CMOS-BioMEMS壓阻式微懸臂樑生物感測器於 腹膜炎治療藥物慶大黴素檢測之研究

A Study of CMOS-BioMEMS Based Piezoresistive Microcantilever Sensor on the Detection of Gentamicin for Patients with Peritonitis

10.6342/NTU201703848

李冠緯

微懸臂樑 ； 慶大黴素 ； 電場操控 ； 微量檢體 ； 低成本封裝 ； Microcantilever ； Gentamicin ； Electric field ； minimization sample ； Low cost package

臺灣大學應用力學研究所學位論文

2017年

碩士

張培仁;顏毅廣

繁體中文

研究證實以CMOS MEMS製程之壓阻式微懸臂樑生醫感測器結合電場操控技術，達成低成本、可攜式、微型化、微量檢體且可定量之小分子藥物感測器。根據實驗結果，壓阻式微懸臂樑生物感測器可針對腹膜炎治療藥物慶大黴素進行定量分析，且此定量分析之線性區域包含慶大黴素有效治療濃度範圍，因此壓阻式微懸臂樑生醫感測器是有絕對的潛力供需要執行療劑監測之病人進行即時檢測慶大黴素在血液中的濃度。 近年來，社會的健康意識逐漸抬頭，諸多檢測逐漸被重視，如食安、環境工程、化工藥劑用量等等，因此對於能夠可攜式且即時檢測之感測器的需求越來越多。其中，以病人為中心的醫療環境也逐漸被重視，所以遠距照護也逐年成長，要達成遠距照護的願景，科技的支援是無可避免的。因此對於可攜式且即時檢測之生醫感測器的需求日益大增，醫生對於病人在藥物的使用上非常的關注，故療劑監測為醫生重視之參考依據，對於病患當下的身體狀況調整藥物的劑量，不僅增加療效外更能避免副作用的發生。但現行醫院儀器的檢測流程繁瑣、耗時，因此在非即時檢測的情況下，醫生容易對病人造成誤判。而本研究以CMOS MEMS製程之壓阻式微懸臂樑生醫感測器應用於治療腹膜炎藥物慶大黴素之檢測。 以台積電已商業化CMOS MEMS 0.35標準製程搭配微機電(MEMS)之後製程和生醫微機電技術(BioMEMS)製作出壓阻式微懸臂樑生醫感測器，並且以低成本的方式針對感測器進行封裝。使之成功定量量測小分子藥物慶大黴素，另外為了提升感測器的靈敏度，更應用了電場操控技術，搭配小分子等電位點之調整，成功增加壓阻式微懸臂樑生醫感測器之靈敏度，同時量測到更低濃度之慶大黴素 本研究之待測藥物慶大黴素和抗體以專一性鍵結的方式鍵結，其鍵結後分子構形的變化在微懸臂樑表面產生表面應力，導致壓阻層訊號發生改變，因此在量測小分子上具有其優勢。實驗上得知在60 V的電場操控比無電場操控之訊號提升量為，於濃度為20 μg/mL的慶大黴素為2.195倍，於濃度為35 μg/mL的慶大黴素為1.954倍，於濃度為50 μg/mL的慶大黴素為，因此在60 V的電場操控下相較未加電場操控平均能夠提升1.823倍的訊號量。最後以60 V定量分析，並且得到微懸臂樑生醫感測器之飽和曲線，其檢測濃度分別為10 μg/mL、20 μg/mL、35 μg/mL、50 μg/mL、150 μg/mL、200 μg/mL，對應其變化量分別為5.030 mΩ、20.912 mΩ、40.854 mΩ、62.087 mΩ、125.796 mΩ、123.391 mΩ，故可知濃度在10 ~50 μg/mL做圖可得到一線性區間，此線性區間便可被療劑監測所用。本研究壓阻式微懸臂樑生醫感測晶片之可偵測最小濃度(LOD)為17.57 μg/mL，可定量最小濃度(LOQ)為23.885 μg/mL，可定量濃度區間為23.885 μg/mL ~50 μg/mL，而慶大黴素在血液中的有效濃度範圍有落在壓阻式微懸臂樑感測晶片之可定量濃度區間內，因此證實本研究之壓阻式微懸臂樑生醫感測器在量測小分子藥物上具有一定的潛力且擁有能針對具有有效治療濃度範圍的藥物進行定量分析之能力。

The study prove that piezoresistive microcantilever can accomplish the low cost, portable, minimization sample and quantitative analysis the small molecule drug which is made by CMOS MEMS process and integrate the technique of electric field at the same time. Accroding to the experiment ruslt, the quantitative analysis curve contain the effective treatment range of Gentamicin which is measured by piezoresistive microcantilever. So piezoresistive microcantilever have the great potential to implement the TDM for patient to monitor the concentration of Gentamicin in blood. In rescent year, there is the growing number of the need of the sensor which can portable and timely detection for lots of detection had been emphasized. The surroundings of medical treatment have being taken seriously. So the need of home care growing year by year. The growing number of the need of protable detection sensor is to fulfill the home care. Adverse drug reaction has received increasing attention recently. For some specific drugs with narrow therapeutic ranges, use of therapeutic drug monitoring (TDM) may reduce the adverse drug reactions for an individual to avoid drug toxicity. Gentamicin, managed in TDM, is a widely used antibiotic drug for patients with renal failure. This drug, composed of a mixture of related gentamicin components and fractions, is used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms. The piezoresistive microcantilever is made by TSMC CMOS MEMS 0.35 process which integrate the MEMS and BioMEMS technique and package by low cost. The piezoresistive microcantilever can detect the small molecule drug and promote the sensitivity by control the electric field to detect more lower concentration By comparing the change of the signals in no electric field of antibody immobilization and 60 volt electric field of antibody immobilization, the result showed that the average singal promote 1.8 times. The quantitative analysis after 60 volt electric field of antibody immobilization show that the different concentration of Gentamicin which contain 10 μg/mL、20 μg/mL、35 μg/mL、50 μg/mL、150 μg/mL、200 μg/mL got the signal indivudlly．5.030 mΩ、20.912 mΩ、40.854 mΩ、62.087 mΩ、125.796 mΩ、123.391 mΩ. according to the result of Gentamicin, it can obtain a quantitative analysis curve of TDM patient.

1. [1] M. A. Cooper and V. T. Singleton, "A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions," J Mol Recognit, vol. 20, pp. 154-84, May-Jun 2007.
   連結：
2. [3] J. Mitchell, "Small Molecule Immunosensing Using Surface Plasmon Resonance," Sensors, vol. 10, p. 7323, 2010.
   連結：
3. [4] Y. Chen, B. Dong, and W. Zhou, "Surface plasmon resonance biosensor modified with multilayer silver nanoparticles films," Applied Surface Science, vol. 257, pp. 1021-1026, 2010/11/15/ 2010.
   連結：
4. [5] J. Fritz, M. K. Baller, H. P. Lang, H. Rothuizen, P. Vettiger, E. Meyer, et al., "Translating biomolecular recognition into nanomechanics," Science, vol. 288, pp. 316-8, Apr 14 2000.
   連結：
5. [7] Y. Zhang, S. P. Venkatachalan, H. Xu, X. Xu, P. Joshi, H.-F. Ji, et al., "Micromechanical measurement of membrane receptor binding for label-free drug discovery," Biosensors and Bioelectronics, vol. 19, pp. 1473-1478, 2004/06/15/ 2004.
   連結：
6. [8] L. S. Huang, Y. Pheanpanitporn, Y. K. Yen, K. F. Chang, L. Y. Lin, and D. M. Lai, "Detection of the antiepileptic drug phenytoin using a single free-standing piezoresistive microcantilever for therapeutic drug monitoring," Biosens Bioelectron, vol. 59, pp. 233-8, Sep 15 2014.
   連結：
7. [9] A. M. Moulin, S. J. O'Shea, and M. E. Welland, "Microcantilever-based biosensors," Ultramicroscopy, vol. 82, pp. 23-31, 2000/02/01/ 2000.
   連結：
8. [11] 廖上智, "腹膜透析感染途徑及細菌的特性與分類," 臺灣腎臟護理學會雜誌, vol. 3, pp. 1-10, 2004.
   連結：
9. [12] F. K. Port, P. J. Held, K. D. Nolph, M. N. Turenne, and R. A. Wolfe, "Risk of peritonitis and technique failure by CAPD connection technique: A national study," Kidney International, vol. 42, pp. 967-974, 1992/10/01/ 1992.
   連結：
10. [14] "Drug Information Handbook," 9th ed, 2001-2002, pp. 55-56, 564-566.
    連結：
11. [16] "Guide to antimicrobial therapy," 32th ed, 2002, pp. 71, 125.
    連結：
12. [17] 張. 陳佩甄, 高雅慧, 蔡慧珊, , "老年人使用每日一次胺基醣苷類抗生素之腎毒性相關危險因子," 台灣老年醫學雜誌, pp. 216-227, 2006.
    連結：
13. [18] 林俊杰, 古貞庭, and 李摩西, "Gentamicin每日單劑量與多劑量療法之成本最低化分析," 台灣醫學, vol. 11, pp. 15-21, 2007.
    連結：
14. [20] U. JA, "Drug interference with warfarin therapy," Clinical Medicine, pp. 20-25, 1970.
    連結：
15. [21] S. E. Moulton, J. N. Barisci, A. Bath, R. Stella, and G. G. Wallace, "Investigation of protein adsorption and electrochemical behavior at a gold electrode," J Colloid Interface Sci, vol. 261, pp. 312-9, May 15 2003.
    連結：
16. [22] Y.-K. Yen, C.-Y. Huang, C.-H. Chen, C.-M. Hung, K.-C. Wu, C.-K. Lee, et al., "A novel, electrically protein-manipulated microcantilever biosensor for enhancement of capture antibody immobilization," Sensors and Actuators B: Chemical, vol. 141, pp. 498-505, 2009/09/07/ 2009.
    連結：
17. [23] R. Berger, E. Delamarche, H. P. Lang, C. Gerber, J. K. Gimzewski, E. Meyer, et al., "Surface Stress in the Self-Assembly of Alkanethiols on Gold," Science, vol. 276, pp. 2021-2024, 1997.
    連結：
18. [24] J. W. Ndieyira, M. Watari, A. D. Barrera, D. Zhou, M. Vogtli, M. Batchelor, et al., "Nanomechanical detection of antibiotic-mucopeptide binding in a model for superbug drug resistance," Nat Nano, vol. 3, pp. 691-696, 11//print 2008.
    連結：
19. [25] C. Vancura, M. Ruegg, Y. Li, C. Hagleitner, and A. Hierlemann, "Magnetically actuated complementary metal oxide semiconductor resonant cantilever gas sensor systems," Anal Chem, vol. 77, pp. 2690-9, May 01 2005.
    連結：
20. [26] C.-W. Huang, H.-T. Hsueh, Y.-J. Huang, H.-H. Liao, H.-H. Tsai, Y.-Z. Juang, et al., "A fully integrated wireless CMOS microcantilever lab chip for detection of DNA from Hepatitis B virus (HBV)," Sensors and Actuators B: Chemical, vol. 181, pp. 867-873, 2013/05/01/ 2013.
    連結：
21. [27] Antibody. Available: http://en.wikipedia.org/wiki/Antibody
    連結：
22. [28] 洪維廷 and W.-T. Hung, "應用壓阻式微懸臂梁生物感測器偵測巴斯德桿菌之研究
    連結：
23. Detection of Photobacterium damselae subsp. Piscicida by a Piezoresistive Microcantilever Biosensor."
    連結：
24. [29] 顏毅廣, "整合式微型壓阻微懸臂樑生物感測器之研究及其應用," 物理, 臺灣大學, 2009.
    連結：
25. [31] H. F. Ji, Y. Zhang, V. V. Purushotham, S. Kondu, B. Ramachandran, T. Thundat, et al., "1,6-Hexanedithiol monolayer as a receptor for specific recognition of alkylmercury," Analyst, vol. 130, pp. 1577-9, Dec 2005.
    連結：
26. [32] H.-F. Ji, E. Finot, R. Dabestani, T. Thundat, G. M. Brown, and P. F. Britt, "A novel self-assembled monolayer (SAM) coated microcantilever for low level caesium detection," Chemical Communications, pp. 457-458, 2000.
    連結：
27. [35] N. V. Lavrik, M. J. Sepaniak, and P. G. Datskos, "Cantilever transducers as a platform for chemical and biological sensors," Review of Scientific Instruments, vol. 75, pp. 2229-2253, 2004/07/01 2004.
    連結：
28. [36] M. J. Tudor, M. V. Andres, K. W. H. Foulds, and J. M. Naden, "Silicon resonator sensors: interrogation techniques and characteristics," IEE Proceedings D - Control Theory and Applications, vol. 135, pp. 364-368, 1988.
    連結：
29. [37] J. Thaysen, R. Marie, and A. Boisen, "Cantilever-based bio-chemical sensor integrated in a microliquid handling system," in Technical Digest. MEMS 2001. 14th IEEE International Conference on Micro Electro Mechanical Systems (Cat. No.01CH37090), 2001, pp. 401-404.
    連結：
30. [38] 辜煜夫, "壓阻式微懸臂梁生化感測系統溫度效應之量測、消除與應用," 物理, 臺灣大學, 2009.
    連結：
31. [39] 張凱峯, "利用電場操控蛋白質佈植於壓阻式生物感測器之藥物治療監測應用," 物理, 臺灣大學, 2012.
    連結：
32. [40] A. M. Moulin, S. J. O'Shea, R. A. Badley, P. Doyle, and M. E. Welland, "Measuring Surface-Induced Conformational Changes in Proteins," Langmuir, vol. 15, pp. 8776-8779, 1999/12/01 1999.
    連結：
33. [41] F. Zhou, W. Shu, M. E. Welland, and W. T. S. Huck, "Highly Reversible and Multi-Stage Cantilever Actuation Driven by Polyelectrolyte Brushes," Journal of the American Chemical Society, vol. 128, pp. 5326-5327, 2006/04/01 2006.
    連結：
34. [43] C. Arnab, J. H. Peter, T. Thomas, and H. Zhiyu, "A piezoresistive microcantilever array for surface stress measurement: curvature model and fabrication," Journal of Micromechanics and Microengineering, vol. 17, p. 2065, 2007.
    連結：
35. [44] A. Loui, F. T. Goericke, T. V. Ratto, J. Lee, B. R. Hart, and W. P. King, "The effect of piezoresistive microcantilever geometry on cantilever sensitivity during surface stress chemical sensing," Sensors and Actuators A: Physical, vol. 147, pp. 516-521, 2008/10/03/ 2008.
    連結：
36. [45] C. L. H. Cheng, Y. C., Hu, W. C., Liao, H. H., Tsai, H. H., Juang, Y. Z. & Lu, Y. W., "Surface Stress on CMOS Cantilever Sensor Induced by Protein Absorption," ISMM, 2012.
    連結：
37. [46] 林隆翊, "壓阻式微懸臂樑生物感測元件於抗癲癇藥物之研究," 物理, 臺灣大學, 2012.
    連結：
38. [47] 李忠憲, "具熱補償設計之標準CMOS製程微懸臂樑於抗癲癇藥物丙戊酸之量測," 物理, 臺灣大學, 2014.
    連結：
39. [48] 吳勝智, "CMOS標準製程之微懸臂樑於抗癲癇藥物丙戊酸之研究," 物理, 臺灣大學, 2015.
    連結：
40. [49] 陳維晢, "利用封裝改良之壓阻式微懸臂樑生物感測器於腹膜炎治療藥物慶大黴素之偵測," 物理, 臺灣大學, 2016.
    連結：
41. [52] Thermo Scientific, Crosslinking Technical Handbook. Thermo SCIENTIFIC, 2012.
    連結：
42. [53] J. HENNER and R. D. SITRIN, "Isoelectric focusing and electrophoretic titration of antibiotics using bioautographic detection," The Journal of antibiotics, vol. 37, pp. 1475-1478, 1984.
    連結：
43. [54] A. Shrivastava and V. Gupta, "Methods for the determination of limit of detection and limit of quantitation of the analytical methods," Chronicles of Young Scientists, vol. 2, pp. 21-21, 2011.
    連結：
44. [55] G. L. Long and J. D. Winefordner, "Limit of detection. A closer look at the IUPAC definition," Analytical Chemistry, vol. 55, pp. 712A-724A, 1983.
    連結：
45. [56] E. Desimoni and B. Brunetti, "About estimating the limit of detection by the signal to noise approach," PHARMACEUTICA ANALYTICA ACTA, vol. 6, pp. 1-4, 2015.
    連結：
46. [57] D. A. Armbruster and T. Pry, "Limit of blank, limit of detection and limit of quantitation," The Clinical Biochemist Reviews, vol. 29, p. S49, 2008.
    連結：
47. [2] W. U. Wang, C. Chen, K.-h. Lin, Y. Fang, and C. M. Lieber, "Label-free detection of small-molecule–protein interactions by using nanowire nanosensors," Proceedings of the National Academy of Sciences of the United States of America, vol. 102, pp. 3208-3212, March 1, 2005 2005.
48. [6] G. Wu, R. H. Datar, K. M. Hansen, T. Thundat, R. J. Cote, and A. Majumdar, "Bioassay of prostate-specific antigen (PSA) using microcantilevers," Nat Biotechnol, vol. 19, pp. 856-60, Sep 2001.
49. [10] 醫學百科-腹膜炎. Available: http://big5.wiki8.com/fumoyan_20378/
50. [13] 张. 刘. 孟. 魏. 王. 张. 曲. 李新建, "培养阴性腹膜透析相关性腹膜炎的实验室指标及预后分析," 中华诊断学电子杂志, vol. 2, pp. 206-209, 2014-08-26 2014.
51. [15] "Aminoglycoside," In: Drugdex Information System, Micromedex, Inc, 2003.
52. [19] J. Murphy, "Clinical Pharmacokinetics Pocket Reference," in Aminoglycoside, T. K. Anderson DM, Ed., 2nd ed, 2001, pp. 23-59.
53. [30] 林世明, 應用生化學課堂講義：晶片蛋白質薄膜製造技術, 2008.
54. [33] H. A. Lee, G. M. Wyatt, S. Bramham, and M. R. Morgan, "Enzyme-linked immunosorbent assay for Salmonella typhimurium in food: feasibility of 1-day Salmonella detection," Applied and Environmental Microbiology, vol. 56, pp. 1541-1546, 1990.
55. [34] K. E. Opheim, M. R. Glick, C. N. Ou, K. W. Ryder, L. C. Hood, V. Ainardi, et al., "Particle-enhanced turbidimetric inhibition immunoassay for theophylline evaluated with the Du Pont aca," Clin Chem, vol. 30, pp. 1870-4, Nov 1984.
56. [42] F. T. Goericke, Simulation, Fabrication and Characterization of Piezoresistive Bio-/chemical Sensing Microcantilevers: Georgia Institute of Technology, 2007.
57. [50] "Preparing Self-Assembled Monolayers (SAMs), A Step-by-Step Guide for Solution-Based Self-Assembly ", ed. SIGMA-ALDRICH.
58. [51] Material Matters, Molecular Self-Assembly vol. 1. SIGMA-ALDRICH, 2006.