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研究生： 李昱廷
Li, Yu-Ting
論文名稱： 金奈米粒子在生物晶片上之修飾及其在去氧核糖核酸及蛋白質親和作用力辨識之應用
Gold Nanoparticle (AuNP) Modification on Biochips and Their Applications for the Detection DNA and Protein Affinity Interactions
指導教授： 陳淑慧
Chen, Shu-Hui
學位類別： 博士
Doctor
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2010
畢業學年度： 98
語文別： 英文
論文頁數： 85
中文關鍵詞： 金奈米粒子 、生物晶片 、血紅素蛋白質 、去氧核糖核酸
外文關鍵詞： heme protein, biochip, gold nanoparticle, DNA
相關次數： 點閱：96  下載：2 
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    • 生物晶片可幫助科學家完成一次大量的篩檢實驗且可減少所需成本，因此它在基因學、蛋白質體學以及疾病診斷研究上已變成一個極為重要的實驗平臺。然而，結合奈米科技的優點更能輔助提升現今生物晶片的表現效能。本篇論文中，將分成三個主題來探討利用金奈米粒子經化學修飾方式去改善實驗偵測的專一性及靈敏度。

    為了提升生物晶片的靈敏度，增加表面積以固定更多的生物探針是最主要的方法，由於金奈米粒子本身具有特殊的光學性質、結構上及官能化後可與生物分子相容之優點。因此在第一個研究中，我們使用金奈米粒子去發展出一個簡便的微流體生物分析晶片，並利用結構改變造成螢光抑制效應，來進行DNA序列的專一性辨識實驗。在這方法中，一個自組裝的金奈米粒子單層先被修飾在微流體晶片管道的管壁上，接著DNA藉由修飾硫醇基的一端接到金奈米子表面，另一端修飾螢光染料也吸附到金奈米子上。這將會在金奈米粒子表面形成拱型結構，進而促使螢光染料的螢光強度被金奈米粒子所抑制。當與互補的DNA進行雜合反應後，探針結構會再度改變，使得螢光團離開粒子表面而讓螢光強度恢復，以此來當作待測基因的感測方法。我們所提出的這個分析方式首先是以一對合成的互補及非互補DNA序列進行測試。後來也將此方式應用到登格熱病毒經PCR放大後的樣品，並以腸病毒來當作對照實驗，結果發現這個分析方式可以非常專一的辨識到待測基因。此外，使用這方法可將去雜合、雜合以及偵測待測樣品的步驟都在晶片的同一個管道內進行。
    在近幾年內，血紅素蛋白質被發現是可以穩定金奈米粒子的連接試劑。因此，在第二個研究中，我們使用血紅素蛋白質來在晶片上製備出三維的多層金奈米粒子，並比較發現它可以比其它聚合物連接劑修飾到更高的密度，並利用在分析應用上。使用ESCA測量，一個較低的Au(0)價氧化態及多個N(1s)化學態在金奈米粒子接到血紅素蛋白後產生明顯的變化，但接到非血紅素蛋白時，卻沒有明顯的改變。因此，我們提出這個穩定金奈米粒子的原因是在於金奈米粒子與血紅素結構的結合所導致。我們也提出這樣的結合是來自於蛋白質的結構改變使得血紅素結構曝露出來，並藉由蛋白質上的其它官能基將金奈米粒子與血紅素結構拉到極近的距離。接著我們將一段雌激素反應元素(ERE，雙股DNA)接到血紅素蛋白修飾過的晶片上。在此我們也利用了雜合誘發螢光恢復的方法來確定雙股探針的形成，並接續的在同一晶片上進行DNA/蛋白質的結合檢測。這個以金奈米粒子接上ERE所發展出來的晶片，針對細胞萃取物中的兩個轉譯因子異構物(雌激素受體α及雌激素受體 β)，藉由ERE的結合而呈現出極佳的靈敏度及專一性，並且減少反應所需的試劑與時間。

    PDMS具有許多的優點，例如光學穿透性、生物相容性以及化學穩定性等，都是讓它成為極具吸引的應用材質。然而，它本質上的疏水性質卻限制了它的應用性。在第三個研究中，我們使用polyelectrolyte的多層修飾方法去將PDMS的表面性質從疏水性改變成親水性，並且經由-NHS官能基與蛋白質上的-NH2形成共價鍵結而製備出多層的Mb-AuNPs。經由水柱角測試，未修飾的PDMS所測得之水柱角約112o， 但經過polyelectrolyte的多層修飾後，其水柱角具減少到10oo以下。接著我們利用固定ERE探針在Mb-AuNPs多層修飾上以製作出一免疫分析工具，並針對雌激素受體α利用酵素結合免疫吸附法在此分析工具上製作出檢量線。此外，我們也設計了兩種競爭性測試去證實雌激素受體與雌激素反應元素間的專一親和性。

    Biochips enable researchers to perform a high throughput screening at low cost and thus, have become an important platform in genomics and proteomics research as well as in disease diagnostics. There are, however, rooms to improve the performance of current biochips by taking useful advantages of nanotechnology. In this dissertation, three topics on the use of gold nanoparticles to improve the detection specificity and sensitivity via chemical modifications will be described.
    Increasing the surface area for probes immobilizing in biochip is an important factor to enhance sensitivity. Due to inherent advantages associated with Au nanoparticles such as their superior optical property, structural and functional compatibility with biomolecules. In the first study, we used colloidal gold nanoparticles to develop a simple microfluidics-based bioassay that is able to recognize and detect specific DNA sequences via conformational change-induced fluorescence quenching. In this method, a self-assembled monolayer of gold nanoparticles was fabricated on the channel wall of a microfluidic chip and DNA probes were bonded to the monolayer via thiol groups at one end and a fluorophore dye was attached to the other end of the probe. The created construct is spontaneously assembled into a constrained arch-like conformation on the particle surface and under which, the fluorescence of fluorophores is quenched by gold nanoparticles. Hybridization of target DNAs results in a conformational change of the construct and then restores the fluorescence, which serves as a sensing method for the target genes. The applicability of proposed assay was first demonstrated by the use of a pair of synthesized complementary and non-complementary DNA sequences. The method was further applied for the detection the PCR product of dengue virus with the use of enterovirus as the negative control and results indicate that the assay is specific for the target gene. Moreover, using this approach, dehybridization, hybridization, and detection of the target genes can be performed in-situ on the same microfluidic channel.

    In the recently year, heme proteins are shown to be an effective linking agent in stabilizing Au Nanoparticles (AuNPs). Therefore, in the second study, we used heme protein to fabricate 3-D AuNP multilayers on a chip, resulting in a higher coating density than those on polymer linker-anchored surfaces for analytical applications. Using Electron Spectroscopy for Chemical Analysis (ESCA) measurements, a lower oxidation state of Au(0) and dramatic changes among multiple chemical states of N(1s) are detected upon coating AuNPs with heme proteins but not detected upon coating AuNPs with non-heme proteins. Thus, we propose that the stabilization power arises from conjugation between AuNPs and the heme group. We also propose that such conjugation must be facilitated by the exposure of the heme group through a conformational change of the protein as well as interactions of other functional groups with AuNPs to bring the heme moiety to a close face-to-face distance with AuNPs. A high density double stranded DNA (dsDNA) composed of a sequence of estrogen response element (ERE) is then fabricated on heme protein- anchored chips. An in-situ hybridization and tracking method is developed based on hybridization-induced fluorescence restoration associated with AuNPs and assists in the subsequent detection of DNA/protein binding on the same chip. The AuNP-ERE chips are shown to have high sensitivity and specificity for quantitative detection of ERE binding with its two transcription factor isoforms, estrogen receptor  and  (ER and ), in cell lysates with reduced reagents and reaction time.

    Poly(dimethylsiloxane) (PDMS) possesses many advantages, like high optical transparency, biocompatibility and chemical stability which makes it an attractive material. However, it has a problem of inherent hydrophobicity which limits its application. In the third study, we use polyelectrolyte multilayers modification to change the surface property of PDMS from hydrophobicity to hydrophilicity and provides –NHS group for fabrication of myoglobin (Mb)-AuNPs multilayers via covalent bond. Using contact angle measurements, the contact angle of native PDMS is about 112o, but the contact angle go further down to 10° or less with polyelectrolyte multilayers modification. Then we utilize the immobilization of ERE on the Mb-AuNPs multilayer to fabricate an immunodevice and make calibration curve of ERα by enzyme-Linked immunosorbent assay (ELISA) in this device. Moreover, we also design two kinds of the competition test to confirm the specific affinity between ER and ERE.

    Abstrate Ⅰ 中文摘要 Ⅳ Acknowledge Ⅵ Table of Contents Ⅶ List of figures Ⅹ List of tables ⅩⅡ Abbreviation ⅩⅢ Chapter 1. Introduction 1.1 DNA Detection in Bioanalysis……………………………………………….2 1.1.1 DNA…………………………………………………………………2 1.1.2 Biological Functions…………………………………………………2 1.1.3 Detection of DNA……………………………………………………3 1.2 Gold Nanoparticles(AuNPs)…………………………………………………3 1.2.1 Definition and Properties of Gold Nanoparticles…………………….3 1.2.2 Synthesis of Gold Nanoparticles…………………………………….. 4 1.2.3 Applications of Gold nanoparticles…………………………………..5 1.3 Fluorescence………………………………………………………………….5 1.3.1 Quantum Yield………………………………………………………..7 1.3.2 Stern-Volmer Equation………………………………………………..7 1.3.3 Fluorescence Resonance Energy Transfer…………………………..9 1.3.4 Applications of Fluorescence Detection……………………………… 9 1.4 Enzyme-Linked Immunosorbent Assay (ELISA)…………………………….10 1.4.1 Types of ELISA……………………………………………………….10 1.5 Biochips………………………………………………………………………..12 1.5.1 The Motives of Developing Biochips………………………………..12 1.5.2 Microfluidic Chips…………………………………………………….12 1.5.3 Microarray Chips………………………………………………………13 1.6 Thesis Organization……………………………………………………………15 1.7 Reference……………………………………………………………………….16 Chapter 2. Gold Nanoparticles (AuNPs) for Microfluidics-Based Biosensing of PCR Products by Hybridization-Induced Fluorescence Quenching 2.1 DNA Hybridization Detection……………………………………………….23 2.2 Experimental Section………………………………………………………….24 2.2.1 Chemicals and Reagents……………………………………………….24 2.2.2 Synthesis and Characterization of Gold nanoparticles……………….25 2.2.3 Fabrication of Colloidal Gold Monolayer on Glass Chips (AuCM)……25 2.2.4 Fabrication of Nano-DNA Probes on the Au surface………………..26 2.2.5 Fluorescence Quenching Measurements………………………………26 2.2.6 Hybridization Assays…………………………………………………27 2.3 Results and Discussions………………………………………………………28 2.3.1 Design of the probe sequence…………………………………………28 2.3.2 Fabrication and characterization of nano-DNA probes……………….28 2.3.3 Quenching Efficiencies………………………………………………..29 2.3.4 Hybridization Detection………………………………………………31 2.4 Conclusions……………………………………………………………………33 2.5 Reference……………………………………………………………………….34 Chapter 3. Heme Protein Assisted Dispersion of Gold Nanoparticle Multilayers on Chips (AuNP-Chips) for Protein/DNA Binding 3.1 DNA-Protein Interaction………………………………………………………45 3.2 Dispersion of Gold Nanoparticle Multilayers on Substrates……………………45 3.3 Heme Protein-assisted Dispersion Stabilization……………………………….46 3.4 Experiment Section……………………………………………………………47 3.4.1 Fabrication of the Protein-Anchored AuNP Multilayer on Glass Chips………………………………………………………………….47 3.4.2 Fabrication of Estrogen Response Element Probes on the Au Surface………………………………………………………………………………………48 3.4.3 Transcription Factor Binding………………………………………….48 3.5 Results and Discussions………………………………………………………49 3.5.1 Comparison of AuNP Modification Efficiency with Different Linkers49 3.5.2 ESCA Test for the Molecular Interaction Between AuNPs and Heme Proteins……………………………………………………………….51 3.5.3 Fabrication of Estrogen Receptor Element Probes on AuNPs Surface.54 3.5.4 Specific Protein Recognition with Enzyme-Linked Immunosorbent Assay…………………………………………………………………55 3.6 Conclusions……………………………………………………………………56 3.7 Reference………………………………………………………………………57 Chapter 4. Quantitative Analysis of Binding Affinity Using AuNPs-Chips 4.1 Estrogen………………………………………………………………………..68 4.2 Estrogen Receptor…………………………………………………………….68 4.3 Detection methods for ER binding ……………………………………………69 4.4 Experimental Section………………………………………………………. …70 4.4.1 Fabrication of the PDMS Chip……………………………………….70 4.4.2 Fabrication of AuNPs-Coated Immunodevice………………………..70 4.4.3 Calibration Curve of ERα……………………………………………..71 4.4.4 Competition Test by ERE…………………………………………….72 4.5 Results and Discussions……………………………………………………….72 4.5.1 Surface Characterization………………………………………………72 4.5.2 ELISA Analysis in the Immunodevice…………………………………73 4.5.3 Detect the Binding Strength between ER and ERE by Competition Test……………………………………………………………………73 4.6 Conclusions…………………………………………………………………..75 4.7 Reference…………………………………………………………………….. 76 Chaper 5. Conclusions 5.1 Overview of the Dissertation…………………………………………………83 5.2 Future works………………………………………………………………….84 List of Figures Figure 1.1 The diagram of atom exciting and releasing: (1) atom is excited from ground state to excitation state by exciting energy (hvex), (2) energy losing from high excitation state (S1’) to low excitation state (S1) by side-reactions, (3) atom is return to the ground state accompanying a emission energy (hvem)……………………………..19 Figure 1.2 Schematic representation of the FRET mechanism. When the emission wavelengths of the FITC and Cy3 (donors) overlaps with the absorption wavelength of Au (acceptor), the fluorescent energy of the FITC or Cy3 are transferred to Au to accomplish the quenching phenomenon.....................................................................20 Figure 1.3 Types of Enzyme-Linked Immunosorbent Assay. (a) Direct ELISA, (b) Indirect ELISA, (c) Sandwish ELISA, (d) Competitive ELISA…………………….21 Figure 2.1 Microchip configuration…………………………………………………36 Figure 2.2 Schematic of the steps involved in the fabrication of the immobilized colloidal Au microfluidic chips. 1. The glass microchannel was functionalized with APTES to provide an amine-terminated surface. 2. Au nanoparticles were conjugated with amine groups for the formation of a monolayer of Au nanoparticles. 3. 3’-thiol, 5’-dye DNA molecules were bound to Au surfaces to form the arch-like constraint that leads to fluorescence quenching. 4. The complementary DNAs were added to the modified microchannel and hybridized with the exposed DNA sequence to release the quenched fluorescence………………………………………………………………………. 37 Figure 2.3 TEM photograph of the synthesized Au nanoparticles (10 nm) in solution…………………………………………………………………………………….38 Figure 2.4 The UV absorbance spectra of Au nanoparticles in different forms. The spectrum A. was measured from the solution; the spectrum B. was measured from the immobilized Au nanoparticles on a glass monolayer; the spectrum C. was measured from the immobilized Au nanoparticles with the attached DNA probe (Cy3-T5-GACAT AGTCT TAGAA CATGG AAGCT GTGTG ACGAC GATGT-T5- SH-3´); and the spectrum D. was measured after adding the complimentary sequence…………………………………………………………………………………….…………………………………….39 Figure 2.5 The Stern Volmer plots constructed for A. Cy3 and B. FITC dyes. The solid plots are for free dyes and the dashed plots are for dye-T15-CCA GTG ACA CTG G-T15-SH-3´ molecules……………………………………………………......40 Figure 2.6 Hybridization detection with the FITC-T15-CCA GTG ACA CTG G-T15-SH-3´ coated microchips before A and after B adding hybridizing solutions indicated in the Figure. The subtracted photograph was shown in C……………….......41 Figure 2.7 Hybridization detection with microchips coated with the probe sequence of Dangue virus (5´-Cy3-T5-GACAT AGTCT TAGAA CATGG AAGCT GTGTG ACGAC GATGT-T5- SH-3´) before (left) and after (right) adding hybridizing solutions indicated in the Figure. To increase the amplification factor, the photos were taken from each channel on the same microchip………………………………………..42 Figure 3.1 Protein-anchored AuNP multilayers (n = 4) and APTESanchored monolayer on glass……………………………………………………………………59 Figure 3.2 SEM and photo images of (a) Mb-anchored AuNP monolayer, (b) Mb-anchored AuNP multilayer (n =4), (c) Casanchored AuNP monolayer, and (d) H-Cas-anchored AuNP monolayer on glass…………………………………..………...60 Figure 3.3 5/2Au and 7/2Au ESCA spectra for (a) Mb-AuNPs, (b) H-Cas-AuNPs, (c) Cas-AuNPs, and (d) aminosilane-functionalized AuNPs…………………..…………61 Figure 3.4 N1s core level of ESCA spectra for (a) Mb, (b) Mb-AuNP, (c) Cas, (d) Cas-AuNP, (e) H-Cas, and (f) H-Cas-AuNP on glass………………………………….62 Figure 3.5 In situ hybridization-induced fluorescence restoration for sensing transcription factor binding on heme protein stabilized AuNP multilayers…………….63 Figure 3.6 Hybridization-induced fluorescence (Cy3) restoration for the fabrication of dsDNAs containing ERE sequences. No fluorescence signal was detected when non- complementary DNA and blank solution were added…….……………….....................64 Figure 3.7 Transcription factor binding assays using Mb-AuNP chips immobilized with ERE dsDNAs. The emission signal for sensing the binding of ERR and ER_ was detected from the control rERR sample as well as 17β-estradiol-treated MCF-7 and A549 cells. Signals detected from three repeated measurements were quantified with their standard deviations indicated by arrow bars……………………………………….65 Figure 4.1 Three kinds of different estrogen structure: (a) Estriol (E1). It has two hydroxyl (-OH) groups which are attached to the D ring (the rightmost ring), (b) Estradiol (E2). It also has two hydroxyl groups that one is attached to the D ring and the other is attached to the A ring (the leftmost ring), (c) Estrone (E3). It has a ketone (=O) group attached to the D ring and a hydroxyl group attached to the A ring……………………………………………………………………………………78 Figure 4.2 The structural changes and dimerization of ER that occur in binding of estrogen, with subsequent binding to estrogen response element (ERE)…………….79 Figure 4.3 Contact angle measurements on differently modified PDMS plates: (A) native PDMS plate; (B) oxidized PDMS plate; (C) polyelectrolyte multilayers coated PDMS plate……………………………………………………………………………….80 Figure 4.4 Calibration curves obtained from immunodevice. The concentration of ERα solution was prepared as 4.06E-9、2.03E-9、1.015E-9、0.203E-9、0.1015E-9 and 0 M………………………………………………………………………………………...81 Figure 4.6 Competition test: (a) ER-binding competitors , (b) non-ER-binding competitors. The volume of competitors was prepared as 0、2、4、8、16 and 32μl and co-incubated with ERα.( 10μl, 2.03nM)……………...…..…………………...…………82 List of Tables Table 2.1 Stern Volmer constants (KQ) of the free and probe labeled dyes measured from the Stern Volmer plots shown in Figure 2.5……………………………………………..43 Table 3.1. Multiple factors for the linker-AuNP interactions……………………………66 Abbreviations APTES : γ-(aminopropyl)triethoxysilane AuNPs : Gold Nanopartices Bp : Base pair CFL : Compact fluorescent lighting Cas : Casein DNA : Deoxyribonucleic acid DMSO : Dimethyl Sulfoxide dsDNA : Double stranded DNA E1 : Estrone E2 : Estradiol E3 : Estriol ESCA : Electron Spectroscopy for Chemical Analysis ERE : Estrogen Response Element ER : Estrogen Receptor EMSA : Electrophoretic Mobility Shift Assay ELISA : Enzyme-Linked Immunosorbent Assay FRET : Fluorescence resonance energy transfer GFP : Green Fluorescent Protein GPER : G protein coupled receptor GPR30 HPLC : High Performance Liquid Chromatography HIV : Human immunodeficiency virus H-Cas : Hemin-β-Casein LEDs : Light-Emitting Diodes MEMS : Mciro electromechanical system μ-TAS : Micro total analysis system MPTS : (3-mercaptopropyl) -trimethoxysilane Mb : Myoglobin NHS : N-hydroxysuccinimide PBS : Phosphate buffered Saline PMT : Photo- Multiplier Tube PEI : Poly(ethyleneimine) PAA : Poly(acrylic acid) PDMS : Poly(dimethyl siloxane) PCR : Polymerase chain reaction SERS : Surface Enhanced Raman Spectroscopy SPR : Surface plasmon resonance SAM : Self-Assemble Monolayer SEM : Scanning Electron Microscopy TLC : Thin-Layer Chromatorgaphy TEM : Transmission Electron Microscopy UV-Vis : Ultraviolet-Visible

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