201219785 - 六、發明說明： . 【發明所屬之技術領域】 本發明是有關於一種晶片，特別是指一種即時電化學 檢測晶片。 【先前技術】 隨著半導體製程技術與微機電技術的蓮勃發展，利用 微機電技術所製作出的微晶片是一新穎而有潛力的產業。 而生物晶片就是最被看好具有未來明星產業之一，生物晶 φ 片是泛指將半導體應用於生物性分析的微小化裝置，生物 晶片通常以矽晶片、玻璃，或高分子為基材，在基材上製 作出結構或是控制電路，用來檢測分析生物性分子或更進 一步的應用。 而目前在檢測DNA上最常用的方法，就是將DNA進201219785 - VI. Description of the Invention: 1. Field of the Invention The present invention relates to a wafer, and more particularly to an instant electrochemical detection wafer. [Prior Art] With the development of semiconductor process technology and MEMS technology, microchips fabricated using MEMS technology are a novel and promising industry. Biochips are one of the most promising future star industries. Biocrystals are generalized devices that use semiconductors for bioanalysis. Biochips are usually based on germanium wafers, glass, or polymers. Structures or control circuits are fabricated on the substrate for detecting biomolecules or further applications. The most common method currently used to detect DNA is to put DNA into it.
行聚 & 連鎖反應（p〇lymerase Chain Reaction, PCR)。PCRP&y; chain reaction (PCR). PCR
主要以三個不斷依序重複之步驟進行，變性（denature，DNAMainly in three steps that are repeated in sequence, denature (denature, DNA
又版为離形成單股黏合（annealing，引子黏合於單股 DNA上的互補位置卜延展（extensi〇n，卩兩個引子為起點合 成新的DNA) ’ PCR完成後進行凝膠電泳來檢測結果。 * -般PCR的循環次數為m而進行凝膠電泳需 要等到PCR完全結束後才能進行，不但耗時，還需要製膠 以及購買許多耗材，並配合大型光學儀器才能判讀，且製 膠的膠粉相當昂責，因此使用凝膠電泳耗時且高成本。 若不採用凝膠電泳，也可以使用毛細電泳，是在 結束後，將PCR產物置於毛細管巾進行分離，㈣此法不 201219785 需製膠，但仍需購買毛細管等耗材，並配合大型光學儀器 才能判讀，同樣耗時與耗費成本。 又，若要採用其他電化學檢測方式，則需待PCR結束 並獲得PCR產物後，加入特定的外切酶進行切割才能進行 檢測，而外切酶同樣相當昂貴，且需要較長的作業時間， 使用上也不理想。 【發明内容】 因此，本發明之目的，即在提供一種可以即時偵測檢 測結果的即時電化學檢測晶片。 於是’本發明即時電化學檢測晶片，包含一反應裝置 、一連通於該反應裝置的第一流速控制裝置、一設置於該 反應裝置下方的加熱裝置，及一設置於該反應裝置上方的 偵測裝置。 該反應裝置包括一基板，及一凹陷形成於該基板上且 重覆彎折延伸的流道，該流道具有一入口，及一相反於該 入口的出口。 該第一流速控制裝置包括一第一流速控制器，及一安 裝於該第一流速控制器上且連通於該流道的第一注射器， 該第一注射器盛裝有檢體與反應試劑。 該加熱裝置包括位於該基板下方且互相間隔的一第一 加熱單元、一第二加熱單元’及一第三加熱單元，該流道 是重覆橫跨於該第一至第三加熱單元上。 該偵測裝置包括一對應蓋設於該基板上的封蓋、多數 個設置於該封蓋上且伸置於該流道内的電極、多數個分別 201219785 設置於所述電極上的探針，及一電連接於所述電極的電流 偵測器。 本發明之功效在於：由該第一注射器將檢體與反應試 劑注入該流道以進行PCR時，檢體内經變性形成的單股 DNA就能直接結合於所述電極上的探針，而改變所述電極 所偵測到的電流值’藉此在PCR進行的過程中就能快速且 直接地獲得PCR的結果。 【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。 參閱圖1、2 ’為本發明即時電化學檢測晶片之較佳實 施例’包含一反應裝置2、一連通於該反應裝置2的第一流 速控制裝置3(只見於圖2)、一設置於該反應裝置2下方的 加熱裝置4、一設置於該反應裝置2上方的偵測裝置5、二 用以將該反應裝置2、加熱裝置4，及偵測裝置5夾置固定 的夾置板6 ’及一連通於該加熱裝置4的第二流速控制裝置 7(只見於圖2)，為了便於說明，於圖2中省略夾置板 該反應裝置2包括一基板21，及一凹陷形成於該基板 21上且重覆彎折延伸的流道22，該流道22具有一入口 221 ，及一相反於該入口 221的出口 222。 如圖1所示，該加熱装置4包括一位於該反應裝置2 之基板21下方且對應該基板21大小的傳熱板41，及位於 s亥基板21下方且互相間隔的一第一加熱單元42、一第二加 201219785 熱單元43，及一第三加熱單元44，該傳熱板41具有一上 表面411、一下表面412，及一由該下表面412凹陷形成的 水流通道413，而位於下方之夾置板6與該傳熱板41配合 使該水流通道413形成封閉態樣。 該第一加熱單元42具有一設置於該傳熱板41下表面 412的第一加熱器421，及一設置於該傳熱板41上表面411 且對應該第一加熱器421的第一導熱件422，該第二加熱單 元43具有一設置於該傳熱板41上表面411且對應該水流通 道413的第二導熱件431，該第三加熱單元44具有一設置 於該傳熱板4丨下表面412的第二加熱器441，及一設置於 該傳熱板41上表面411且對應該第二加熱器441的第三導 熱件442 ’而該水流通道413是位於該第一、二加熱器421 、441之間。該流道22是重覆彎折並橫跨於該第一、二、 三導熱件422、4S1、料2上而被加熱。 該偵測裝置5包括一對應蓋設於該基板2丨上的封蓋5 i 、多數個設置於該封蓋51上且伸置於該流道22内的電極 52、多數個分別設置於所述電極52上的探針（pr〇be，由於 抓針非肉眼可見，不予以繪示），及一電連接於所述電極52 的電流偵測器53。 如圖2所不，該第一流速控制裝置3包括一第一流速 控制器3 1 ’ A *裝於該第—流速控制器3 i上且連通於該 流道22的第-注射器32,該第—注射器32盛裝有檢體與 反應試劑。該第二流速控制裝置7包括一第二流速控制器 71 ’及-安裝於該第二流速控制器71上並盛裝有液體，且 201219785 . 連通於該水流通道413(見圖1)的第二注射器72。 於本實施例中，分別控制該第一加熱器421及第三加 熱器，使該第一導熱件422的溫度為95°C，該第三導熱件 442的溫度為72°C，並控制該第二流速控制器71，使該第 二注射器72内的液體以一定的流速流入該水流通道413, 讓該第一、二加熱器422、442產生的熱能被該水流通道 413内的液體吸收而使其溫度上升至55°C。要說明的是， 上述溫度只是本實施例所設定之實驗條件，可以視各種不 • 同的實驗而改變該第一、二加熱器422、442的溫度，來調 整該第一導熱件422及第三導熱件442的溫度，而該第二 導熱件431的溫度，則藉由改變該液體的流速來控制，當 流速越快，溫度越低，流速越慢，溫度越高。 - 以下則配合實驗的進行說明本發明的使用方式，於本 ' 實施例中，令該第一導熱件422、第二導熱件43 1、第三導 熱件442的溫度分別為95°C、55°C、72°C，而該流道22是 重覆彎折並重覆橫跨於該第一、二、三導熱件422、43 1、 φ 442上，因此將該流道22區分成多個位於第一導熱件422 上的變性區223(denaturation)、多個位於第二導熱件431上 的黏合區224(annealing)，及多個位於第三導熱件442上的 延展區225(extension)，該流道22的入口 221是位於第一個 變性區223，而出口 222是位於最後一個延展區225，檢體 通過該變性區223、黏合區224，及延展區225為聚合酶連 鎖反應（Polymerase Chain Reaction, PCR)的一次循環。 首先，確認一段能與PCR產物互補的片段，並將此片 201219785 段固定於所述電極52上作為探針，接下來確認所述電極52 要設置在PCR第幾次循環的位置，於本實施財，所述電 極52是分別設置於第5循環、帛12循環，及第18循環的 變性區223與黏合區224之間，由於設置探針的方式為熟 知該項技藝者所能輕易實施，不予以贅述，且所述電極52 的設置位置是可依實驗條件不同而有改變，不以本實施例 所揭露的内容為限。 接著，將要進行PCR的檢體以及反應試劑混合形成一 反應物後置於該第一注射器32内，並啟動該第一流速控制 器31，將該反應物由該流道22的入口 221注入後，再使該 反應物以設定的速度於該流道22内移動。反應物在變性區 223、黏合區224,及延展區225的時間比例通常趨近於j :1 : 2 ’因此在設計上可以加大該基板的面積，而使該 流道22的延展區225的長度較長，也可以是使流道22的 延展區225具有較多的彎折而讓延展區225的路徑較長， 上述兩者都是可以實施的方法，不以此為限。而該第一流 速控制器31的速度控制，可使反應物在每完成一次變性區 223—黏合區224->延展區225的反應後，會以較快的速度 由該延展區225前進到下一個變性區223，以使反應物進行 下一次變性區223—黏合區224—延展區225的循環。 透過上述設計’當PCR產物在流經變性區223而使得 雙股DNA分解成單股DNA時，由於所述電極52是設置於 變性區223與黏合區224之間，因此單股DNA若有與探針 互補的片段，就會與探針結合，隨著單股DNA結合於探針 201219785 • 上的數目越多，則該電流偵測器53所偵測到對應電極52 . 的電流量就會越低。 藉 不仁在PCR反應的過程中就能即時彳貞測pcr的 產物的生成量’不需等到PCR反應完成，能大幅節省實驗 時間，另外，直接以電流強度進行檢測結果的判讀，不需 使用大量或昂貴的耗材，也不需額外添加外切酶能降低 實驗成本。 综上所述，透過PCR過程中檢體内單股的dna能直接 • 結合於所述電極52上的探針，而改變所述電極52所债測 到的電流值’藉此能快速且直接地獲得pcR的結果，不需 要再製膠、購買大量或昂貴的耗材，也不需要配合大型光 學儀器才能判讀，而能節省實驗成本，故確實能達成本發 明之目的。 准以上所述者，僅為本發明之較佳實施例而已，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 範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。 【圖式簡單說明】 圖1疋iL體刀解圖，說明本發明即時電化學檢測晶 片之較佳實施例；及 圓2是一俯視圖’輔助說明圖1，為了便於說明，圖中 省略部分構件。 201219785 【主要元件符號說明】 2 反應裝置 42 第一加熱單元 21 基板 421 第一加熱器 22 流道 422 第一導熱件 221 入口 43 第二加熱單元 222 出口 431 第二導熱件 223 變性區 44 第三加熱單元 224 黏合區 441 第二加熱器 225 延展區 442 第三導熱件 3 第一流速控制裝置 5 偵測裝置 31 第一流速控制器 51 封蓋 32 第一注射器 52 電極 4 加熱裝置 53 電流偵測器 41 傳熱板 6 夾置板 411 上表面 7 第二流速控制裝置 412 下表面 71 第二流速控制器 413 水流通道 72 第二注射器 10The other version is to form a single-strand bond (annealing, the primer binds to the complementary position of the single-stranded DNA) (extensi〇n, the two primers are used to synthesize new DNA). After PCR, the gel electrophoresis is performed to detect the result. * The average number of cycles of PCR is m, and gel electrophoresis needs to wait until the PCR is completely finished. It is not only time-consuming, but also requires glue and many consumables to be read, and can be read with large optical instruments. Powder is quite expensive, so the use of gel electrophoresis is time consuming and costly. If gel electrophoresis is not used, capillary electrophoresis can also be used. After the end, the PCR product is placed in a capillary tube for separation. (4) This method is not required for 201219785. Gluing, but still need to buy consumables such as capillaries, and can be read with large optical instruments, which is also time consuming and costly. Also, if other electrochemical detection methods are used, it is necessary to add specific PCR to the PCR product. The exo-enzyme is cleaved for detection, and the exonuclease is also quite expensive, and requires a long working time, which is not ideal for use. Therefore, the object of the present invention is to provide an instant electrochemical detection wafer capable of detecting the detection result in real time. Thus, the instant electrochemical detection wafer of the present invention comprises a reaction device and a first communication device. a flow rate control device, a heating device disposed under the reaction device, and a detecting device disposed above the reaction device. The reaction device includes a substrate, and a recess is formed on the substrate and repeatedly bent and extended. a flow path, the flow item has an inlet, and an outlet opposite to the inlet. The first flow rate control device includes a first flow rate controller, and a first flow rate controller is mounted on the first flow rate controller and connected to the flow path a first injector, the first injector containing a sample and a reagent. The heating device includes a first heating unit, a second heating unit, and a third heating unit located below the substrate and spaced apart from each other. Repeatingly spanning the first to third heating units. The detecting device includes a cover corresponding to the substrate, and a plurality of covers An electrode disposed on the cover and extending into the flow path, a plurality of probes respectively disposed on the electrodes of 201219785, and a current detector electrically connected to the electrodes. The effect of the present invention is: When the sample and the reaction reagent are injected into the flow channel by the first syringe to perform PCR, the single-stranded DNA formed by denaturation in the sample can be directly bonded to the probe on the electrode, and the electrode is detected. The measured current value 'to thereby obtain the result of the PCR quickly and directly during the process of PCR. [Embodiment] The foregoing and other technical contents, features and effects of the present invention are described below with reference to the drawings. A detailed description of a preferred embodiment will be apparent. Referring to Figures 1 and 2', a preferred embodiment of the instant electrochemical detection wafer of the present invention comprises a reaction device 2, a communication device 2 a first flow rate control device 3 (only seen in FIG. 2), a heating device 4 disposed below the reaction device 2, a detecting device 5 disposed above the reaction device 2, and two for heating the reaction device 2 Loading 4, and the detecting device 5 sandwiches the fixed clamping plate 6' and a second flow rate controlling device 7 (see Fig. 2) connected to the heating device 4, and the clamping plate is omitted in Fig. 2 for convenience of explanation. The reaction device 2 includes a substrate 21, and a flow channel 22 formed on the substrate 21 and recessed and extended. The flow channel 22 has an inlet 221 and an outlet 222 opposite to the inlet 221. As shown in FIG. 1, the heating device 4 includes a heat transfer plate 41 located below the substrate 21 of the reaction device 2 and corresponding to the size of the substrate 21, and a first heating unit 42 located below the S-substrate 21 and spaced apart from each other. a second plus 201219785 thermal unit 43, and a third heating unit 44, the heat transfer plate 41 has an upper surface 411, a lower surface 412, and a water flow channel 413 formed by the lower surface 412, and is located below The sandwiching plate 6 cooperates with the heat transfer plate 41 to form the water flow passage 413 in a closed state. The first heating unit 42 has a first heater 421 disposed on the lower surface 412 of the heat transfer plate 41, and a first heat conducting member disposed on the upper surface 411 of the heat transfer plate 41 and corresponding to the first heater 421. 422, the second heating unit 43 has a second heat conducting member 431 disposed on the upper surface 411 of the heat transfer plate 41 and corresponding to the water flow passage 413. The third heating unit 44 has a heat conducting plate 4 disposed under the heat transfer plate 4 a second heater 441 of the surface 412, and a third heat conducting member 442' disposed on the upper surface 411 of the heat transfer plate 41 and corresponding to the second heater 441, and the water flow channel 413 is located at the first and second heaters Between 421 and 441. The flow path 22 is repeatedly bent and heated across the first, second, and third heat conducting members 422, 4S1, and 2. The detecting device 5 includes a cover 5 i correspondingly disposed on the substrate 2 , and a plurality of electrodes 52 disposed on the cover 51 and extending in the flow channel 22 , and a plurality of electrodes 52 are respectively disposed in the cover The probe on the electrode 52 (pr〇be, which is not visible to the naked eye, is not shown), and a current detector 53 electrically connected to the electrode 52. As shown in FIG. 2, the first flow rate control device 3 includes a first flow rate controller 3 1 'A* mounted on the first flow rate controller 3 i and connected to the flow path 22 of the first syringe 32. The first syringe 32 contains a sample and a reagent. The second flow rate control device 7 includes a second flow rate controller 71' and - mounted on the second flow rate controller 71 and containing liquid, and 201219785. a second connected to the water flow channel 413 (see Fig. 1) Syringe 72. In this embodiment, the first heater 421 and the third heater are respectively controlled such that the temperature of the first heat conducting member 422 is 95 ° C, the temperature of the third heat conducting member 442 is 72 ° C, and the The second flow rate controller 71 causes the liquid in the second syringe 72 to flow into the water flow channel 413 at a certain flow rate, so that the heat energy generated by the first and second heaters 422, 442 is absorbed by the liquid in the water flow channel 413. The temperature was raised to 55 °C. It should be noted that the above temperature is only the experimental condition set in this embodiment, and the temperature of the first and second heaters 422 and 442 can be changed according to various experiments to adjust the first heat conducting member 422 and the first The temperature of the third heat conducting member 442, and the temperature of the second heat conducting member 431, is controlled by changing the flow rate of the liquid. The faster the flow rate, the lower the temperature, the slower the flow rate, and the higher the temperature. - The following is a description of the use of the present invention in conjunction with the experiment. In the present embodiment, the temperatures of the first heat conducting member 422, the second heat conducting member 43 1 and the third heat conducting member 442 are respectively 95 ° C, 55. °C, 72 ° C, and the flow channel 22 is repeatedly bent and repeated across the first, second, and third heat conducting members 422, 43 1 , φ 442, thus dividing the flow channel 22 into a plurality of a denaturation zone 223 on the first heat conducting member 422, a plurality of bonding regions 224 on the second heat conducting member 431, and a plurality of extensions 225 on the third heat conducting member 442. The inlet 221 of the flow channel 22 is located in the first denaturation zone 223, and the outlet 222 is located in the last extension zone 225. The sample passes through the denaturation zone 223, the adhesion zone 224, and the extension zone 225 is a polymerase chain reaction (Polymerase). One cycle of Chain Reaction, PCR). First, a segment complementary to the PCR product is confirmed, and the segment 201219785 is fixed on the electrode 52 as a probe, and it is confirmed that the electrode 52 is to be placed at the position of the PCR cycle. The electrodes 52 are disposed between the fifth cycle, the 帛12 cycle, and the denaturation region 223 of the 18th cycle and the bonding region 224, respectively. Since the manner of providing the probe is easily implemented by those skilled in the art, It is not described in detail, and the position of the electrode 52 is changed depending on the experimental conditions, and is not limited to the content disclosed in the embodiment. Next, the sample to be subjected to PCR and the reaction reagent are mixed to form a reactant, which is placed in the first syringe 32, and the first flow rate controller 31 is activated, and the reactant is injected from the inlet 221 of the flow channel 22. The reactant is then moved within the flow channel 22 at a set rate. The ratio of the time of the reactants in the denaturation zone 223, the adhesion zone 224, and the extension zone 225 generally approaches j:1:2' so that the area of the substrate can be increased in design so that the extension zone 225 of the flow channel 22 The length of the extension region 225 of the flow channel 22 is relatively large, and the path of the extension region 225 is long. Both of the above methods are applicable, and are not limited thereto. The speed control of the first flow rate controller 31 allows the reactants to advance from the extension zone 225 to the faster speed after each reaction of the denaturation zone 223 - the adhesion zone 224 - the extension zone 225 is completed. The next denatured zone 223 is such that the reactants undergo a cycle of the next denatured zone 223 - the bond zone 224 - the extended zone 225. Through the above design, when the PCR product is decomposed into single-stranded DNA by flowing through the denatured region 223, since the electrode 52 is disposed between the denatured region 223 and the adhesive region 224, if the single-stranded DNA is present, The fragment complementary to the probe will bind to the probe. As the number of single strands of DNA bound to the probe 201219785 is increased, the amount of current detected by the current detector 53 corresponding to the electrode 52 will be The lower. In the process of PCR reaction, the amount of PCR product can be detected immediately. It does not need to wait until the PCR reaction is completed, which can greatly save the experimental time. In addition, the interpretation of the detection result is directly performed by the current intensity, without using a large amount of Or expensive consumables, without the need for additional exozymes to reduce the cost of the experiment. In summary, the single-stranded DNA in the in vivo can be directly and indirectly coupled to the probe on the electrode 52, and the current value measured by the electrode 52 can be changed, thereby enabling rapid and direct As a result of obtaining the pcR, it is not necessary to make a glue, purchase a large amount of expensive or expensive consumables, and it is not necessary to cooperate with a large optical instrument to interpret it, and the experiment cost can be saved, so that the object of the present invention can be achieved. The above is only the preferred embodiment of the present invention, and the scope of the invention is not limited thereto, that is, the simple equivalent changes and modifications made by the scope of the invention and the description of the invention are All are still within the scope of the patent of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a schematic diagram of a body blade illustrating a preferred embodiment of the instant electrochemical detection wafer of the present invention; and a circle 2 is a top view of the auxiliary description FIG. 1, and for the sake of convenience, some components are omitted in the drawing. . 201219785 [Description of main component symbols] 2 Reaction device 42 First heating unit 21 Substrate 421 First heater 22 Flow path 422 First heat conduction member 221 Entrance 43 Second heating unit 222 Exit 431 Second heat conduction member 223 Denaturation region 44 Third Heating unit 224 Bonding zone 441 Second heater 225 Extension zone 442 Third heat conduction member 3 First flow rate control device 5 Detection device 31 First flow rate controller 51 Cover 32 First syringe 52 Electrode 4 Heating device 53 Current detection Heater plate 6 sandwich plate 411 upper surface 7 second flow rate control device 412 lower surface 71 second flow rate controller 413 water flow channel 72 second syringe 10