200815276 九、發明說明： 【發明所屬之技術領域】 本發明係有關一種具三維微結構之生物微粒抓取器及 其製造方法，特別是指一種利用介電泳電極所產生之介電 泳力（DEP Force)來捕捉生物微粒於預期之穴井内的抓取 裔及其製造方法，該抓取器係包含有上層體、下層體及微 流管道，於上、下層體各設有電極，使上、下層體電極之 電場方向與微流管體之流場方向相垂直而形成縱向不均勻 _ 電極，可迅速有效捕捉浮流於微流管道中的生物微粒至下 層體所设之穴井内，可增進抓取效能與解析度，可避免重 疊與聚集的缺點真正達到單細胞或單顆粒的抓取能力。本 $明特點是將三維結構之概念應用於介電泳生物晶片，此 介電冰晶片主要目的在抓取與固定生物微粒例如細胞、功 能性乳膠粒子、奈米顆粒、基因等。並且由於微結構的效 應可使細胞於吸附後，與穴井凹槽表面之真空吸附力及微 、、Ό構的側支撐力固定細胞，而無須繼續提供介電泳力所需 •之交流電源’可大幅增加細胞於晶片中的存活率。 【先前技術】 由於細胞的尺度介於微米等級，近年來微機電系統製 程技術（MEMS)的發展，促使操控單一細胞與量測之目 的得以實現，其中以介電泳力應用在細胞型生物晶片最為 廣泛’目前已知介電泳力應用在細胞型生物晶片之模組包 含下列幾種功能：細胞固定、細胞傳輸、細胞分離、細胞 對位、細胞量測、細胞分類等。介電泳驅動的機制為當細 200815276 胞處於一非均勻交流電場（AC non-uniform electric Held )， 由於細胞的介·電性質會在其表面誘發電荷而產生與外加電 %方向相同或相反的電偶極矩（electric dipole)，進而因電 場的非均勻性而受正介電泳力移動到強電場密度區或受負 介電泳力作用而集中至弱電場區，故可藉由電極的設計讓 細胞因電場梯度而驅動並使其固定在所設計的區域。習知 方式有者係利用柱狀、交趾狀（inter(jigital)、與點狀等不 同型式的電極設計成功將細胞定位，但是其缺點為細胞所 _ 受到的介電泳作用力限於表面移動，且所捕捉到的細胞個 數亦無法有效控制在單一細胞，這是由於細胞與細胞之間 所受誘發之電何，會因庫倫力而吸引最後造成細胞相互吸 附形成串珠狀（pead chain)，此現象被稱為相互介電泳力 (mutual dielectrophoresis )。 ' 目前介電泳之電極設計多以平面2D電極為主，如美國 專利 NO.6,989,086 ; 6,764,583 ; 6,835,552及 6,875,329 等， 雖然其製程簡單，但其缺點在於所產生的電場梯度較小， φ 捕捉細胞的效率較低。 另方面，細胞晶片重點在於能捕捉或是操控單一細胞 之目標，但2D的介電泳電極所造成的電場分佈無法達到僅 捕捉單一細胞之精度，前揭美國專利案即是存有此項缺 失。本發明人即是有感於習知介電泳晶片在電極設計上仍 存有缺失，無法增進捕捉或是操控單一細胞之效能，乃潛 心研究’期能克服習知之缺失，M經多次努力I終乃發展 出本發明。 6 200815276 【發明内容】 緣是： 本發明之主要目的，其係提供一種具三維微結構之生 物微粒抓取器，該抓取器係包含有上層體、下層體及位於 上、下層體間之微流管體，上、下層體均設有電極，使上'、 下層體之電極的電場方向與微流管體内之流場方向相垂直 而足以產生垂直的電場梯度，可增加捕捉微粒細胞的效率。 本發明之另-主要目的，其係提供一種具三維微結構 •之生物微粒抓取器，其係利用介電冰效應在三維穴井結構 中，因上、下層電極的佈局與介電泳之非均勻電場的效應， 使細胞附著於穴井凹槽内，吸附後細胞與凹槽表面之直空 吸附力及微結構的側支撐力足以固定細胞，此時可調降電 場強度並利用，流道沖刷介電泳電極區域，讓其他細胞得 以清除，僅存單一細胞於預設的三維穴井中。 本發明之再-主要目的，其係提供一種具三維微結構 之生物微粒抓取器’當細胞因介電泳效應被捕捉至穴井凹 •槽後，即使在沒有任何電場的作用下，單一細胞仍可固定 於預設穴井位置内，並且在一定的流速以下，細胞仍舊固 定於穴井内，不被流速所沖走，除非流速大岭該細胞所捕 捉的介電泳力。 本發明之又一主要目的，其係提供一種具三維微結構 之生物微粒抓取器，因此，本發明將以一符合細胞尺寸之 二維微結構輔助介電泳力捕捉細胞，以達到單細胞之精 度’並S又a十陣列式細胞陷阱同時擁有單細胞之精度外還可 以取得群體的結果與單一細胞各種生化反應的電性量測。 200815276 本發明之復一主要目的，其係提供一種具三維微結構 之生物微粒抓取器的製造方法，該方法可利用準分子雷射 加工、以熱壓製程法及以SU-8厚膜光阻來製造三維穴井詰 構單細胞型之介電泳捕捉晶片及其所屬之下層體，並結合 上層體、微流管道體，可簡易地完成製造出抓取器者。 為使貴審查員進一步瞭解本發明，茲佐以圖示詳加 說明如后。 【實施方式】 請參考第一至三圖，本發明生物微粒抓取器係包含 有··上層體⑴、微流管體(2)及下層體(3)，於上層體⑴設 有上電極(11)及入口（12)、出口（13)，下層體(3)亦設有下電 極(31)，前述入口（12)即是提供流體進入抓取器而在微流管 道(21)内流通，並由出口（13)流出抓取器。前述下層體（3) 另設有矩陣式穴井(4)。本發明最主要特徵即是上、下層體 (1)、（3)之電極的電場方向與微流管道(21)内之流體的流場 方向相垂直而形成縱向不均勻電極，可迅速有效捕捉浮流 於微流管道(2)中的生物微粒（5)至下層體（3)所設之穴井(4) 内。 本發明抓取器在下層體（3)所完成之穴井（4)的立體結 構大小，其係可依據所欲捕捉之微粒子的大小而設定，矩 陣式穴井中之每相鄰穴井（4)間之間距亦可依需要性而選 擇其間距長度。進一步而言，穴井(4)可如半球型（如第三 圖），亦气如第四圖所示之矩型體穴井(41)。 200815276 本啦明鈿述之下層體除了可設計求介電泳下電極(31) 外，在三維穴井結構之·層體⑶上另可增减測電極⑹， 如第五圖所示，其目的在於抓取單一微粒細胞後，經由電 壓的切換，可對單—細胞進行各種生化反應之電性量測以 提供從事藥物篩選的功能。 本發明抓取器之下層體(3)係由玻璃（7i)、suj厚膜光 阻(72)、金(73)所組成，其中金(73)即是為下層體(3)内所設 之下電極(31)，該下層體(3)以準分子雷射加工後形成矩陣 • 型之穴井(4);微流管體(2)係由PDMS(70)所製成；上層體(1) 則是由導電玻璃ITO Glass(74)所構成；其整體即可如第六 圖所不者。本發明為達成前述具三維微結構之生物微粒抓 取器，可利用準分子雷射加工製作三維穴井結構單細胞型 之介電泳捕捉晶片。以準分子雷射加工，可加工出三維穴 井微結構，以便更能確實捕捉微粒以及細胞。準分子雷射 不僅加工出穴井結構，並且可將蒸鍍在su_8上的電極去 除，因此可得到週期性的電極及介電泳力所造成的非均勻 # 電場現象，可將微粒與細胞捉取至穴井結構内，如第六圖 所示。 請參考第三圖所示之抓取器，前述下層體(3)係由玻璃 (75)、PDMS(76)及金(77)所組成，其中金(77)即是為下層體 (3)内所設之下電極(31)，該下層體(3)係以熱壓製程法製作 出二維穴井結構之介電泳捕捉晶片，即可形成穴井(4);微 流管體(2)係由PDMS(78)所製成；上層體(1)則是由導電玻 璃ITO Glass(79)所構成；其整體即可如第三圖所示者。 200815276 請參考第四圖所示之抓取器，下層體（3)係 (8〇)、金⑽及SU-8(82)所組成，其中金 = ⑶内所設—之下電極⑼，該下層體(3)係以利用黃 程製作出二維穴井結構之介電泳捕捉晶片，即可形成= 體穴井(41);微流管體⑺係由PDMS(83)所製成；_^層體⑴ 則是由導電破璃iTQ Glass(84)所構成；其整體即可二 圖所示者。 請參考第五圖所示之抓取器，前述下層體(3)係由玻璃 φ (85)、金(86)、SU-8(87)及金(88)所組成，其中金(88)即是為 下層體(3)内所設之下電極(31)，而金（86)即是為感測電極 (6)，該下層體(3)係利用準分子雷射加工出三維穴井之介電 泳捕捉晶片，即可形成穴井(4);微流管體(2)係由pDMS(89) 所製成；上層體⑴則是由導電玻璃IT0 Glass(9〇w構成； 其整體即可如第五圖所示者。 本發明抓取器在下層體設有矩陣型三維穴井，該等穴 井係可利用雷射加工、熱壓製程及微影製程等方法予以完 φ 成’進而可和上層體、微流管體相結合成一具有三維穴井 之抓取器。請參考第七圖，先以玻璃、SU-8、金等材料相 結合成一平面狀下層體，復以準分子雷射加工成具有三維 六井之下層體；該下層體進而與微流管體、上層體相結合 成具三維穴井之抓取器。 請參考第八圖，以PU材料經準分子雷射加工成具三維 穴井之基材，其後經微電鍍槽電鍍一層金屬膜以形成具有 三維穴井之金屬凸模，該凸模與下層體經熱壓製程即可完 成一具有三維穴井之下層體，進而與ITO導電玻璃之上層體 200815276 及微流管體共同結合成本發明具三維穴井之抓取器。 本發明亦可利用黃光微影製程來完成，請參考第九 圖，以玻璃、金、SU-8相結合成一平面狀下層體，其經黃 光微影製程即可形成具有三維六井之下層體，進而與上層 體、微流管體相結合成本發明具三維穴井之抓取器。 統觀前論，本發明利用上、下層體各設有電極，使上、 卞層體電極之電場方向與微流管體之管道内的流場方向相 垂直，可形成縱向不均勻電極，可迅速有效捕捉浮流於微 φ 流管道中的生物微粒至下層體所設之穴井内，可增進抓取 效能與解析度，可避免重疊與聚集的缺點真正達到單細胞 或單顆粒的抓取能力。 本發明之設計異於習知利用電場方向平行於流體方向 所設計之抓取器，本發明顯然具其新穎性，況乃本發明更 具有抓取微粒細胞之功效，另具進步性，顯然允合發明專 利之要件，乃提出專利申請。200815276 IX. Description of the Invention: [Technical Field] The present invention relates to a biological particle grabber having a three-dimensional microstructure and a method of manufacturing the same, and more particularly to a dielectrophoretic force generated by a dielectrophoretic electrode (DEP Force) a grasping body for capturing biological particles in a desired hole well and a manufacturing method thereof, the grasping device comprising an upper layer body, a lower layer body and a micro flow tube, wherein electrodes are provided on the upper and lower layers to make the upper and lower layers The electric field direction of the body electrode is perpendicular to the flow field direction of the microfluidic tube to form a longitudinal non-uniform electrode, which can quickly and effectively capture the biological particles floating in the microfluidic pipeline to the wells provided in the lower layer body, thereby improving the grasping Performance and resolution, avoiding the disadvantages of overlap and aggregation, truly achieve the ability to capture single cells or single particles. This feature is based on the application of the concept of three-dimensional structure to dielectrophoretic biochips. The main purpose of this dielectric ice wafer is to capture and immobilize biological particles such as cells, functional latex particles, nanoparticles, genes, and the like. And because of the effect of the microstructure, the cells can be fixed after adsorption, and the vacuum adsorption force on the surface of the well hole and the side support force of the micro- and Ό structures can be maintained without the need to continue to provide the dielectrophoretic force. Significantly increase the survival rate of cells in the wafer. [Prior Art] Since the size of cells is in the micron range, the development of microelectromechanical system technology (MEMS) in recent years has promoted the purpose of manipulating single cells and measuring, and the application of dielectrophoretic force to cell type biochips is the most A wide range of currently known dielectrophoretic applications in cell-based biochip modules include the following functions: cell fixation, cell transport, cell separation, cell alignment, cell measurement, cell sorting, and the like. The mechanism of dielectrophoresis driving is that when the fine 200815276 cells are in a non-uniform electric Held, the dielectric properties of the cells will induce charges on the surface to produce the same or opposite electrical directions as the applied electricity. The electric dipole, which is moved by the positive dielectrophoretic force to the strong electric field density region or concentrated by the negative dielectrophoretic force to the weak electric field region due to the non-uniformity of the electric field, can be made by the design of the electrode Driven by the electric field gradient and fixed in the designed area. Conventional methods use columnar, interdigitated (inter (jigital), and dot-like electrode types to successfully locate cells, but the disadvantage is that the dielectrophoretic force to which the cells are subjected is limited to surface movement, and The number of captured cells cannot be effectively controlled in a single cell. This is because the induced electricity between the cells and the cells is attracted by the Coulomb force and finally causes the cells to adsorb each other to form a pead chain. The phenomenon is called mutual dielectrophoresis. 'At present, the electrode design of dielectrophoresis is mainly based on planar 2D electrodes, such as US Patent Nos. 6,989,086; 6,764,583; 6,835,552 and 6,875,329, etc., although its process is simple, its disadvantages The generated electric field gradient is small, and φ captures cells with low efficiency. On the other hand, the cell wafer focuses on the target of capturing or manipulating a single cell, but the electric field distribution caused by the 2D dielectrophoretic electrode cannot achieve a single capture. The accuracy of the cell, the former US patent case is the lack of this. The inventor is aware of the traditional dielectric There is still a defect in the design of the electrode, which does not improve the performance of capturing or manipulating a single cell. It is a painstaking study to overcome the lack of conventional knowledge. M has developed the invention after many efforts. 6 200815276 [Summary content The main purpose of the present invention is to provide a three-dimensional microstructured biological particle grasper, the grasper comprising an upper layer body, a lower layer body and a microfluidic tube between the upper and lower layers. The upper and lower layers are respectively provided with electrodes, so that the electric field direction of the electrodes of the upper and lower layers is perpendicular to the flow field direction in the microfluidic tube, which is sufficient to generate a vertical electric field gradient, which can increase the efficiency of capturing the microparticle cells. The other main purpose is to provide a three-dimensional micro-structured biological particle grabber that utilizes the dielectric ice effect in a three-dimensional hole well structure due to the layout of the upper and lower electrodes and the non-uniform electric field of dielectrophoresis. The effect is that the cells are attached to the well of the well, and the direct adsorption force of the cells and the surface of the groove after adsorption and the lateral support force of the microstructure are sufficient to fix the cells, and the adjustable electric field is strong at this time. And use, the flow channel washes the dielectrophoretic electrode area, allowing other cells to be removed, and only a single cell is stored in the preset three-dimensional hole well. The re-main purpose of the present invention is to provide a three-dimensional microstructure of biological particles When the cells are captured by the dielectrophoresis effect into the wells and grooves, even without any electric field, the single cells can be fixed in the preset well position, and below a certain flow rate, the cells are still fixed. In the well, it is not washed away by the flow rate unless the flow rate is high. The other object of the present invention is to provide a biological particle grabber having a three-dimensional microstructure, and therefore, the present invention The cell will be captured by a two-dimensional microstructural assisted dielectrophoretic force that meets cell size to achieve single cell precision' and S and a ten array cell traps with single cell precision can also achieve population results with single cells Electrical measurements of various biochemical reactions. 200815276 The main object of the present invention is to provide a method for manufacturing a biological particle grabber having a three-dimensional microstructure, which can utilize excimer laser processing, hot pressing method and SU-8 thick film light The three-dimensional well-well single-cell type dielectrophoresis capture wafer and its underlying layer are combined, and the upper layer and the micro-flow tube body are combined to easily manufacture the gripper. In order for the examiner to further understand the present invention, the following is a detailed description of the present invention. [Embodiment] Referring to Figures 1 to 3, the biological particle grabber of the present invention comprises an upper layer body (1), a microfluidic tube body (2) and a lower layer body (3), and an upper electrode (1) is provided with an upper electrode. (11) and inlet (12), outlet (13), the lower layer (3) is also provided with a lower electrode (31), the inlet (12) is to provide fluid into the gripper and in the microfluidic conduit (21) Circulate and flow out of the gripper by the outlet (13). The aforementioned lower layer body (3) is additionally provided with a matrix type well (4). The most important feature of the present invention is that the electric field direction of the electrodes of the upper and lower layers (1) and (3) is perpendicular to the flow field direction of the fluid in the microfluidic tube (21) to form a longitudinal non-uniform electrode, which can be quickly and effectively captured. The floating stream flows from the biological particles (5) in the microfluidic conduit (2) to the wells (4) provided in the lower layer (3). The size of the three-dimensional structure of the hole well (4) completed by the gripper in the lower layer body (3) can be set according to the size of the microparticles to be captured, and each adjacent hole in the matrix type well (4) The distance between the distances can also be selected according to the needs. Further, the hole well (4) may be a hemispherical type (as shown in the third figure), and also a rectangular body well (41) as shown in the fourth figure. 200815276 In addition to the design of the dielectrophoresis lower electrode (31), the layer can be increased or decreased (3) on the layer body (3) of the three-dimensional hole well structure, as shown in the fifth figure. After grasping a single microparticle cell, the electrical measurement of various biochemical reactions can be performed on the single cell by voltage switching to provide a function for drug screening. The layer body (3) of the gripper of the present invention is composed of glass (7i), suj thick film photoresist (72) and gold (73), wherein gold (73) is provided for the lower layer body (3). The lower electrode (31), the lower layer body (3) is processed by excimer laser to form a matrix type hole (4); the microfluidic tube body (2) is made of PDMS (70); the upper layer body ( 1) It is composed of conductive glass ITO Glass (74); the whole can be as shown in the sixth figure. In order to achieve the foregoing three-dimensional microstructure microparticle grasper, the present invention can utilize a quasi-molecular laser processing to fabricate a three-dimensional well structure single cell type dielectrophoresis capture wafer. By excimer laser processing, a three-dimensional well micro-structure can be processed to more accurately capture particles and cells. The excimer laser not only processes the well structure, but also removes the electrode evaporated on the su_8, thus obtaining a non-uniform electric field phenomenon caused by periodic electrodes and dielectrophoretic forces, which can capture particles and cells to Inside the hole well structure, as shown in the sixth figure. Please refer to the gripper shown in the third figure. The lower layer body (3) is composed of glass (75), PDMS (76) and gold (77), wherein gold (77) is the lower layer (3). The lower electrode (31) is provided, and the lower layer body (3) is formed by a thermocompression method to prepare a dielectrophoretic capture wafer of a two-dimensional hole structure, thereby forming a hole well (4); the microfluidic tube body (2) It is made of PDMS (78); the upper layer (1) is made of conductive glass ITO Glass (79); the whole can be as shown in the third figure. 200815276 Please refer to the grabber shown in the fourth figure, the lower layer body (3) is composed of (8 〇), gold (10) and SU-8 (82), where gold = (3) is set - the lower electrode (9), The lower layer body (3) is formed by dielectrophoresis of a two-dimensional well structure using a yellow process to form a cavity (41); the microfluidic body (7) is made of PDMS (83); The body (1) is composed of conductive glass iTQ Glass (84); the whole can be as shown in the two figures. Please refer to the gripper shown in the fifth figure. The lower layer body (3) is composed of glass φ (85), gold (86), SU-8 (87) and gold (88), of which gold (88) That is, the lower electrode (31) is provided in the lower layer body (3), and the gold (86) is the sensing electrode (6), and the lower layer body (3) is processed by excimer laser to process the three-dimensional hole well. Electrophoresis captures the wafer to form a well (4); the microfluidic tube (2) is made of pDMS (89); the upper layer (1) is made of conductive glass IT0 Glass (9〇w; The figure is shown in the fifth figure. The gripper of the present invention is provided with a matrix type three-dimensional hole well in the lower layer body, and the hole well system can be completed by using a laser processing, a hot pressing process and a lithography process, and then can be combined with the upper layer. The body and the microfluidic tube are combined into a grabber with a three-dimensional hole well. Please refer to the seventh figure, firstly combine the glass, SU-8, gold and other materials into a planar lower layer body, and then process the excimer laser into a The utility model has a three-dimensional six-well lower layer body; the lower layer body is further combined with the micro-flow tube body and the upper layer body to form a three-dimensional hole well grabber. Please refer to the eighth figure to divide the PU material by standard. The laser is processed into a substrate with a three-dimensional hole well, and then a metal film is plated through the micro-plating tank to form a metal punch having a three-dimensional hole well, and the punch and the lower layer are completed by a hot pressing process to complete a layer having a three-dimensional hole well The body is further combined with the ITO conductive glass upper layer body 200815276 and the microfluidic tube body to form a gripper with a three-dimensional hole well. The invention can also be completed by using a yellow light lithography process, please refer to the ninth figure, with glass, gold, SU-8 combines into a planar lower layer body, which can form a three-dimensional six-well lower layer body through the yellow light lithography process, and then combine with the upper layer body and the micro-flow tube body to invent the three-dimensional hole well grabber. According to the present invention, the upper and lower layers are respectively provided with electrodes, so that the electric field direction of the upper and lower layer electrodes is perpendicular to the flow field direction in the pipe of the microfluidic tube body, and a longitudinal uneven electrode can be formed, which can quickly and effectively capture The biological particles floating in the micro-φ flow pipe to the hole wells provided in the lower layer can improve the gripping efficiency and resolution, and avoid the disadvantages of overlap and aggregation. Or the grasping ability of a single particle. The design of the present invention is different from the conventional grasper designed to use the direction of the electric field parallel to the direction of the fluid, and the present invention obviously has the novelty, and the present invention has the effect of grasping the particulate cells, and It is progressive, and it is obvious that the requirements of the invention patent are granted, and the patent application is filed.
11 200815276 【圖式簡單說明】 第-圖：係本發明抓取器部份透視之立體圖。 第二圖：係本發明抓取器之外觀立體圖。 第三、四、五、六圖：係本發明抓取器之構造剖視圖。 第七、八、九圖：係本發明抓取器製造方法之流程圖。 【主要元件符號說明】 (1) 上層體 (11) 上電極 (12) 入口 (13) 出口 (2) 微流管體 (21)微流管道 (3) 下層體 (31)下電極 (4) 穴井 (41)矩型體穴井 (5) 生物微粒 (6) 感測電極11 200815276 [Simple description of the drawings] Fig.: A perspective view of a partial perspective view of the gripper of the present invention. Second drawing: is a perspective view of the appearance of the gripper of the present invention. Third, fourth, fifth, and sixth figures: A cross-sectional view showing the structure of the gripper of the present invention. Seventh, eight, and nine figures: A flow chart of the method for manufacturing the gripper of the present invention. [Main component symbol description] (1) Upper layer (11) Upper electrode (12) Entrance (13) Exit (2) Micro flow tube (21) Micro flow tube (3) Lower layer (31) Lower electrode (4) Hole well (41) rectangular body well (5) biological particles (6) sensing electrode
(70) PDMS (71) 玻璃 C72)SU-8厚膜光阻 (73) 金 (74) 導電玻璃ITO Glass (75) 玻璃(70) PDMS (71) Glass C72) SU-8 Thick Film Resist (73) Gold (74) Conductive Glass ITO Glass (75) Glass
(76) PDMS (77) 金(76) PDMS (77) Gold
(78) PDMS (79) 導電破璃ITO Glass (80) 玻璃 (81) 金 (82) SU_8(78) PDMS (79) Conductive ITO Glass (80) Glass (81) Gold (82) SU_8
(83) PDMS (84) 導電玻璃ITO Glass (85) 玻璃 (86) 金 (87) SU-8 (88) 金(83) PDMS (84) Conductive Glass ITO Glass (85) Glass (86) Gold (87) SU-8 (88) Gold
(89) PDMS (90) 導電玻璃ITO Glass 12(89) PDMS (90) Conductive Glass ITO Glass 12