1308131 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 器及其製造方法’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 ，體所設之穴井内，可增進抓取效能與解析度，可避免重 豐與聚集的缺點真正達到單細胞或單顆粒的抓取能力。本 發明特點是將三維結構之概念應用於介電泳生物晶 介雷ϋ θ μ i ^ 月b性礼膠粒子、奈米顆粒 應可 # 知除 HtA 、奈米顆粒、基因等。並且由於微結構的效1308131 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 gripper for capturing biological particles in a desired hole well and a method for manufacturing the same. The gripper includes an upper layer body, a lower layer body and a micro flow tube, and electrodes are provided on the upper and lower layers to make the upper and lower layers The direction of the electric field of the body electrode is perpendicular to the direction of the flow field 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 lower part of the well, and can enhance the grasping The efficiency and resolution can avoid the disadvantages of heavy and aggregate and truly achieve the single cell or single particle grabbing ability. The invention is characterized in that the concept of the three-dimensional structure is applied to the dielectrophoretic biocrystals, the Thunder θ μ i ^ month b ritual particles, and the nano granules can be used to eliminate HtA, nano particles, genes and the like. And due to the effectiveness of the microstructure
At 日月主要目的在抓取與固定生物微粒例如細胞、功 【先前技術】The main purpose of At-Sun is to capture and fix biological particles such as cells and work. [Prior Art]
介電泳驅冑的機制為當細 1308131 胞處於一非均勻交流電場（AC non-uniform electric field )， 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 場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（interdigital )、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 所受誘發之電荷，會因庫倫力而吸引最後造成細胞相互吸 附形成串珠狀（pearl 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， 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’歷經多次努力而終乃發展 出本發明。 1308131 【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。 本發明之另一主要目的，其係提供一種具三維微結構 鲁之生物微粒抓取器，其係利用介電泳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真空 吸附力及微結構的側支撐力足以固定細胞，此時可調降電 場強度並利用微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，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，除非流速大於該細胞所捕 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 三維微結構輔助介電泳力捕捉細胞，以達到單細胞之精 度，並設計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。 1308131 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結 構單細胞型之”電冰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。口 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（1)、微流管體(2)及下層體（3)，於上層體（1)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 _ 方向相垂直而开> 成縱向不均勻電極，可迅速有效捕捉浮流 於微流管道(2)中的生物微粒至下層體(3)所設之穴井 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），亦可如第四圖所示之矩型體穴井（41)。 1308131 本發明前狀下層體除了可設計成介電泳下電極⑼ 外在三維穴井結構之下層體(3)上另可增加感測電極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，可對單一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（71)、su_8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 圖所示者。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，以便更能破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 φ 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(ή)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 出三維穴: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。 1308131 請參考第四圖所示之抓取器，下層體（3)係由玻璃 (80)、金(81)及SU-8(82)所組成，其中金(81)即是為下層體 (3)内所設之下電極(31) ’該下層體(3)係以利用黃光微影製 程製作出三維穴井結構之介電泳捕捉晶片，即可形成矩型 體穴井(41);微流管體(2)係由PDMS(83)所製成；上層體（1) 則是由導電玻璃ITO Glass(84)所構成；其整體即可如第四 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 所製成；上層體（1)則是由導電玻璃ITO Glass(90)所構成. 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#& 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 穴井之下層體；該下層體進而與微流管體、上層體相梦人 成具三維穴井之抓取器。 σ 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臈以形成具有 三維穴井之金屬凸模，該凸模與下層體經熱壓製程即可完 成一具有三維六井之下層體，進而與ΙΤΟ導電玻璃之上層$ 1308131 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穴井之下層體，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 | 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。The mechanism of dielectrophoresis is that when the fine 1308131 cell is in an AC non-uniform electric field, the dielectric property of the cell induces a charge on its surface to produce a galvanic couple with the same or opposite direction to the applied electric field. 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 due to the non-uniformity of the electric field, can be made by the design of the electrode. The electric field gradient is driven and fixed in the designed area. Conventional methods use cylindrical, interdigital, and dot-like electrode designs to successfully position cells, but the disadvantage is that the dielectrophoretic force to which the cells are subjected is limited to surface movement and is captured. The number of cells cannot be effectively controlled in a single cell. This is due to the induced charge between the cells and the cells, which is attracted by the Coulomb force and finally causes the cells to adsorb each other to form a pearl chain. This phenomenon is called Mutual dielectrophoresis ° At present, the electrode design of dielectrophoresis is mainly based on planar 2D electrodes, such as U.S. Patent Nos. 6,989,086; 6,764,583; 6,835,552 and 6,875,329, etc., although the process is simple, the disadvantage is that the generated electric field The gradient is small and the efficiency of capturing cells is low. On the other hand, the cell wafer focuses on the ability to capture or manipulate a single cell, but the electric field distribution caused by the 2D dielectrophoretic electrode cannot achieve the accuracy of capturing only a single cell. The US patent case has this deficiency. The inventor is aware that the conventional dielectrophoresis wafer still has a defect in the electrode design, which is unable to enhance the performance of capturing or manipulating a single cell, and is devoted to the study of the period to overcome the lack of conventional knowledge. The present invention has been developed. 1308131 [Invention] The main purpose of the present invention is to provide a three-dimensional microstructure of a biological particle grabber, the grasper comprising an upper layer body, a lower layer body and a microscopic structure between the upper and lower layers The flow tube body is provided with electrodes on the upper and lower layers, 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. . Another main object of the present invention is to provide a three-dimensional micro-structured Lu bio-grass grabber which utilizes a dielectrophoretic 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 vacuum adsorption force of the cell and the groove surface after adsorption and the side support force of the microstructure are sufficient to fix the cells, and the electric field strength can be adjusted and the microelectrofluid is used to wash the dielectrophoretic electrode. The area allows other cells to be removed, leaving only a single cell in a preset 3D hole. A further main object of the present invention is to provide a biological particle grabber having a three-dimensional microstructure. When a cell is captured by a dielectrophoretic effect to a well, a single cell is still in the absence of any electric field. It can be fixed in the preset well position, and below a certain flow rate, the cells are still fixed in the well and are not washed away by the flow rate unless the flow rate is greater than the dielectrophoretic force captured by the cell. Another main object of the present invention is to provide a biological particle grabber having a three-dimensional microstructure. Therefore, the present invention will capture cells with a three-dimensional microstructure assisted dielectrophoretic force in accordance with cell size to achieve single cell precision. And designing array-type cell traps while possessing the accuracy of single cells can also achieve electrical measurements of the population's results with various biochemical reactions of a single cell. 1308131 A primary 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 photoresist. The three-dimensional well-well structure single-cell type "electric ice capture wafer and its underlying layer body, combined with the upper layer body and the micro-flow pipe body, can easily complete the manufacture of the gripper. The mouth is to enable the examiner to further understand this The invention is described in detail below with reference to the drawings. _ [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 the lower layer body (3), the upper layer body (1) is provided with an upper electrode (11), an inlet (12), and an outlet (13), and the lower layer body (3) is also provided with a lower electrode (31), and the inlet (12) That is, the fluid is supplied into the gripper and flows in the microfluidic conduit (21), and flows out of the gripper by the outlet (13). The lower layer body (3) is further provided with a matrix type well (4). The characteristics are the electric field direction of the electrodes of the upper and lower layers (1), (3) and the microfluidic tube (21) The flow field of the body _ is perpendicular to the direction and becomes a longitudinal non-uniform electrode, which can quickly and effectively capture the biological particles floating in the micro-flow pipe (2) to the hole wells provided in the lower layer body (3). The size of the three-dimensional structure of the hole well (4) completed by the lower layer body (3) can be set according to the size of the particles to be captured, and the distance between each adjacent hole (4) in the matrix type hole well can also be The pitch length is selected according to the necessity. Further, the hole well (4) may be a hemispherical type (as shown in the third figure) or a rectangular body well (41) as shown in the fourth figure. 1308131 The lower layer body can be designed as a dielectrophoretic lower electrode (9). The sensing electrode (6) can be further added to the layer body (3) under the three-dimensional hole well structure, as shown in the fifth figure. The purpose is to capture a single particle cell and pass the voltage. The switch can perform electrical measurement of various biochemical reactions on a single cell to provide a function for drug screening. The lower layer of the grasper of the present invention (3) is made of glass (71), su_8 thick film photoresist (72) And gold (73), in which gold (73) is the lower layer (3) The lower electrode (31) is provided, and the lower layer body (3) is processed by excimer laser to form a matrix type hole (4); the micro flow tube body (2) is made of PDMS (70); The body (1) is composed of conductive glass ITO Glass (74); the whole can be as shown in Fig. 6. The invention can realize the above-mentioned biological particle grabber with three-dimensional microstructure, and can utilize the excimer mine The single-cell type dielectrophoresis capture wafer is fabricated by three-dimensional hole well structure. The excimer laser processing can process the three-dimensional hole well microstructure to better capture the particles and cells. The excimer laser not only processes the well structure. Moreover, the electrode evaporated on the su_8 can be removed, so that a non-uniform φ electric field phenomenon caused by periodic electrodes and dielectrophoretic forces can be obtained, and the particles and cells can be captured into the 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 (ή) is the lower layer (3). The lower electrode (31) is provided, and the lower layer body (3) is formed by a hot pressing process to form a three-dimensional hole: a dielectrophoresis capture wafer of a well structure, thereby forming a hole well (4); a 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. 1308131 Please refer to the gripper shown in the fourth figure. The lower layer (3) is composed of glass (80), gold (81) and SU-8 (82), of which gold (81) is the lower layer ( 3) The lower electrode (31) is set in the lower layer body (3) to form a rectangular body cavity well (41) by using a yellow light lithography process to fabricate a three-dimensional hole well structure dielectrophoresis capture wafer (41); a microfluidic tube body (2) is made of PDMS (83); the upper layer (1) is made of conductive glass ITO Glass (84); the whole can be as shown in the fourth figure. 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. The electrophoresis capture wafer can form a well (4); the microfluidic tube (2) is made of; the upper layer (1) is made of conductive glass ITO Glass (90). The figure shown. The gripper of the invention is provided with a matrix type three-dimensional hole well in the lower layer body, and the #& well system can be completed by using a laser processing, a hot pressing process and a lithography process, and can be combined with the upper layer body and the micro flow tube. The body is combined into a gripper with a three-dimensional hole well. Please refer to the seventh figure, firstly combine glass, SU-8, gold and other materials into a planar lower layer body, and then process the excimer laser into a layer body with a three-dimensional hole well; the lower layer body and the micro flow tube body, The upper layer of the dreamer is a gripper with a three-dimensional hole well. σ Please refer to the eighth figure, the PU material is processed into a three-dimensional hole well substrate by excimer laser, and then a metal crucible is electroplated through the micro-plating tank to form a metal punch with a three-dimensional hole well, the punch and the lower layer The hot-pressing process can complete a layered body with a three-dimensional six wells, and then combined with the upper layer of the conductive glass of the first layer of $1308131 and the microfluidic tube 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, combining glass, gold and SU-8 into a planar lower layer body, which can form a layer body with a three-dimensional hole well through a yellow light lithography process, and then The upper layer body and the micro-flow tube body are combined to form a gripper having a three-dimensional hole well. In 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. Quickly and effectively capture the biological particles in the micro-flow pipeline to the wells located in the lower layer, which can improve the grasping efficiency and resolution, and avoid the shortcomings of overlap and aggregation to truly achieve the single-cell or single-particle grasping ability. 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. The invention obviously has the novelty, and the invention has the effect of grasping the microparticle cells, and is further advanced, and obviously allows the invention. The patent requirement is a patent application.
11 1308131 【圖式簡單說明】 弟圖.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。11 1308131 [Simple description of the drawing] A drawing 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.
【主要元件符號說明】 (1) 上層體 (Π)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[Description of main component symbols] (1) Upper layer (Π) 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 (72) SU-8厚膜光阻 (73) 金 (74) 導電玻璃ITO Glass (75) 玻璃 (76) PDMS ㈤金(70) PDMS (71) Glass (72) SU-8 Thick Film Resist (73) Gold (74) Conductive Glass ITO Glass (75) Glass (76) PDMS (V) Gold
(78) PDMS (79) 導電玻璃ITO Glass (80) 玻璃 (81) 金 (82) SU-8(78) PDMS (79) Conductive Glass 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(89) PDMS (90) Conductive Glass ITO Glass