簡易檢索 / 詳目顯示
| 研究生： |
李文斌 Lee, Wen-Bin |
|---|---|
| 論文名稱： |
整合型微流體系統應用於C反應蛋白之自動化快速檢測 An Integrated Microfluidic System for Fast Detection of C-Reactive Proteins in an Automatic Format |
| 指導教授： |
李國賓
Lee, Gwo-Bin |
| 學位類別： |
碩士 Master |
| 系所名稱： |
工學院 - 工程科學系 Department of Engineering Science |
| 論文出版年： | 2010 |
| 畢業學年度： | 98 |
| 語文別： | 英文 |
| 論文頁數： | 64 |
| 中文關鍵詞： | C反應蛋白 、微流體晶片 、適合體 、心血管疾病 、生醫微機電 、疾病診斷 |
| 外文關鍵詞： | aptamer, C- reactive protein, micropump, micro-injector, MEMS, microfluidic, human serum, point-of-care |
| 相關次數： | 點閱：186 下載：6 |
| 分享至: |
| 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 |
C反應蛋白(C-reactive protein)為一種在人體血液裡常見的蛋白質，其在血液中的濃度已普遍被當作人體發炎反應的指標。而在西元2003年，美國疾病管制局(CDC)及美國心臟協會(AHA)指出，人類血清裡的C反應蛋白在低濃度時(約小於10 mg/L)亦與心血管疾病的罹患率呈正相關，其中分為三種等級:高危險群為3 mg/L以上；中危險群為1~3mg/L；低危險群則不超過1mg/L，因此在高靈敏度偵測下之C反應蛋白(high sensitive C-reactive protein, hs-CRP)亦可視為心血管疾病之風險評估因子。
本研究為一種自動化之整合型微流體晶片系統平台，包含自行設計之多功能微流體晶片及自製冷光偵測儀器，藉由磁珠上本研究團隊自製之具有與C反應蛋白高親和力且高特異性之單股去氧核醣核酸適合體(CRP-specific single strand DNA aptamer)，進行磁珠適合體免疫分析法檢測流程(aptamer-based CRP detection)，用以偵測C反應蛋白濃度的冷光值，進而評估人類罹患心血管疾病之風險率。
利用微流體晶片系統平台體積小、自動化、反應快速、及靈敏度高等優點，可將高靈敏度C反應蛋白 (hs-CRP) 檢測縮小化於一晶片上，此晶片包含微型幫浦、微型漩渦式混合器、微型閥門以及微型注射器等元件，並加以整合達到自動化傳輸流體、混合、偵測等功能，便可應用於磁珠適合體免疫分析法之C反應蛋白定量分析。
實驗結果顯示出，本研究成功的將自動化傳輸流體、混合、偵測整合於一片多功能微流體晶片上，偵測極限可達到0.0125 mg/L，且藉由內有光電倍增管(Photomultipliers, PMT)、小型空壓機(Air compressor)及電磁閥(Electronic magnet valve, EMV) 之自製冷光偵測裝置，便可將整個檢測流程完全自動化進行，無需額外手動添加檢體及取出偵測，僅僅只需手動轉動握柄切換磁座區及偵測區，且整個檢測流程可在30分鐘以內完成；所需之檢體量也僅僅只有5μL，遠小於大型商用儀器所需之量。
此研究更深入進行臨床檢驗，以18組未知濃度之人類血清驗證此系統平台之穩定性與可靠性，並由實驗結果與數據分析後可得知，經由微流體系統平台所獲得之C反應蛋白濃度與由大型商用儀器檢測之值近乎吻合，皆在可容許的誤差範圍內，證明其可應用於臨床檢驗中使用，且檢測時不需將血清進行稀釋，增加了其方便性。期望此檢測C反應蛋白之微流體晶片系統平台能夠成為往後居家醫療的實用工具，希冀達到增進人類福祉的目的。
C-reactive protein (CRP) is a well-known inflammation marker for humans. In 2003, CRP with low concentration (< 10mg/L) in human serums was identified as an independent predictor for cardiovascular diseases, such as heart attack and stroke, by the United States Center for Disease Control (CDC) and the American Heart Association (AHA). A CRP concentration below 1.0 mg/L represents low risk; a range from 1.0 to 3.0 mg/L represents medium risk; and a measurement over 3.0 mg/L represents high risk.
Taking advantage of microfluidic systems, such as miniature dimensions, rapid reaction rates, automated reactions, and high sensitivities, traditional magnetic bead-based enzyme immunoassay can be shrunk into a chip which contains pneumatic micropumps, a vortex-type micromixer, a pneumatic micro-injector and several microvalves. The multi-functional microfluidic chip is designed to automatically perform the entire process including sample transportation, incubation between target CRP and anti-CRP antibody, washing process, development process, and detection. In addition, the chemiluminescent signal was measured by using a lab-made optical system which consists of a photomultiplier tube, a portable air compressor and eight electronic magnet valves to quantify the concentration of CRP.
Experiment results show that a new microfluidic system platform integrated with optical detection devices for automatic measurement of CRP, incorporated with aptamer which was developed by our group and has high affinity and specificity with CRP, was successfully developed. The entire immunoassay process is fully automated without manually adding the reagents, and is accomplished by only turning the handle to switch the detection zone and magnetic zone. When compared to previous works, not only can the new chip perform the entire process by integrating a new micro-injector and new micropumps, but a novel CRP-specific DNA aptamer screened by our group was also used for CRP measurement. Experimental data show that the developed system can automate the entire process within 30 minutes with a detection limit of 0.0125 mg/L, which is superior to previous works.
Moreover, this study also researched the clinical application by testing 18 groups of unknown concentrations of human serum to verify stability and reliability of the developed microfluidic system. The results of the experiment and data analysis indicate that the concentration of CRP by using the microfluidic system platform approximates the value of using the bench-top system, which demonstrates its applicability in clinical laboratories. We therefore anticipate that this microfluidic chip system platform could become the point-of-care machine of the future for CRP detection.
[1]N. Maluf, “An Introduction to Microelectromechanical Systems Engineering,” Artech House, Boston, 1, 2000.
[2]H.A. Stone, A.D. Stroock and A. Ajdari, “Engineering Flows in Small Devices: Microfluidics Toward a Lab-on-a-Chip,” Annu. Rev. Fluid Mech., 36, 2004, 381–411.
[3]T. Vilkner, D. Janasek and A. Manz, ”Micro Total Analysis Systems. Recent Developments,” Anal. Chem., 76, 2004, 3373–3386.
[4]J. Khandurina, T. E. McKnight, S. C. Jacobson, L. C. Waters, R. S. Foote, and J. M. Ramsey “Integrated System for Rapid PCR-Based DNA Analysis in Microfluidic Devices,” Anal. Chem., 72, 2000, 2995–3000.
[5]S. J. Haswell, V. Skelton, “Chemical and biochemical microreactors,” Trac - Trend Anal. Chem., 19(6), 2000, 389–395.
[6]P. S. Dittrich, K. Tachikawa, A. Manz, “Micro Total Analysis Systems. Latest Advancements and Trends,” Anal. Chem., 78, 2006, 3887–3908.
[7]C.D. Chin, V. Linder, S. K. Sia, “Lab-on-a-chip devices for global health: Past studies and future opportunities,” Lab Chip, 7, 2007, 41–57.
[8]L. Gervais and E. Delamarche, “Toward one-step point-of-care immunodiagnostics using capillary-driven microfluidics and PDMS substrates,” Lab Chip, 9, 2009, 3330–3337.
[9]P. Yager, T. Edwards, E. Fu, K. Helton, K. Nelson, M. R. Tam and B. H. Weigl, “Microfluidic diagnostic technologies for global public health,” Nature, 442, 2006, 412–418.
[10]B.Weigl, G. Domingo, P. LaBarre and J. Gerlach, “Towards non- and minimally instrumented, microfluidics-based diagnostic devices,” Lab Chip, 8, 2008, 1999–2014.
[11]Abbott Point-of-Care inc., i-STAT, http://www.abbottpointofcare.com/, accessed July 2010.
[12]Biosite Inc., Cardiovascular Diseases, http://www.biosite.com/products/cardio.aspx, accessed July 2010.
[13]F. S. Apple, R. H. Christenson, R. Valdes, A. J. Andriak, A. Berg, S. H. Duh, Y. J. Feng, S. A. Jortani, N. A. Johnson, B. Koplen, K. Mascotti and A. H. Wu, “Simultaneous rapid measurement of whole blood myoglobin, creatine kinase MB, and cardiac troponin I by the triage cardiac panel for detection of myocardial infarction,” Clin. Chem., 45, 1999, 199–205.
[14]Åmic AB, 4Castchip, http://www.amic.se/, accessed July 2010.
[15]C. Jonsson, M. Aronsson, G. Rundstrom, C. Pettersson, I. Mendel- Hartvig, J. Bakker, E. Martinsson, B. Liedberg, B. MacCraith, O. Ohman and J. Melin, “Silane–dextran chemistry on lateral flow polymer chips for immunoassays,” Lab Chip, 8, 2008, 1191–1197.
[16]W. S. Tillett and T. Francis, “Serological reactions in pneumonia with a non-protein somatic fraction of Pneumococcus,” The Journal of Experimental Medicine, 52, Copyright, 1930, The Rockefeller Institute for Medical Research New York, 561–571.
[17]C. M. MacLeod and O. T. Avery, “The occurrence during acute infections of a protein not normally present in the blood protein,’’ The Journal of Experimental Medicine, 73, Copyright, 1941, The Rockefeller Institute for Medical Research New York, 191–200.
[18]J. E. Volanakis, “Human C-reactive protein: expression, structure, and function,” Mol. Immunol., 38, 2001, 189–197.
[19]M. B. Pepys, G. M. Hirschfield, “C-reactive protein: a critical update,” J. Clin. Invest., 111, 2003, 1805–1812.
[20]D. Thompson, M. B. Pepys, S.P. Wood, “The physiological structure of human C-reactive protein and its complex with phosphocholine,” Structure Fold. Des., 7, 1999, 169–177.
[21]M. B. Pepys and M. L. Baltz, “Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein,” Adv. Immunol., 34, 1983, 141–212.
[22]M. Pourcyrous, H. S. Bada, S. B. Korones, V. Baselski, S. P. Wong, “Significance of Serial C-Reactive Protein Responses in Neonatal Infection and Other Disorders,” Pediatrics, 92(3), 1993, 431–435.
[23]B. Shine, F. C. de Beer and M. B. Pepys, “Solid phase radio immunoassays for C-reactive protein,” Clin. Chim. Acta., 117, 1981, 13–23.
[24]D. M. Steel, A. S. Whitehead, “The major acute-phase reactants - C-reactive protein, serum amyloid-P component and serum amyloid-A protein,” Immunol. Today, 15, 1994, 81–88.
[25]T. A. Pearson, G. A. Mensah, R. W. Alexander, J. L. Anderson, R. O. Cannon, III, M. Criqui, Y. Y. Fadl, S. P. Fortmann, Y. Hong, G. L. Myers, N. Rifai, S. C. Smith, K. Taubert, R. P. Tracy, F. Vinicor, “AHA/CDC Scientific Statement : Markers of Inflammation and Cardiovascular Disease- Application to Clinical and Public Health Practice A Statement for Healthcare Professionals From the Centers for Disease Control and Prevention and the American Heart Association,” Circulation, 107, 2003, 499–511.
[26]G. L. Myers, N. Rifai, R. P. Tracy, W. L. Roberts, R. W. Alexander, L. M. Biasucci, J. D. Catravas, T. G. Cole, G. R. Cooper, B. V. Khan, M. M. Kimberly, E. A. Stein, K. A. Taubert, G. R. Warnick and P. P. Waymack, “CDC/AHA Workshop on Markers of Inflammation and Cardiovascular Disease: Application to Clinical and Public Health Practice: Report From the Laboratory Science Discussion Group,” Circulation, 110, 2004, e545–e549.
[27]A.D. Ellington, J.W. Szostak, “In vitro selection of RNA molecules that bind specific ligands,” Nature, 346, 1990, 818–822.
[28]C. Tuerk, L. Gold, Science, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase,” Science, 249, 1990, 505–510.
[29]Y. Xu, J. A. Phillips, J. Yan, Q. Li, Z. H. Fan, and W. Tan , “Aptamer-Based Microfluidic Device for Enrichment, Sorting, and Detection of Multiple Cancer Cells,” Anal. Chem., 81, 2009, 7436–7442.
[30]K. M. You, S. H. Lee , A. Im, S.B Lee,. “Aptamers as functional nucleic acids: in vitro selection and biotechnological applications,” Biotechnol. Bioprocess. Eng., 8, 2003, 64–75.
[31]E. Luzi, M. Minunni, S. Tombelli, M. Mascini, “New trends in affinity sensing: aptamers for ligand binding,” Trends Anal. Chem., 22, 2003, 810–818.
[32]M. Khati, “The future of aptamers in medicine,” J. Clin. Pathol., 63, 2010,480–487.
[33]W. L. Roberts, R. Sedrick, L. Moulton, A. Spencer, and N. Rifai, “Evaluation of Four Automated High-Sensitivity C-Reactive Protein Methods: Implications for Clinical and Epidemiological Applications,” Clin. Chem., 46(4), 2000, 461–468.
[34]T.B. Ledue and N. Rifai, “High Sensitivity Immunoassays for C-Reactive Protein: Promises and Pitfalls,” Clin. Chem. Lab Med., 39(11), 2001, 1171–1176.
[35]H. Y. Tsai, C. F. Hsu, I. W. Chiu, C. B. Fuh, “Detection of c-reactive protein based on immunoassay using antibody-conjugated magnetic nanoparticles,” Anal. Chem., 79, 2007, 8416–8419.
[36]F. Ibraimi, D. Kriz, M. Lu, L. O. Hansson, K. Kriz, “Rapid one-step whole blood C–reactive protein magnetic permeability immunoassay with monoclonal antibody conjugated nanoparticles as superparamagnetic labels and enhanced sedimentation,’’ Anal. Bioanal. Chem., 384, 2006, 651–657.
[37]T. G. Henaresa, F. Mizutania, H. Hisamoto, “Current development in microfluidic immunosensing chip,” Anal. Chim. Acta., 611, 2008, 11–30.
[38]A. H. C. Ng, U. Uddayasankar, A. R. Wheeler, “Immunoassays in microfluidic systems,” Anal. Bioanal. Chem., 397, 2010, 991–1007.
[39]A. Bhattacharyya, C. M. Klapperich, “Design and testing of a disposable microfluidic chemiluminescent immunoassay for disease biomarkers in human serum samples,” Biomed. Microdevices, 9, 2007, 245–251.
[40]N. Christodoulides, S. Mohanty, C. S. Miller, M. C. Langub, P. N. Floriano, P. Dharshan, M. F. Ali, B. Bernard, D. Romanovicz, E. Anslyn, P. C. Foxf and J. T. McDevitt “Application of microchip assay system for the measurement of C-reactive protein in human saliva,” Lab Chip, 5, 2005, 261–269.
[41]M. H. F. Meyer, M. Hartmann, H. J. Krause, G. Blankenstein, B. M. Chorus, J. Oster, P. Miethe, M. Keusgen “CRP determination based on a novel magnetic biosensor,” Biosens. Bioelectron., 22, 2007, 973–979.
[42]M. H. F. Meyer, M. Hartmann, M. Keusgen, “SPR-based immunosensor for the CRP detection-A new method to detect a well known protein,” Biosens. Bioelectron., 21(10), 2006, 1987-1990.
[43]H. C. Kim, S. K. Lee, W. B. Jeon, H. K. Lyu, S. W. Lee, S. W. Jeong “Detection of C-reactive protein on a functional poly(thiophene) self-assembled monolayer using surface plasmon resonance,” Ultramicroscopy, 108, 2008, 1379– 1383.
[44]Md. S. Islam, H. G. Lee, J. Choo, J. M. Song, S. H. Kang, “High sensitive detection of C-reactive protein by total internal reflection fluorescence microscopy on rapidly making nanoarray protein chip,” Talanta, 81, 2010, 1402–1408.
[45]C. Albrecht, N. Kaeppel, G. Gauglitz, “Two immunoassay formats for fully automated CRP detection in human serum,” Anal. Bioanal. Chem., 391, 2008, 1845-1852.
[46]F. Baldinia, A. Carloni, A. Giannetti, G. Porro, C. Trono “An optical PMMA biochip based on fluorescence anisotropy: Application to C-reactive protein assay,” Sens. Actuators B: Chem., 139, 2009, 64–68.
[47]S. Centi, L. B. Sanmartin, S. Tombelli, I. Palchetti, M. Mascini, “Detection of C Reactive Protein (CRP) in Serum by an Electrochemical Aptamer-Based Sandwich Assay,” Electroanalysis, 21(11), 2009, 1309-1315.
[48]S. C. Shiesh, T. C. Chou, X. Z. Lin, P. C. Kao, “Determination of C-reactive protein with an ultra-sensitivity immunochemiluminometric assay,” J. Immunol. Methods, 311, 2006, 87–95.
[49]J. Pultar, U. Sauer, P. Domnanich, C. Preininger “Aptamer–antibody on-chip sandwich immunoassay for detection of CRP in spiked serum,” Biosens. Bioelectron., 24, 2009, 1456–1461.
[50]A. Bini, S. Centi, S. Tombelli, M. Minunni, M. Mascini, “Development of an optical RNA-based aptasensor for C-reactive protein,” Anal. Bioanal. Chem., 390, 2008, 1077–1086.
[51]Y. N. Yang, H. I. Lin, J. H. Wang, S. C. Shiesh, G. B. Lee, “An integrated microfluidic system for C-reactive protein measurement,” Biosens. Bioelectron., 24, 2009, 3091-3096.
[52]C. J. Huang, H .I. Lin, S. C. Shiesh, G. B. Lee, “Integrated microfluidic system for rapid screening of CRP aptamers utilizing systematic evolution of ligands by exponential enrichment (SELEX),” Biosens. Bioelectron., 25, 2010, 1761-1766.
[53]A. J. Cocuzza, F. W. Hobbs, R. J. Zagursky, N. A. Straus, “Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products,” US Patent 5,484,701, 1996.
[54]Y. N. Yang, S. K. Hsiung, G. B. Lee, “A pneumatic micropump incorporated with a normally closed valve capable of generating a high pumping rate and a high back pressure,” Microfluid. Nanofluid., 6, 2009, 823-833.
[55]S. Y. Yang, J. L. Lin and G. B. Lee, “A vortex-type micromixer utilizing pneumatically driven membranes,” J. Micromech. Microeng., 19, 2009, 035020.
[56]J. M. Bland, D. G. Altman, “Measuring agreement in method comparison studies,” Stat. Meth. Med. Res., 8(2), 1999, 135-160.
[57]H. Passing, W. Bablok, “New Biometrical Procedure for Testing the Equality of Measurements from Two Different Analytical Methods,” J. Clin. Chem. Biochem., 21(11), 1983, 709-720.
校內：2013-08-30公開校外：2013-08-30公開