201113524 六、發明說明： 【發明所屬之技術領域】 本案所屬的技術涵蓋微機電、微流體系統、表面改質以及生化檢測範 疇。本案提出一被動式檢測生化流體的晶片：在微流道中，生化流體藉由 毛細力而自發性地傳輸至檢測區，此時，生化流體内之生化物質與檢測區 表面之生化探針的反應與否，影響此檢測區的表面可濕性，進而決定生化 流體是否往後端移動。經由生化流體在檢測區反應完後的移動與否，可達 到檢測的目的。使用本專利概念所製作的生化檢測晶片具有自發性驅動生 化流體、可重複使用、可同時檢測多種生化物質、可用於定量生化物質的 總含量以及操作簡單、容易判定等特點，此晶片可為單一元件或是微流體 網絡之組成。 【先前技術】 隨著微機電(micro-electro-mechanical systems, MEMS)製程發展的一日 千里’掀起微流體(microfluidics)相關研究之熱潮，微流體生化晶片便是此 思維下的產物，左右未來生醫產業的動向。簡言之，微流體生化晶片係利 用微流體特質結合跨領域之技術，期望達成檢體的任意操控、快速處理、 低樣品用量、精確檢測以及低成本的目的。微流體生化晶片之相關研究的 議題’於近幾十年以指數型態成長、擴張，相關的研究論文與專利申請如 雨後春筍般逐漸湧出，Manz ¢/ α/·，公/isws 1990 提出這類晶片的核心理念，具體將樣品處理、生化反應以及檢測分析等流 程集約化’而Burns β a/·，SaV/ice，1998實現微型生化系統於一晶片上， 201113524 試圖取代以往費時、耗人力、繁雜的樣品處理流程；樣品處理包含分離、 混合、篩選、純化、收集等步驟。Helmut Kaiser顧問諮詢公司的市場報告 預測’在2010年，微流體生化晶片的產值佔生化晶片產業總市值的27〇/〇， 至2020年，其佔的比例將逐年增加至39%。 先前技術(中華民國專利證書號：1314162)發明一微流式檢測裝置。適 用於檢測一液態待測物，包含電極與檢測模組。液態待測物之流動是藉由 電極產生電％’形成液體介電泳現象(liquid dielectrophoresis)，以驅動液態 待測物之流動；檢測模組具有發光元件及分析單元，由發光元件發出檢測 光源激發液態待測物中具發光能力的檢體發光，藉此用以檢測於流動區域 中流動之液態待測物。這類型的檢測裝置可任意操控檢測之樣品流體，然 而’必須外加驅動源(電場)來操控樣品流體的移動，而且裝置本體的構件相 對複雜，大大降低此裝置的實用價值。另一先前技術(中華民國專利證書號： 1289666)¼出一無動力驅動檢體液珠的檢測裝置，將待測的檢體液珠與反應 試劑液珠放置於-具有微結構的表面上，這魏株將自發性地移動至反應 區，兩者反應後所產生的訊號由後端的檢測裝置檢測。這一類型的裝置靠 著表面張力的概念驅動液珠移動，實屬被動式驅動的手段，然而，此裝置 的後端檢測必齡著外加的❹彳器純感測樣品的反應减，肋達成檢 測的目的’此外，藉域結構趨動樣品液珠的_，包含幾項潛在的缺點： 第-、微結構的製作過程相對複雜。製作過程包含微影、侧與翻製等步 驟，在翻製上’翻製好的結構表面仍需執行表面改質以舰珠於表面上的 驅動；第二、樣品液珠的蒸發與污染。液珠所處的移動空間為一開放系統， 不免有污U發之疑慮，第二、以居家檢測、纟帛檢的觀點來看檢測過 201113524 程的人事物白難以草控，如果樣品液珠於微結構表面移動時，此表面 為非水平狀態，將嚴重影響液珠移動行走的路徑，大幅降低檢測的成功率。 近幾年來的數量與日倶增，居家快速、方便的檢測 f吣㈣，PGQ鶴得相當重要，㈣目紐雜纽^⑽展必須 符。帛樣_«_體的被動式驅動。無需外加額外的驅動源來驅動樣品 移動，可大幅降低成本與操作的複雜度。第二、樣品的平行處理，時 檢測數種樣品，加速檢測的流程與縮短時i第三、可重複使用。絕大多 •數的生化晶片都是可拋棄式的’然而有些生化晶片的成價格昂貴，因此發 展可重複使用之晶片可降低成本。第四、操作方便、簡單且快逮判定結果。 有鑑於此，本發明提出一符合上述幾項優點的晶片，極具進步、新賴以及 實用之價值。 【發明内容】 本專利案提出一種具有自發驅動流體移動之特性的生醫檢測晶片，此晶 籲 #疋以微機電製程製作出微流道，該微流道内壁至少有-面是親水性表 面，利用表面可濕性改質技術，將蚊的探針分子修飾於微流道内 壁之部份區域，此區域的可濕性因而降低(即親水性降低、疏水性增強），是 為檢測區。由於麵尺度下主要的側力之—就是分界祕力因此將生 化流體置在晶片的微流道入口處’此流體會受到不平衡的分界面張力而被 吸入微流道内部，當流體前進至表面改質的區域時(檢親），因為所受到的 不平衡分界面張力變小而停留在此區域。當生化流體内的目標㈣et)生化 分子與檢測區的探針分子反應後’表面的可濕性增強(即親水性增強、疏水 201113524 性降低），流體即可通過檢測區；若生化流體内未含有目標生化分子時，則 由於檢測區的表面特性未改變，生化流體將繼續停留在此區域，因此可藉 由流體是否能通過檢測區來檢測出特定的生化分子。 下文藉由具體實施例配合所附的圖式詳加說明，更容易瞭解本發明的目 的、技術内容、特點及其所達成的功效。 【實施方式】 本發明提出-製作被動式生化檢測晶片的作法，以下以圖丨來說明檢 測區製作之方法’本實施綱使關制區可應麟DNA之雜上，另外 以圖2來說明檢測晶片的整體型態及其檢測原理。 如圖la所示’使用親水性之石夕晶圓做為基材(1〇〇)，由於dna無法直 接與石夕晶圓表面接合’ @此在表面上部份區域(1〇la)先以十一碳稀二甲基氯 矽烷（10-undecenyldimethylchlorosilane)製作分子自組 | 仲 、、*^^·早 膜 (self-assembled m_layerXl〇2) ’此薄膜的官能基為烯基(CH2)，無法直接與 DNA接合。所以利用過錳酸鹽過碟酸鹽溶液加職啊站㈣遍攸 sohUion)將烯基酸化成羧基(COOH)，由於羧基可與氨基(NH)接合，因此先 將探針DNA(pr〇be DNAK103)的其中-端修飾成氨基；由於dna本身為親 水性，為了製作疏水性的檢測區，故在探針DNA的另一端上修飾氣類化合 物(1〇4)。讓兩端都修飾好的探針DNA與第—層自組裝薄膜上的絲反應， 即可產生第二層的分子自組裝薄膜(1〇3㈣句，由於項端的官能基為_ 化合物，所以此區域表面呈疏水性(可濕性較低）。 由於DNA具有互漏交的特性，探針職只會與特定序列的dna 201113524 雜交’也就是說只會與目標DNA(105)接合。特意將探針DNA Μ段的長度 設計成比目標DNA的長度短，當制區上的探針DNA與目標dna互補 雜交後，結果就會如圖lb所示，目標驗的一端裸露在表面上，由於目 標DNA沒有修飾疏水分子，故互補雜交後表面特性將變成親水性。 如圖2a所示，利用標準的微機電製程，以聚雙曱基矽氧烷 (pdydimethylsiloxane，PDMS)製作具有溝槽的蓋板(2〇2)，將此蓋板經由氧 電漿處理後，與上述具有檢腦晶圓基材_)接合後形成一微流 • 道，此即為本案所提出的生化流體檢測裝置。其檢測原理如圖2b與2c所 示，首先將内含DNA(204a&204b)的流體(2〇3a& 203b)滴在流道的入口端 (右端)’由於毛細力的作用，流齡被吸入流道内，當流體向左端前進時(2〇5) 會碰到疏水性·，越在此處所受到的毛細力降低，就會無法繼續 前進。當流體停留在此區域時，流體内的目標DNA會與表面的探針DNA 產生互補雜交，此時檢測區就會變成親水性表面(2〇lb)，流體就會通過(圖 2c)。若待測的生化流體内不具有目標DNA，則流體將保持無法通過的狀態 •(即保持圖2b所示之狀態）’因此可藉由流體的通過與否，檢測出流體内是 否含有目標DNA。 备檢測完生化流體後，可將本晶片加熱處理(約95。〇，在此溫度狀態 下不會破壞第一層與第二層的自組裝薄膜結構與PDMS蓋板，只會將已經 互補雜交後的雙股DNA分離(denature)，使檢測區從親水性(圖lb的狀態） 恢復成疏水性(圖la的狀態）’也就是說本晶片具有重複使用的特性。 接下來以兩個例子來說明本發明的具體應用及其他特點。圖3所示之 檢測晶片的基材上(300)具有四個並排的檢測區(3〇2a〜3〇2d),四個檢測區分 201113524 別有四種不同序列的探針DNA，由PDMS所製作的蓋板㈣在左端同樣也 分成四個微流道，分麟制四働砸。當生化趙進人流勒時，若 含有與探針DNA 1(3()la)互獅DNA諸時(目標DNA)麟體可通過第一 個檢顺(301a) ’若不含目標DNA則無法通過，依此類推，其他三個檢測 區(301b~301d)具有同樣的檢測原理，故本實施例所示範的檢測晶片可同時 檢測四種不同的DNA，也就是說本案所提出的檢測裝置具有同時檢測多種 生化物質的功能。 圖4所不之檢測晶片的基材上(4〇〇)具有三個檢測區(4〇2&〜4〇2c)，此三 _ 個檢測區的探針DNA完全相同。當生化流體(4〇3)進入流道内時，因内含有 目標DNA(404a) ’故先與第一個檢測區反應’將其改質為親水性，使流體 繼續别進碰觸到第二個檢測區(4〇2b)，生化流體内的目標DNA因為與第一 個檢測區的探針DNA互補雜交，其總數已經減少，當生化流體碰觸到第二 個檢測區時，目標DNA數量如果已經被第一個檢測區消耗到不足以改變第 二個檢測區的親疏水性時則無法通過第二個檢測區（圖4所示的狀況）。反 之’如果生化流體内所含的目標DNA夠多時，則可將第二個檢測區轉變成鲁 親水性而通過此區。依此齡，流體能㈣過第三個檢職取決於它内部 所含的目標DNA數量。本實施例所示範的檢測晶片可藉由生化流體通過檢 測區的數量來觸频⑧NA _數4，樣找树騎糾的概念可 以用來定量生化物質的含量。 不論是上述的方法、手段、想法，甚至於相關概念的延伸，皆屬本專 利之範圍。 8 201113524 【圖式簡單說明】 第一圖 DNA於矽晶圓表面積基材上自組裝示意圖 第二圖檢測晶片不意圖 第三圖多種DNA同步檢測示意圖 【主要元件符號說明】 100 矽晶圓基版(親水性表面）； 101a 檢測區(疏水性）；201113524 VI. Description of the invention: [Technical field to which the invention pertains] The technology to which the present invention pertains covers microelectromechanical, microfluidic systems, surface modification, and biochemical detection. In this case, a passive detection biochemical fluid wafer is proposed: in the microchannel, the biochemical fluid is spontaneously transmitted to the detection zone by capillary force. At this time, the biochemical substance in the biochemical fluid reacts with the biochemical probe on the surface of the detection zone. No, it affects the surface wettability of this detection zone, which in turn determines whether the biochemical fluid moves to the back end. The movement of the biochemical fluid after the reaction in the detection zone can achieve the purpose of detection. The biochemical detection wafer fabricated by using the patent concept has the characteristics of spontaneously driving biochemical fluid, reusable, simultaneous detection of various biochemical substances, total content of quantitative biochemical substances, simple operation and easy judgment, and the wafer can be single. The component or the composition of the microfluidic network. [Prior Art] With the development of micro-electro-mechanical systems (MEMS) processes, microfluidic biochips are the product of this thinking, and the future biomedicine Industry trends. In short, microfluidic biofilms combine microfluidic traits with cross-domain technologies to achieve any manipulation, rapid processing, low sample throughput, accurate detection, and low cost. The topic of research on microfluidic biochemical wafers has grown and expanded exponentially in recent decades, and related research papers and patent applications have sprung up, Manz ¢/α/·, public/isws 1990 The core concept of the wafer specifically intensifies the processes of sample processing, biochemical reactions, and detection and analysis. While Burns β a/·, SaV/ice, 1998 realizes the micro biochemical system on a wafer, 201113524 attempts to replace the time-consuming, labor-intensive, A cumbersome sample processing procedure; sample processing includes steps such as separation, mixing, screening, purification, and collection. Helmut Kaiser Consulting's Market Report Predicts that in 2010, the output value of microfluidic biochemical wafers accounted for 27% of the total market value of the biochemical wafer industry, and by 2020, its proportion will increase to 39% year by year. The prior art (Republic of China Patent Certificate No.: 1314162) invented a microfluidic detection device. It is suitable for detecting a liquid analyte, including an electrode and a detection module. The liquid sample is flowed by the electrode to generate liquid dielectrophoresis to drive the flow of the liquid analyte; the detection module has a light-emitting element and an analysis unit, and the light-emitting element emits a detection light source to excite The light-emitting ability of the liquid to be tested is illuminated, thereby detecting the liquid analyte flowing in the flow region. This type of detecting device can arbitrarily manipulate the detected sample fluid, but the driving source (electric field) must be applied to manipulate the movement of the sample fluid, and the components of the device body are relatively complicated, greatly reducing the practical value of the device. Another prior art (Republic of China patent certificate number: 1289666) 1⁄4 out a non-powered test liquid bead detection device, the test liquid bead and the reaction reagent liquid bead are placed on the surface with a microstructure, this Wei The strain will spontaneously move to the reaction zone, and the signal generated by the reaction between the two will be detected by the detection device at the rear end. This type of device drives the liquid bead movement by the concept of surface tension, which is a passive driving method. However, the back end detection of this device is dependent on the reaction of the externally-sensed pure sensing sample, and the rib is tested. The purpose of 'in addition, the use of the domain structure to drive the sample liquid droplets _, contains several potential drawbacks: The first -, the microstructure of the production process is relatively complex. The production process includes lithography, side and flipping steps. The surface of the restructured structure is still subjected to surface modification to drive the ship's beads on the surface. Second, the evaporation and contamination of the sample beads. The moving space where the liquid bead is located is an open system, and it is inevitable that there is a suspicion of filth. Secondly, from the point of view of home inspection and inspection, it is difficult to control the whiteness of the person who has tested the process of 201113524. When the surface of the microstructure moves, the surface is in a non-horizontal state, which will seriously affect the path of the liquid bead moving and greatly reduce the success rate of detection. In recent years, the number and the number have increased, and the home is fast and convenient to detect f吣(4). The PGQ crane is very important, and (4) the target is mixed.帛 _ « _ body passive drive. No additional drive source is required to drive sample movement, which significantly reduces cost and operational complexity. Second, the parallel processing of the samples, the detection of several samples, the process of accelerating the detection and the shortening of the third, reusable. Most biofilms are disposable. However, some biochemical wafers are expensive, so developing reusable wafers can reduce costs. Fourth, the operation is convenient, simple and quick to arrest the judgment result. In view of this, the present invention proposes a wafer that meets the above several advantages, which is extremely advanced, new and practical. SUMMARY OF THE INVENTION The present patent application proposes a biomedical detection wafer having the characteristics of spontaneously driving fluid movement, and the crystal micro-electromechanical process produces a micro-flow channel having at least a surface-hydrophilic surface. The surface wettable modification technique is used to modify the probe molecules of the mosquito to a part of the inner wall of the microchannel, and the wettability of the region is thus reduced (ie, the hydrophilicity is lowered and the hydrophobicity is enhanced), which is the detection area. . Because of the main side force at the surface scale—the boundary force is so the biochemical fluid is placed at the microchannel inlet of the wafer. This fluid is subjected to unbalanced interfacial tension and is drawn into the interior of the microchannel. When the fluid advances to When the surface is modified (inspection), it stays in this area because the unbalanced interface tension is small. When the target (4) et biochemical molecule in the biochemical fluid reacts with the probe molecule in the detection zone, the surface wettability is enhanced (ie, the hydrophilicity is enhanced, the hydrophobicity is reduced to 201113524), and the fluid can pass through the detection zone; if the biochemical fluid is not When the target biochemical molecule is contained, since the surface characteristics of the detection zone are not changed, the biochemical fluid will continue to stay in this region, so that the specific biochemical molecule can be detected by whether the fluid can pass through the detection zone. The objects, technical features, features and effects achieved by the present invention will be more readily understood from the following detailed description of the embodiments. [Embodiment] The present invention proposes a method for producing a passive biochemical detection wafer, and the following describes a method for fabricating a detection zone by using a diagram of the present invention, which can be used to illustrate the detection of the DNA in the closure zone. The overall shape of the wafer and its detection principle. As shown in Figure la, 'the hydrophilic stone wafer is used as the substrate (1〇〇), because the DNA cannot be directly bonded to the surface of the Shixi wafer. @@上在上上 partial area (1〇la) Molecular self-assembly made of 10-undecenyldimethylchlorosilane | secondary, *^^· early film (self-assembled m_layerXl〇2) 'The functional group of this film is alkenyl (CH2), It is not possible to directly bind to DNA. Therefore, using the permanganate over-dissolved acid solution, the station (4) is used to acidify the alkenyl group into a carboxyl group (COOH). Since the carboxyl group can be bonded to the amino group (NH), the probe DNA is first (pr〇be). The DNA-endion of DNAK103) is modified to an amino group; since DNA itself is hydrophilic, in order to prepare a hydrophobic detection region, a gas compound (1〇4) is modified on the other end of the probe DNA. The probe DNA modified at both ends is reacted with the silk on the first layer self-assembled film to produce a second layer of molecular self-assembled film (1〇3(4) sentence, since the functional group at the terminus is a compound, so this The surface of the area is hydrophobic (lower wettability). Due to the mutual leakage of DNA, the probe position will only hybridize to the specific sequence of DNA 201113524, which means that it will only bind to the target DNA (105). The length of the probe DNA segment is designed to be shorter than the length of the target DNA. When the probe DNA on the region hybridizes to the target DNA, the result is as shown in Figure lb, and the end of the target is exposed on the surface due to The target DNA is not modified with hydrophobic molecules, so the surface properties will become hydrophilic after complementary hybridization. As shown in Figure 2a, a trench with a trench is fabricated using a standard microelectromechanical process with pdydimethylsiloxane (PDMS). The plate (2〇2), after the cover plate is treated by the oxygen plasma, is combined with the above-mentioned cerebral wafer substrate _) to form a microfluidic channel, which is the biochemical fluid detecting device proposed in the present invention. The detection principle is shown in Figures 2b and 2c. First, the fluid containing DNA (204a & 204b) (2〇3a & 203b) is dropped on the inlet end (right end) of the flow channel. In the suction passage, when the fluid advances to the left end (2〇5), it will encounter hydrophobicity. As the capillary force received here decreases, it will not continue. When the fluid stays in this area, the target DNA in the fluid will hybridize with the probe DNA on the surface, and the detection zone will become a hydrophilic surface (2〇lb) and the fluid will pass (Fig. 2c). If the biochemical fluid to be tested does not have the target DNA, the fluid will remain unpassable (ie, maintain the state shown in Figure 2b). Therefore, whether the fluid contains the target DNA can be detected by the passage of the fluid. . After the biochemical fluid has been tested, the wafer can be heat treated (about 95 〇, at this temperature state, the self-assembled film structure of the first layer and the second layer and the PDMS cover plate are not destroyed, and only the complementary hybridization will be performed. The latter double-strand DNA is denatured, and the detection zone is restored from hydrophilic (state of Figure lb) to hydrophobic (state of Figure la). That is to say, the wafer has reusable characteristics. Next, two examples are given. To illustrate the specific application and other features of the present invention, the substrate (300) of the test wafer shown in FIG. 3 has four side-by-side detection zones (3〇2a~3〇2d), and four detection zones 201113524 have four A different sequence of probe DNA, the cover plate made of PDMS (4) is also divided into four micro-flow channels at the left end, and is divided into four micro-channels. When biochemical Zhao Jinren flows, if it contains DNA with probe 1 ( 3()la) Mutual lion DNA time (target DNA) can pass the first test (301a) 'If the target DNA is not available, and so on, the other three detection areas (301b~301d) With the same detection principle, the detection wafer demonstrated in this embodiment can simultaneously detect four different DNA, that is to say, the detection device proposed in the present invention has the function of simultaneously detecting a plurality of biochemical substances. The substrate on the detection wafer of Fig. 4 (4〇〇) has three detection areas (4〇2&~4〇2c) The probe DNA of the three detection zones is identical. When the biochemical fluid (4〇3) enters the flow channel, it contains the target DNA (404a) and therefore reacts with the first detection zone. The hydrophilicity allows the fluid to continue to touch the second detection zone (4〇2b). The target DNA in the biochemical fluid has been reduced by complementary hybridization with the probe DNA of the first detection zone. When the biochemical fluid touches the second detection zone, the target DNA amount cannot pass through the second detection zone if it has been consumed by the first detection zone to change the hydrophilicity of the second detection zone (Fig. 4). Conversely, 'If the target DNA contained in the biochemical fluid is sufficient, the second detection zone can be converted into Lu hydrophilicity and pass through this zone. According to this age, the fluid can (4) pass the third inspection. Depending on the amount of target DNA contained in it, it is demonstrated by this embodiment. The detection wafer can be touched by 8NA _ number 4 by the number of biochemical fluids passing through the detection zone, and the concept of finding a tree ride can be used to quantify the content of biochemical substances. Whether the above methods, means, ideas, or even related concepts The extension of this patent belongs to the scope of this patent. 8 201113524 [Simple description of the diagram] The first figure shows the self-assembly of DNA on the surface area of the wafer surface area. The second picture shows the detection of the wafer. Component symbol description] 100 矽 wafer base (hydrophilic surface); 101a detection area (hydrophobic);
101b 與目標DNA雜交後親水性增加之檢測區； 102 自組裝於基材表面的十一碳烯二甲基氯矽烷； 103 自組裝於十一碳烯二甲基氣矽烷上之探針DNA ; 104 修飾於DNA端點之氟類化合物； 105 目標 DNA; 200 矽晶圓基版； 201a 檢測區(疏水性）； 201b 與目標DNA雜交後變成親水性之檢測區； 202 PDMS製作之蓋板； 203a 流道内部含DNA之生化流體； 203b 流道入口處含DNA之生化流體； 201113524 204a 204b 205 300 301 302a 302b 302c 302d 400 401 402a 402b 402c 403 404a 404b g 標 DNA ; 非目標DNA ; 液體在流道内受到毛細力移動之方向； 矽晶圓基版； PDMS製作之蓋板； 含探針DNA1之檢測區； 含探針DNA 2之檢測區； 含探針DNA 3之檢測區； 含探針DNA 4之檢測區； ♦晶圓基版， PDMS製作之蓋板； 第一個檢測區(與目標DNA雜交故呈親水性）； 第二個檢測區(疏水性）； 第三個檢測區(疏水性）； 含DNA之生化流體； 目標DNA ; 非目標DNA ;a detection region in which 101b is hybridized with a target DNA, and has a hydrophilicity; 102 self-assembled on the surface of the substrate; undecene dimethyl chlorodecane; 103 self-assembled probe DNA on undecene dimethyl decane; 104 a fluorine compound modified at the DNA end point; 105 target DNA; 200 矽 wafer basis; 201a detection area (hydrophobicity); 201b hybridization to the target DNA to become a hydrophilic detection zone; 202 PDMS made cover; 203a biochemical fluid containing DNA inside the channel; 203b biochemical fluid containing DNA at the entrance of the channel; 201113524 204a 204b 205 300 301 302a 302b 302c 302d 400 401 402a 402b 402c 403 404a 404b g standard DNA; non-target DNA; The direction in which the capillary is moved by the capillary; the wafer base plate; the cover plate made of PDMS; the detection zone containing the probe DNA1; the detection zone containing the probe DNA 2; the detection zone containing the probe DNA 3; 4 detection area; ♦ wafer base plate, PDMS production cover; first detection area (hybrid with hybridization of target DNA); second detection area (hydrophobic); third detection area (hydrophobic) Sex); biochemical fluid containing DNA; The DNA; non-target the DNA;