整合型微流體系統平台於分子診斷技術之應用

Applications of integrated microfluidic systems for molecular diagnosis

張文馨(Wen-Hsin Chang)

指導教授 ： 李國賓

國立清華大學/工學院/動力機械工程學系/博士(2014年)

https://doi.org/10.6843/NTHU.2014.00212

摘要

隨著社會的進步，個體間的距離越來越近，許多快速傳播的感染病因而出現。這些感染病不僅威脅人類健康甚鉅，亦造成嚴重經濟損失。早期偵測及診斷是遏止傳染疾病擴散的關鍵因素。因此許多研究學者投身於開發既正確又快速的偵測及診斷工具。在所有偵測工具中，分子診斷已被廣泛地應用於感染病偵測因為其具有高靈敏性和專一性。分子偵測可提供正確且靈敏的偵測結果，這些結果可作為處置方法之參考。然而，複雜的程序及操作者所造成的誤差和汙染使得分子診斷尚無法成為一良好的田間試驗或即時醫療工具。 為了解決以上問題，本論文提出四種整合式微流體系統，分別可以直 由魚類，蘭花及人類關節液樣本中對細菌及病毒等病原體以分子診斷的方式進行偵測。這些整合式微流體系統借助生物微機電系統的知識，將數個微流體元件整合至單一生物晶片上而達成晶片實驗室的想法。此外，整合式微流體系統可自動化執行傳統生醫實驗中所有手動步驟，不需操作者介入而省下時間金錢，因此可避免操作者所造成的誤差和汙染。 本論文針對這些整合式微流體系統的效能進行確效，結果顯示其具有高度專一性，且不同系統之靈敏度分別為20條DNA拷貝數(恆溫環狀擴增法及螢光偵測)，35皮克質體去氧核糖核酸(恆溫環狀擴增法及濁度偵測)，200菌落形成單位(聚合酶鏈鎖反應及螢光偵測)及100個菌落形成單位(奈米金探針偵測)。此外，本研究以具有溫度控制模組，液體傳輸模組及光學偵測模組的整合式控制系統自動化地於短時間內完成包含分離去氧核糖核酸/核糖核酸/細菌，利用聚合酶鏈鎖反應或恆溫環狀擴 增進行核酸增幅，奈米金探針偵測或光學偵測之所有實驗流程。實驗結果顯示這些微型全分析系統可於不遠的將來作為田間試驗或即時醫療之有用工具。

關鍵字

微流體 ； 恆溫環狀擴增法 ； 聚合酶鏈鎖反應 ； 奈米金探針 ； 水產養殖病原菌 ； 蝴蝶蘭 ； 關節周邊組織感染 ； 微機電系統 ； 微型全分析系統

並列摘要

As the society progresses, many rapidly-spread infectious diseases emerge because the distance between individuals is getting closer and closer. Not only do these infectious diseases threaten people health significantly but they also cause huge economic loss. The key factor to cease the spread of infectious diseases is early detection and diagnosis.Researchers have devoted to develop detection and diagnostic tools which are accurate and rapid. Among all detection tools, molecular diagnosis has been extensively employed in infectious disease detection because of its high sensitivity and high
specificity. It may provide accurate and sensitive detection results which could be references for making proper treatment decisions. However, complicated processes,human errors and contamination prevent its potential to be an in-filed or point-of-care
tool.
To overcome the above-mentioned disadvantages, four different integrated microfluidic systems were developed to detect pathogens including bacteria and viruses
from aquaculture, orchids and human joint fluidic sample directly are presented in this dissertation. By means of the knowledge from bio-Micro-Electro-Mechanical-System
(Bio-MEMS), the integrated microfluidic systems integrated several microfluidic components into one single biochip to realize to concept of Lab-on-a-Chip (LOC).Furthermore, the integrated control systems can be used to perform all manual steps in a traditional biomedical experiment automatically with less human intervention to save labor and cost. With Bio-MEMS, human error and contamination can also be reduced. The performances of the integrated microfluidic systems were validated in this dissertation and the results showed that the purposed microfluidic systems were highly specific with sensitivities as low as 20 copies of plasmid deoxyribonucleic acid (DNA) for loop-mediated isothermal amplification (LAMP) with fluorescent detection, 35 pg of plasmid DNA for LAMP with turbidity detection, 200 colony formation units (CFU) for polymerase chain reaction (PCR) with fluorescent detection and 100 CFU for nanogold probe detection, respectively. Moreover, in this study, the whole
experimental procedures including DNA/RNA/bacteria isolation, nucleic acid amplification by PCR or LAMP, nanogold probes detection or optical detection can
be performed on an integrated control system which consisted of a temperature control module, a liquid transportation module and an optical detection module
automatically within a short period of time. Based on the experiment data, these micro-total-analysis-systems (μTAS) are promising tools for in-field diagnosis or
point-of-care in the near future.

並列關鍵字

microfluidics ； loop-mediated isothermal amplification (LAMP) ； polymerase chain reaction (PCR) ； nanogold probe ； aquaculture pathogens ； Phalaenopsis orchid ； periprosthetic joint infection ； Micro-Electro-Mechanical-Systems (MEMS) ； micro-total-analysis-system (μTAS)

參考文獻

Escherichia coli, 2012, 31, 523-528.

50. M. Safavieh, M. U. Ahmed, M. Tolba and M. Zourob, Biosens Bioelectron,

68. A. K. Reddy, P. K. Balne, R. K. Reddy, A. Mathai and I. Kaur, J Clin

38. X. E. Fang, Y. Y. Liu, J. L. Kong and X. Y. Jiang, Anal Chem, Loop-Mediated

66. Y. Q. Wang, Z. H. Kang, Y. L. Gao, L. T. Qin, L. Chen, Q. Wang, J. K. Li, H.