應用於快速生化檢測的三維圖案化多功能晶片

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研究生：	李璧伸 Lee, Bi-Shen
論文名稱：	應用於快速生化檢測的三維圖案化多功能晶片 Rapid Biochemical Mixture Screening by Three-Dimensional Patterned Multifunctional Substrate
指導教授：	嚴大任 Yen, Ta-Jen
口試委員:	鄭兆珉 Cheng, Chao-Min 朱治偉 Chu, Chih-Wei 朱仁佑 Chu, Jen-You 林鼎晸 Lin, Ding-Zheng
學位類別：	博士 Doctor
系所名稱：	工學院 - 材料科學工程學系 Materials Science and Engineering
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	92
中文關鍵詞：	表面增強拉曼效應、超薄層層析法、金屬輔助濕式蝕刻、局部表面電漿共振、三維圖案化多功能晶片
外文關鍵詞：	Surface enhanced Raman scattering, Ultra thin layer chromatography, Metal assisted chemical etching, localized surface plasmon resonance, three-dimensional patterned multifunctional substrate
相關次數：	點閱：2 下載：0
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近年來，越來越多的人工添加物，使食品安全成為了世界上重要的議題之一，因此，開發出一套具有高靈敏度的快篩檢測技術是迫切需要的。至今，傳統的檢測技術，如層析法、質譜法、或是免疫蛋白測定法，上述方法大多相當昂貴且耗時，且往往是針對單一特定待測物進行設計，離實際快篩的應用還有一段距離。
就我們所知，拉曼光譜是一種強而有力的分析技術，能夠提供分子的結構資訊，因此拉曼光譜又被稱為分子的指紋。但在實際檢測中，複雜的環境背景，會使拉曼訊號被減弱甚至是無法辨別。因此，發展一個簡單的前處理技術再搭配拉曼散射的高靈敏度檢測，漸漸成為近年來研究的顯學。在此論文中，我們提出了一種多功能的快篩檢測晶片，此晶片同時具有表面增強拉曼效應與超薄層層析法兩種功能。在進行檢測複雜的真實樣品時，可先利用層析法將混和物進行分離，使不同種類的分子散布在晶片的不同位置之上，接著再利用表面增強拉曼的效應，分別對於不同區域的待測物測量並分析拉曼光譜，最終來達成真實複雜樣品的高靈敏度檢測。

此多功能快篩檢測晶片，是由矽奈米線陣列作為基底，在其表面覆蓋金屬奈米顆粒所構成。此矽奈米陣列可以藉由簡易且低成本的濕式蝕刻製程來完成，接著，透過化學還原法或是電子束蒸鍍，在矽奈米結構之上生成金屬奈米顆粒。透過矽基版所具有的獨特拉曼特徵峰(位於520 cm-1)，我們可以進一步的對量測結果進行校正，達到更低的標準差，進一步提高晶片的訊號穩定性。而藉由金屬奈米結構的局部表面電漿共振效應，可在特定區域形成數量眾多的”熱點”，增強拉曼散射的訊號強度。此外，為了更進一步優化此晶片，我們也透過了簡易的光刻過程，設計了數種不同的三維圖案在所開發的多功能晶片中，透過特殊設計的三維圖案所提供的流道限制與分子集中的效果，可使此晶片原所具有的校能顯著的被增強，達到更佳的檢測極限。

在分析檢測上，我們選用了多種不同種類的分子，包含了苯硫醇、孔雀石綠、三聚氰胺以及多種染料來展示此晶片所具有的不同的功能。例如將具有顏色的染了混合物作為待測物，透過超薄層層析法之後，不同顏色的染料出現在晶片上的不同位置，形成不連續的顏色線條，也驗證了層析分離的概念。而透過三聚氰胺與牛奶混合物的實驗，我們成功的將混合在牛奶中的三聚氰胺透過此晶片分離出來，並且透過表面增強拉曼訊號，達到了一個符合世界法規標準的檢測極限，證實了此晶片可用於日常真實的樣品檢測。因此，我們相信，所開發的低成本高靈敏度，結合分離與檢測技術多功能晶片，將有機會被實際應用在快速生化檢測的領域當中。

Currently, food safety is a serious issue worldwide due to the increasing artificial additives and thus there appears an urgent demand to develop a rapid screening method that requires an excellent separation and sensitivity for mixtures. To date the typical analytical techniques such as high-performance liquid chromatography (HPLC), liquid chromatography/mass spectroscopy (LC/MS), and immune-assays, are time-consuming and pricy. Also, they are designed for specific analysis instead of ultimate solution for fast screening and trace detection. Raman spectrum is a powerful analytical technique, providing the information of molecule structures and revealing the fingerprint of molecules. However, in real applications on sensing mixtures, the signal from targets is easily overwhelmed or interfered by complicated background signals. Therefore, developing a simple pretreatment route with highly sensitivity Raman scattering becomes a hot ticket research in recent years. Here, we proposed a multifunctional chip of ultra-thin layer chromatography (UTLC) and surface enhanced Raman scattering (SERS) which enables analytes separation and label-free detection simultaneously to allow biochemical mixture detection with excellent sensitivity.

This multifunctional chip is comprised of silicon nanowires array (SiNWA), decorated with metal nano-particles. The SiNWA is fabricated by a facile and low-cost metal assisted chemical etching (MaCE) process and the metal nano-particles are then decorated onto SiNWA by a chemical reduction process or an electron-beam evaporation process. By applying the intrinsic Raman peak sited at 520 cm-1 of our silicon-based substrate, the Raman signals from the targets can be calibrated to obtain a lower standard error deviation. This intrinsic peak acts as an inner mark in Raman measurement to ease the burden of externally normalizing the performance of substrate. The lower deviation represents the outstanding uniformity and stability of developed multifunctional chip. On the other hand, with the localized surface plasmon resonance (LSPR) from metal nano-structure, the “hot spots” are formed at specific regions, lifting the intensity of Raman signals. In order to further optimize our developed multifunctional chip, we designed certain three-dimensional patterns on developed multifunctional chip by a facile photolithographic process. The designed patterns acts as a channel, confining and directing the migration of analyte molecules in the UTLC separation process. Furthermore, the target molecule is relocated to specific region due to the concentrated effect of pattern. Benefited from the confinement and concentrated effects, we expect that the integrated performance of UTLC separation and SERS detection will be substantially improved.

In the end, for the applications, we choose many molecules, including thiophenol, malachite green, melamine, and colorful dyes to demonstrate the functions of developed multifunctional chip. For example, after UTLC development process, the dyes with different colors appear at different regions onto the substrate. The discontinuous color-bands verify the function of separation process. On the other hand, by sensing the toxic melamine and milk mixtures, the melamine was successfully separated and detected from milk. Furthermore, by the UTLC and SERS effects of our chip, we reached a melamine detection limit of 2.5 ppm that satisfies the Taiwan and EU’s regulation. Therefore, we believe that the developed low-cost, high sensitivity multifunctional chip possess the potential to practical applications in biochemical assays, food safety and other fields.

摘要....................................................................................................................I
Abstract...........................................................................................................III
Acknowledgement............................................................................................V
Contents..........................................................................................................VI
List of Figures...............................................................................................VIII
List of Tables...............................................................................................XVI
Chapter 1 Introduction
1    Introduction to Surfaced-Enhanced Raman Scattering (SERS).................1
2    Introduction to Ultra-Thin Layer Chromatography (UTLC).......................4
3    Dissertation Organization.........................................................................5
Chapter 2 Literature Review
1 Raman Spectroscopy..................................................................................................7
1.1 Raman Scattering (RS).........................................................................7
1.2 Surfaced-Enhanced Raman Scattering (SERS)...................................10
1.3 Different SERS Substrates and Applications......................................11
2 Chromatography............................................................................................17
2.1 Thin Layer Chromatography (TLC).....................................................17
2.2 Ultra-Thin Layer Chromatography (UTLC).........................................20
3 Commercial Product of SERS substrates and TLC plates........................22
4 Combination of TLC and SERS Techniques.............................................23
Chapter 3 A Low-cost, Highly-stable SERS Substrate by Si Nanowire Arrays Decorated with Au Nanoparticles and Au Backplate
1 Introduction....................................................................................................26
2 Design of Experiment...............................................................................28
2.1 Fabrication of SiNWA via MaCE..........................................................28
2.2 Formation of AuNPs and AuMBP onto the SiNWA scaffold................30
2.3 Design of Experiment by Taguchi Method.........................................32
3 Measurements and Discussions..............................................................35
3.1 SERS spectra of Different Fabricated Substrates..............................35
3.2 Demonstration of Electrical Field Distribution by FDTD.....................37
3.3 Quantitative Measurement of Malachite Green.................................39
Chapter 4 A High-performance Multifunctional Substrate of Ultra-Thin Layer Chromatography (UTLC) and Surface Enhanced Raman Scattering (SERS) for Rapid Biochemical Mixture Screening
1 Introduction...................................................................................................43
2 Methods and Experimental Details..........................................................45
2.1 Fabrication of AgNDs@SiNWA Substrate Via MaCE..........................45
2.2 Optimization of SERS Effect by Different Reaction Time to Form AgNDs............................................................................................................47
2.3 Optimization of UTLC Effect by Different Depths of SiNWA..............50
2.4 Enhancement of Electrical Field Intensity by The Optimized AgNDs.52
3 Results and Discussions..........................................................................56
3.1 Quantitative Measurement of Melamine in Milk Mixtures...................56
3.2 Stability Test of Developed UTLC-SERS Substrate...........................60
Chapter 5 Rapid Biochemical Mixture Screening by Three-Dimensional Patterned Multifunctional Substrate with Ultra-Thin Layer Chromatography (UTLC) and Surface Enhanced Raman Scattering (SERS)
1 Introduction...................................................................................................62
2 Materials and Methods............................................................................64
2.1 Fabrication of Three-Dimensional Patterned UTLC-SERS Multifunctional Substrate..............................................................................64
2.2 Design of 3DP-AgNDs@SiNWA UTLC-SERS Multifunctional Substrate......................................................................................................66
2.3 Process Flow of UTLC-SERS Measurement Process........................67
3 Results and Discussions.........................................................................69
3.1 Morphology and Surface Property of Fabricated UTLC-SERS Multifunctional Substrate..............................................................................69
3.2 Separation and Detection of Dye Mixtures by 3DP-AgNDs@SiNWA Multifunctional Substrate..............................................................................70
3.3 Comparison between Un-patterned AgNDs@SiNWA and Different Kinds of 3DP-AgNDs@SiNWA UTLC-SERS Multifunctional Substrate.........72
3.4 Wafer scale fabrication of 3DP-AgNDs@SiNWA UTLC-SERS Multifunctional Substrate.............................................................................75
Chapter 6 Conclusions
1 Conclusions............................................................................................79
2 Future Prospects....................................................................................82
References...................................................................................................84
Appendix
Metal-assisted Chemical Etching (MaCE)....................................................90
ANOVA table for S/N ratio............................................................................91
Publication List.............................................................................................92

                                

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