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研究生:陳姿穎
研究生(外文):Tzu-YingChen
論文名稱:交流電動力進行血液中致病菌濃縮與其應用於快速檢測金黃色葡萄球菌
論文名稱(外文):AC Electrokinetic Concentration of Pathogen from Blood for Rapid Detection of Staphylococcus aureus
指導教授:張憲彰
指導教授(外文):Hsien-Chang Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:奈米科技暨微系統工程研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:74
中文關鍵詞:非均勻極化交流電滲介電泳拉曼光譜金黃色葡萄球菌
外文關鍵詞:asymmetric polarization AC electroosmosis (A-P ACEO)dielectrophoresis (DEP)Raman spectroscopyStaphylococcus aureus
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近年來,致病菌的分離和濃縮技術在臨床樣本檢測的重要性逐漸增加。根據美國疾病與預防控制中心之國家院內感染監視系統及流行病原監控的統計資料,顯示金黃色葡萄球菌(Staphylococcus aureus; S. aureus)在醫院中是最常見的血管通道感染的微生物,故快速檢測成為治療上的關鍵點。然而現階段傳統的分離方法不僅耗時且需要繁複的操作流程,因此在這研究中,我們發展以微流體為平台的濃縮晶片,其結合介電泳與非均勻極化交流電滲流來誘導溶液產生對流以達到在數分鐘內從血球中快速、選擇性與擴大範圍地濃縮細菌,以便於後續的檢測與分析。實驗結果顯示,在一個理想的電場梯度(直流偏壓0.5 Vpp提供在外圈電極所施加的交流電壓12 Vpp上與內圈電極所施加的交流電壓10 Vpp上)與最佳頻率1.3 kHz的條件,使金黃色葡萄球菌在交流電滲流產生的阻力與正介電泳力交互作用下,可以從紅血球中被分離出來並且濃縮在中央電極的停滯點,具有最佳的分離濃縮效果。反之,紅血球被誘導出強的負介電泳力,且當此力大於交流電滲流誘導出的阻力時,紅血球將被排斥在電極邊緣。藉由此方法,金黃色葡萄球菌可以被捕捉在中央電極的表面,以利於拉曼光譜的檢測,而紅血球則會被排斥在電極外以減少檢測細菌上的干擾,使得細菌濃度為5×10^6 CFU/mL與血球混合後在拉曼光譜中1521 cm-1這個峰值仍然可以被辨識出來。此電動力學之晶片成功於數分鐘內將金黃色葡萄球菌從血球中分離出來並同時濃縮在檢測區。此結果與需數天培養的傳統法相比較,我們的檢測平台可以快速(6-10分鐘)地分離濃縮致病菌,相信本技術平台亦可延伸至其它微生物(如大腸桿菌等)檢測等應用。
Recently, separation and concentration of pathogens before identifying them in clinical settings becomes increasingly important. According to the statistics of National Nosocomial Infection Surveillance System in Centers for Disease Control and Prevention and Surveillance and Control of Pathogens of Epidemiologic Importance, Staphylococcus aureus (S. aureus) is the most common microbial infection of bloodstream in hospitals. Rapid detection is the key point for treatment, but traditional separation methods are time-consuming and require several complicated steps. Therefore, in this research, we developed a microfluidic platform for rapid, selective and long-range concentration of bacteria by combining dielectrophoresis (DEP) with asymmetric polarization AC electroosmosis (A-P ACEO) to selectively concentrate pathogens from blood cells for follow-up detection and analysis. The experimental results show that S. aureus can be selectively separated from blood cells and concentrated at the stagnation point on the central electrode. This is done by coupling ACEO and positive DEP under an optimal electric field gradient as a DC biased voltage of 0.5 V was added to AC voltages of 12 Vpp (outer) and 10 Vpp (inner) at an optimal frequency of 1.3 kHz after diluting blood cells 100-fold. In contrast, a strong negative DEP force was induced in blood cells. If this induced negative DEP force is larger than the ACEO flow induced drag force, blood cells would be excluded at the edge of the electrode. By this way, S. aureus can be trapped on the central electrode surface for the detection using Raman spectroscopy and blood cells can be excluded to reduce the interference in bacterial detection. Raman spectra with a peak at 1521 cm-1 can also be identified using the S. aureus concentration of 5×10^6 CFU/mL for a mixture of the bacteria and blood cells. The AC electrokinetic chip successfully separates S. aureus from blood cells and S. aureus is simultaneously concentrated; thereby, the waiting time is reduced. Compared to traditional selective culture, our simple chip demonstrated an extremely fast (6-10 min) and selective isolation and concentration capability that decreased the bio-target detection time from days to minutes. Our AC electrokinetic chip can be extended to other bacterial detection and identification.
Abstract I
摘要 II
誌謝 III
Contents IV
List of Figures VI
Chapter 1 Introduction 1
1.1 Background and Motivation 2
1.2 Traditional Detection 4
1.2.1 Gram Stain 5
1.2.2 Biochemical Detection 7
1.2.3 Fatty Acid Profiles 9
1.2.4 Immunoassay Technique 9
1.2.5 Real-Time PCR 11
1.2.6 Flow Cytometry 12
1.3 Raman Spectroscopy 14
1.4 Micro-electro-mechanical System Technology 18
1.5 Theory of Electrokinetics 20
1.5.1 AC/DC Electroosmotic Flow (AC/DC EOF) 23
1.5.2 Dielectrophoresis 29
1.6 Literature Review 31
1.7 Research Configuration 36
Chapter 2 Materials and Methods 38
2.1. The Working Principle of AC Electrokinetic Chip (ACEK-chip) 38
2.2. Chip Design and Micro-Fabrication 39
2.3. Sample Preparation 42
2.4. System Configuration 44
Chapter 3 Results and Discussions 46
3.1. AC Electrokinetic Concentration of Particles 46
3.2. AC Electrokinetic Concentration of Staphylococcus aureus 51
3.3. Optimal Conditions of AC Electrokinetic Concentration from Blood 55
3.4. Detection Limit of Staphylococcus aureus for Raman Signal 58
Chapter 4 Conclusion 65
References 67
Appendix 71
Personal Information 74
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