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Specific Recognition Force, Dissociation and Thermodynamics of Single-pair Antibody-Antigen Interaction Using Atomic Force Microscopy
No full text生物分子藉由彼此間的辨識及鍵結過程在生命體中進行訊息傳遞、酵素催化及免疫保護等生化反應過程。為探討生物分子間的交互作用，本論文利用原子力顯微術進行免疫球蛋白及其抗體與升糖素及其抗體在不同酸鹼值、環境溫度及解離速度等條件下量測其解離力、分子間解離動力常數及熱力學參數。此結果可提供直接的證據去解釋分子間在更接近事實且不同環境下的行為。
本研究將免疫球蛋白及其抗體分別共價鍵結於原子力顯微鏡之探針與玻片並利用力曲線進行解離力在不同酸鹼值溶液下檢測，其結果顯示解離力在中性溶液擁有最佳鍵結強度。隨著增加或降低溶液之pH值皆會使生物分子間交互作用力下降。該結果可歸因於生物分子之構形在不同酸鹼值溶液下會產生不同形變，而造成分子間不完全的接合。此外，生物分子之官能基與生物功能皆會隨著溶液酸鹼值改變而改變，進而影響其抗體之辨識。而在分析的解離速率與自由能也證實了生物分子在中性溶液具有低解離速率及高能障等特點，使生物分子間能穩定的鍵結相較於在酸性或鹼性溶液下。在環境溫度變化對免疫球蛋白間鍵結的實驗結果中顯示分子間之交互作用力會隨著度增加而降低，此可歸因於溫度變化會引起分子構形改變造成生物分子間無法緊密皆合及溫度所造成的布朗運動碰撞分子間的鍵結，使鍵結強度降低。藉由改變環境溫度可檢測生物分子之解離熵及解離焓，其結果顯示解離熵及解離焓均會隨著溫度上升而增加，此說明了溫度會造成分子結構鬆散進而影響分子間的交互作用。
在升糖素及其抗體的研究中也顯示了生物分子間鍵結強度會隨著環境酸鹼值的增加或降低而下降，此結果說明了分子結構會受溶液中之離子影響進而使分子間交互作用力下降。此結果也可從所估算之自由能強度及解離速率得到印證。在溫度變化之實驗中發現分子間鍵結力會隨著溫度上升而線性下降。此可歸因於升糖素為構形簡單之胺基酸長鏈所組成，因此與溫度會產生強烈之關聯性。此外，在熱動力參數發現升糖素及其抗體在35 0C會急劇上升，此說明了分子構形及鍵結性質均在35 0C產生變化。
本論文已成功地利用原子力顯微術進行生物分子間鍵結強度、解離速率、活化能、解離熵及解離焓之量測。其結果可被應用在生物檢測晶片之生物分子解離環境及探討生命科學之依據。Molecular recognition and intermolecular binding are essential for implementing many biochemical and biological processes in living organisms. To further understand the molecular binding mechanisms, this study used atomic force microscopy as a force-based senor for investigation of the force strength between antibody and antigen complex in a single pair level with varied physiological (pH and temperature) and physical (loading rate) conditions. The results provided direct evidence of unbinding force, dissociation rate and thermodynamic parameters that explained intermolecular behavior of human IgG1/anti-human IgG1 complex and glucagon/anti-glucagon IgG complexes, respectively.
Mean measured forces of human IgG1 and its specific antibody system with pH-varied liquid environments showed a sharp decrease with a decrease of pH value (acidic environment), and a gradual decrease with an increase of pH value (alkaline environment) from a reference level at neutrality. This could have corresponded to the pH-induced change in conformational change and outer functional groups of amino acids which are protonated. As a result of change in pH environment of human IgG1/anti-human IgG1 complex, surface protonated properties and conformation weakened intermolecular force. Molecular dynamic behavior and free energy change were also contributed to a high probability of bonds breaking and a low magnitude of energy barrier when molecules were immersed in acidic or alkaline solution. Temperature-dependent unbinding force experiments were also carried out. The results showed that the unbinding forces decreased with an increase of environmental temperatures. This could be largely due to temperature-induced conformational change in volume expansion and strong Brownian motion. As a result, interaction forces decreased. Estimated dynamic behavior and thermodynamic parameters also showed weak interactions under high temperatures. This could have corresponded to looser molecular structure and weaker intermolecular interaction in high temperature, thereby increasing entropy and enthalpy.
The interaction between glucagon and anti-glucagon IgG with pH- varied liquid environment exhibited weak interaction force, low energy barriers and high dissociation rates under acidic and alkaline solutions. This indicated that molecular interactions turned out weak forces when pH values increased or decreased away from neutrality. Force measurement as a function of temperature exhibited a nearly linear decrease of force strength with an increase of temperature. This could have been attributed to molecular charge-free conformational changes, resulting in incomplete binding. The thermodynamic enthalpy and entropy for interaction showed an increase with increasing temperature.
Specific interactions of human IgG1/anti-human IgG1 pairs and glucagon/anti-glucagon IgG pairs have been successfully investigated by the atomic force microscope. With the use of an extended Bell and Evans model, the molecular dynamic behavior, free energy change and thermodynamic parameters can be obtained by varying physiological (pH and temperature) and physical (loading rate) conditions. The results provided directly evidence to explain the biological interactions.CONTENTS
ABSTRACT ............................................................................................I
摘 要 …………………………………………………………..III
謝 誌 ……………………………………………………………V
CONTENTS ………………………………………………………….VII
LIST OF TABLE X
LIST OF FIGURE XI
NOMENCLATURES XXII
CHAPTER 1 INTRODUCTION 1
1-1 Background 1
1-2 Literature survey for the molecules 5
1-2-1 Immunoglobulin molecule 6
1-2-2 Glucagon molecule 8
1-3 Literature survey for AFM in Biologists 9
1-3-1 Avidin-biotin interaction 9
1-3-2 Antibody-antigen interaction 11
1-3-3 Chemicals interaction 16
1-3-4 Other interaction 21
1-4 Motivation 31
CHAPTER 2 MATERIAL AND METHODS 34
2-1 Introduction of atomic force microscopy 34
2-1-1 AFM probe for sensing tip-sample interactions 36
2-1-2 Piezoelectric scanner for moving samples 39
2-1-3 Laser and photodiode for detecting cantilever deflection 41
2-2 AFM tip holder for liquid measurement 45
2-3 A novel temperature-controlled liquid cell system 46
2-4 In-situ temperature-detected biochip 48
2-5 Functional operations of AFM 52
2-5-1 Contact mode 52
2-5-2 Non-contact mode 55
2-5-3 Tapping mode 56
2-6 Calibration of cantilever sensitivity 59
2-7 Calibration of cantilever spring constant 60
2-8 Bio-molecule sample 63
2-8-1 Human IgG1 antigen and its anti-IgG1 antibody 63
2-8-2 Glucagon antigen and its anti-glucagon antibody 67
2-9 AFM tip functionalized with bio-samples 69
2-10 Force-distance curve 72
CHAPTER 3 ANALYSIS OF INTERMOLECULAR PARAMETERS 75
3-1 Discrimination of specific and nonspecific force curve 75
3-2 Dynamic model of intermolecular dissociation 81
3-3 Standard free energy for intermolecular dissociation 85
3-4 Enthalpy and entropy for Intermolecular dissociation 90
CHAPTER 4 HUMAN IgG1 — ANTI- HUMAN IgG1 INTERACTIONS 96
4-1 Dynamic response of IgG1/anti-IgG1 pairs to pH 96
4-1-1 pH-induced changes in the intermolecular forces 96
4-1-2 pH-induced changes in the dissociation rate and free energy 111
4-2 Temperature-induced changes of intermolecular behavior 116
4-2-1 Temperature-induced changes in the intermolecular forces 116
4-2-2 Temperature-induced changes in the dissociation rate and free energy 125
4-2-3 Temperature-induced changes in the thermodynamic parameters 128
CHAPTER 5 GLUCAGON — ANTI-GLUCAGON INTERACTIONS 135
5-1 Dynamic response of glucagon/anti-glucagon pairs to pH 135
5-1-1 pH-induced changes in the intermolecular forces 135
5-1-2 pH-induced changes in the dissociation rate and free energy 145
5-2 Temperature-induced changes of intermolecular behaviors 151
5-2-1 Temperature-induced changes in the intermolecular forces 151
5-2-2 Temperature-induced changes in the dissociation rate and free energy 160
5-2-3 Temperature-induced changes in the thermodynamic parameters 164
CHAPTER 6 CONCLUSION 172
6-1 Conclusion 172
6-2 Recommendation 172
REFERENCES 17
Force measurement of specific antibody-antigen interactions in pH-varied liquid environments
No full textA Free-Space Optical Communication System using Digital Signal Processors
No full text無線光通訊具有易架設、高傳輸速度、不易竊聽、不需經過頻譜申請等優點。本論文設計一個光收發模組來展示無線光通訊的鏈結。光收發模組包含一個發射器以及一個接收器，發射器是由雷射驅動電路、Thorlabs 850 nm 10mW 的雷射光源以及對準器所組成，接收器是由GaAs PIN 感光二極體、轉阻放大器和取決電路所組成。在傳送端，數位訊號經由電腦的編碼再經由類比數位轉換器的轉換來調變雷射光源。在接收端，光訊號由GaAs PIN 感光二極體所接收再經由類比數位轉換器的處理以還原傳送資訊。
本論文提出一個修正的調變及編碼方式來改善傳輸效能。利用前置位元以及漢明碼來達成同步與錯誤更正以改善位元串的正確性以及可靠性。本論文使用德州儀器定點TMS320C6416T 的數位訊號處理器以及高速數位類比轉換器AED-101提供80 Msps 的取樣率。本論文以紅外線通訊協定為基礎，距離一公尺、資料發射率1.152 Mbps，展示一無線光通訊的觀念。There are several advantages with optical communications in free space such as high data rate, dedicated communication and being difficult to eavesdrop. Free-space optical communications is an alterative to short-distance communcations. In this work, optical transceiver modules are designed to demonstrate an optical communications link. An optical transceiver module consists of a tansmitter and a receiver. The transmitter is composed of a driver circuit
and a Thorlabs L850P010 850 nm 10 mW diode laser with a monitor-diode feedback loop and focused by an external collimation tube. The receiver is composed of a TMC-8D41-000
GaAs PIN plus pre-amplifier photodiode and decision circuits. Digital signals from PC are encoded and sent to a digital-to-analog converter (DAC) to modulate the diode laser. At the receiver side, the light is received by the GaAs PIN photodiode, then sent to an analog-todigital
converter (ADC) for post processing. We propose a revised protocol to improve the performance in the conventional encoding scheme. The revised protocol has the capability
of synchronization and error correction. The preamble code and Hamming encoding scheme are used to improve the accuracy and reliability of the bit stream. The TI TMS320C6416T fixed-point digital signal processor (DSK) with an analog expansion daughterboard (AED-101) are used to provide maximum sampling rate up to 80 Msps. In this work, IrDA data rate of 1.152 Mbps is chosen as a reference for a communications link over one meter.Abstract i
Table of Contents ii
List of Figures iv
1 Introduction 1
1.1 Lightwave Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Optical Communication Systems . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Issues of Free-Space Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.5 Thesis Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2 Optical System 18
2.1 Laser Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2 Transmitter Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.3 Properties of PIN Photodiodes . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.4 ReceiverDesign . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.5 Hardware Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3 Digital Signal Processing 38
3.1 Hardware Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.2 Conventional Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.3 Revised Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4 Demonstration 53
5 Conclusions 5
Study on the Application of Micro-Bubble Drag Reduction Technique in ship Model
No full text節能減碳是目前世界各國的重要課題，在世界80%貿易運輸
依賴海運的情況下，若能減少船舶阻力，提高能源使用效率，對
節能減碳便能有不小貢獻。本研究為在船模上開發具有潛力與潔
淨的微泡減阻技術研究，開發出可應用於船模的微泡減阻技術。
本研究首先使用三種微泡產生方式來進行實驗： (1)針孔微
泡產生方式、(2)滷化技術及(3)透氣材微泡產生方式。針孔微泡
產生方式會產生氣體堆積現象，沒有使用於減阻實驗。將滷化技
術及透氣材於拖曳水槽中進行平板減阻實驗，實驗結果發現透氣
材噴氣時的平板減阻效果明顯優於滷化技術。在拖曳速度3.6m/s
時有最佳減阻效果31.6%。因此本研究選擇了透氣材來應用在船
模上，在船模的底部分別裝設三塊透氣材。實驗的結果顯示三塊
透氣材共同噴氣時有最佳減阻效果7.2%。為了增加微泡覆蓋面
積，增設了球艏噴氣裝置，實驗結果發現球艏噴氣裝置與船底透
氣材共同噴氣有最佳減阻效果9.6%， 證明了增加微泡覆蓋面積
可增加減阻效果。Energy saving and carbon reduction is one of the most important
research in the world. With more than 80% of international trade in
goods being carried by sea, reducing the resistance and raising the
efficiency of ship propulsion is a great contribution to Energy
saving and carbon reduction. The major target of the research is to
develop the most promising and cleaning micro-bubble drag reduction
technique in ship model. It is expected to develop a micro-bubble drag
reduction technique which can be applied to the full scale ship.
There are three micro-bubble generated methods used in this
research. It includes pinhole, air/water mixer and porous plates.
The pinhole method was not used in the drag reduction test because
the air injected by pinhole is accumulated and form large bubble.
The air/water mixer and aluminum porous plates were used to
reduce the drag of flat-plate in towing tank. The drag reduction rate
of aluminum porous plates was better than air/water mixer as
shown from the test results. The 31.6% drag reduction was obtained
by the micro-bubble generated by Aluminum porous plates when
the model velocity is 3.6m/s.
Thus the aluminum porous plates have been selected to apply
on the ship model to verify the drag reduction effect. There are
three aluminum porous plates have been installed on a flat bottom
of ship model. The best drag reduction rate of 7.2% was found in
this test. For better micro-bubble coverage, the bulb bow injector
was set up on the ship model. The 9.6% drag reduction was
obtained when both of bulb bow injector and aluminum porous
plates operated at the same time. The test results prove that the
better micro-bubble coverage bring the better drag reduction rate
Atomic force microscopy: Determination of unbinding force, off rate and energy barrier for protein–ligand interaction
No full textAtomic force microscopy: Determination of unbinding force, off rate and binding energy for protein-ligand interaction.
No full textCalculation of BR([overline B]0-->Lambdac++[overline p]) in the perturbative QCD approach
No full text
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