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研究生:廖佳真
研究生(外文):Chia-Chen Liao
論文名稱:利用QCM生物感測器偵測生物素濃度之研究
論文名稱(外文):Measurement of the Biotin Concentration Using QCM Biosensors
指導教授:張耀仁張耀仁引用關係
指導教授(外文):Yaw-Jen Chang
學位類別:碩士
校院名稱:中原大學
系所名稱:機械工程研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:69
中文關鍵詞:電鍍免疫感測器石英微量天秤(QCM)自聚性金屬蛋白質晶片硝化纖維晶片
外文關鍵詞:ElectroplatingImmunosensorsSelf-assembled metallic protein chipNitrocellulose (NC) chipQuartz crystal microbalance (QCM)
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石英微量天秤(Quartz Crystal Microbalance)是利用質量負載(Mass-Loaded)原理,藉由生物辨識層的薄膜,可以將被偵測的蛋白質(抗原或抗體),利用分析儀器準確辨識出濃度。本論文主要研究出一新型的QCM生物感測器,利用電鍍技術將鎳鈷合金沉積於基頻為10MHz的AT-cut切角的石英晶體表面上。由於鎳鈷金屬會與修飾過His-tagged後的蛋白質產生專一性的親和性吸附,運用此免疫機制,將生物素(Biotin)固定於QCM鎳鈷金屬層上,經由阻抗分析儀器量測頻率的結果,可以發現不同的生物素濃度對應到不同的頻率下降量。除此之外,在螢光檢測方面利用硝化纖維薄膜(NC membrane)與QCM生物感測器以免疫學分析方法以及高靈敏度LSIV機台檢測螢光影像,並驗證此QCM 生物感測器確實具有生物素吸附之效果。
此生物感測器的優點包含以下幾點: (1)檢測物質不受限制,偵測方式主要根據QCM 振盪器之原理,進行頻率量測; (2) 減少檢體的浪費,本研究每點樣本僅需6μl即可進行實驗; (3) QCM 可以重複使用,使用經稀釋過後的3%硝酸可將鎳鈷金屬層移除掉,可以進行重複實驗; (4) 成本低廉,使用現今純熟的電鍍技術將可大量製造生物感測器,以減少成本支出。
Quartz crystal microbalance (QCM) which makes use of the mass-load theorem by biologic recognition component can detect protein concentration accurately. This paper presents a novel Ni-Co alloy coated QCM biosensor fabricated by electroplating nickel and cobalt on the surface of 10-MHz AT-cut QCM. The biotin was immobilized on the coated surface by the affinity mechanism of Ni-Co alloy. Then, the immunoreactions can be performed on the QCM biosensors. The results show that the Ni-Co alloy coated QCM biosensor can provide specific binding with His-tagged biotin. For different biotin concentrations, the QCM produced different frequency shifts. In addition, fluorescent detection was also performed to show that the coated Ni-Co film has better nonspecific binding ability than nitrocellulose (NC) membrane to immobilize His-tagged proteins for target analyses. The advantages of this biosensor include (1) label-free detection based on the principle of QCM resonator; (2) minimal sample consumption (6 l of biotin each drop used in this study); (3) QCM is reusable the coated Ni-Co alloy can be removed by 3% nitrate acid, so that the QCMs can be re-electroplated as biosensors; (4) inexpensive fabrication process, the electroplating technique is well-developed and the equipment and materials are very cheap. Hence, the mass production of QCM biosensors would be available.
摘要 I
Abstract II
誌謝 III
Contents IV
List of Figures Ⅵ
List of Tables Ⅸ
1 Introduction 1
1-1 Introduction to Biochip 1
1-2 Technique of Immunoassay 2
1-3 Purpose of Research 3
1-4 Structure of Research 3
1-5 Overview 5
2 Literature Review and Preliminaries 6
2-1 Literature Review 6
2-1-1 Development of Biosensors 7
2-1-2 Types of Biosensors 7
2-2 Quartz Crystal Microbalance 9
2-2-1 The Piezoelectric Effect of Quartz Crystal Microbalance 11
2-2-2 The Principle of Quartz Crystal Microbalance 16
2-2-3 Applications of QCM Sensors 19
2-3 Self-assembly Metallic Chip 21
2-3-1 Theory of Self-assembly Metallic Chip 22
2-3-2 Applications of Self-assembly Metallic Chip 24
3 Manufacturing Procedure of QCM Biosensors 25
3-1 Materials and Equipment 25
3-1-1 Materials 25
3-1-2 Equipment 27
3-2 Nickel and Cobalt Electroplating 28
3-2-1 Fundamental Principles 28
3-2-2 Deposition Efficiency of Plating 29
3-2-3 Manufacturing of Self-assembly Metallic Layer 30
3-2-4 Manufacturing the Electrode of Nickel and Cobalt 31
3-3 Preparation of Nitrocellulose Solution 36
4 Experimental Results and Discussions 40
4-1 The Principle and Method with Measurement of Protein 40
4-1-1 The Method of QCM Measurement 40
4-2 Immunofluorescence Detection 43
4-2-1 QCM Immunofluorescence 43
4-2-2 Nitrocellulose Membrane Immunofluorescence 45
4-3 Results and Discussions 46
5 Conclusions and Future Prospects 56
5-1 Conclusions 56
5-2 Future Prospects 57
Reference 58



List of Figures
Figure 1.1: The flow chart of investigative of organization ....................................4
Figure 1.2: The flow chart of QCM biosensors .......................................................4
Figure 1.3: The flow chart of immunoreactions ......................................................4
Figure 2.1: Shows schematic layout of a biosenosr .................................................6
Figure 2.2: The Schematic about various types of sensors sensitivity ....................8
Figure 2.3: Photograph of a quartz crystal microbalance (QCM) .........................10
Figure 2.4: A variety of quartz cutting angle ......................................................... 11
Figure 2.5: The temperature characteristic of quartz .............................................12
Figure 2.6: The stress in different directions will cause the direction of electric
dipole change .......................................................................................14
Figure 2.7: Polarization phenomenon of resonance ...............................................15
Figure 2.8: Additional electric field and quartz vibration ......................................15
Figure 2.9: The effect of mass-loaded ...................................................................19
Figure 2.10: Metal chelate compound combined with His-tag ..............................23
Figure 3.1: Electroplating reaction by electrochemical principle ..........................30
Figure 3.2: The structures of commercial QCM products .....................................31
Figure 3.3: The flow chart of QCM oscillator cleaning steps ................................32
Figure 3.4: The procedures of anodic process .......................................................33
Figure 3.5: The flow chart of biochemical experimentation and fabrication
process..................................................................................................34
Figure 3.6: Electroplating equipment ....................................................................35
Figure 3.7: GeneTAC LSIV scanner .....................................................................35
Figure 3.8: The schematic of mixture after adding GPTS solution .......................37
Figure 3.9: The schematic of mixture (NC powders 0.15g) adding into the
acetone and GPTS solution ..................................................................38
Figure 3.10: Programmable spin-coater .................................................................38
Figure 3.11: Programmable oven ...........................................................................39
Figure 4.1: The flow chart of experimental measurement .....................................40
Figure 4.2: Schematic diagram of experimental apparatus ....................................42
Figure 4.3: Schematic diagram of the actual measurement ...................................42
Figure 4.4: The instrument fixture of QCM ...........................................................43
Figure 4.5: Immunoassay experiment on biotin-dropped QCM biosenosr ............44
Figure 4.6: The flow chart of QCM immunofluorescence steps ...........................44
Figure 4.7: Immunoassay experiment of biotin-dropped NC chip ........................46
Figure 4.8: The flow chart of NC chip immunofluorescence steps .......................46
Figure 4.9: The adsorption concentration and frequency shift of
5×His-biotin product ..........................................................................47
Figure 4.10: The resonance frequency at 5×His-biotin diluted in 10×
measured by Impedance Analyzer (before) .......................................48
Figure 4.11: The resonance frequency at 5×His-biotin diluted in 10×
measured by Impedance Analyzer (after) ..........................................48
Figure 4.12: The resonance frequency at 5×His-biotin diluted in 50×
measured by Impedance Analyzer (before) .......................................49
Figure 4.13: The resonance frequency at 5×His-biotin diluted in 50×
measured by Impedance Analyzer (after) ........................................49
Figure 4.14: The resonance frequency at 5x His-biotin diluted in 100x
measured by Impedance Analyzer (before) .......................................50
Figure 4.15: The resonance frequency at 5x His-biotin diluted in 100x
measured by Impedance Analyzer (after) ..........................................50
Figure 4.16: The resonance frequency at 5x His-biotin diluted in 150x
measured by Impedance Analyzer (before) .......................................51
Figure 4.17: The resonance frequency at 5x His-biotin diluted in 150x
measured by Impedance Analyzer (after) ..........................................51
Figure 4.18: The resonance frequency at 5x His-biotin diluted in 200x
measured by Impedance Analyzer (before) .......................................52
Figure4.19: The resonance frequency at 5x His-biotin diluted in 200x
measured by Impedance Analyzer (after) ..........................................52
Figure 4.20: The NC chips .....................................................................................53
Figure 4.21: The QCM biosensors .........................................................................53
Figure 4.22: Comparison of fluorescence intensities at 5×His-biotin
experiments between QCM biosenosrs and NC chips .......................54
Figure 4.23: The image of fluorescent scanning ....................................................54


List of Tables
Table 2.1: The characteristics of transducer mode ...........................................9
Table 2.2: The equivalent of metals ...............................................................22
Table 2.3: Types of metal affinity chromatography .......................................23
Table 2.4: Binding capacity and selectivity of metals ...................................23
Table 3.1: The composition of plating bath ...................................................26
Table 3.2: The coating condition of NC solution ...........................................37
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