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研究生：	鄧宇倫 Deng, Yu-Luen
論文名稱：	介電泳微晶片在生醫診斷與生質燃料生產的應用:病原體與微藻的篩選及利用表面增強拉曼散射分析 Dielectrophoresis microchip for applications in medical diagnosis and biofuel production: Screening and SERS detection of pathogens and microalgae
指導教授：	莊怡哲 Juang, Yi-Je
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	85
中文關鍵詞：	微流體晶片、介電泳、表面增強拉曼散射、微藻、經皮藥物輸送、微針
外文關鍵詞：	microfluidic chips, dielectrophoresis, surface enhanced Raman scattering (SERS), microalgae, transdermal drug delivery, microneedle
相關次數：	點閱：98 下載：9
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在最近幾年間快速的生醫檢測分析需求在多個領域例如醫藥研究、病原體偵測、生質能生產、環境監控與本土安全均有顯著的成長。透過使用微機電技術製作出的微流體晶片具有少量樣品需求、更加快速的分析速度、較高的樣品負載量、複合式的檢測功能、相對簡單的樣品前處理程序以及良好的可攜帶性等優點，因此能夠更加貼近不同領域的需求。在本論文中，我們提出並且發展了數種微流體技術以應對上述各種領域的需求。在生質能源生產的部份我們製作出批次與連續流動式介電泳晶片來即時的分辨以及篩選出在培養過程中具有較高含油量的微藻，並且探討不同含油量的微藻細胞在非均勻的交流電場下的介電泳行為機制。在病原偵測的部份我們建立整合微針陣列的微流體系統，在交流電場的作用下菌血症病原之一的金黃色葡萄球菌成功與紅球分離並且聚集於微針尖端，加上尖端結構良好的拉曼散射增強效應，只需要稀少的細菌量即可確認感染菌種的拉曼特徵光譜。表面增強拉曼散射的機制與所謂的”熱點”數量有密切的相關性，我們使用感應耦合離子電漿蝕刻技術製作出不同尺度與密集程度的黑矽基板來進行相關的探討。當單位面積內所包含的黑矽尖錐結構越多則熱點的的數量也隨之增加，搭配適當厚度的金屬蒸鍍層來最佳化拉曼增強效果，我們能夠有效的檢測出10fM Rhodamine 6G分子的拉曼訊號。最後，我們提出一種相對快速、簡單而且可靠的製作技術來蝕刻出以polydimethyl siloxane為材質的微針結構進而應用在製作微針整合的微流體晶片與經皮藥物輸送領域。

In recent years, the need of rapid bio-detection and analysis in many territories such as medical research, pathogen detection, biomass production, environmental monitoring, homeland security have been increasing drastically. The microfluidic devices constructed by exploiting the MEMS (Micro-Electro-Mechanical System) technology can well meet the demands because they possess several advantages such as small amount sample required, faster analysis, high throughput, multiplexing, relatively simple procedure for sample pretreatment and increased portability. In this study, we addressed and developed some of microfluidic techniques for the applications in the abovementioned areas. To effectively select and identify the algal strains with high-lipid content and potentially monitor the cultivation process in biofuel production, we constructed the batch and continuous dielectrophoresis (DEP) microchip to better understand the flow behavior of microalgae with different lipid contents under the non-uniform AC electric field and utilize this information to perform continuous sorting and detection of microalgae. For fast analysis of pathogens, the microfluidic chips with microneedle array were fabricated and performed under the non-uniform AC electric field. By combining both the effects of dielectrophoresis and surface enhanced Raman spectroscopy (SERS), the Staphylococcus aureus (S. aureus) was first effectively separated from the red blood cells (RBCs) and concentrated at the tips of the microneedle array, followed by analysis through SERS. For better heterogeneous SERS detection, one of the important parameters is the amount of the “hot spots” on the SERS substrates. To realize the effect of “hot spots” on SERS detection, several black silicon (BS) surfaces consisting of different tip densities which correspond to different amount of “hot spots” were constructed via inductively coupled plasma etching and gold evaporation. It was concluded that the SERS spectra of rhodamine 6G molecules at solution concentration as low as 10 fM can be obtained by using the optimized BS surface. Finally, a relative rapid, simple and reliable technique to fabricate microneedles by 3D polydimethylsiloxane (PDMS) etching was proposed and developed, which can be used to construct the microfluidic chip with microneedle array and in the applications of transdermal drug delivery.

Table of contents
Chapter 1 Introduction	1
1.1	Overview of microelectro mechanical systems (MEMS) for bio-detection	1
1.2 Motivations and objectives	2
1.4 Topics arrangement	5
Chapter 2 Electrokinetic trapping and SERS detection of biomolecules using optofluidic device integrated with a microneedles array	8
2.1 Introduction	8
2.2 Experimental	11
2.2.1 Device fabrication	11
2.2.2 Sample preparation	13
2.2.3 DEP separation of latex beads with different sizes	13
2.2.4 DEP separation of S. aureus/RBCs	14
2.2.5 Raman detection of R6G and S. aureus using micro needles array	14
2.3 results and discussion	15
2.3.1 SEM images of silicon micro needles array	15
2.3.2 DEP separation of latex beads with different sizes	17
2.3.3 Raman detection of R6G at the tips	18
2.3.4 On-chip separation of S. aureus/RBCs and SERS analysis of S. aureus	19
Chapter 3 Black silicon SERS substrate: Effect of surface morphology on SERS detection and application of single algal cell analysis	23
3.1 Introduction	23
3.2 Experimental	26
3.2.1 Fabrication of black silicon SERS substrates	26
3.2.2 Sample preparation	27
3.2.3 Raman measurement and estimation of “hot spots” on SERS substrate	27
3.3 Results and discussion	28
3.3.1 Fabrication of black silicon with different tip densities	28
3.3.2 SERS effect on the black silicon with different tip density	30
3.3.3 The effect of the thickness of gold layer on SERS	34
3.3.4 SERS measurement of single algal cell	38
Chapter 4 Polydimethyl siloxane wet etching for 3D fabrication of microneedle array and high-aspect-ratio micropillars	41
4.1 Introduction	41
4.2 Experimental	44
4.2.1 Fabrication of PDMS microneedle array	44
4.2.2 Fabrication of PVP microneedle array	45
4.3 Results and discussion	45
Chapter 5 Separation of microalgae with different lipid contents by dielectrophoresis	52
5.1 Introduction	52
5.2 Experimental	54
5.2.1 Sample preparation	54
5.2.2 Fabrication of DEP microfluidic device	55
5.2.3 Separation of microalgae by DEP	57
5.3 Results and discussion	58
5.3.1 Effect of solution conductivity on crossover frequency	58
5.3.2 DEP separation of microalgae with 11wt% and 45wt% lipid contents	61
5.3.3 Characterization of crossover frequencies of microalgae with various lipid contents and perform continuous separation of microalgae	62
Chapter 6 Conclusions and future applications	68
6.1 Summary	68
6.2 Future applications	70
Reference	73



List of the tables and figures
Figure 2-1 Schematic and photograph of DEP microfluidic device	12
Figure 2-2 (a) The effect of patterned area on etching uniformity. Left: large patterned area (2*2 cm2). Right: small patterned area (0.5*0.5 cm2). (b) Time-lapsed images of microneedles fabricated during the etching process.	17
Figure 2-3 Time-lapsed images of DEP separation of latex beads with different sizes. The dashed circles indicate the region surrounding the tips of microneedles.	18
Figure 2-4 Raman detection of R6G molecules on the gold coated substrate and substrate with microneedles array.	19
Figure 2-5 (a)DEP motions of S. aureus and RBCs at 170 kHz and 80 μS/cm (b)Time-lapsed images of DEP separation of S. aureus and RBCs.	21
Figure 2-6 SERS spectra of S. aureus acquired from the gold coated substrate and substrate with microneedles array.	22

Figure 3-1 The black silicon morphology obtained at the etching conditions (a) 100-9-50 (BS1), (b) 100-12-30 (BS2), (c) 100-12-40 (BS2), and (d) 25-35-3 (BS3). xx-xx-xx refers to SF6 flow rate- O2 flow rate-etching time.	30
Figure 3-2 (a) The Raman spectra obtained by using the black silicon substrate with different tip densities. (b) The relationship between the tip density and the peak intensity at different Raman shift.	32
Figure 3-3 The SEM pictures of the black silicon substrates (a) BS1, (b) BS2, (c) high aspect ratio BS2, and (d) BS3 deposited with 100 nm gold layer. Bridging between cone-like structure by the deposited gold layer was indicated by the red circles.	33
Figure 3-4 The SEM pictures of the BS3 substrate deposited with (a) 100 nm, (b) 200 nm, (c) 400 nm, and (d) 600 nm thick gold layer.	35
Figure 3-5 (a) SERS detection of 10-7 M R6G on the BS3 substrate deposited with different thickness of gold layer (b) The Raman spectra for 10-9 M to 10-14 M R6G on the BS3 substrate deposited with 400 nm thick gold layer.	36
Figure 3-6 (a) The Raman intensity mapping (200 μm point space) and (b) the line scanning (50 μm point space) of the BS3 substrate deposited with 400 nm thick gold layer. The concentration of R6G solution is 0.1 nM.	37
Figure 3-7 The Raman intensities at 612 cm-1 measured at five different dates for different sample concentrations.	38
Figure 3-8 SERS detection of a single algal cell by using the BS3 substrate deposited with 400 nm gold layer. The inset are the images of the algal cells dispersed at the substrates taken from the optical microscope.	39
Figure 3-9 The Raman spectra obtained from five randomly selected algal cells.	40

Figure 4-1 (a) The initial micropillar with 400 μm in diameter and 700 μm in height. The optical images of PDMS micropillars at the etching time (b) 15, (c) 27 and (d) 32 minutes in the etching process without agitation. (e) The etched PDMS microneedle array. (f) The cross sectional view of the PDMS negative mold. (g) The PVP microneedle array. The scale bar in the inset of (f) and (g) is 300 μm.	46
Figure 4-2 The relationship of (a) height and (b) aspect ratio between the initial PDMS micropillar and the etched PDMS MNs.	48
Figure 4-3 (a) The initial PDMS micropillars with 300 μm in diameter and 800 μm in height. The optical images of PDMS micropillars at the etching time (b) 9, (c) 22 and (d) 30 minutes in the etching process with agitation (rotation speed of 500 rpm).	49
Figure 4-4 casted PVP micropillars with aspect ratio of 13	50
Figure 4-5 PDMS micropillars after 35-minute etching under agitation	51

Table 5-1 DEP motion collection of microalgae with different lipid content	63

Figure 5-1 The schematic of (a) top and (b) side view of the DEP microfluidic device. (c) Image of the fabricated device.	56
Figure 5-2 (a) The schematic of the experimental setup. (b) The proposed scheme for separation of microalgae with different lipid content. (c) The photo of the continuous flow DEP microfluidic chip.	57
Figure 5-3 The DEP motion of the microalgae with 11 wt% lipid content under different operating frequencies at the solution conductivity (a) 2.45, (b) 2.95 and (c) 1.4 mS/cm.	60
Figure 5-4 The DEP motion of the microalgae with 45 wt% lipid content under different operating frequencies at the solution conductivity (a) 2.95 and (b) 1.4 mS/cm.	61
Figure 5-5 The movement of the microalgae with 11wt% (transparent dots) and 45wt% (bright dots) lipid contents. (a) before and (b) after AC was turned on.	62
Figure 5-6 Calculation of Re(fcm) for microalgae with different lipid content	64
Figure 5-7 The alignment of the microalgae (a) before and (b) after turning on the AC function generator. (c) The sequential images for separation of the microalgae with 24% and 35% lipid contents. The electrodes (dashed lines) and the edge of the microchannel (solid sline) are outlined for clarity.	66
Figure 5-8 The measured fluorescence intensity of the microalgae with different lipid contents at rest and in motion.	67

Figure 6-1 Schematic of ACEO flow type on 3D micorneedle electrode	71
Figure 6-2 0.92 μm particles are driven in tip-centered symmetric circulation by ACEO flow and trapped at tips as they move close to microneedles.	72
                                    

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