臺灣博碩士論文加值系統

本論文分為兩個部分，第一部分是關於應用在光遺傳學的上轉換奈米粒子 (UCNPs)，第二部分是關於二維奈米材料應用於生物感測。
(i) 以上轉換奈米粒子進行光遺傳學之研究
我們藉由biotin和neutravidin間分子的作用，將UCNP和表現光敏感性之通道蛋白─channel rhodopsin(ChR2)的活細胞結合，並藉由細胞裂解和活細胞的實驗(免疫點試驗和蛋白質體外结合實驗)確認UCNP和ChR2的專一性作用。在這項研究中，和UCNP結合之ChR2只需要低能量的近紅外光即可被激發，並藉由量測通過連接通道的離子電流和細胞內上升之鈣離子濃度而證實。我們的設計降低了UCNP和光敏感性蛋白間之距離至分子等級，因此不只縮小了所需的近紅外光能量，也在光遺傳學之應用中提供了可引導專一性結合的方法。
(ii) 論文的第二部分是關於二維奈米材料應用於生物感測。由於所要感測的生物樣品中含有複雜多樣的基質，這些基質的干擾對於生物感測器在生理或真實樣品中的應用是一個巨大的挑戰。為了解決這個問題，本研究著重在生物感測系統中引入聚乙二醇 (PEG)， 二維奈米片(2D-NSs)被聚乙二醇修飾後，可避免二維奈米片的聚集，增強其穩定性和偵測靈敏度，同時能夠減少複雜基質的干擾。生成三元過渡金屬硫化物(Mn0.02Ta3S6 和 Fe0.65Ta3S6)單晶後，將其剝離成二維奈米片，並製備聚乙二醇修飾的二維奈米片。接著，基於聚乙二醇修飾的二維奈米片具有高螢光淬滅能力和核酸適體對待測生化分子的親和力，可達到在不同介質（1倍PBS 緩衝液、牛奶和人血清）中對黃麴毒素 B1 和黃麴毒素 M1 的多重檢測。黃麴毒素是一種真菌毒素，它不僅會污染食物，食用後還會對人體健康產生不利的影響，並可作為肝癌和其他可怕疾病的生物標誌物。所設計的生物感測器具備超高的多重檢測靈敏度（偵測極限可達到pM）、廣泛的線性工作範圍（≥ 5 個分析物濃度的數量級）、目標選擇性、快速檢測和成本效益。因此，本研究所設計的方法在食品工業、生理環境中眾多疾病生物標誌物的臨床診斷應用上非常有希望，對實際醫療保健和基礎研究是一個重大的里程碑和並且廣泛的影響。

The thesis contains two parts. The first part of thesis is related to upconversion nanoparticles (UCNPs) for optogenetics applications.
Optogenetics is an innovative technology adopted by neuroscientist, where the expression of a light-sensitive protein in the specific cells and the activation of the light-sensitive protein-conjugated ion channels in those cells via illumation. However, most of optogenetic light-sensitive proteins are activated by ultraviolet or visible light, which has a limitation to penetrate tissue. This problem could be solved by upconversion nanoparticles (UCNPs), a fluorescent nanoparticle that absorb near-infrared (NIR) light and then emit light with shorter wavelengths. In this study, we selectively conjugated the UCNPs with channelrhodopsin-2 (ChR2) to show the target selectivity. The extracellular N-terminal of ChR2 (V5-ChR2m) has been tagged by V5 epitope and NeutrAvidin was functionalized on the surface of UCNP (NAv-UCNPs). As a result of the biotinylated antibody against V5 adhering to the V5-ChR2m expressed in the plasma membrane of living HEK293T cells, our data demonstrated that the NAv-UCNPs were specifically bound to the membrane of cells expressing V5-ChR2m. However, without the V5 epitope or the NAv alteration, no binding of UCNPs to the cell membrane was seen. In the cells expressing V5-ChR2m and associated with NAv-UCNP, 488 nm illumination and the upconverted blue fluorescence from UCNP by 980 nm excitation both generated an inward current and increased the intracellular Ca2+ concentration. By shortening the distance between UCNP and the light-sensitive protein to a molecular level, our study reduces the amount of NIR energy needed. Hence, this strategy provides a pathway for the specific binding for optogenetics applications.
The second part of thesis is related to two-dimensional nanomaterials for biosensing. The application of the biosensor in physiological or real samples is a big challenge because of the interference from the complex matrix of sensing medium. To solve this issue, the present study focuses on the introduction of polyethylene glycol (PEG) in the biosensing system. The PEG modification enhances the stability and detection sensitivity by preventing the aggregation of 2-dimensional Nanosheets (2D-NSs) and minimizing the interferences in the complex matrix. Ternary transition metal sulfides (Mn0.02Ta3S6 and Fe0.65Ta3S6) single crystals have been grown, exfoliated to NSs and PEGylated NSs were prepared. It has been demonstrated that PEGylated NSs improves the detection sensitivity ~ 248 times better than non-PEGylated NSs. Then, multiplexed detection of aflatoxins B1 and aflatoxins M1 in diverse mediums (PBS buffer (1×), milk, and human serum) based on the high fluorescence quenching abilities and affinities towards the aptamers was demonstrated via PEG@NSs. Aflatoxins are fungal toxins which not only contaminate food, but also adversely affect human health if consumed and function as biomarkers for liver cancer and other dreadful diseases. The designed sensor demonstrates ultrahigh multiplexed detection sensitivity (limit of detection ~ pM), wide span of linear working range (≥ 5 orders of analyte concentrations), target selectivity, quick detection and cost-effective method. Consequently, the designed strategy is highly promising not only for the food industry, but also for the clinical diagnosis of numerous disease biomarkers in physiological environments.

Table of Contents
Acknowledgements......………………………………………………….…………..…….ii
摘要…………..……...………………………………………………….…………...….. iv
Abstract…….……………....………………………………………….…………………vi
Table of Contents…..……..…………………………………………….…………….....viii
List of Figures…….………..………………………………………….……………........xii
List of Tables…………………..………………………………………………………...xiv
Chapter 1 Upconversion nanoparticle……………..……………………………………1
1.1 Introduction of Upconversion nanoparticles………………………….…….....1
1.2 Upconversion mechanism………………………..…..………………………..2
1.2.1. Excited State Absorption (ESA)………………..…………………………..3
1.2.2. Energy Transfer Upconversion (ETU)………...…………...………………4
1.3 Dopant and Host Selection Criteria…………….……………………………..5
1.4 Upconversion Nanoparticle Synthesis……….………………………………..8
1.4.1. Hydrothermal/solvothermal method….…..……………….…………….…8
1.4.2. Coprecipitation method ……………………………………………………8
1.4.3. Thermal decomposition…….………………………………………………8
1.5 Methods for surface modification of UCNP…………………………………………8
1.5.1. Ligand Exchange…..……………………………………………………….9
1.5.2. Ligand Removal…...….……..……………………………………………..9
1.5.3. Ligand Oxidation ……...........…………..…………………………………9
1.5.4. Layer-by-Layer Assembly .………………………..………………………9
1.5.5. Surface Silanization ………........................................................................9
1.5.6. Amphiphilic Polymer Coating….………….……………………………..10
1.6 Biocompatability test of UCNP……………………………………………...10
1.7 Various Applications of Upconversion nanoparticles………...……………..10
1.7.1 Bioassay….……....……………………………………….……………….10
1.7.2 High contrast Bioimaging…………………………………………………11
1.7.3 Cellular Imaging …….……………………………………………………11
1.7.4 Drug delivery………….…………………………………………………..12
1.7.4 (i) Hydrophobic pockets.………....……………………………………….12
1.7.4 (ii) Mesoporous silica shells……..……..…………………………………13
1.7.5 In Vitro and In Vivo Photoactivations…………………………………….13
1.7.6 Upconversion-Guided Photothermal Therapy…………………………….13
1.8 Summary……………………………………………………………………..14
1.9 References……………………………………………………………………15
Chapter 2 Two-Dimensional Nanomaterials…..…….…………...……………………22
2.1 Introduction of Two-Dimensional Nanomaterials…...………………………22
2.2 Growth of 2D materials…………….…………………..…………………….22
2.2.1 Chemical Vapor Transport ……………..…………………………………23
2.2.2 Bridgman Technique…………..….……………………………………….23
2.2.3. Mechanical Exfoliation…………………………………………………...24
2.2.4. Liquid Exfoliation....………………………………………………….…..25
2.3 Biocompatibility of 2D TMDs ………………………………………………26
2.4 Biosensing Applications Based on 2D materials…………………………….26
2.5 Summary…………………………………..…………………………………28
2.6 References……………………………………………………………………29
Chapter 3 Material Characterizations and Instrumentation….….………………….32
3.1 X-ray Diffraction.…….………………………………………………………32
3.2 Transmission Electron Microscopy.……….…………………………………34
3.3 Scanning Electron Microscopy and Energy Dispersive X-ray analysis….….37
3.4 Zeta potential………..……………………………………………………….38
3.5 Atomic Force Microscopy.....…..……………………………………………39
3.6 Confocal Microscopy….………..……………………………………………41
3.7 Fluorescence Spectroscopy……..……………………………………………42
3.8 References….……………………..………………………………………….44
Chapter 4 Aim and Scope………...……..………..…………………………………….45
4.1 Optogenetics study using upconversion nanoparticles in living cells………..47
4.2 Multiplexed detection of aflatoxin B1 and aflatoxin M1 using PEGylated
2Dimensional Nanosheets…………...……………………………….……....47
4.3 References…..……..…………………………………………………………50
Chapter 5 Optogenetics study using upconversion nanoparticles in living cells..…..54
5.1 Introduction……..……………………………………………………………54
5.2 Experimental Section….……………………………………………………..57
5.3 Results and Discussions.……..………………………………………………64
5.3.1 UCNP characterizations and Experimental design………………..……….64
5.3.2 V5-ChR2m localizes in the cell membrane…………..……………………71
5.3.3a Specific binding of NAv-UCNP to V5-ChR2m in cell lysate…….……...72
5.3.3b Specific binding of NAv-UCNP to V5-ChR2m in live cells……………..72
5.3.4 Optogenetic activation of ChR2…...………………………………………77
5.3.4 (a) Optogenetic activation of ChR2 validated by electrophysiological
Measurements…………………………………………………………..77
5.3.4 (b) Optogenetic activation of ChR2 confirmed by monitoring [Ca2+]i ……79
5.4 Summary……………………………………………………………………..82
5.5 References...………………………………………………………………….83
Chapter 6 Multiplexed detection of aflatoxin B1 and aflatoxin M1 using
PEGylated two-dimensional nanosheets……………….…………............88
6.1 Introduction…………………………………………………………………..88
6.2 Experimental Section…..…………………………………………………….90
6.3 Results & discussions……….…………………………………………….. 100
6.3.1. Characterization of Mn0.02Ta3S6 and Fe0.65Ta3S6 single crystals and
NSs…………………………………………………………………....…100
6.3.2. Characterization of PEG@Mn0.02Ta3S6 and PEG@Fe0.65Ta3S6 NSs..…..102
6.3.3. Optimization of experimental conditions .……………………..………..107
6.3.4a. Mechanism for Fluorescence Sensing of AFB1…………….………….118
6.3.4b. Selectivity test of Tam-Apt-B1 and Fam-Apt M1…………….......……120
6.3.5. Detection of AFB1…………………………………………….…………123
6.3.6. Multiplexed detection of AFM1 and AFB1………...……………………126
6.3.6a. Factors for multiplexed detection……………………………………….126
6.3.6b. Control experiments before multiplexed detection………...…..……….128
6.3.6c. Multiplexed detection of AFM1 and AFB1 in PBS buffer, milk and
human serum……………………………..………………….…………..129
6.4 Summary……………………………………………………………………135
6.5 References…………………………………………………………….……136
Chapter 7 Conclusions and Future Scope.….……….……………………………….141
7.1 Significant contribution……………………………………………………141
7.2 Future scope………………………………………………………………..142
Awards and conferences….……...………….……………………………..143
List of publications ………………………..………………. ……………..144

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