1274075 九、發明說明 【發明所屬之技術領域】 本發明是有關於一種基因轉殖技術，特別是適用於電脈衝 胞膜穿孔技術之裝置與方法。 【先前技術】 基因轉殖（gene transfer)應用係把一段外來的基因或核 酸片斷，經過外在力量（如注射、通電、粒子槍或化學藥劑 麵^等）迫使它進入配子、胚胎或體細胞的細胞核或細胞質内， 好讓外來基因能在胚胎或細胞中繼續複製，進而在個體上表 現該段外來基因的特色。 試管試驗（In-Vitro)的基因轉殖方法中，可以將外來基 -因導入配子、胚胎或體細胞的細胞核或細胞質内的方法有很 -多’如鈣離子法，微脂粒法、病毒感染法、胚幹細胞法、顯 庄射法 電脈衝胞膜穿孔法（Electroporation)、雷射誘導 法、基因槍法及精子載體法。1274075 IX. Description of the Invention [Technical Field] The present invention relates to a gene transfer technique, and more particularly to an apparatus and method suitable for electroporation membrane perforation. [Prior Art] Gene transfer applications force a foreign gene or nucleic acid fragment into a gamete, embryo, or somatic cell by external force (such as injection, energization, particle gun, or chemical agent). Within the nucleus or cytoplasm, so that the foreign gene can continue to replicate in the embryo or cell, and then the characteristics of the foreign gene in the individual. In the In-Vitro gene transfer method, there are many methods for introducing a foreign group-into the nucleus or cytoplasm of a gamete, embryo or somatic cell, such as a calcium ion method, a liposome method, and a virus. Infection method, embryonic stem cell method, electro-acoustic perforation method, electron inducing method, laser-inducing method, gene gun method and sperm carrier method.
…、、而，使用病毒法進行基因轉殖，有免疫和基因重組驾 等的剎作用及疑慮。而使用基因搶雖然有極高的轉殖率，令 是受限於器材必須直接與轉殖區直接接觸，且基因搶法的 體微粒製備手婧臂：# Μ十 躓繁雜儲存時間短，還需要有投入昂貴的1 備。置於微注射法，目丨# ° — 1僅適用於單一細胞之上，無法同時勒 4丁於多個細胞。而宜 /、他方法如，微脂粒法、雷射誘導法、参 離子法荨技術也存在 本高#皮敏 轉 圍有限、儀器設備昂責、操作成 本间或私序繁雜等缺點。 1274075 乂下，電脈衝胞膜穿孔法’利用脈衝電壓施加於目 ::…吏細胞膜上的微小孔洞張開，將霞4列或藥物 傳遞：入細胞之中’操作程序較為單純。然而，傳統電脈衝 胞膜穿孔法操作規模γg γ 缺點暑-m 的耗材試劑；且最大的 缺點疋间達百伏的電壓產生大量的 死亡影響轉殖效率。 、、、耳熱，-成部份細胞的 因此有必要提供一種轉殖效率高、消耗樣本少、操作成 低的電脈衝胞膜穿孔方法與裝置。 ’、 【發明内容】 明的目的就是在提供一種轉殖效率高、消耗樣;..., and, using the virus method for gene transfer, there are brakes and doubts about immunity and genetic reorganization. Although the use of gene robbing has a very high conversion rate, it is limited by the fact that the equipment must be directly in contact with the transgenic area, and the gene granules are prepared by the body particles: # Μ 踬 踬 杂 杂 杂 杂Need to have expensive inputs. Placed in the microinjection method, the target # ° - 1 is only applicable to a single cell and cannot be simultaneously multiplied by multiple cells. However, he/methods such as the microlipid method, the laser-induced method, and the ion-inducing method also have the disadvantages of limited height, limited equipment, high equipment, and complicated operation. 1274075 Underarm, electric pulse membrane perforation method uses a pulse voltage to apply to the tiny pores on the cell membrane of the ...:吏 cell membrane to open the column 4 or the drug: into the cell' operation procedure is relatively simple. However, the conventional electric pulse cell perforation method operates a scale γg γ defect heat-m consumable reagent; and the biggest disadvantage is that a voltage of up to one hundred volts during the day produces a large number of deaths affecting the transfer efficiency. ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, The content of the invention is to provide a high efficiency and consumption sample;
低的電脈衝胞膜穿孔裝置與方法。其特徵在W 膜穿？轉殖法相結合，在電胞臈穿^ DNA? I / β供個預濃縮的電場力’來提高所要傳遞4 =Α序列或藥物在目標細胞周圍的局部濃度；並且縮小操.Low electrical pulse cell perforation device and method. Its characteristics are worn in the W film? The combination of transgenic methods, in the cell 臈 ^ ^ DNA? I / β for a pre-concentrated electric field force 'to increase the local concentration of the 4 = Α sequence or drug around the target cells; and reduce the operation.
:積，降低操作樣本與試劑之消耗，減少電能轉換生㈣“ 私細胞的損#，藉此增進轉殖效率降低成本。 … 在本發明的一些實施例之， 衝胞膜穿孔裝置，至少包括：至少二、二：預濃：式電朋 果# 一電極、第三電極以及一電源供應裝 /基材之平滑表面結合，藉以定義出相互連 區。第一電極與第二電極位於第-區之 :第且[=極與第一電極之間至少具有-距離。第三電極位 於第一區之内，具有大於哕筮 °亥第一區之-半徑範圍。電源供應裝 1274075 置貝]刀別&供苐一電極、電二電極以及電二電極所需的工作電 壓。 在本發明的較佳實施例之中，提供一工作容量小於〇 〇5ml 之電脈衝胞膜穿孔裝置。其中，此一裝置之電源供應裝置可以 輸出一交握電壓，藉以在電胞膜穿孔反應之前或之間提供一 個預濃縮的電場力，來提高目標細胞周圍DNA序列或藥物的 局部濃度。而且第一電極與該第二電極具有小於3mm之一距 離，兩者之間的有效工作電可降低至12v以下，以減少電脈衝 %胞膜穿孔反應中，因為電能轉換所產生之熱能對細胞的破壞。 因此根據以上述，本發明確實可以提供一種轉殖效率高、 消耗樣本少、操作成低的電脈衝胞膜穿孔裝置，以達到本發 明之目的。 •【實施方式】 本發明的目的就是在提供一種電脈衝胞膜穿孔裝置與方 法。係利用電泳原理提供一個預濃縮的電場力與電脈衝胞膜 &穿孔轉殖法相結合，並使用微機電製程結合微流體設計 (Microfiuidics )，改善一般電脈衝胞膜穿孔技術的高電位問 題，提供-電轉殖效率高、消耗樣本少、操作成低的電泳預 濃縮式電脈衝胞膜穿孔基因轉殖裝置。此一裝置至少包括：一 基材、第-電極電場、第二電極電場、以及電源供應裝置。其 中第一電場位於提供目標細胞生存所需的培養基之中，第一電 場可以促使細胞膜上之微小孔洞張開，使培養基中之帶電荷 之分子可以傳遞進入該細胞之中；第二電場包圍於該第一電場 1274075 之外，藉以在第一電場形成之前或形成之間提供一個濃縮帶 電荷分子的電場；而電源供應裝置則分別提供第一電場以及電 二電場所需之工作電壓。 在本發明的較佳實施例之中，電泳預濃縮式電脈衝胞膜穿 -孔裝置係由一電泳預濃縮式電脈衝胞膜穿孔晶片1〇〇(以下簡稱 反應晶片100)以及一電源供應裝置所組成。其中反應晶片1〇〇 係=供電脈衝胞膜穿孔作用之反應空間，電源供應裝置，係分 別提仏電冰預濃縮電場以及電脈衝胞膜穿孔反應所需之工作電 壓。 、在本發明的一些實施例之中，電源供應裝置係一脈衝電 源供應系統，包括一電脈衝強度（amplitude)控制裝置、一 脈衝維持時間（duration time )控制裝置以及一脈衝數目控 制。在本發明的-較佳實施例之中，係以單晶片微處理器作 -為脈衝電源供應系統，提供一交握電壓的輸出。 晴參照第1圖，第1圖係根據本發明之一較佳實施例所 繪示之電泳預濃縮式電脈衝胞膜穿孔晶片1〇〇的俯視圖。在本 月㈤。實施例之中，反應晶片至少包括基材工Μ、殼體結 構104、第一電極106、第二電極1〇8以及第三電極ιι〇。 其中基材1G2係作為承載基礎，同時具有生物相容性， 使細胞可以生長，因此至少具有一平滑表面。構成基材ι〇2的 材質至少包括-玻璃基板，在本發明的一些較佳實施例之中， 基材102係76口26 mm之載玻片。 - μ乡’、、、第2圖’第2圖係根據本發明之—較佳實施例所 繪示之殼體結構的透視圖。在本發明之中，殼體結構ι〇4至少 1274075 :括：中空結構’例如，四方體形中空結構、圓柱體形中空結 ★ ^夕邊柱狀體形中空結構。在本發明的較佳實施例之中， 殼體結構係一四方方體形中 R + / τ工、、Ό稱，尺寸大約為15mmx 15mm x 2mm 〇 ^ 在本發明之中，殼體結構副與基材102平滑表面相連接， ，以定義出相互連通之第—區域2〇1以及第二區域2〇3，有效容 量大約小於〇.〇5ml。 >在本t明的一些實施例之中，第一區2〇 1係殼體結構1 、與該基材102戶斤定義之内層空間。此一内層空間可以是，例如 圓柱體形、四方方體形或多邊形柱狀體。在本發明的較佳實施 例之中，殼體104之四方體形中空結構與該基材1〇2所定義出 的内層空間（第一區201)係為一圓柱體形；此一圓柱體具有小於 2mm之半徑，以及小於2mm之深度。在本發明之中，第一區域 -201，可容納細胞培養時所需的培養基，係培養細胞的位置， 也是胞膜穿孔的反應範圍。 在本發明之中，殼體結構1〇4與該基材1〇2所定義之空間更 包括一外層空間（第二區203)與内層空間相連通。外層空間（第二 區203)至少包括一凹室2〇5位於第一區2〇1之側壁周圍。在本 發明的一些實施例之中，凹室2〇5係由包圍第一區2〇1之中空 結構與基材所定義而成，其中，包圍第一區2〇 1之中空結構係 以一側壁與第一區201相隔，並以此一側壁與第一區201相互 連通。在本發明的另一些實施例之中，凹室205係位於第一區 .201側壁上，並與第一區2〇丨相互連通之一凹槽。在本發明的較 佳實施例之中，如第2圖所繪示，第二區2〇3係包圍於第一區 1274075 201之外侧’並與基材所共同定義而成之-U字型 室205内層側壁上方與第一區2〇1相互連通。 ，凹 根據本發明的較佳實施例，殼體結構1〇4之材質 有化學成分安定’無溶劑揮發等特性 有而，、 佳，不易傷害基板與電極，組裝方便加工容易貝材有質= 便實驗觀察等優點。其中較佳材質至少包括聚二甲 (Polydimethylsiloxane ^ PDMS) 〇 凡Accumulate, reduce the consumption of operating samples and reagents, reduce the conversion of electrical energy (4) "loss of private cells, thereby increasing the efficiency of translocation and reducing costs. ... In some embodiments of the invention, the perforating device of the cell membrane includes at least : at least two or two: pre-concentration: type electric pong fruit # one electrode, the third electrode and a smooth surface of a power supply device / substrate to define a mutual connection. The first electrode and the second electrode are located at - Zone: first and [= at least - distance between the pole and the first electrode. The third electrode is located within the first zone, having a radius larger than the first zone of the 哕筮 ° Hai. Power supply package 1274075] The operating voltage required for the electrode, the electric electrode, and the electric electrode. In a preferred embodiment of the present invention, an electric pulse cell perforating device having a working capacity of less than ml5 ml is provided. The power supply device of the device can output a handshake voltage to provide a pre-concentrated electric field force before or between the cell membrane perforation reaction to increase the local concentration of the DNA sequence or drug around the target cell. And the first electrode and the second electrode have a distance of less than 3mm, and the effective working electric power between the two can be reduced to less than 12v, so as to reduce the electric pulse% in the perforation of the membrane, because the thermal energy generated by the electric energy conversion to the cell Therefore, according to the above, the present invention can provide an electric pulse cell perforation device which has high transfer efficiency, low sample consumption, and low operation, and achieves the object of the present invention. It is to provide an electric pulse cell perforation device and method. The electrophoresis principle is used to provide a pre-concentrated electric field force combined with an electric pulse membrane & perforation method, and a microelectromechanical process combined with a microfluidic design (Microfiuidics). The utility model improves the high potential problem of the general electric pulse cell perforation technology, and provides an electrophoresis preconcentration electric pulse cell perforation gene transduction device with high electrotransfer efficiency, low consumption sample and low operation. The device includes at least: a base Material, a first electrode electric field, a second electrode electric field, and a power supply device, wherein the first electric field is located to provide a target cell Among the required mediums, the first electric field can cause the tiny pores on the cell membrane to open, so that the charged molecules in the medium can be transferred into the cells; the second electric field is surrounded by the first electric field 1274075, The electric field of the concentrated charged molecules is provided before or during the formation of the first electric field; and the power supply device provides the operating voltages required for the first electric field and the electric two electric fields, respectively. In a preferred embodiment of the invention The electrophoresis preconcentration type electric pulse cell membrane perforation device comprises an electrophoresis preconcentration type electric pulse cell perforated wafer 1 (hereinafter referred to as reaction wafer 100) and a power supply device. Department = power supply pulse cell membrane perforation reaction space, power supply device, is to increase the electric ice pre-concentration electric field and the electrical pulse cell membrane perforation reaction required operating voltage. In some embodiments of the invention, the power supply device is a pulsed power supply system including an electrical pulse amplitude control device, a pulse duration control device, and a pulse number control. In a preferred embodiment of the invention, a single wafer microprocessor is used as the pulsed power supply system to provide an output of the handshake voltage. Referring to Figure 1, Figure 1 is a plan view of an electrophoretic preconcentrated electrically pulsed cell membrane perforated wafer 1 according to a preferred embodiment of the present invention. In this month (five). In the embodiment, the reaction wafer includes at least a substrate process, a casing structure 104, a first electrode 106, a second electrode 1〇8, and a third electrode ιι. Among them, the substrate 1G2 is used as a bearing foundation, and at the same time has biocompatibility, so that the cells can grow, and thus at least have a smooth surface. The material constituting the substrate ι 2 includes at least a - glass substrate. In some preferred embodiments of the present invention, the substrate 102 is a 76 mm glass slide of 26 mm. - Fig. 2, Fig. 2 is a perspective view of the housing structure according to the preferred embodiment of the present invention. In the present invention, the casing structure ι 4 is at least 1274075: including: a hollow structure 'e.g., a tetragonal hollow structure, a cylindrical hollow structure, and a hollow cylindrical structure. In a preferred embodiment of the present invention, the housing structure is a square shape of R + /τ, nickname, and has a size of about 15 mm x 15 mm x 2 mm. In the present invention, the casing structure is It is connected to the smooth surface of the substrate 102 to define a first region 2〇1 and a second region 2〇3 which are in communication with each other, and the effective capacity is less than about 〇5〇. > In some embodiments of the present invention, the first zone 2〇1 is a shell structure 1 and an inner layer space defined by the substrate 102. This inner layer space may be, for example, a cylindrical shape, a rectangular parallelepiped shape or a polygonal columnar body. In a preferred embodiment of the present invention, the rectangular hollow structure of the casing 104 and the inner layer space (the first region 201) defined by the substrate 1〇2 are cylindrical; the cylinder has a smaller A radius of 2 mm and a depth of less than 2 mm. In the present invention, the first region -201 can accommodate the medium required for cell culture, the position of the cultured cells, and the reaction range of perforation of the membrane. In the present invention, the housing structure 1〇4 and the space defined by the substrate 1〇2 further include an outer space (second region 203) in communication with the inner layer space. The outer space (second zone 203) includes at least one recess 2〇5 located around the side wall of the first zone 2〇1. In some embodiments of the present invention, the recess 2〇5 is defined by a hollow structure surrounding the first region 2〇1 and a substrate, wherein the hollow structure surrounding the first region 2〇1 is The side wall is spaced apart from the first region 201 and communicates with the first region 201 by a side wall. In other embodiments of the invention, the recess 205 is located on the side wall of the first zone 201 and is in communication with one of the first zones 2 凹槽. In a preferred embodiment of the present invention, as shown in FIG. 2, the second region 2〇3 is surrounded by the outer side of the first region 1274074 and is defined by the substrate-U-shaped The upper side wall of the inner portion of the chamber 205 communicates with the first region 2〇1. According to a preferred embodiment of the present invention, the material of the casing structure 1〇4 has the characteristics of chemical composition stability, such as solvent-free volatilization, and is good, and it is not easy to damage the substrate and the electrode, and the assembly is convenient and easy to process. Then experiment and observe and other advantages. Among them, the preferred material includes at least polydimethylsiloxane (PDMS) 〇 凡
請參照第3圖1 3圖係根據本發明之—較佳實施例所 繪不之電脈衝胞膜穿孔晶片丨⑽的電路圖。在本發明之中 -電極H)6與第二電⑸08位於第一 @ 2〇1之内；分別 脈衝胞膜穿孔反應之正極與負極。第三電極係位於第二區：〇3 之中，係用來提供-電泳預濃縮電場。在本發明的_些實 之中第-電極1〇6、第二電極1〇8以及第三電極ιι〇係分別由不 同製程所形成之電極；1第一電極、第二電極以及第三電極不 共平面。但在本發明較佳實施例之中，第一電極1〇6、第二電極 108以及第三電極110係、由同一製程所同時形成之一電路圖配 置’其中第-電極106、第二電極1〇8以及第三電極n〇係同時 形成於該基材102之該平滑表面。（如第3圖所繪示）。 在此一電路圖配置中，第一電極1〇6與第二電極1〇8兩者 都具有線寬至少小於6G # m之指又（Intei*digitated)狀結構，且此 一指叉狀結構父互排列，彼此並不相連接。其中第一電極i % 與該第二電極108之指又狀結構之間的距離至少小於3mm。 第二電極11 0至少包括一環形電極，在本發明的較佳實施 例之中第二電極11 〇係一環形電極，位於該基材之該平滑表面 10 1274075 之上’且位於弟一區203之中’環繞第一電極1〇6與第二電極 108之指叉結構。第三電極11〇之半徑大於第一區2〇1之半徑範 圍，大於3mm小於5mm，在電脈衝胞膜穿孔反應區周圍形成一 環燒電場。在電脈衝胞膜穿孔反應之前或之間，當電源供應 •裝置輸出一交握電壓時，第三電極110可以提供一個預濃縮的 電場力，來提高目標細胞周圍DNA序列或藥物的局部濃度。 請參照第4圖，第4圖係根劇本發明之較佳實施例所繪 不之電脈衝胞膜穿孔晶片之製程分解圖。根據本發明的較佳 %實施例，本發明的第一電極106、第二電極1〇8或第三電極ιι〇 係同時形成於該基材102之該平滑表面之電路。材質較佳係一 金屬，例如金、銀、鈦、鋁、銅、以及上述物質之任意組合。 在本發明的較佳實施例之中，第一電極106、第二電極108與第 -三電極U0係由一鈦金屬層401以及金（Au)質層4〇3採用微機電 .方法所形成。由於鈦金屬與玻璃基材1〇2之附著力較佳，因此 以欽金屬層4〇1作為金質層403與基材1〇2之間的黏著層。由 於金具有良好的導電率及生物相容性，不容易產生電化學反 、應’可作為電脈衝胞膜穿孔之反應電極層。首^先玻璃基 板，然後利用，例如蒸鍍法將鈦金屬沉基於基材1〇2之上。 再於鈦金屬層4〇1上蒸鍍一層金質薄臈。之後，進行一連串 光罩、姓刻製程以完成如第3圖麟示之電路結構。由於微 機電製程係習知技術在此不作贅述。 «以上說明書所述之實施例’本發明不僅利用電泳原理 •提供一預濃縮的電場力，可以將目標細胞周目dna序列或藥 物的局部濃度提高，增加反應濃度極反應效率；且整合微機 1274075 電製程與電泳原理，將工作電壓範圍控制在12V以下，減少電 能轉換對細胞的破壞。#以提一種轉殖效率冑、消耗樣本少、 操作成低的電脈衝胞膜穿孔裝置。 —雖然本發明已以一較佳實施例揭露如上，然其並非用以限 ，定本發明，任何熟習此技藝者，在不脫離本發明之精神和範圍 内，當可作各種之更動與潤飾，因此本發明之保護範圍當視後 附之申請專利範圍所界定者為準。 >【圖式簡單說明】 透過以下說明書之詳細描述，並配合附圖之說明，讀者將會 對本發明所揭露之内容有更多的了解。必須強調的是，各個二 圖之中相同（似）的元件具有相同的元件參照號碼，且為了清楚地 —說明起見，所有的附圖均未按照比例尺繪圖，不同的附圖會依 ♦據表達的重點，加以擴大或縮小。 第1圖係係根據本發明之一較佳實施例所繪示之電泳預 濃縮式電脈衝胞膜穿孔晶片的俯視圖。 \ 第2圖係根據本發明之—較佳實施例所繪示之殼體結構 的透視圖。 第3圖係根據本發明之-較佳實施例所繪示之電脈衝胞 膜穿孔晶片的電路圖。 第4圖係根劇本發明之較佳實施例所繪示之電脈衝胞膜 •穿孔晶片電極之製程分解圖。 【主要元件符號說明】 12 1274075 100 ： 反應晶片 102 104 ： 殼體結構 106 108 ： 第二電極 110 201 ： 第一區域 203 205 ： 凹室 401 403 ： 金質層 基材 第一電極 第三電極 第二區域 鈦金屬層Referring to Figure 3, there is shown a circuit diagram of an electrically pulsed cell membrane perforated wafer crucible (10) according to a preferred embodiment of the present invention. In the present invention - the electrode H) 6 and the second electric (5) 08 are located within the first @ 2〇1; respectively, the positive and negative electrodes of the pulsed membrane perforation reaction. The third electrode is located in the second zone: 〇3 and is used to provide an electrophoretic preconcentration electric field. In the present invention, the first electrode 1, the second electrode 1 〇 8 and the third electrode ιι are respectively formed by electrodes of different processes; 1 first electrode, second electrode and third electrode Not coplanar. However, in the preferred embodiment of the present invention, the first electrode 1〇6, the second electrode 108, and the third electrode 110 are formed by a circuit diagram in which the first electrode 106 and the second electrode 1 are simultaneously formed by the same process. The tantalum 8 and the third electrode n are simultaneously formed on the smooth surface of the substrate 102. (as shown in Figure 3). In this circuit diagram configuration, both the first electrode 1〇6 and the second electrode 1〇8 have an Intei*digitated structure with a line width of at least less than 6G #m, and the finger-shaped structure is a parent. They are arranged to each other and are not connected to each other. The distance between the first electrode i% and the finger-like structure of the second electrode 108 is at least less than 3 mm. The second electrode 110 includes at least one ring electrode. In the preferred embodiment of the present invention, the second electrode 11 is a ring electrode located on the smooth surface 10 1274075 of the substrate and located in the 203 area. Among them, an interdigitated structure surrounding the first electrode 1〇6 and the second electrode 108. The radius of the third electrode 11〇 is larger than the radius of the first region 2〇1, and is greater than 3 mm and less than 5 mm, and a ring-burning electric field is formed around the electroporation perforation reaction zone. The third electrode 110 can provide a pre-concentrated electric field force to increase the local concentration of the DNA sequence or drug surrounding the target cell before or during the electrical pulse cell perforation reaction. Referring to Fig. 4, Fig. 4 is a process exploded view of the electrospray cell perforated wafer which is not depicted in the preferred embodiment of the invention. According to a preferred embodiment of the present invention, the first electrode 106, the second electrode 1 〇 8 or the third electrode ι of the present invention is simultaneously formed on the smooth surface of the substrate 102. The material is preferably a metal such as gold, silver, titanium, aluminum, copper, and any combination of the foregoing. In a preferred embodiment of the present invention, the first electrode 106, the second electrode 108, and the third-electrode U0 are formed by a titanium metal layer 401 and a gold (Au) layer 4〇3 by a microelectromechanical method. . Since the adhesion between the titanium metal and the glass substrate 1〇2 is preferred, the metal layer 4〇1 is used as the adhesion layer between the gold layer 403 and the substrate 1〇2. Since gold has good electrical conductivity and biocompatibility, it is not easy to produce an electrochemical reaction, which can be used as a reaction electrode layer for electroporation. First, the glass substrate is first placed on the substrate 1 〇 2 by, for example, evaporation. Further, a layer of gold thin crucible is evaporated on the titanium metal layer 4〇1. After that, a series of masks and surname processes are performed to complete the circuit structure as shown in FIG. The prior art of the MEMS process is not described here. «Examples described in the above description' The present invention not only utilizes the principle of electrophoresis, but also provides a pre-concentrated electric field force, which can increase the local concentration of the target cell cycle dna sequence or drug, increase the reaction concentration and the reaction efficiency; and integrate the microcomputer 1274075 The electro-process and electrophoresis principle controls the operating voltage range below 12V to reduce the damage of cells by electrical energy conversion. #To propose an electric pulse cell perforation device with a low efficiency, low consumption of samples, and low operation. </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Therefore, the scope of the invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS [0009] The reader will be more aware of the disclosure of the present invention by the following detailed description of the specification and the accompanying drawings. It must be emphasized that the same (like) elements in the respective two figures have the same element reference numbers, and for the sake of clarity - description, all the drawings are not drawn according to the scale, and different drawings will be The focus of expression is expanded or reduced. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a top plan view of an electrophoretic preconcentrated electrically pulsed cell membrane perforated wafer in accordance with a preferred embodiment of the present invention. Figure 2 is a perspective view of a housing structure in accordance with a preferred embodiment of the present invention. Figure 3 is a circuit diagram of an electropulsing cell membrane perforated wafer in accordance with a preferred embodiment of the present invention. Figure 4 is a diagram showing an exploded view of a pulsed cell electrode of a preferred embodiment of the invention. [Main component symbol description] 12 1274075 100 : Reaction wafer 102 104 : Housing structure 106 108 : Second electrode 110 201 : First region 203 205 : Alcove 401 403 : Gold layer substrate First electrode Third electrode Two-region titanium metal layer
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