200932916 九、發明說明： 【發明所屬之技術領域】 本發明是有關於一種生物晶片，特別是指一種用於快 速純化DNA與進行聚合酶連鎖反應之微流體生物晶片。 【先前技術】 傳統遺傳醫學應用檢測中，臨床檢體之前處理、去氧 核糖核酸（deoxyribonucleic acid，DNA)以及核糖核酸 (ribonucleic acid, RNA)的萃取與純化尤其複雜，往往耗費 許多的處理時間與人力成本，且其繁複的操作與步驟更容 易增加檢體之損耗與檢測之準確性。例如使用酚-氣仿 (phenol and chloroform)法，除了需要花數十小時處理的時 間且過程相當繁瑣外，更會使用到有機化學毒性試劑，而 會增加實驗過程中的危險性，且會產生有毒廢液，而生物 樣品之大量耗損，也都造成潛在成本與資源之浪費。 目前的DNA萃取試劑盒使用的方法，包括使用固相吸 附（solid-phase adsorption)，蛋白質和 DNA相繼沉殿 (sequential protein and DNA precipitation)，微磁珠吸附 (magnetic bead adsorption)。其中使用 DNA 沉澱 (precipitation)法，常會受限於時間的浪費，且整體過程中 需要繁瑣之人為操作程序與離心步驟，而不易自動化；若 選用管柱親合性（column affinity)法雖然相對於沉澱法較易 自動化，但對於體積小的檢體萃取效果不佳。此外，將 DNA萃取完後，其後段的遺傳性疾病檢測，諸如聚合酶連 鎖反應（polymerase chain reaction，PCR)，更是耗時費力。 200932916 隨著近年來微機電製程技術之成熟，在許多不同的生 物領域中有顯著的發展，尤其以微小化流體快速生物醫學 分析晶片’更具發展潛力與市場價值。藉由微機電製程技 術所生產之微流體生醫檢測晶片，其具有高檢測靈敏度、 可拋棄式、可攜帶性、低樣品及檢體消耗量、低耗能、體 積小以及成本低等優點，相較傳統分析檢測技術下，有著 犬破性的發展價值，有利於發展用以純化DNa與快速利用200932916 IX. DESCRIPTION OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to a biochip, and more particularly to a microfluidic biochip for rapidly purifying DNA and performing a polymerase chain reaction. [Prior Art] In the traditional genetic medicine application, the extraction and purification of pre-treatment of clinical samples, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are particularly complicated, and often take a lot of processing time and Labor costs, and its complicated operations and procedures are more likely to increase the loss of the specimen and the accuracy of the detection. For example, using the phenol and chloroform method, in addition to the time required to process for tens of hours, and the process is quite cumbersome, organic chemical toxicity reagents are used, which increases the risk during the experiment and generates Toxic waste, and the large loss of biological samples, also cause potential costs and waste of resources. Current methods of DNA extraction kits include the use of solid-phase adsorption, sequential protein and DNA precipitation, and magnetic bead adsorption. The use of DNA precipitation method is often limited by the waste of time, and the cumbersome human operation procedure and centrifugation step are required in the whole process, and it is not easy to automate; if the column affinity method is selected, The precipitation method is easier to automate, but it does not work well for small volume samples. In addition, after DNA extraction, genetic disease detection in the latter stage, such as polymerase chain reaction (PCR), is time consuming and labor intensive. 200932916 With the maturity of MEMS process technology in recent years, significant developments have been made in many different biotech fields, especially with the rapid development of micro-chemical fluid biomedical analysis wafers. Microfluidic biomedical test wafers produced by MEMS process technology have high detection sensitivity, disposable, portability, low sample and sample consumption, low energy consumption, small size and low cost. Compared with the traditional analytical detection technology, it has the development value of dog breaking, which is conducive to the development of purification DNa and rapid utilization.
❹ DNA進行PCR反應，而可應用於遺傳性疾病檢測的晶片。 【發明内容】 因此，本發明之目的，即在提供一種可用於快速純化 DNA與進行聚合酶連鎖反應之生物晶片。 於疋，本發明生物晶片，適用於搭配微磁珠使用，而 可用於快速純化DNA等生物分子與進行聚合酶連鎖反應， 該生物晶片包含一晶片本體，及分別設置於晶片本體上之 -電磁吸附器、一電磁溫控器與一溫度感測器。該晶片本 體包括由下往上依序疊接之__第—板層一第二板層、一 第三板層與一第四板層，且該等板層相配合界定出一混合 槽帛反應槽、一第二反應槽與-廢液槽，及一介於 其中二相接合板層間之微流道，該微流道具有一連通混合 槽與第一反應槽之第—流道段、—連通第二反應槽與廢液 槽之第-流道段’及一連通該等反應槽之第三流道段該 晶片本體還包括-可被充氣驅動而在第三流道段中產生可 雙向，制液體流動方向之幫浦作用的雙向幫浦機構、一可 被充氣驅動而驅使混合槽内之液體產生擾動的震盪機構， 200932916 及二可分別被充氣驅動而分別用以塞封第一流道段與第二 流道段之閥門機構。 該電磁吸附器是安裝固定於第一反應槽下方，並可被 通電驅動而於第一反應槽中產生將微磁珠吸附固定之磁力 。該電磁溫控器是設置於第二反應槽下方，並可被通電驅 動而於第二反應槽中產生將微磁珠吸附固定之磁力及改變 第二反應槽内之液體溫度。溫度感測器是設置於晶片本體 上且可對應第二反應槽之溫度變化而輸出一電訊號。 【實施方式】 有關本發明之前述及其他技術内容、特點與功效，在 以下配合參考圖式之一個較佳實施例的詳細說明中，將可 清楚的呈現。 如圖1〜3所示’本發明生物晶片的較佳實施例，適用 於搭配多數第一種微磁珠（粒徑4 5 # m，圖未示）來對生 物檢體中之特定生物細胞進行萃取純化，並以多數第二種 φ 微磁珠（粒徑2·8 ，圖未示）來萃取純化上述細胞之 DNA並了搭配DNA聚合酶（DNA polymerase)以及高專一 性之引子（primer)，來進行聚合酶連鎖反應（p〇lymerase chain reaction，PCR)，而可以快速進行遺傳性疾病之檢測 其中，β亥等第一種微磁珠表面接種有可與白企球結合之 CD15/CD45抗體，而第二種微磁珠會因所處溶液的ρΗ值不 同，而改變其表面之電性，當溶液ρΗ<6 〇時第二種微磁 珠表面會帶正電荷，而可吸附溶液中之DNA，當溶液 8.5時，第二種微磁珠表面會帶負電荷，進而會排斥釋放所 200932916 面接種的抗體種 吸附之DNA。但實施時’第一種微磁珠表 類與檢體之細胞種類皆不以此為限。 2，及分別設置於晶片本體 6 —薄膜狀微型電磁溫控 8。在本實施例中，由於晶 且该等薄膜狀微型電磁吸 該生物晶片包含一晶片本體 2上之一薄膜狀微型電磁吸附器 器7與一薄膜狀微型温度感測器 片本體2上的結構都相當微小，❹ DNA is subjected to a PCR reaction and can be applied to wafers for hereditary disease detection. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a biochip that can be used to rapidly purify DNA and perform a polymerase chain reaction. In the present invention, the biochip of the present invention is suitable for use with micromagnetic beads, and can be used for rapidly purifying biomolecules such as DNA and performing polymerase chain reaction. The biochip comprises a wafer body and electromagnetic fields respectively disposed on the wafer body. An adsorber, an electromagnetic thermostat and a temperature sensor. The wafer body includes a second plate layer, a third plate layer and a fourth plate layer which are sequentially stacked from bottom to top, and the plate layers cooperate to define a mixing groove. a reaction tank, a second reaction tank and a waste liquid tank, and a micro flow passage between the two phase joint plates, the micro flow prop has a communication mixing tank and a first flow passage section of the first reaction tank, The first flow channel section connecting the second reaction tank and the waste liquid tank and the third flow passage section communicating with the reaction tanks further comprise - can be inflated to generate a bidirectional in the third flow passage section a two-way pumping mechanism for the pumping action of the liquid flow direction, an oscillating mechanism that can be driven by the air to drive the liquid in the mixing tank to be disturbed, 200932916 and the second can be respectively driven by the air to respectively seal the first flow path The valve mechanism of the segment and the second channel segment. The electromagnetic adsorber is mounted and fixed under the first reaction tank, and can be driven by electric current to generate a magnetic force for adsorbing and fixing the micromagnetic beads in the first reaction tank. The electromagnetic thermostat is disposed under the second reaction tank and can be driven by the electric current to generate a magnetic force for adsorbing and fixing the micromagnetic beads in the second reaction tank and changing the temperature of the liquid in the second reaction tank. The temperature sensor is disposed on the wafer body and can output a signal corresponding to a temperature change of the second reaction tank. The above and other technical contents, features, and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments. As shown in Figures 1 to 3, the preferred embodiment of the biochip of the present invention is suitable for use with a plurality of first micromagnetic beads (particle size 4 5 # m, not shown) for specific biological cells in a biological sample. Extracting and purifying, and extracting and purifying the DNA of the above cells with a plurality of second φ micromagnetic beads (particle size 2·8, not shown) and combining with DNA polymerase and high specific primer (primer) ), to carry out polymerase chain reaction (PCR), and can quickly detect hereditary diseases. Among them, the first microbeads such as βHai are inoculated with CD15/CD45 antibody which can bind to white globules. The second microbead will change the electrical properties of the surface due to the difference in the value of the solution. When the solution is ρΗ<6 〇, the surface of the second microbead will be positively charged, and it can be adsorbed in the solution. The DNA, when the solution is 8.5, the surface of the second micromagnetic beads will be negatively charged, which will repel the DNA adsorbed by the antibody seeded by the 200932916 surface. However, the cell type of the first micromagnetic bead and the sample are not limited to this. 2, and respectively disposed on the wafer body 6 - film-like micro electromagnetic temperature control 8 . In this embodiment, the biofilm comprises a film-like micro-electromagnetic adsorber 7 on a wafer body 2 and a structure on a film-shaped micro temperature sensor sheet body 2 due to the crystal and the film-like micro-electromagnetic absorption. They are quite small,
附器6、電磁溫控器7與溫度感測器8的結構亦非常小，為 方便了解，在以下各圖式中之各構件比例皆為原結構之放 大示意圖，所以實施時，該等構件大小比例不以圖式所示 比例為限。 該晶片本體2包括由下往上依序疊接之一第一板層21 、一第二板層22、一第三板層23與一第四板層24，該等 板層21〜24相配合界定出由右往左依序排列且開口分別朝 上之一混合槽200、一第一反應槽201、一第二反應槽2〇2 與一廢液槽203、一介於第二板層22與第三板層23間之微 流道204、一介於第三板層23與第四板層24間之雙向幫浦 機構3、一介於第一板層21與第二板層22間之震盪機構4 ’及一介於第三板層23與第四板層24間之閥門機構5。 在本實施例中’第一板層21為玻璃材質，第二〜第四 板層22〜24材質為PDMS，但實施時，該等板層21〜24之材 質不以此為限。 該混合槽200、第一反應槽201與第二反應槽202，是 分別由第三板層23與第四板層24上之貫孔230、24〇和該 第二板層22相配合所構成。該微流道204是左右延伸地凹 200932916 a史於第二板層23底面，祐1女 並具有一連通混合槽200與第一反 應槽201之第—流道段2Q5、 、 ,^ ^ _、 連通第二反應槽202與廢液 槽203之第一流道段206，β 、* 够一 及—連通該等反應槽201、202 之弟二流道段2 0 7，兮笛=、去、# 2〇1 . °"二〜道段207具有二分別與該等反 應槽201、202連通之連通邱 °卩208，及一連通於該等連通部 208間之圓環狀幫浦部2〇9。 該雙向幫浦機構3包括一位於Μ β 位於第二板層23上且界定出 第三流道段207之幫浦邱々 Ο ^ 209頂緣的彈性幫浦膜31、四個 凹设於第四板層24底面並沿兮暂 w 田亚，口該幫浦膜31對稱排列成環狀 之絮浦軋至32、四分別連通於相 艰仏相鄰兩幫浦氣室32間且孔徑 較小而可延遲氣體於兩幫浦氣室32間流動的㈣㈣… 及二分別與左右間隔對稱之兩幫浦氣室Κ連通之進氣孔％ ’且該等幫浦氣室32分別涵蓋局部幫浦膜31頂面。 、為方便區別，以下將該等幫浦氣室Μ由右往左依序區 幫錢室32、上下對稱且分別透過延遲溝槽η與 ©第一幫浦氣室32連通之第-餮、、者名 弟一幫浦乳室32，及一與該等第二 幫浦氣室32連通之第三幫浦氣宮 屬席虱至32，而該等進氣孔34是 分別和第一與第三幫浦氣室32連通。 如圖1、2、4所示，該震湯嫩姓1 展盈機構4包括一成型於第二 板層22上且界定出該混合槽2〇〇麻絲^卿 置U底緣之彈性震盪膜41、一 凹设於第二板層22底面且局部涵篆兮 . 1 '由盍該震盪膜41底面之震 盪氣室42’及二分別連通於該震盪 辰屋軋至42兩相反端之充氣 孔43 ’該震盡氣室42具有多數柏鬥眩 另夕致相間隔且分別局部涵蓋該震 I膜41之氣室部421，及多數分 夕数刀別連通於相鄰兩氣室部 200932916 421間且孔徑較小的延遲部422，該等延遲部422可延遲氣 體於兩氣室部421間之流動，該等充氣孔43是成型於第二 〜第四板層22〜24上，且分別與最兩端之氣室部421連通。 該閥門機構5包括二分別位於第三板層23上且分別界 定出第一與第二流道段205、206頂緣之彈性閥門膜51、二 凹設於第四板層24底面且分別涵蓋該等閥門膜51頂面之 閥門氣室52，及二分別與該等閥門氣室52連通之閥門氣孔 53。為方便區別，以下將設置於第一流道段2〇5處之閥門 膜51與閥門氣室52分別定義為第一閥門膜5 1與第一閥門 氣室52，而將設置於第二流道段206處之閥門膜5 1與閥門 氣室52分別定義為第二閥門膜51與第二閥門氣室52。 如圖1、2、5所示’該微型電磁吸附器6、電磁溫控器 7與溫度感測器8是分別電鍍成型於第一板層21頂面，其 中，該電磁吸附器6具有一對應位於第一反應槽201下方 之線圈部61，及二分別與該線圈部61兩相反端電連接之電 極部62，該線圈部61是由一條往復彎折延伸之薄膜狀金屬 線所構成。該電磁溫控器7具有二間隔位於第二反應槽202 下方並分別由一往復彎折延伸之金屬線所構成之線圈部71 ，及四分別電連接於該等線圈部71兩相反端的電極部72。 該溫度感測器8是設置於該等線圈部71間，並具有一前後 延伸且間隔位於該等線圈部71相向側間的細長感測部81。 在本實施例中，該等線圈部61、71可分別被通電驅動 而分別於該等反應槽201、202中產生往下吸附固定微磁珠 的磁力，其中，電磁溫控器7之該等線圈部71還會對第二 10 200932916 反應槽202内之液賴知叙 及體加熱，而改變液體溫度，該溫度感測 器8之感測部81則可對應第二反應槽202之溫度變化，而 產生輸出一電訊號。 ，生物晶片使用時’會將該等進氣孔34、充氣孔43與 閥門，孔53分別連通組接_空麼機（圖未示），而可經由 ”亥等礼孔34、43、53對該等氣室32、42、52灌注高壓氣 體。且會將該電磁吸附器6與電磁溫控器7之該等電極部The structure of the applicator 6, the electromagnetic thermostat 7 and the temperature sensor 8 is also very small. For the convenience of understanding, the proportions of the components in the following figures are enlarged views of the original structure, so when implemented, the components are The size ratio is not limited to the ratio shown in the figure. The wafer body 2 includes a first plate layer 21, a second plate layer 22, a third plate layer 23 and a fourth plate layer 24, which are sequentially stacked from bottom to top, and the plate layers 21 to 24 Cooperating to define a mixing tank 200 arranged in a sequence from right to left and having openings respectively upward, a first reaction tank 201, a second reaction tank 2〇2 and a waste liquid tank 203, and a second layer 22 a microchannel 204 between the third ply 23, a bidirectional pumping mechanism 3 between the third ply 23 and the fourth ply 24, and a concussion between the first ply 21 and the second ply 22 The mechanism 4' and a valve mechanism 5 between the third ply 23 and the fourth ply 24. In the present embodiment, the first layer 21 is made of glass, and the second to fourth layers 22 to 24 are made of PDMS. However, the material of the layers 21 to 24 is not limited thereto. The mixing tank 200, the first reaction tank 201 and the second reaction tank 202 are respectively formed by the cooperation of the third plate layer 23 and the through holes 230, 24A of the fourth plate layer 24 and the second plate layer 22, respectively. . The micro flow channel 204 is a left and right extending recess 200932916 a history on the bottom surface of the second ply 23, and has a communicating mixing tank 200 and a first flow channel section of the first reaction tank 201 2Q5, , , ^ ^ _ And connecting the second reaction tank 202 and the first flow path section 206 of the waste liquid tank 203, β, * are sufficient to communicate with the second flow passage section of the reaction tanks 201, 202, 2, 7, flute =, go, # 2〇1. °" nd to section 207 has two communication ports respectively communicating with the reaction tanks 201, 202, and a ring-shaped pump portion 2〇9 communicating between the communication portions 208 . The two-way pump mechanism 3 includes an elastic pump film 31 located on the second plate layer 23 and defining a top edge of the third flow path segment 207, and four recesses. The bottom surface of the four-layer layer 24 is along the w temporary w Tianya, and the pump film 31 is symmetrically arranged into a ring-shaped floc-rolled to 32, and four are respectively connected to the adjacent two puffer chambers 32 and the aperture is relatively (4) (4) which is small and delays the flow of gas between the two plenums 32, and two intake vents which are respectively connected to the two plenums which are symmetrically spaced from each other, and the pump chambers 32 respectively cover the partial gangs The top of the membrane 31. In order to facilitate the difference, the pump chambers are arranged from the right to the left to help the money chamber 32, and the first and second symmetry and respectively communicate with the first pump chamber 32 through the delay groove η. The famous younger brother, the Pu milk room 32, and the third gang of the Puqi Palace, which are connected to the second puddle chamber 32, belong to the 32, and the air intake holes 34 are respectively the first and the first The third pump chamber 32 is connected. As shown in Figures 1, 2, and 4, the shocking surname 1 exhibiting mechanism 4 includes a resiliently oscillating shape formed on the second ply 22 and defining the bottom of the mixing trough 2 The film 41 is recessed on the bottom surface of the second plate layer 22 and partially covered. 1 'The oscillating gas chamber 42' and the second portion of the bottom surface of the oscillating film 41 are respectively connected to the oscillating chamber to the opposite ends of the 42. The inflating hole 43' has a plurality of ventilating chambers 42 which are spaced apart from each other and partially cover the chamber portion 421 of the seismic membrane 41, and a plurality of circadian valves are connected to the adjacent two chamber portions. 200932916 421 and a small aperture delay portion 422, the delay portion 422 can delay the flow of gas between the two gas chamber portions 421, the inflatable holes 43 are formed on the second to fourth plate layers 22 to 24, And communicating with the gas chamber portion 421 at the most ends. The valve mechanism 5 includes two elastic valve films 51 respectively located on the third plate layer 23 and defining the top edges of the first and second flow path segments 205, 206, respectively, and two recesses on the bottom surface of the fourth plate layer 24 and respectively covering Valve plenums 52 on the top surface of the valve membranes 51, and valve vents 53 communicating with the valve plenums 52, respectively. For convenience of distinction, the valve film 51 and the valve plenum 52 disposed at the first flow path section 2〇5 are defined as the first valve film 51 and the first valve plenum 52, respectively, and will be disposed in the second flow path. The valve membrane 51 and the valve plenum 52 at the section 206 are defined as a second valve membrane 51 and a second valve plenum 52, respectively. As shown in Figures 1, 2, and 5, the micro-electromagnetic adsorber 6, the electromagnetic thermostat 7 and the temperature sensor 8 are respectively electroplated on the top surface of the first ply layer 21, wherein the electromagnetic adsorber 6 has a Corresponding to the coil portion 61 located below the first reaction tank 201 and the electrode portion 62 electrically connected to opposite ends of the coil portion 61, the coil portion 61 is formed by a film-like metal wire extending reciprocally. The electromagnetic thermostat 7 has two coil portions 71 which are spaced apart from the second reaction tank 202 and are respectively formed by a reciprocatingly bent metal wire, and four electrode portions electrically connected to opposite ends of the coil portions 71, respectively. 72. The temperature sensor 8 is disposed between the coil portions 71 and has an elongated sensing portion 81 extending forward and backward and spaced apart between opposite sides of the coil portions 71. In this embodiment, the coil portions 61 and 71 are respectively driven by the electric current to generate magnetic forces for adsorbing and fixing the micromagnetic beads in the reaction tanks 201 and 202 respectively, wherein the electromagnetic thermostats 7 are The coil portion 71 also heats the liquid in the second 10 200932916 reaction tank 202 to change the liquid temperature, and the sensing portion 81 of the temperature sensor 8 can correspond to the temperature change of the second reaction tank 202. And produce an output signal. When the biochip is used, the air intake holes 34, the inflation holes 43 and the valves 53 and the holes 53 are respectively connected to each other (not shown), and can be passed through the "Hai et al. 34, 43, 53 The gas chambers 32, 42, 52 are filled with high pressure gas, and the electromagnetic adsorber 6 and the electrode portions of the electromagnetic thermostat 7 are
62 72 ’及該溫度感測$ 8分別電連接至一控制裝置（圖 未示）。該晶>1崎DNA之萃取純化與遺傳疾病檢測的步 驟如下： 步驟（一）混合微磁珠與臨床檢體。先經由該等閥門 氣孔53對該等閥門氣室52灌注高壓氣體，迫使該等閥門 膜51往下彈性突伸入第—與第二流道段2〇5、2〇6中而 分別塞封第一與第二流道段2〇5、2〇6。接著，將修飾有 CD15/CD45抗體的微磁珠與臨床血液檢體—起置人該混合 槽200中。 然後，啟動該震盪機構4,經由其中一充氣孔43對該 震盪氣室42灌注高壓，使高壓氣體經由該等延遲部422而 依序充滿各氣室部421，當氣室部421内被充滿高壓氣體時 ，會迫使其所涵蓋之震盪膜41部位往上彈性突伸入該混合 槽200中’而造成混合槽2〇〇内之液體產生擾動因此， 可藉由該等氣室部421依序地被充填高壓氣體的方式，驅 使震盈膜41之各部位分別往上突伸入混合槽2〇0中，而可 在混合槽200中產生使微磁珠和血液檢體充分混合之擾流 200932916 ，使血液檢體中之白血球與微磁珠上之CD15/CD45抗體結 合。實施時，並可透過控制由任一充氣孔43進行充氣的方 式’來改變擾流形式。 待微磁珠與血液檢體充分混合後，停止驅動震盪機構4 ’並釋放第一閥門氣室52之氣體，使第一閥門膜51彈性 復位而開啟第一流道段205。然後，再啟動該雙向幫浦機構 3 ’經由進氣孔34間歇地對第一幫浦氣室32灌注高壓氣體 ，並藉由該等孔徑較小之延遲溝槽33設計，延遲氣體進入 並填滿相鄰幫浦氣室32的時間，使第一幫浦氣室32、第二 幫浦氣室32與第三幫浦氣室32依序被灌注高壓氣體，依 序將所涵蓋之幫浦膜31部位往下彈性擠推突伸入第三流道 段207之幫浦部209中’而在該幫浦部209中產生由右往 左輸送液體之蠕動式幫浦作用，進而逐漸將混合槽2〇〇内 之液體經由第一流道段205而吸引輸送至第一反應槽20 ][ 中。然後再次驅使第一閥門膜51塞封第一流道段205，並 停止驅動雙向幫浦機構3。 步驟（二）萃取純化白血球。對該電磁吸附器6施加 預疋電流’使該線圈部61於第一反應槽201内產生之磁 力’將結合有白血球之微磁珠往下吸附固定於第二板層22 頂面。然後，於該混合槽200中注入細胞洗滌緩衝液，並 開啟第一流道段205與第二流道段206，且驅使雙向幫浦機 構3產生往左輸送液體之蠕動式幫浦作用，驅使細胞洗滌 緩衝液流經第一反應槽201，而將檢體中未被吸附之其他血 球沖洗至廢液槽203中。最後再封閉第一與第二流道段205 12 200932916 、206，並停止驅動雙向蠕動式幫浦機構3，便完成白血球 之萃取與純化。 步驟（三）萃取純化DNA。將用以進行DNA萃取純化 之第二種微磁珠與細胞裂解液（lysis buffer )混合注入該混 合槽200中，並開啟第一流道段205，且停止驅動電磁吸附 器6，再經由進氣孔34開始對第三幫浦氣室32灌注高壓氣 體，使第三、第二與第一幫浦氣室32依序被充氣，而使該 幫浦膜31在該幫浦部209中產生往右輸送液體之蠕動式幫 浦作用，而將結合有白血球之第一種微磁珠溶液自第一反 應槽201輸送回混合槽200中，然後關閉第一流道段205。 接著，再次驅動震盪機構4，驅使白血球、細胞裂解液 與第二種微磁珠充分混合，使白血球裂解而釋出DNA，藉 由控制第二種微磁珠所處之液體的pH值，使DNA被吸附 於第二種微磁珠表面。然後，再開啟第一與第二流道段205 、206，並啟動雙向幫浦機構3，將混合槽200中之液體全 部輸送至第二反應槽202中，且對該電磁溫控器6施加預 定電流，使吸附有DNA之第二種微磁珠被電磁溫控器6產 生之磁力往下吸附固定於第二板層22頂面。再將洗滌緩衝 液注入混合槽200中，並驅使雙向幫浦機構3作動而將洗 滌缓衝液輸送經過該第二反應槽202，而將白血球碎片與其 他雜質沖洗至廢液槽203中，此時，便完成高品質DNA的 萃取與純化。 若只需做大量的DNA萃取與純化，只需再以充提液（ elution buffer)將DNA自第二種微磁珠表面溶解出來。但 13 200932916 如要進行遺傳性疾病之偵測，則需透過PCR反應，來將某 一特定遺傳性基因片段進行訊號放大處理。進行PCR反應 時，可於第二反應槽202中加入DNA聚合酶（DNA polymerase)及高專一性之引子（primer)，並對該電磁溫控 器7施加預定電流，驅使該電磁溫控器7之該等線圈部71 • 調變第二反應槽202内之溫度，同時藉由該溫度感測器8 - 對應第二反應槽202溫度變化所產生之訊號，控制施加於 電磁溫控器7之電流大小，進而可精確控制第二反應槽202 ❹ 内之溫度。由於PCR反應過程並非本發明之創作重點，因 此不再詳述。 在本實施例中’是以200 " 1的全血檢體進行DNA之 萃取與純化，同時將所純化之DNA量和傳統DNA萃取試 劑盒與手動操作式微磁珠萃取法進行比較，並針對 methylenetetrahydrofolate reductase (MTHFR) C677T 之基 因片段進行PCR反應，且與傳統DNA試劑法所萃取之 φ DNA經大型PCR儀器分析的結果進行比較。 由表1可知，本發明晶片所萃取純化之DNA量可達 15 _ 1 8 ng/ // 1 ’明顯優於傳統DNA萃取法。 如圖 6 所示，lane L 為 l〇〇-bp DNA Maker，lane 1-2 為 傳統DNA試劑法萃取並經大型PCR儀器分析之結果， lane3-4為於本發明晶片上進行萃取與PCr反應分析之結果 ’由實驗結果顯示，於本發明晶片進行DNA萃取與PCR反 應分析之結果相同於傳統PCR儀器。因此，本發明晶片可 用以取代傳統DNA萃取方法與傳統PCR儀器。 14 200932916 ---_ 表 1 生物檢體 傳統DNA萃 手動操作式微 本發明快速DNA純 取试劑盒 磁珠萃取法 化與遺傳性疾病檢 須J晶片 主血 (u 1) 200 200 200 佩權琢歎重 (微磁珠/ml) 3xl〇7 3χ1〇7 DNA濃度 —. ------- (ng/w η 10.25 31.69 15.18 歸納上述’透過該晶片本體2所構成之微流道'可 ❹ 雙向㈣流體㈣方向之雙向幫浦機構3，及可有效促使混 合槽200内之液體擾動而進行混合的震盈機構4等結構設 計，並配合設置於晶片本體2上之微型電磁吸附器6、㈣ 電磁溫控器7與微型溫度感測器8等結構設計，使得該生 物晶片可用以决速萃取與純化生物檢體中之特定生物分子 與DNA ’並可用以進行pcR反應分析，而可用以快速檢測 遺傳性疾病。整個萃取純化與PCR反應過程中，都是在晶 片本體2上進行，且不需使用危險的有機化學試劑，僅需 〇 利用極J量之成本與樣品損耗，便能達到優於傳統萃取技 術的萃取純化效果，再加上簡便操作程序可大幅縮短傳 統技術上之操作時間，並降低生物檢體被污染之風險更 可減少人為操作的不穩定性。因此，確實可達到本發明之 目的。 准以上所述者’僅為本發明之一較佳實施例而已，當 不能以此限定本發明實施之範圍，即大凡依本發明申請專 利耗圍及發明說明内容所作之簡單的等效變化與修飾，皆 仍屬本發明專利涵蓋之範圍内。 15 200932916 * 【圖式簡單說明】 圖1是本發明生物晶片之立體分解圖； 圖2是該較佳實施例之組合俯視示意圖，說明四板層 相疊接所界定出之各構件之相對位置； 圖3是該較佳實施例的第四板層之局部俯視放大圖； - 圖4是該較佳實施例之第二板層的局部俯視放大圖； - 圖5是該較佳實施例之第一板層之俯視放大圖；及 圖6是該較佳實施例之萃取之DNA進行PCR反應之平 板膠電泳之測試結果。62 72 ' and the temperature sensing $ 8 are electrically connected to a control device (not shown). The steps of extracting and purifying the DNA >1 S. DNA and detecting the genetic disease are as follows: Step (1) Mixing the micromagnetic beads with the clinical sample. The valve chambers 52 are first filled with high-pressure gas through the valve vents 53 to force the valve membranes 51 to project downwardly into the first and second flow passage sections 2〇5, 2〇6 to be respectively sealed. The first and second flow path segments 2〇5, 2〇6. Next, the micromagnetic beads modified with the CD15/CD45 antibody and the clinical blood sample are placed in the mixing tank 200. Then, the oscillating mechanism 4 is activated to inject a high pressure into the oscillating plenum 42 via one of the inflation holes 43 so that the high pressure gas sequentially fills the plenum portions 421 via the delay portions 422, and when the plenum portion 421 is filled When the high-pressure gas is forced, the portion of the oscillating film 41 that is covered by the swelled film is forced into the mixing tank 200, and the liquid in the mixing tank 2 is disturbed. Therefore, the ventilating portion 421 can be The method of sequentially filling the high-pressure gas drives the respective portions of the seismic film 41 to protrude into the mixing tank 2〇0, respectively, and the interference between the micro-magnetic beads and the blood sample can be generated in the mixing tank 200. Flow 200932916, the white blood cells in the blood sample are bound to the CD15/CD45 antibody on the micromagnetic beads. In practice, the form of the spoiler can be changed by controlling the manner in which it is inflated by any of the inflatable holes 43. After the microbeads are sufficiently mixed with the blood sample, the driving of the oscillating mechanism 4' is stopped and the gas of the first valve plenum 52 is released, and the first valve film 51 is elastically reset to open the first flow path section 205. Then, the two-way pump mechanism 3' is again activated to inject high-pressure gas into the first pumping chamber 32 intermittently via the air inlet 34, and the delay groove 33 is designed to delay the gas entering and filling. When the adjacent pump chamber 32 is full, the first pump chamber 32, the second pump chamber 32 and the third pump chamber 32 are sequentially filled with high-pressure gas, and the covered pumps are sequentially arranged. The portion of the film 31 is squeezing downwardly into the pumping portion 209 of the third flow path section 207, and a peristaltic pumping action of transporting liquid from right to left is generated in the pumping portion 209, thereby gradually mixing the mixing tank 2 The liquid in the crucible is suctioned and transported to the first reaction tank 20 through the first flow path section 205. Then, the first valve film 51 is again driven to seal the first flow path section 205, and the driving of the two-way pump mechanism 3 is stopped. Step (2) extracting and purifying white blood cells. The electromagnetic current is applied to the electromagnetic adsorber 6 to cause the magnetic force generated by the coil portion 61 in the first reaction tank 201 to adsorb and fix the micromagnetic beads combined with the white blood cells downward to the top surface of the second plate layer 22. Then, the cell washing buffer is injected into the mixing tank 200, and the first flow path section 205 and the second flow path section 206 are opened, and the bidirectional pump mechanism 3 is driven to generate a peristaltic pumping action for transporting the liquid to the left to drive the cells. The washing buffer flows through the first reaction tank 201, and the other blood cells that are not adsorbed in the sample are flushed into the waste liquid tank 203. Finally, the first and second flow path segments 205 12 200932916, 206 are closed, and the driving of the two-way peristaltic pump mechanism 3 is stopped, thereby completing the extraction and purification of the white blood cells. Step (3) extract and purify the DNA. The second micromagnetic beads for DNA extraction and purification are mixed with a lysis buffer to be injected into the mixing tank 200, and the first flow path section 205 is opened, and the electromagnetic adsorber 6 is stopped to be driven, and then the air is driven. The hole 34 begins to infuse the third pumping chamber 32 with high pressure gas, so that the third, second and first pumping chambers 32 are sequentially inflated, so that the pumping membrane 31 is generated to the right in the pumping portion 209. The peristaltic pumping action of the liquid is carried, and the first micromagnetic bead solution combined with the white blood cells is transported from the first reaction tank 201 back to the mixing tank 200, and then the first flow path section 205 is closed. Then, the oscillating mechanism 4 is driven again to drive the white blood cells, the cell lysate and the second microbeads to be thoroughly mixed, so that the white blood cells are lysed to release the DNA, and by controlling the pH of the liquid of the second microbeads, The DNA is adsorbed on the surface of the second microbead. Then, the first and second flow path sections 205 and 206 are further turned on, and the two-way pump mechanism 3 is activated to transfer all the liquid in the mixing tank 200 to the second reaction tank 202, and the electromagnetic temperature controller 6 is applied. The current is predetermined so that the second microbeads to which the DNA is adsorbed are adsorbed and fixed to the top surface of the second ply 22 by the magnetic force generated by the electromagnetic thermostat 6. The washing buffer is injected into the mixing tank 200, and the bidirectional pumping mechanism 3 is driven to transport the washing buffer through the second reaction tank 202, and the white blood cell debris and other impurities are flushed into the waste liquid tank 203. The extraction and purification of high quality DNA is completed. If only a large amount of DNA extraction and purification is required, it is only necessary to dissolve the DNA from the surface of the second micromagnetic beads with a elution buffer. However, 13 200932916 For the detection of hereditary diseases, a specific genetic gene fragment is amplified by a PCR reaction. When the PCR reaction is carried out, a DNA polymerase (DNA polymerase) and a high specific primer can be added to the second reaction tank 202, and a predetermined current is applied to the electromagnetic thermostat 7, and the electromagnetic thermostat 7 is driven. The coil portion 71 • modulates the temperature in the second reaction tank 202, and controls the application to the electromagnetic thermostat 7 by the signal generated by the temperature sensor 8 - corresponding to the temperature change of the second reaction tank 202 The magnitude of the current, in turn, allows precise control of the temperature within the second reaction tank 202. Since the PCR reaction process is not the focus of the present invention, it will not be described in detail. In the present embodiment, DNA extraction and purification are carried out by using a whole blood sample of 200 " 1, and the amount of purified DNA is compared with a conventional DNA extraction kit and a manually operated micromagnetic bead extraction method, and The methylenetetrahydrofolate reductase (MTHFR) C677T gene fragment was subjected to a PCR reaction and compared with the results of large-scale PCR instrument analysis of φ DNA extracted by a conventional DNA reagent method. As can be seen from Table 1, the amount of DNA extracted and purified by the wafer of the present invention can be as high as 15 _ 18 ng / // 1 ', which is superior to the conventional DNA extraction method. As shown in Fig. 6, lane L is l〇〇-bp DNA Maker, lane 1-2 is extracted by conventional DNA reagent method and analyzed by large-scale PCR instrument, and lane 3-4 is subjected to extraction and PCr reaction on the wafer of the present invention. The results of the analysis 'The results of the experiments show that the results of DNA extraction and PCR reaction analysis of the wafer of the present invention are the same as those of the conventional PCR instrument. Therefore, the wafer of the present invention can be used in place of conventional DNA extraction methods and conventional PCR instruments. 14 200932916 ---_ Table 1 Biological sample traditional DNA extraction manual operation micro-inventive rapid DNA pure extraction kit magnetic bead extraction and hereditary disease detection J wafer main blood (u 1) 200 200 200琢 重 重 (micromagnetic beads / ml) 3xl 〇 7 3 χ 1 〇 7 DNA concentration -. ------- (ng / w η 10.25 31.69 15.18 summarized above 'micro flow channel formed by the wafer body 2' The two-way (four) fluid (four) direction bidirectional pump mechanism 3, and the structure of the shock mechanism 4, which can effectively promote the mixing of the liquid in the mixing tank 200, and the micro electromagnetic adsorption device disposed on the wafer body 2 6. (4) The electromagnetic thermostat 7 and the micro temperature sensor 8 are designed such that the biochip can be used to extract and purify specific biomolecules and DNA in the biological sample at a constant rate and can be used for PCR reaction analysis. It can be used to quickly detect hereditary diseases. The whole process of extraction and purification and PCR reaction are carried out on the wafer body 2, and no dangerous organic chemical reagents are needed, only the cost and sample loss of the electrode J need to be utilized. Can achieve better than The extraction and purification effect of the extraction technology, together with the simple operation procedure, can greatly shorten the operation time of the conventional technology, reduce the risk of contamination of the biological sample, and reduce the instability of human operation. Therefore, the present invention can be achieved. The above is only a preferred embodiment of the present invention, and the scope of the present invention cannot be limited thereto, that is, the simpleness of the patent application and the description of the invention according to the present invention. </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The relative positions of the members defined by the four layers are illustrated; FIG. 3 is a partial enlarged plan view of the fourth layer of the preferred embodiment; FIG. 4 is the second layer of the preferred embodiment. FIG. 5 is a top plan view of the first plate layer of the preferred embodiment; and FIG. 6 is a plate gel electrophoresis for PCR reaction of the extracted DNA of the preferred embodiment. Test Results.
16 200932916 【主要元件符號說明】 2....... ....晶片本體 21 —第一板層 22 .·.·· —第二板層 23 .···· •…第二板層 230···· ....貫孔 24 ..... —第四板層 240..·· ....貫孔 200.... ....混合槽 201.... ....第一反應槽 202.... ··.第二反應槽 203·... ....廢液槽 204..·· ....微流道 205.... ···.第一流道段 206··.· ....第二流道段 207.… ….第=流道段 208···· ....連通部 209.... ....幫浦部 3....... ....雙向幫浦機構 31 ..... ....幫浦膜 32 ....幫浦氣室16 200932916 [Description of main component symbols] 2............. wafer body 21 - first ply 22 . . . . . - second ply 23 . . . . Layer 230····....through hole 24 ..... — fourth plate layer 240..·....through hole 200...... mixing groove 201.... ....first reaction tank 202....··. second reaction tank 203·.... waste liquid tank 204..·....micro flow passage 205.... ···. First flow passage section 206····..Second flow passage section 207......th=flow passage section 208····....communication section 209.... .Pump Department 3.........Two-way pumping mechanism 31 ..... .... pump membrane 32 .... pump chamber
33 .........延遲溝槽 34 .........進氣孔 4 ...........震盪機構 41 .........震盪膜 42 .........震盪氣室 421 ........氣室部 422 ........延遲部 43 .........充氣孔 5 ...........閥門機構 51 .........閥門膜 52 .........閥門氣室 53 .........閥門氣孔 6 ...........電磁吸附器 61 .........線圈部 62 .........電極部 7 ...........電磁溫控器 71 .........線圈部 72 .........電極部 8 ...........溫度感測器 81 .........感測部 1733 ......... delay groove 34 ... ... intake hole 4 ... ..... oscillating mechanism 41 ......... Oscillation membrane 42 ... oscillates air chamber 421 ..... air chamber portion 422 ..... delay portion 43 ... ... inflatable hole 5 ...........valve mechanism 51 .... valve membrane 52 ... ... valve chamber 53 ... ... valve Air hole 6 ...........electromagnetic adsorber 61 .... coil portion 62 ... electrode portion 7 ......... .. electromagnetic thermostat 71 ... ... coil portion 72 ... ... electrode portion 8 ..... ... temperature sensor 81 ... ...the sensing part 17