-probe Silica Protection Layer Thickness 58
4-1-5 SERS-probe Lyophilization 59
4-1-6 SERS-probe Antibody Surface Coverage 61
4-1-7 SERS-probe Raman Spectrum Signal 64
4-1-8 Raman Reporter Selection 65
4-2 Raman Reporter Mixture Signal Demultiplexing 66
4-3 Microfluidics 69
4-3-1 Microfluidics Design 69
4-3-2 Laser cutting and Thermal Bonding 70
4-3-3 Dean Flow COMSOL Simulation 70
4-3-4 Microfluidics System Setup 71
4-4 Cell Surface Biomarker Labeling with SERS-probes 71
4-5 Exosome (sEV) Capturing and Labeling 74
4-5-1 TEM Imaging of Dynabead-captured Exosome 74
4-5-2 SEM Imaging of Polystyrene Bead-captured sEV 74
4-5-3 Dynabead-captured sEV Staining with PKH-26 staining 75
4-5-4 SERS-probes Raman Spectrum on Dynabead-captured sEV 76
Chapter 5. Discussion 76
Chapter 6. Conclusion 78
Chapter 7. Future Work 79
Chapter 8. References 80

參考文獻

[1] Palacín-Aliana, I.; García-Romero, N.; Asensi-Puig, A.; Carrión-Navarro, J.; González-Rumayor, V.; Ayuso-Sacido, Á. Clinical Utility of Liquid Biopsy-Based Actionable Mutations Detected via ddPCR. Biomedicines 2021, 9, 906. https://doi.org/10.3390/biomedicines9080906
[2] Boguszewska, K., Szewczuk, M., Urbaniak, S. et al. Review: immunoassays in DNA damage and instability detection. Cell. Mol. Life Sci. 76, 4689–4704 (2019). https://doi.org/10.1007/s00018-019-03239-6
[3] Wang, Y. W., Reder, N. P., Kang, S., Glaser, A. K., & Liu, J. T. C. (2017). Multiplexed Optical Imaging of Tumor-Directed Nanoparticles: A Review of Imaging Systems and Approaches. Nanotheranostics, 1(4), 369–388. https://doi.org/10.7150/ntno.21136
[4] Meeseon Lee. A Raman Study of CeO2 Nanomaterials with Different Morphologies. Friedrich-Alexander University Erlangen-Nürnberg. Master Thesis. November 2017.
[5] Zong, C., Xu, M., Xu, L. J., Wei, T., Ma, X., Zheng, X. S., Hu, R., & Ren, B. (2018, April 11). Surface-Enhanced Raman Spectroscopy for Bioanalysis: Reliability and Challenges. Chemical Reviews, 118(10), 4946–4980. https://doi.org/10.1021/acs.chemrev.7b00668
[6] Alhalaby, H.; Principe, M.; Zaraket, H.; Vaiano, P.; Aliberti, A.; Quero, G.; Crescitelli, A.; Di Meo, V.; Esposito, E.; Consales, M.; et al. Design and Optimization of All-Dielectric Fluorescence Enhancing Metasurfaces: Towards Advanced Metasurface-Assisted Optrodes. Biosensors 2022, 12, 264. https://doi.org/10.3390/bios12050264
[7] Khnykina, K. A., Baranov, M. A., Babaev, A. A., Dubavik, A. Y., Fedorov, A. V., Baranov, A. V., & Bogdanov, K. V. (2021, July 1). Comparison study of Surface-enhanced Raman spectroscopy substrates. Journal of Physics: Conference Series, 1984(1), 012020. https://doi.org/10.1088/1742-6596/1984/1/012020
[8] Guerrini, L., Pazos-Perez, N., Garcia-Rico, E. et al. Cancer characterization and diagnosis with SERS-encoded particles. Cancer Nano 8, 5 (2017). https://doi.org/10.1186/s12645-017-0031-3
[9] Campbell, J. M., Balhoff, J. B., Landwehr, G. M., Rahman, S. M., Vaithiyanathan, M., & Melvin, A. T. (2018). Microfluidic and Paper-Based Devices for Disease Detection and Diagnostic Research. International journal of molecular sciences, 19(9), 2731. https://doi.org/10.3390/ijms19092731
[10] Hou, H., Warkiani, M., Khoo, B. et al. Isolation and retrieval of circulating tumor cells using centrifugal forces. Sci Rep 3, 1259 (2013). https://doi.org/10.1038/srep01259
[11] Navier-Stokes Equation. Glenn Research Center, NASA. May 13th, 2021. Retrieved from https://www.grc.nasa.gov/www/k-12/airplane/nseqs.html
[12] Azizipour, N., Avazpour, R., Rosenzweig, D.H., Sawan, M., Ajji, A. Evolution of Biochip Technology: A Review from Lab-on-a-Chip to Organ-on-a-Chip. Micromachines 2020, 11, 599. https://doi.org/10.3390/mi11060599
[13] Kolliopoulos, P., Kumar, S. Capillary flow of liquids in open microchannels: overview and recent advances. npj Microgravity 7, 51 (2021). https://doi.org/10.1038/s41526-021-00180-6
[14] Johnston, I.D., McDonnell, M.B., Tan, C.K.L. et al. Dean flow focusing and separation of small microspheres within a narrow size range. Microfluid Nanofluid 17, 509–518 (2014). https://doi.org/10.1007/s10404-013-1322-6
[15] Force balancing model creating true understanding on particle interaction within industrial processes. JOA Air Solutions. (n.d.). Retrieved from https://aus.libguides.com/apa/apa-no-author-date
[16] Sun, C., Hsieh, YP., Ma, S. et al. Immunomagnetic separation of tumor initiating cells by screening two surface markers. Sci Rep 7, 40632 (2017). https://doi.org/10.1038/srep40632
[17] Keller, P.J., Lin, A.F., Arendt, L.M. et al. Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines. Breast Cancer Res 12, R87 (2010). https://doi.org/10.1186/bcr2755
[18] Gligorich, K.M., Vaden, R.M., Shelton, D.N. et al. Development of a screen to identify selective small molecules active against patient-derived metastatic and chemoresistant breast cancer cells. Breast Cancer Res 15, R58 (2013). https://doi.org/10.1186/bcr3452
[19] Maginnis, M. S., Forrest, J. C., Kopecky-Bromberg, S. A., Dickeson, S. K., Santoro, S. A., Zutter, M. M., Nemerow, G. R., Bergelson, J. M., & Dermody, T. S. (2006). Beta1 integrin mediates internalization of mammalian reovirus. Journal of virology, 80(6), 2760–2770. https://doi.org/10.1128/JVI.80.6.2760-2770.2006
[20] Cell Signaling Technology. EpCAM (D9S3P) Rabbit mAb (BSA and Azide Free) #55725. 2023. Retrieved from https://www.cellsignal.com/products/primary-antibodies/epcam-d9s3p-rabbit-mab-ihc-preferred-bsa-and-azide-free/55725
[21] Biorbyt. CD44 antibody. Retrieved from https://www.biorbyt.com/cd44-antibody-orb135213.html
[22] Aviva Systems Biology. CD81 Antibody - C-terminal region (ARP63231_P050). 2023. Retrieved from https://www.avivasysbio.com/cd81-antibody-c-terminal-region-arp63231-p050.html
[23] Yokoi, A., & Ochiya, T. (2021). Exosomes and extracellular vesicles: Rethinking the essential values in cancer biology. Seminars in cancer biology, 74, 79–91. https://doi.org/10.1016/j.semcancer.2021.03.032
[24] Gao, Y., Qin, Y., Wan, C., Sun, Y., Meng, J., Huang, J., Hu, Y., Jin, H., & Yang, K. (2021, February 1). Small Extracellular Vesicles: A Novel Avenue for Cancer Management. Frontiers. https://doi.org/10.3389/fonc.2021.638357
[25] Khushman M, Bhardwaj A, Patel GK, Laurini JA, Roveda K, Tan MC, Patton MC, Singh S, Taylor W, Singh AP. Exosomal Markers (CD63 and CD9) Expression Pattern Using Immunohistochemistry in Resected Malignant and Nonmalignant Pancreatic Specimens. Pancreas. 2017 Jul;46(6):782-788. doi: 10.1097/MPA.0000000000000847. PMID: 28609367; PMCID: PMC5494969.
[26] Andreu, Z., & Yáñez-Mó, M. (2014). Tetraspanins in extracellular vesicle formation and function. Frontiers in immunology, 5, 442. https://doi.org/10.3389/fimmu.2014.00442
[27] Zi Yu, Liao. Encodable Functional SERS Tag for Highly Sensitive and Multiplex Biomolecule Detection. National Central University. Master Thesis. June 2022.
[28] Stöber, W., Fink, A.I., & Bohn, E. (1968). Controlled growth of monodisperse silica spheres in the micron size range. Journal of Colloid and Interface Science, 26, 62-69.
[29] Surve, Milind Laxman. Silica coating of gold nanoparticles. University of Missouri-Rolla. Master Thesis. 2003. https://scholarsmine.mst.edu/masters_theses/2390
[30] Kimling, J., Maier, M., Okenve, B., Kotaidis, V., Ballot, H., & Plech, A. (2006). Turkevich method for gold nanoparticle synthesis revisited. The journal of physical chemistry. B, 110(32), 15700–15707. https://doi.org/10.1021/jp061667w
[31] PLS_Toolbox Advanced Chemometrics Software for use with MATLAB®. Eigenvector Research, Inc. (2023). Retrieved from https://eigenvector.com/software/pls-toolbox/
[32] Alluxa. Flow Cytometry Filters. 2023. Retrieved from https://www.alluxa.com/optical-filter-applications/flow-cytometry-filters/
[33] Olivieri, A.C. (2018). The Classical Least-Squares Model. In: Introduction to Multivariate Calibration. Springer, Cham. https://doi.org/10.1007/978-3-319-97097-4_2
[34] Darweesh, R. S., Ayoub, N. M., & Nazzal, S. (2019). Gold nanoparticles and angiogenesis: molecular mechanisms and biomedical applications. International journal of nanomedicine, 14, 7643–7663. https://doi.org/10.2147/IJN.S223941
[35] Prof. Tom O′Haver. (August, 2021) Curve fitting B: Multicomponent Spectroscopy. TerpConnect, Division of Information Technology, University of Maryland. Retrieved from https://terpconnect.umd.edu/~toh/spectrum/CurveFittingB.html
[36] He, Y. Q., Liu, S. P., Kong, L., & Liu, Z. F. (2005). A study on the sizes and concentrations of gold nanoparticles by spectra of absorption, resonance Rayleigh scattering and resonance non-linear scattering. Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 61(13-14), 2861–2866. https://doi.org/10.1016/j.saa.2004.10.035
[37] Sánchez-Purrà, M., Roig-Solvas, B., Rodriguez-Quijada, C., Leonardo, B. M., & Hamad-Schifferli, K. (2018). Reporter Selection for Nanotags in Multiplexed Surface Enhanced Raman Spectroscopy Assays. ACS Omega, 3(9), 10733–10742. https://doi.org/10.1021/acsomega.8b01499
[38] McFarland, A. D., Young, M. A., Dieringer, J. A., & Van Duyne, R. P. (2005). Wavelength-scanned surface-enhanced Raman excitation spectroscopy. The journal of physical chemistry. B, 109(22), 11279–11285. https://doi.org/10.1021/jp050508u
[39] Hue, D. T., Thu Huong, T. T., Thu Ha, P. T., Trang, T. T., Ha Lien, N. T., and Xuan Hoa, V., “The dependence of medium refractive index on optical properties of gold nanorods and their SERS application”. AIP Advances, vol. 11, no. 5, 2021. doi:10.1063/5.0052882.
[40] Moenad. (2020, September 8). COMSOL Tutorial | Dean Flow or Secondary Flow in a Curved Channel | Dean Vortices Arrow Plot [Video]. YouTube. https://www.youtube.com/watch?v=W_WRiO-MsYE
[41] Cong, L., et al. (2022). Microfluidic Droplet-SERS Platform for Single-Cell Cytokine Analysis via a Cell Surface Bioconjugation Strategy. Analytical Chemistry, 94(29), 10375–10383. https://doi.org/10.1021/acs.analchem.2c01249
[42] Martel, J. M., & Toner, M. (2014). Inertial focusing in microfluidics. Annual review of biomedical engineering, 16, 371–396. https://doi.org/10.1146/annurev-bioeng-121813-120704
[43] Hampitak, P., Melendrez, D., Iliut, M., Fresquet, M., Parsons, N., Spencer, B., Jowitt, T. A., & Vijayaraghavan, A. (2020, September). Protein interactions and conformations on graphene-based materials mapped using a quartz-crystal microbalance with dissipation monitoring (QCM-D). Carbon, 165, 317–327. https://doi.org/10.1016/j.carbon.2020.04.093

指導教授

黃貞翰(Chen-Han Huang)

審核日期

2023-8-21

推文