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  • 學位論文

生物感測分析系統之開發研究---應用於微生物與病毒之檢測

Biological sensing system of Research and Development---Application microbiology and virus detection

指導教授 : 陳建源

摘要


本論文整合三種不同機制的感測元件(表面聲波、石英微質量天平、表面電漿子共振),應用高分子聚合物 (聚對二甲苯;parylene)改質基材或是電極表面,以獲得具有低介電、高阻抗、化學安定性佳、具生物相容性、低液相插入損失、可進行化學修飾與具有做為封裝介面層潛力等特性的表面。分別於其電極表面固定具有專一性免疫反應的生物分子做為感測元件(分別為PSAW、PQCM、PSPR),並成功應用於微生物與病毒等之偵測。 PSAW於電極上鍍聚對二甲苯(厚度1微米)系統震頻達314.5MHz的工作靈敏區。利用PSAW直接於液態培養基中量測E.coli生長,可以順利量測其生長趨勢。達生長平緩期時PSAW約有3000Hz移差(O.D=2.5)。固定抗體分子時100 ng μl-1附載造成約120Hz移差,可以抓附600~800 ng μl-1二次抗體,其頻率移差約500Hz。 PQCM具有較傳統QCM高約三倍的質量靈敏度(mass sensitivity)。PQCM應用於腸病毒71型較ELISA法靈敏度高約105倍,與CB3型偵測較ELISA法靈敏度高約104倍,其特異性訊號對非特異性訊號比值約4倍。 應用PQCM偵測微生物生長時,因parylene具有高阻抗特性,PQCM可以直接浸入於液體環境中進行偵測工作。且溶液特性(酸鹼值、導電度、黏滯度等)之影響性遠小於(小於百分之一)菌體附著於電極表面所引起之質量附載效應。 PQCM對菌體最小靈敏度為102 cells ml–1(移差約 2 × 103 Hz.)直接運用於微生物生長偵測(大腸桿菌生長O.D 2.5 時移差約300KHz)。適合於液相環境中進行微生物生長之長期監測。 兩步驟自組裝單層法改質之PSPR表層,表面具有之–NH2官能基能夠以電性吸引力抓附奈米金顆粒成為單層膜。其SPR特性吸收峰 (中心峰值為518-523 nm) 與均勻分散於液相中之奈米金粒子相比完全重合(中心峰值517-523 nm)。原子力顯微鏡分析證實其為均勻分散狀態, PSPR製程比化學法有更佳的單顆粒分散狀態。經由電漿氣體混合比例來控制奈米金顆粒之間距離,取代目前主流的共聚合法。與PDMS所構成之流道進行封裝,使整個環境一直維持於液相中,注入奈米金顆粒進行自組裝可以避免氧化發生,且製程適合整併於微機電製程中,能夠同時提供封裝與感測器需求。

並列摘要


The PSAW、PQCM and PSPR sensor was fabricated by depositing di-para-xylene (parylene) over the entire surface of a SAW、QCM、SPR sensor through a chemical vapor deposition (CVD) process. The parylene coated sensor exhibites the characteristics of good chemical stability, low insertion loss and frequency drift the sensor with silver or aluminum electrodes are usable, and the capability of immobilizing chemicalligand. A two-step biological functionalized process was designed for the parylene coated sensor to immobilize antibody. The surface of electrodes were fixed with affinity biological molecules as sensing elements, and successfully applied to detect microbes and viruses. When the coating thickness of the PSAW sensor reach 1 μm, the frequency of system was level-up to 314.5 MHz of the sensitive work areas. The operation of the PSAW sensor in the liquid environment, the resonance frequency of the sensor set in the medium of a cultivation flask shifted in response to the microbial population(3000 Hz at stationary phase). The Immuno-globin G fixed at PSAW surface produce the frequency shift of 120 Hz at concentration around 100 ng μl-1, can capture secondary antibody of 600~800 ng μl-1 (the frequency shift of around 500 Hz). The PQCM exhibites a sensitivity of around 3 times than the basal QCM. The sensitivity achieved on enterovirus measurement using this approach is approximately five-folds greater than the ELISA method, The specific signal of this approach is around 4 times of the non-specific ones. An electrically insulated film of parylene on the QCM sensor enabled the operation of the sensor in the liquid environment, and the resonance frequency of the pQCM sensor set in the medium of a cultivation flask shifted in response to the microbial population. The effects of pH, conductivity, and viscosity of the medium on the frequency shift of the pQCM sensor were investigated. Ignorable responses (less than 1 % at 103 cells) were obtained during an incubation cycle. The detection limit of the pQCM sensor was identified as 102 cells ml–1 with a frequency shift of around 2 × 103 Hz. The cell numbers of E. coli cultivated in both the YEM medium and whole milk were detected. A satisfactory correlation (r2 = 0.95) was obtained between the cell number and the response of the pQCM sensor. Experimental results suggest that the pQCM described here is applicable to the continuous long-term detection of microbial populations during a fermentation process. Two step self assembled monolayer of the PSPR sensor, with the surface laminated functional groups able to grasp power of attraction of nanometer gold particles become a single-layer film. Absorption peak of its SPR (Centre for peak 518-523) and uniformly dispersed in the liquid phase of the nano-gold compared to complete coincidence (center wavelength 517-523). Atomic force microscope analysis confirmed its status as evenly dispersed. The results was demonstrated, the PSPR sensor has better mono-dispersion state than chemical process. By the mixed gas flow ratio of plasma to control the distance between nano-gold particles, replace the current mainstream copolymer process. The flow channel(PDMS)is packaged with the PSPR sensor, cause that the whole environment of sensor surface has been maintained in the liquid. Then, the nano-particles has injected into flow channel to become SAM layer, avoided oxidize of gold nano-particle. The whole process of PSPR is suitable integrated into MEMs process.

參考文獻


1 Allara, D.L. (1995). Critical issues in applications of self-assembled monolayers. Biosens. Biolectron. ,10, 771-783.
2 Amador, S.M., Pachence, J.M.,Fischetti, R., McCauley, Jr. J.P., Smith, III, A.B. & Blasie, J.K.(1993). Use of self-assembled monolayers to covalently tether protein monolayers to the surface of solid substrates. Langmuir, 9, 812-817.
3 Frisbie, C.D., Rozsnyai, L.F., Noy, A., Wrighton, M.S. & Lieber, C.M. (1994). Functional group imaging by chemical force microscopy. Science, 265, 2071-2074.
4 Florin, E.-L., Moy, V.T. & Gaub, H.E. (1994). Adhesion forces between individual ligand-receptor pairs. Science, 264, 415-417.
5 Gopel, W. (1989). Solid state chemical sensors: Atomistic models and research trends. Sens. Actuators,16, 167-193.

被引用紀錄


林宗煌(2010)。多通道生醫電阻抗分析系統研製〔碩士論文,淡江大學〕。華藝線上圖書館。https://doi.org/10.6846%2fTKU.2010.01102
史佩珊(2008)。使用經聚對二甲苯修飾之表面電漿子共振系統開發A型流感病毒免疫感測器之研究〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2008.10447
劉怡妏(2008)。使用經聚對二甲苯修飾之壓電石英晶體微天平系統開發肺結核分枝桿菌核酸感測器偵測之研究〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2008.10432

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