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研究生:李淨倫
研究生(外文):Li, Jinglun
論文名稱:整合型微流體系統應用於基於雙層適配體的 糖化血色素之快速檢測
論文名稱(外文):Rapid and sensitive measurement of glycated hemoglobin for diabetes monitoring by using a two-aptamer assay on an integrated microfluidic system
指導教授:李國賓李國賓引用關係
指導教授(外文):Lee, Gwo-Bin
學位類別:碩士
校院名稱:國立清華大學
系所名稱:動力機械工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:73
中文關鍵詞:糖尿病適配體微流體系統糖化血色素
外文關鍵詞:Diabetes MellitusAptamerMicrofluidicsHbA1c
相關次數:
  • 被引用被引用:0
  • 點閱點閱:321
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  • 下載下載:10
  • 收藏至我的研究室書目清單書目收藏:0
摘要
糖尿病是目前世界上十分常見的一種新陳代謝異常造成的疾病。它常常導致視網膜疾病以及相關的其他眼部疾病,腎衰竭以及其併發症,以及非創傷性的手足截肢。糖化血色素可以反映過去三個月以來的血糖及相關參數的平均值,因此可以作為檢測控制和風險評測糖尿病的重要指標。和另一個檢測糖尿病的指標,血糖相比,糖化血色素的應用減少了由於進食多寡和個體生理差異導致的干擾因素影響。本研究中,著重介紹和開發一個具有快速檢測糖化血色素功能的整合型微流體晶片。它可以利用兩條與糖化血色素具有高度親和力的適配體和糖化血色素進行特異性結合的方式進行對糖尿病的快速檢測。相較於本團隊之前利用兩條特異性抗體和糖化血色素進行反應的方式,本實驗中介紹的新型系統更加快速,靈敏並且更加具有經濟性,因為基於適配體的檢測方式相較之下更加便宜。本研究中所開發的晶片,其檢測時間全程僅需三十分鐘,此外,本微流體系統可以在一次實驗中一個晶片同步完成血色素和糖化血色素的檢測,因此它可以同時完成對糖化血色素和血色素的檢測。基於本系統可以自動化快速完成糖化血色素/血色素比例的偵測過程,糖尿病患者很有望利用該系統隨時隨地進行自我檢測和看護,這將大大減少檢測的時間成本和經濟成本。因此,這種具有檢測快捷,經濟實惠雙重優點的集成晶片檢測系統非常有望成為未來可能的病人自我檢測和家庭檢測的工具。

Abstract
Diabetes mellitus (DM), one of the most common metabolic diseases worldwide, is the leading cause of blindness, renal failure and non-traumatic foot/leg amputations. Blood glycated hemoglobin (HbA1c), which reflects an average glucose concentration over past three months, is an important indicator for the diagnosis, monitoring and risk assessment of diabetes. Compared to another common indicator, i.e. blood glucose, the measurement of HbA1c has less biological variations and does not need fasting. In this study, a new integrated microfluidic system using a novel two-aptamer assayc apable of rapid and accurate measurement of HbA1c in blood was developed. It can automate the entire diagnostic process of the two-aptamer assay on a magnetic bead which exhibits high affinity and specificity to HbA1c. Compared to our previous work using a two-antibody assay to measure HbA1c, the new microflui dicsystem is faster, more cost-effective, more sensitive and more reliable since DNA-based aptamers are more environmentally stable and cheaper when compared to antibodies. Furthermore, the microfluidic system has been extended to parallel processing such that multiple assays can be realized on a single chip. The entire process could be reduced to 30 minuites and the measurement of HbA1c and Hb could be performed simultaneously on a single chip. It can serve as a quick and easy means for diabetes patients to carry out point-of-care diagnosis whenever and wherever possible. Combining the advantages of microfluidics and aptamers, this developed microfluidic system may be promising for providing a home-care device for diabetes patients in the near future.

Keywords: Diabetes Mellitus, Aptamer, Microfluidics, HbA1c

Table of Contents
Abstract I
摘要 III
Table of Contents IV
List of Figures VI
Abbreviations X
Nomenclature XII
Chapter 1 Introduction 1
1.1 Diabetes and HbA1c 1
1.2 Background and literature survey 4
1.2.1 HbA1c as a clinical indicator 4
1.2.2 Diagnostic methods of diabetes 5
1.3 Comparisons of aptamer and antibody 7
1.4 Microfluidics and Bio-MEMS 10
1.5 Working principle of microfluidic control systems 11
1.5.1 Fluid mechanics theory of laminar flow 11
1.5.2 Membrane activation theory 12
1.5.3 Mixing efficiency 14
1.6 Motivation and novelty 14
Chapter 2 Materials and methods 16
2.1 Design of microfluidic systems 16
2.1.1 Design of detection chip 16
2.1.2 Experimental protocols 18
2.2 Fabrication of the microfluidic systems 26
2.2.1 Formation of the microfluidic control module 26
2.2.2 PDMS casting 26
2.2.3 Microfluidic chip bonding 28
2.3 Sample preparation 29
2.3.1 Preparation of the Hemoglobin-specific Aptamer 29
2.3.2 Preparation of the HbA1c - specific Aptamer 31
2.3.3 Preparation of the HbA1c-specific Aptamer-Conjugated Magnetic Beads 33
2.3.4 Preparation of the Hb- specific Aptamer-Conjugated Magnetic Beads 35
2.3.5 Preparation of the whole blood sample 36
Chapter 3 Results and discussion 36
3.1 Microfluidic chip characterization 36
3.1.1 Pumping rate of micropump 36
3.1.2 Mixing Index of micromixer 37
3.2 Characterization of the developed standard curve 38
3.2.1 Developed standard curve of HbA1c 39
3.2.2 Developed standard curve of Hb 40
3.3 Double-blind test based on developed standard curve 42
3.4 Specificity test of HbA1c and Hb aptamers 43
3.4.1 Developed standard curve of HbA1c 43
3.4.2 Developed standard curve of Hb 44
3.5 Comparison of the SELEX for traditional methods and the microfluidic system 45
Chapter 4 Conclusions and Future Perspectives 48
4.1 Review of the Thesis 48
4.2 Future Perspectives 49

References 50
Publication list 56


References
1. Kim, K. S., Kim, S. K., Lee, Y. K., Park, S. W. and Cho, Y. W., Diagnostic value of glycated haemoglobin (HbA1c) for the early detection of diabetes in high‐risk subjects. Diabetic Medicine, 2008, 25: p.997-1000.
2. Monnier, L., et al, Contributions of fasting and postprandial plasma glucose increments to the overall diurnal hyperglycemia of type 2 diabetic patients variations with increasing levels of HbA1c. Diabetes care, 2003, 26: p. 881-885.
3. Saudek, C. D., et al., A new look at screening and diagnosing diabetes mellitus. The Journal of Clinical Endocrinology & Metabolism, 2008, 93: p. 2447-2453.
4. Pirart, J., et al., Diabetes mellitus and its degenerative complications: a prospective study of 4,400 patients observed between 1947 and 1973. Diabetes care, 1978, 1: p.168-188.
5. Ellington, A.D. and J.W. Szostak, In vitro selection of RNA molecules that bind specific ligands. Nature, 1990, 346: p. 818-822.
6. Schweizer, A., et al, Comparison between vildagliptin and metformin to sustain reductions in HbA1c over 1 year in drug‐naïve patients with Type 2 diabetes. Diabetic Medicine, 2007, 24: p. 955-961.
7. Maluf, N., An introduction to microelectromechanical systems engineering. Measurement Science and Technology, 2002, 13: p. 229.
8. Herr, J. K., et al., Aptamer-conjugated nanoparticles for selective collection and detection of cancer cells. Analytical Chemistry, 2006, 78: p. 2918-2924.
9. Woerle, H. J., et al., Impact of fasting and postprandial glycemia on overall glycemic control in type 2 diabetes: importance of postprandial glycemia to achieve target HbA1c levels. Diabetes research and clinical practice, 2007, 77: p. 280-285.
10. Corn, R.M., et al., Detection of protein biomarkers using RNA aptamer microarrays and enzymatically amplified surface plasmon resonance imaging. Analytical Chemistry. 2007, 79: p. 1082-1088.
11. Bonora, E., et al., Plasma Glucose Levels Throughout the Day and HbA1c Interrelationships in Type 2 Diabetes Implications for treatment and monitoring of metabolic control. Diabetes care, 2001, 24: p.2023-2029.
12. West, S.D., Effect of CPAP on insulin resistance and HbA1c in men with obstructive sleep apnoea and type 2 diabetes. Thorax, 2007, 62: p. 969-974.
13. Yang, X., et al., Immunofluorescence assay and flow‐cytometry selection of bead‐bound aptamers. Nucleic acids research, 2003, 31(10): p. e54-e54.
14. German, I., Aptamers as ligands in affinity probe capillary electrophoresis. Analytical Chemistry, 1998, 70: p. 4540-4545.
15. Berezovski, M., et al., Nonequilibrium capillary electrophoresis of equilibrium mixtures: a universal tool for development of aptamers. Journal of the American Chemical Society, 2005, 127: p.3165-3171.
16. Qian, J., et al., Generation of highly specific aptamers via micromagnetic selection. Analytical Chemistry, 2009, 81: p. 5490-5495.
17. Dittrich, P.S., et al., Microfluidics in drug discovery, Nature Reviews Drug Discovery. Lab-on-a-chip, 2006, 5: p. 210-218.
18. Squires, T.M., et al., Microfluidics: Fluid physics at the nanoliter scale. Reviews of modern physics, 2005, 77: p.977.
19. Davis, I.W., MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic acids research, 2004, 32: p.W615-W619.
20. Engelgau, M. M., et al., Comparison of fasting and 2-hour glucose and HbA1c levels for diagnosing diabetes: diagnostic criteria and performance revisited .Diabetes care, 1997, 20: p. 785-791.
21. Rohlfing, C. L., et al., Defining the relationship between plasma glucose and HbA1c analysis of glucose profiles and HbA1c in the Diabetes Control and Complications Trial. Diabetes care, 2002, 25: p. 275-278.
22. Mari, A., et al., A model-based method for assessing insulin sensitivity from the oral glucose tolerance test, Diabetes care, 2001, 24: p. 539-548.
23. Unwin, N., et al., Comparison of the current WHO and new ADA criteria for the diagnosis of diabetes mellitus in three ethnic groups in the UK. Diabetic medicine, 1998, 15: p. 554-557.
24. Stone, H., et al., Engineering flows in small devices: microfluidics toward a lab-on-a-chip. Annu. Rev. Fluid Mech., 2004, 36: p. 381-411.
25. Lim, C., et al., Bead-based microfluidic immunoassays: the next generation. Biosensors and Bioelectronics, 2007, 22: p. 1197-1204.
26. Liss M, Petersen B, Wolf H, An aptamer-based quartz crystal protein biosensor ,Analytical Chemistry, 2002, 74: p. 4488-4495.
27. Bruno, J.G., et al., In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection. Biosensors and Bioelectronics, 1999, 14: p. 457-464.
28. Wang, J., et al., Nucleic-acid immobilization, recognition and detection at chronopotentiometric DNA chips. Biosensors and Bioelectronics, 1997, 12: p. 587-599.
29. Osborne, S.E., et al., Aptamers as therapeutic and diagnostic reagents: problems and prospects. Current opinion in chemical biology, 1997, 1: p. 5-9.
30. Mairal T, Özalp V C, Sánchez P L, Aptamers: molecular tools for analytical applications, Analytical and bioanalytical chemistry, 2008, 390: p. 989-1007.
31. Lou, X., Micromagnetic selection of aptamers in microfluidic channels. Proc Natl Acad Sci U S A, 2009, 106: p. 89-94.
32. Oh, S.S., Generation of highly specific aptamers via micromagnetic selection. Anal Chem, 2009, 81: p. 54-59.
33. Brody, E.N. and L. Gold, Aptamers as therapeutic and diagnostic agents. Reviews in Molecular Biotechnology, 2000, 74: p. 5-13.
34. Mann, D., In vitro selection of DNA aptamers binding ethanolamine. Biochemical and biophysical research communications, 2005, 338: p. 1928-1934.
35. Khan H A, Sobki S H, Khan S A. Association between glycaemic control and serum lipids profile in type 2 diabetic patients: HbA1c predicts dyslipidemia, Clinical and experimental medicine, 2007, 7: p. 24-29.
36. Whitesides G M. The origins and the future of microfluidics. Nature, 2006, 442: p. 368-373.
37. Jayasena S D, Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clinical chemistry, 1999, 45: p.1628-1650.
38. T. Fujii., PDMS-based microfluidic devices for biomedical applications. Microelectronic Engineering, 2002, 61: p. 907-914.
39. Ochsner, M., et al., Dimensionality Controls Cytoskeleton Assembly and Metabolism of Fibroblast Cells in Response to Rigidity and Shape. Plos One, 2010, 5: e9445.
40. H. Y. Tseng, C. H. Wang, W. Y .Lin, G. B. Lee, “Membrane-activated microfluidic rotary devices for pumping and mixing,” Biomedical Microdevices, 2007, 9: p. 545-554.
41. Yang, S.Y., et al., A vortex-type micromixer utilizing pneumatically driven membranes. Journal of Micromechanics and Microengineering, 2009,19: 035020.
42. Ching. C. Wu., et al., A Rapid Screening For Hemoglobin-Specific Aptamers By Using A Continuous Microfluidic System. Micro-TAS, 2012, Okinawa, Japan.2.1
43. Zheng, R. B., et al., Development of a highly specific amine-terminated aptamer functionalized surface plasmon resonance biosensor for blood protein detection. Biomedical optics express, 2011, 2: p. 2731-2740.
44. Lin, H. I., et al., Selection of aptamers specific for glycated hemoglobin and total hemoglobin using on-chip SELEX. Lab on chip, 2015, 15:p.486-494.
45. Wu, C. C., et al., Measurement of glycated hemoglobin levels using an integrated microfluidic system.Microfluidics and Nanofluidics,2015, 10:p.1007-1014.

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