晶片載具型即時偵測同步定量聚合酶連鎖反應器開發

Development of Q-PCR on a Chip System for DNA Detection and Quantification

陳煌仁(Huang-Jen Chen)

指導教授 ： 李達生

國立臺北科技大學/機電學院/能源與冷凍空調工程系碩士班/碩士(2008年)

摘要

即時偵測同步定量聚合酶連鎖反應(Real-Time PCR)技術，為近年來最重要的分子生物學的重要技術之一。此技術，主要需結合溫控機構(Thermal Cycler)與螢光分析儀(Fluorimeter)，在溫控過程進行DNA體外增生，同時監控增生過程中DNA嵌合螢光(Labeling Dye)螢光強度的變化，進行定量分析，實現此技術之儀器被稱為即時偵測同步定量聚合酶連鎖反應器(Real-time PCR machine)。本研究嘗試結合晶片實驗室(Lab on a chip)與即時偵測同步定量聚合酶連鎖反應器，希望提高定量分析實驗精確度或減少分析的時間。
而本研究的PCR單區晶片是採用微機電系（Micro-Electro-Mechanical-System）以研發微小型的加熱器與溫度感測器，以應用於微小型PCR 系統中，並成功地設計出一可搭配微製程晶片的溫度控制系統，以便實際應用於DNA的複製與檢測 上。並配合ANSYS 工程軟體分析反應腔室內檢體溫度場和不同體積下的熱傳遞分析，使得反應腔室內的檢體能快速達到均溫，以提高 PCR的放大效率和成功率。
另外，本研究開發螢光分析儀是利用商用規格之分光儀，配合共焦光學檢測設計，經測試其檢測下限與動態範圍均超過國際大廠出產即時偵測同步定量聚合酶連鎖反應器配置之螢光檢測儀，且此一架構可實現400~800 nm連續頻譜螢光檢測。並針對晶片機構定位問題再進行改善，並以70度斜角收光的設計來改善雜光干擾問題，以提高其檢測性能。而聚光器（Compound Parabolic Concentrator）的新聚光技術設計，可以更有效提高螢光分析儀的螢光偵測值。螢光分析儀初步達到HBV DNA螢光偵測分析，並能有效收集頻譜數據，用於定量分析，未來將進行可靠度評估,以利發展出低成本高規格的商用機台。

關鍵字

即時偵測同步定量聚合酶連鎖反應 ； 螢光分析儀 ； 晶片實驗室

並列摘要

Real-Time Polymerase Chain Reaction (PCR) technique is one of the most important technologies for detecting biomolecμles in recent years. The technique is requires a thermal cycling process to amplify target DNA strand in vitro, intercalation of labeling into the amplified DNA strand, and detection of the variation of fluorescence intensity of labeling dye during the amplification of DNA strand a Fluorimeter. Then, the concentration of target DNA strand can be determined from the variation of fluorescence intensity. An instrument that performs such a technique is called a real-time PCR machine. This project aims to develop a real-time PCR machine that can amplify DNA strand in a microfluidic chip and simμltaneously detect the variation of fluorescence intensity of sample solution in chip for quantitatively determining the concentration of target DNA strand. Since the amplification of DNA strand is conducted in a microfluidic chip, it coμld improve the sensitivity of quantitative determination of target DNA concentration and reduce the duration for performing such a real-time PCR process.
The PCR chips of this study is to develop micro temperature sensors and heaters, which are combined with a suitable control circuit to be used in micro PCR system, based on MEMS(Micro-electro-mechanical system) technologies. The integrated system including the PCR chip by MEMS and the control circuit applies for amplification and detection of DNA. Subsequently, we analyzed the temperature field of the reaction chamber by numerical analyses software, ANSYS. Two of advantages in this technique are displayed. One is the analysis of heating transference in different volume and the other is increasing the speed of cooling. Taken together, our study can significantly improve the efficiency and the success of PCR.
Otherwise, this reaserch exploit the flurimeter which a commercial spectrometer with a confocal design for optic path. After performing a calibration test, the detection sensitivity and range of spectrometer outperforms the Fluometer used in the real-time PCR machine provided by major pharmaceutical companies. The proposed spectrometer can perform the continuous detection of fluorescence of which the wavelength ranges from 400 to 800 nm. And focus on the improvement of detection sensitivity of platform by modifying the positioning mechanism of chip and by reducing the background optical noise through a 700 alignment between light source and the light receiving fiber. the design of Compound Parabolic Concentator coμld enhance the flourencence detecting value more efficacious.Here a new technique was established in this study.The outcome of this study has constructed a real time pcr machine for DNA amplification and quantification.In the future, this instrument can be commercialezed for a low cost and high accuracy DNA quantification solution.

並列關鍵字

Real-Time PCR ； Fluorimeter ； Lab on a chip

參考文獻

2. R. K. Saiki, S. Scharf, F. Faloona, K. B. Mμllis, G. T. Horn, H. A. Erlich and N. Arnheim, ”Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia,” Science, Vol. 230, pp. 1350-1354,1985.

4. M. A. Northrup, C. Gonzalez, D. Hadley, R. F. Hills, P. Landre, S. Lehew, R. Saiki, J. J. Sninsky, R. Watson and R. Watson, “A MEMS-based miniature DNA analysis system,” Proceedings of the 8th International Conference on Solid-State Sensors and Actuators and Eurosensors IX Stockholm, Sweden, pp. 25-29, 1995.

5. N. I. Maluf, G. T. A. Kovacs and D. Gee, “Recent Advances in Medical Applications of MEMS,” WESCON/96, pp. 60-63, 1996.

8. A. M. Chaudhari, T. M. Woudenberg, M. Albin and K. E. Goodson, “Transient Liquid Crystal Thermometry of Microfabricated PCR Vessel Arrays,” Journal of Microelectromechanical Systems, Vol. 7, No. 4, 1998.

9. Z. Zhan, C. Dafu, Y. Zhongyao and W. Li, “Biochip of PCR Amplification in silicon,” 1st Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine and Biology, United Kingdom, pp. 25-28, 2000.