除了血液及尿液等一般常見檢體，女性生殖道的分泌物亦為重要的檢體之一，子宮頸及子宮內膜的分泌物，皆匯集於子宮頸與陰道相接處的陰道窟窿，若在早期診斷時收集此處的體液檢測其各項生理週期或病變相關之分子，除了反應局部的發炎、癌前病變或癌症變化乃至排卵生理週期的改變，未來將可提供篩檢或判斷子宮內膜癌或癌前病變，提供內膜癌病患個人化治療的參考，但目前缺乏快速且便利的收集方法。
本計畫目的為 (1)開發並測試婦女生殖道分泌物之檢體採樣工具：開發生殖道分泌物採集器，以便快速且精確提供可運用之檢體供後續研究使用。將邀請465名20歲以上婦女於進行婦科檢查或抹片前，利用我們開發的棉棒清潔子宮頸表面並收集分泌物，進行採樣。(2)開發紙基檢測平台，將所採集之檢體應用於紙基材檢測平台，利用偵測pH值、乳酸及肝醣之檢測試片以進行初步陰道炎的檢測驗證。 (3)利用採集器所採樣出的檢體，進行癌症病患常見的生物標記IL-6, PRTN-3, VEGF檢測，並同時與胞外囊泡的濃度和粒徑大小差異比較，用來作為子宮內膜癌及癌前病變的檢測。
因此本計畫將開發能過濾黏液、大分子等之生殖道分泌物採集器，以便快速且精確提供可運用之檢體供後續研究使用。

Vaginal fluid plays an important role in the detection of many female genital diseases, but the lack of suitable collection devices in the market severely challenges test success rate. Appropriate clinical sampling devices for vaginal fluid collection would help physicians detect diseases and states of diseases more rapidly, efficiently, and accurately. The objective of this study is to develop a readily applicable sampling collection device that would eliminate macromolecular interference and accurately provide specimens for further studies. This study is designed to develop an effective device to collect vaginal fluid from women with symptoms of endometrial lesions, women attending a routine Papanicolaou smear in the clinic, and/or women seeking a routine gynecologic checkup. In this thesis, paper-based assay, ELISA, and qNano were used to provide accurate diagnoses. A total of 465 patients successfully used the developed device to collect vaginal fluid. Some of the collected specimens were used to detect glycogen, lactate, and pH for determining pathogen infection. Other specimen samples were tested for the presence of female genital cancer by comparing IL-6, PRTN-3, and VEGF concentration and microvesicle concentration. We proposed a non-invasive screening test for the diagnosis of female genital diseases using a dual-material collection device. The outer, nonwoven fabric portion of this device was designed to filter macromolecules, and the inner cotton portion was designed to absorb vaginal fluid.

摘要 III
ABSTRACT IV
LIST OF FIGURES VII
LIST OF TABLES X
CHAPTER 1: INTRODUCTION 11
1.1 Vaginal Fluid Secretion and Collection 12
1.1.1 Application of Collecting Body Fluids 12
1.1.2 Development of a Sampling Collection Method 13
1.2 Introduction to Female Genital Tract Disease 17
1.2.1 Vaginitis 17
1.2.2 Endometrial Cancer 22
1.3 Introduction to Paper-Based Diagnostic Platform 25
1.3.1 The Use of Lactate Assay in Vaginitis 26
1.3.2 The Use of pH Assay in Vaginitis 27
1.3.3 The Use of Glycogen Assay in Vaginitis 29
1.4 Introduction to Molecular Diagnostic 30
1.4.1 The Use of ELISA in Molecular Diagnostics 32
1.4.2 Interleukin 6 (IL-6) 34
1.4.3 Proteinase 3 (PRTN-3) 35
1.4.4 Vascular Endothelial Growth Factor (VEGF) 36
1.5 Introduction to Extracellular Vesicle 37
1.5.1 The Importance of Microvesicles 39
1.5.2 Common Techniques for Detecting Nanoparticles 40
1.5.3 Principle of Tunable Resistive Pulse Sensing (TRPS) 43
1.6 Motivation and Research Outline 44
CHAPTER 2: EXPERIMENTAL DESIGN 47
2.1 Materials 47
2.2 Development of Novel Collection Method: Vaginal Swab 48
2.2.1 Collection of Clinical Sample 49
2.2.2 Recovery of the Collection Sample 49
2.3 Development of Paper-Based Diagnostic Platform 50
2.3.1 Preparation of µPADs for Bioanalytes 51
2.3.2 Quantitative Detection of Analytes using µPADs 52
2.4 Detection of Biomarkers by Sandwich ELISA Assay 52
2.5 Detection of Microvesicles 53
2.5.1 Preparation of Instrument and Standard Solution 53
2.5.2 Measurement of Microvesicles 54
2.5.3 Modification and Fabrication of Paper-based Device 54
2.5.4 Scanning Electron Microscopy Image 55
CHAPTER 3: RESULTS AND DISCUSSION 56
3.1 Prototyping of Vaginal Swabs 56
3.2 Quantitative Detection of Molecules in Vaginal Fluid by µPADs 60
3.2.1 Standard Curve 60
3.2.2 Comparison of Healthy Women and Diagnosed Patients with Vaginitis 63
3.3 Quantitative Detection of Proteins in Vaginal Fluid by ELISA 64
3.4 Correlation between Microvesicles and Endometrial Cancer 67
3.4.1 Detection of Microvesicles Morphology 67
3.4.2 Comparison between Different Body Fluid 69
3.4.3 Particle Size and Concentration 71
CHAPTER 4 CONCLUSION 74
CHAPTER 5 FUTURE WORK 75
5.1 Statistics of the Correlation for all Molecules 75
5.2 Verification of Microvesicles in Vaginal Fluid 75
CHAPTER 6 REFERENCE 76

1. Egan, M.E. and M.S. Lipsky, Diagnosis of vaginitis. American family physician, 2000. 62(5): p. 1095-1104.
2. Watelet, J.-B., et al., Collection of nasal secretions for immunological analysis. European Archives of Oto-Rhino-Laryngology and Head & Neck, 2004. 261(5): p. 242-246.
3. Giampaoli, S., et al., Molecular identification of vaginal fluid by microbial signature. Forensic Science International: Genetics, 2012. 6(5): p. 559-564.
4. Fleming, R.I. and S. Harbison, The use of bacteria for the identification of vaginal secretions. Forensic Science International: Genetics, 2010. 4(5): p. 311-315.
5. Sakurada, K., et al., Expression of statherin mRNA and protein in nasal and vaginal secretions. Legal Medicine, 2011. 13(6): p. 309-313.
6. How do you collect vaginal secretions. Clinical Diagnostic Research, 2007.
7. Tong, S.-W., et al., Methods of collecting and transporting vaginal discharge for detection of infectious organisms and to facilitate cervical cancer screening. 2009, Google Patents.
8. Carozzi, F., et al., New Technologies for Cervival Cancer Screening (NTCC) Working Group Use of p16-INK4A overexpression to increase the specificity of human papillomavirus testing: a nested substudy of the NTCC randomised controlled trial. Lancet Oncol, 2008. 9: p. 937-945.
9. Willyard, C., pH paper trumps expensive kits in measuring acidity. Nature medicine, 2007. 13(10): p. 1128-1129.
10. Contente, A., B.F. Rose, and R.C. Potter, Vaginal discharge collection device and intravaginal drug delivery system. 1994, Google Patents.
11. Sweet, R.L., et al., Infectious diseases of the female genital tract. 2002: Lippincott Williams & Wilkins Philadelphia, PA.
12. Carr, P.L., D. Felsenstein, and R.H. Friedman, Evaluation and management of vaginitis. Journal of general internal medicine, 1998. 13(5): p. 335-346.
13. Sobel, J., Vulvovaginitis in healthy women. Comprehensive therapy, 1999. 25(6-7): p. 335-346.
14. Hillier, S.L., Diagnostic microbiology of bacterial vaginosis. American journal of obstetrics and gynecology, 1993. 169(2): p. 455-459.
15. Hill, G.B., The microbiology of bacterial vaginosis. American journal of obstetrics and gynecology, 1993. 169(2): p. 450-454.
16. Kent, H.L., Epidemiology of vaginitis. American journal of obstetrics and gynecology, 1991. 165(4): p. 1168-1176.
17. Horowitz, B.J., D. Giaquinta, and S. Ito, Evolving pathogens in vulvovaginal candidiasis: implications for patient care. The Journal of Clinical Pharmacology, 1992. 32(3): p. 248-255.
18. Sobel, J.D., Vaginitis. New England Journal of Medicine, 1997. 337(26): p. 1896-1903.
19. Petrin, D., et al., Clinical and microbiological aspects ofTrichomonas vaginalis. Clinical microbiology reviews, 1998. 11(2): p. 300-317.
20. Lossick, J.G. and H.L. Kent, Trichomoniasis: trends in diagnosis and management. American journal of obstetrics and gynecology, 1991. 165(4): p. 1217-1222.
21. Sexuality, Fertility, and Sexually Transmitted Infections (Maternal and Newborn Nursing) http://what-when-how.com/nursing/sexuality-fertility-and-sexually-transmitted-infections-maternal-and-newborn-nursing-part-5/].
22. Defining Cancer. National Cancer Institute., 2014.
23. Endometrial Cancer Treatment National Cancer Institute, 2014.
24. Kong, A., et al., Adjuvant radiotherapy for stage I endometrial cancer. The Cochrane Library, 2012.
25. What You Need To Know: Endometrial Cancer. National Cancer Institute, 2014.
26. Amant, F., et al., Endometrial cancer. Lancet, 2005. 366(9484): p. 491-505.
27. Johnson, K. Brachytherapy Boosts Survival in Inoperable Endometrial Cancer. 2015 April 27]; http://www.medscape.com/viewarticle/843771].
28. Kurman, R.J., P.F. Kaminski, and H.J. Norris, The behavior of endometrial hyperplasia. A long-term study of" untreated" hyperplasia in 170 patients. Obstetrical & Gynecological Survey, 1986. 41(1): p. 58-61.
29. Costales, A.B., et al., Clinically significant endometrial cancer risk following a diagnosis of complex atypical hyperplasia. Gynecologic oncology, 2014. 135(3): p. 451-454.
30. Gungorduk, K., et al., A Novel Preoperative Scoring System for Predicting Endometrial Cancer in Patients with Complex Atypical Endometrial Hyperplasia and Accuracy of Frozen Section Pathological Examination in This Context: A Multicenter Study. Gynecologic and obstetric investigation, 2015. 79(1): p. 50-56.
31. 癌症防治組, 衛生福利部國民健康署公布2011年新發生癌症人數及排名. 2014.
32. Martinez, A.W., et al., Patterned paper as a platform for inexpensive, low‐volume, portable bioassays. Angewandte Chemie International Edition, 2007. 46(8): p. 1318-1320.
33. Bruzewicz, D.A., M. Reches, and G.M. Whitesides, Low-Cost Printing of Poly(dimethylsiloxane) Barriers To Define Microchannels in Paper. Analytical Chemistry, 2008. 80(9): p. 3387-3392.
34. Martinez, A.W., et al., Simple Telemedicine for Developing Regions: Camera Phones and Paper-Based Microfluidic Devices for Real-Time, Off-Site Diagnosis. Analytical Chemistry, 2008. 80(10): p. 3699-3707.
35. Martinez, A.W., et al., FLASH: A rapid method for prototyping paper-based microfluidic devices. Lab on a Chip, 2008. 8(12): p. 2146-2150.
36. Martinez, A.W., S.T. Phillips, and G.M. Whitesides, Three-dimensional microfluidic devices fabricated in layered paper and tape. Proceedings of the National Academy of Sciences, 2008. 105(50): p. 19606-19611.
37. Abe, K., K. Suzuki, and D. Citterio, Inkjet-printed microfluidic multianalyte chemical sensing paper. Analytical chemistry, 2008. 80(18): p. 6928-6934.
38. Cheng, C.-M., et al., Millimeter-scale contact printing of aqueous solutions using a stamp made out of paper and tape. Lab on a Chip, 2010. 10(23): p. 3201-3205.
39. Fenton, E.M., et al., Multiplex lateral-flow test strips fabricated by two-dimensional shaping. ACS Appl Mater Interfaces, 2009. 1(1): p. 124-9.
40. Nie, Z., et al., Electrochemical sensing in paper-based microfluidic devices. Lab on a Chip, 2010. 10(4): p. 477-483.
41. Lu, Y., et al., Rapid prototyping of paper‐based microfluidics with wax for low‐cost, portable bioassay. Electrophoresis, 2009. 30(9): p. 1497-1500.
42. Fu, E., et al., Transport in two-dimensional paper networks. Microfluidics and nanofluidics, 2011. 10(1): p. 29-35.
43. Osborn, J.L., et al., Microfluidics without pumps: reinventing the T-sensor and H-filter in paper networks. Lab on a Chip, 2010. 10(20): p. 2659-2665.
44. Struss, A., et al., Paper strip whole cell biosensors: a portable test for the semiquantitative detection of bacterial quorum signaling molecules. Analytical chemistry, 2010. 82(11): p. 4457-4463.
45. Carrilho, E., et al., Paper microzone plates. Analytical chemistry, 2009. 81(15): p. 5990-5998.
46. Pérez, S. and E. Fàbregas, Amperometric bienzymatic biosensor for L-lactate analysis in wine and beer samples. Analyst, 2012. 137(16): p. 3854-3861.
47. Owen, D.H. and D.F. Katz, A vaginal fluid simulant. Contraception, 1999. 59(2): p. 91-95.
48. Caillouette, J.C., et al., Vaginal pH as a marker for bacterial pathogens and menopausal status. American Journal of Obstetrics and Gynecology, 1997. 176(6): p. 1270-1277.
49. Donders, G., et al., Predictive value for preterm birth of abnormal vaginal flora, bacterial vaginosis and aerobic vaginitis during the first trimester of pregnancy. BJOG: An International Journal of Obstetrics & Gynaecology, 2009. 116(10): p. 1315-1324.
50. El Nahhal, I.M., et al., Thin film optical BTB pH sensors using sol–gel method in presence of surfactants. International Nano Letters, 2012. 2(1): p. 1-9.
51. Bueno, C., et al., The Excited‐State Interaction of Resazurin and Resorufin with Aminesin Aqueous Solutions. Photophysics and Photochemical Reaction¶. Photochemistry and photobiology, 2002. 76(4): p. 385-390.
52. Moller, B. and P. Kaspersen, Vaginitis and vaginosis. 1992, Wiley-Liss, New York.
53. Kontula, K., et al., Binding of progestins to the glucocorticoid receptor: correlation to their glucocorticoid-like effects on in vitro functions of human mononuclear leukocytes. Biochemical pharmacology, 1983. 32(9): p. 1511-1518.
54. Kristiansen, M., et al., Identification, synthesis, and characterization of new glycogen phosphorylase inhibitors binding to the allosteric AMP site. Journal of medicinal chemistry, 2004. 47(14): p. 3537-3545.
55. Roach, P.J., Glycogen and its metabolism. Curr Mol Med, 2002. 2(2): p. 101-20.
56. Childs, R.E. and W.G. Bardsley, The steady-state kinetics of peroxidase with 2, 2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen. Biochem. J, 1975. 145: p. 93-103.
57. Bateman Jr, R.C. and J.A. Evans, Using the glucose oxidase/peroxidase system in enzyme kinetics. Journal of Chemical Education, 1995. 72(12): p. A240.
58. Yang, S. and R.E. Rothman, PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. The Lancet Infectious Diseases, 2004. 4(6): p. 337-348.
59. Marsh, P. and D.N. Cardy, Molecular Diagnostics, in Genomics, Proteomics, and Clinical Bacteriology, N. Woodford and A. Johnson, Editors. 2004, Humana Press. p. 167-189.
60. Raux, G., et al., Molecular diagnosis of autosomal dominant early onset Alzheimer’s disease: an update. Journal of Medical Genetics, 2005. 42(10): p. 793-795.
61. Molecular Diagnostics Market Expected to Reach USD 8.7 Billion through 2019: TMR. transparency market research, 2014.
62. Grody, W.W., et al., Molecular diagnostics: techniques and applications for the clinical laboratory. 2009: Academic Press.
63. Espy, M., et al., Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clinical microbiology reviews, 2006. 19(1): p. 165-256.
64. Paik, S., et al., A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. New England Journal of Medicine, 2004. 351(27): p. 2817-2826.
65. Handyside, A.H., et al., Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. New England Journal of Medicine, 1992. 327(13): p. 905-909.
66. Lemna, W.K., et al., Mutation analysis for heterozygote detection and the prenatal diagnosis of cystic fibrosis. New England Journal of Medicine, 1990. 322(5): p. 291-296.
67. Tang, Y.-W. and C.-Y. Ou, Past, present and future molecular diagnosis and characterization of human immunodeficiency virus infections. Emerg Microbes Infect, 2012. 1: p. e19.
68. Singer, J.M. and C.M. Plotz, The latex fixation test: I. Application to the serologic diagnosis of rheumatoid arthritis. The American Journal of Medicine, 1956. 21(6): p. 888-892.
69. Berson, S.A. and R.S. Yalow, Quantitative aspects of the reaction between insulin and insulin-binding antibody. Journal of Clinical Investigation, 1959. 38(11): p. 1996.
70. Gan, S.D. and K.R. Patel, Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. J Invest Dermatol, 2013. 133(9): p. e12.
71. Overview of ELISA. Thermo Fisher Scientific Inc.
72. Simpson, R.J., et al., Interleukin-6: structure-function relationships. Protein Sci, 1997. 6(5): p. 929-55.
73. Petersen, A.M.W. and B.K. Pedersen, The anti-inflammatory effect of exercise. Journal of applied physiology, 2005. 98(4): p. 1154-1162.
74. Bellone, S., et al., High serum levels of interleukin-6 in endometrial carcinoma are associated with uterine serous papillary histology, a highly aggressive and chemotherapy-resistant variant of endometrial cancer. Gynecologic oncology, 2005. 98(1): p. 92-98.
75. Friedenreich, C.M., et al., Case–control study of inflammatory markers and the risk of endometrial cancer. European Journal of Cancer Prevention, 2013. 22(4): p. 374-379.
76. Rao, N.V., et al., Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties. J Biol Chem, 1991. 266(15): p. 9540-8.
77. Sturrock, A., et al., Characterization and localization of the genes for mouse proteinase-3 (Prtn3) and neutrophil elastase (Ela2). Cytogenetic and Genome Research, 1998. 83(1-2): p. 104-108.
78. Kamat, A.A., et al., Clinical and biological significance of vascular endothelial growth factor in endometrial cancer. Clinical Cancer Research, 2007. 13(24): p. 7487-7495.
79. Peng, X., et al., [Clinical significance of vascular endothelial growth factor in sera of patients with gynaecological malignant tumors]. Ai zheng= Aizheng= Chinese journal of cancer, 2002. 21(2): p. 181-185.
80. Xu, H., et al., Anti-angiogenic effects of green tea catechin on an experimental endometriosis mouse model. Human reproduction, 2009. 24(3): p. 608-618.
81. Becker, C.M., et al., A novel noninvasive model of endometriosis for monitoring the efficacy of antiangiogenic therapy. The American journal of pathology, 2006. 168(6): p. 2074-2084.
82. McLaren, J., et al., Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Human Reproduction, 1996. 11(1): p. 220-223.
83. Agrawal, R., et al., Serum vascular endothelial growth factor (VEGF) in the normal menstrual cycle: association with changes in ovarian and uterine Doppler blood flow. Clinical endocrinology, 1999. 50(1): p. 101-106.
84. Raposo, G. and W. Stoorvogel, Extracellular vesicles: exosomes, microvesicles, and friends. The Journal of cell biology, 2013. 200(4): p. 373-383.
85. György, B., et al., Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and molecular life sciences, 2011. 68(16): p. 2667-2688.
86. Ratajczak, J., et al., Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication. Leukemia, 2006. 20(9): p. 1487-1495.
87. Théry, C., L. Zitvogel, and S. Amigorena, Exosomes: composition, biogenesis and function. Nature Reviews Immunology, 2002. 2(8): p. 569-579.
88. Janowska‐Wieczorek, A., et al., Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer. International journal of cancer, 2005. 113(5): p. 752-760.
89. Millimaggi, D., et al., Tumor Vesicle—Associated CD147 Modulates the Angiogenic Capability of Endothelial Cells. Neoplasia, 2007. 9(4): p. 349-357.
90. Al-Nedawi, K., et al., Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR. Proceedings of the National Academy of Sciences, 2009. 106(10): p. 3794-3799.
91. Diaconu, A., Demersuri teoretico-aplicative pentru construcția și implementarea unor strategii de utilizare a feedback-ului formativ în școală. 2013.
92. Goldburg, W., Dynamic light scattering. American Journal of Physics, 1999. 67(12): p. 1152-1160.
93. Carr, B. and M. Wright, Nanoparticle tracking analysis. Innovations in Pharmaceutical Technology, 2008. 26: p. 38-40.
94. Schuck, P., Sedimentation analysis of noninteracting and self-associating solutes using numerical solutions to the Lamm equation. Biophysical Journal, 1998. 75(3): p. 1503-1512.
95. Van Der Pol, E., et al., Optical and non‐optical methods for detection and characterization of microparticles and exosomes. Journal of Thrombosis and Haemostasis, 2010. 8(12): p. 2596-2607.
96. Burg, T.P. and S.R. Manalis, Suspended microchannel resonators for biomolecular detection. Applied Physics Letters, 2003. 83(13): p. 2698-2700.
97. Platt, M., G.R. Willmott, and G.U. Lee, Resistive pulse sensing of analyte‐Induced multicomponent rod aggregation using tunable pores. Small, 2012. 8(15): p. 2436-2444.
98. Coumans, F.A.W., et al., Reproducible extracellular vesicle size and concentration determination with tunable resistive pulse sensing. 2014, 2014.
99. Chen, C., et al., Paper-based immunoaffinity devices for accessible isolation and characterization of extracellular vesicles. Microfluidics and Nanofluidics, 2014. 16(5): p. 849-856.
100. Wan, C.-Y. and T.A. Wilkins, A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical biochemistry, 1994. 223(1): p. 7-12.
101. Kealey, D., Quantitative reflectometry—I: Principles and scope. Talanta, 1972. 19(12): p. 1563-1571.
102. Analysing Extracellular Vesicles using TRPS. IZON Science, 2015