M373489 五、新型說明： 【新型所屬之技術領域】 本創作係關於一種生物晶片，特別係指一種可溫控之微型生物分子反 應晶片及其應用。 【先前技術】 生物晶片（biochip)係應用分子生物學、基因資訊、分析化學、生物 資訊等原理進行設計，以石夕晶圓、玻璃或高分子為基材，配合微小化技術 整合微機電、光電、化學、生化及醫學工程等領域，用以執行醫療檢驗、 環境檢測、食品檢驗、新藥開發、基礎研究等用途的精密微小化設備。M373489 V. New description: [New technology field] This work is about a biochip, especially a temperature-controlled micro biomolecule reaction wafer and its application. [Prior Art] Biochips are designed using molecular biology, genetic information, analytical chemistry, and biological information. They are based on Shixi wafers, glass or polymers, and micro-electromechanical integration with miniaturization technology. Precision micro-devices for medical inspection, environmental testing, food testing, new drug development, basic research, etc. in the fields of optoelectronics, chemistry, biochemistry and medical engineering.
目前發展中之生物晶片可約略分成：基因晶片（gene chip or DNA microarray)、蛋白晶片（protein chip)與實驗室晶片（Lab-on-a-chip)三類。 基因晶片是所有不同種類之生物晶片中發展最快的一種。指的是在數平方 公分之面積上安裝數千或數萬個核酸探針，經由一次測驗，即可提供大量 基因序列相關資訊。實驗室晶片的功能則依不同的需求進行設計，在微晶 . 片上進行不同的反應’目前已知可在微縮實驗室晶片上進行的生化實驗包 · 括具有基罔增幅功能的聚合酶鏈鎖反應(polymerase chain reaction，PCR)、 核酸的定序反應、微流體操作、電泳(electrophoresis)、質譜分析(MS，mass spectrograph)、抗原-抗體結合或一般的酵素反應等。 聚合酶鍵鎖反應(polymerase chain reaction，PCR)已廣泛地被應用於生 醫檢測之前處理過程上，其DNA複製增加量可提高生醫檢測之靈敏度。傳 統之PCR裝置，係將樣本置於塑膠管中，放入大型PCR裝置中，產生特定 4 M373489 - 溫度 '時間及次數之循環，複製出大量之DNA並進行後續檢測以提高生 醫檢測鑑別度。但是因為所需樣本量大，耗費時間往往都超過三小時；相 - 反地，以微機電製程所製造的微型生醫晶片即可改善上述缺點》 另外’近來常以即時定量PCR裝置(real_time quantitative pcR)，用以即 時檢測生物分子(如DNA)之含量，不同於傳統PCR裝置，即時定量PCR -裝置除了可進行PCR反應外，搭配光學檢測系統，可即時偵測反應中生物 ·分子之含量。進行即時定量PCR反應時，需將樣品至於一毛細管中，放入 春大型即時定量PCR裝置中，於該裝置產生特定溫度、時間及次數之循環， 藉由加熱、昇溫、降溫以促使樣品中之酵素進行反應，進而複製大量之 DNA，且樣品巾含有勞光物f，故可用以即時細生物分子之含量。然而， 與傳統之PCR裝置相同地，即時uPCR裝置係以傳統加溫方式藉由熱 傳導將毛細管中之分子樣品加溫以進行反應，故該裝置係具高耗能、體積 大、成本高及不可攜帶性等缺點。 另，常見之生物分子溫控培養裝置，如細胞培養箱；或f見之生物分 鲁·子培養監控裝置，如於共輛焦顯微鏡搭配一小型生物分子培養箱，皆藉由 -加熱器提供-熱能於培養箱中，使培養箱達到定溫度，促使培養箱 内之培養皿中之生物分子可進行生長及觀察。然而，相同於傳統pcR裝置， 此類生物分子雜培養裝置，鶴由线之鱗導方式，以提供樣品熱能， 故反應溫度不準確、反應耗時、裝置龐大且不易微型操作等缺點。 由此可見，上述習用PCR裝置、溫控培養裝置仍有諸多缺失及困難， 實非一良善之設計者，而亟待加以改良。 IT〇導電玻璃(indium tin oxide glass)係將不導電的玻璃基板上，梦上— [S) M373489 層可導電的金>1材料，該金屬材質為可轉電的氧化滅（indium tin oxide，ITO)，故稱ITO玻璃。ΠΌ玻璃是現今製造液晶顯示器面板重要的 關鍵零組件之一，且ΙΤΟ可能的應用在近年來已變得更加普遍。雖然如此， 在生物分子分析之應用上至今仍未被提出。 是以，本案創作人鑑於上述習用PCR裝置、溫控培養箱所衍生的各項 缺點及困難，乃亟思加以改良創新，並經多年苦心孤詣潛心研究後，終於 成功研發完成本件一種可溫控之微型生物分子反應晶片。 【新型内容】 本創作之目的即在於提供一種可溫控之微型生物分子反應晶片，其構 成簡易’故製備該晶片之方法極為簡易。 本創作之次一目的即在於提供一種可溫控之微型生物分子反應晶片， 該晶片由於製程簡便，故為可拋棄式設計。 本創作之另一目的係在於提供一種可溫控之微型生物分子反應晶片之 應用，該晶片可搭配一光學檢測系統，便可於生物分子反應之時，同時進 行生物分子含量、或生物分子狀態之檢測。 本創作之又一目的係在於提供一種可溫控之微型生物分子反應晶片， 係在於其可準確調控溫度，用以進行各類需精確控溫之反應。 可達成上述創作目的之可溫控之微型生物分子反應晶片，至少包括有： 一導電基板，係具有一與生物分子直接接觸之反應表面，且該導電基板具 電傳導性及導熱性； 反應框體’該框體具有第一表面及第二表面，其中該第一表面為黏貼面， 6 M373489 係將反應框體之黏貼面黏貼於導電基板之反應表面上’藉此形成一可裝載 生物分子樣品之反應槽。 前述之可溫控之微型生物分子反應晶片可進一步包含一蓋體，係用以 遮蓋反應槽。 前述之可溫控之微型生物分子反應晶片可進一步搭配一電源供應器、 一控制單元及一溫度感測單元，係用以控制該晶片之溫度達到所欲之設定 值。 前述之可溫控之微型生物分子反應晶片可進一步搭配一光學檢測單 元’係用以檢測反應槽内生物分子之含量。 前述之可溫控之微型生物分子反應晶片，其中該反應框體可進一步為 一「田」字構型，可與導電基板形成四個反應槽，可供四個反應同時進行 及偵測。 【實施方式】 本創作係觸m控之微型生齡子反應“，其諸彡優點與特 徵將從下述詳細說明並配合圖式得到進一步的瞭解。 吻參閱圖-、圖—及圖八麻’為本創作之可溫控之娜生物分子反 應晶片1各元件結構圖及立體圖，主要包括有： —導電基板u ’储有-與生物分子直接闕之反絲面m，且該導 電基板11具電傳導性及導熱性； ——反應框體12 ’該框體12具有第一表面121及第二表面122，其中該 第一表面12!為黏貼面，係將反應框體12之第—表面12_於導電基板 M373489 11之反應表面111上，藉此形成一可裝載生物分子樣品之反應槽丨3。 前述之導電基板11為一導體，且該導體可為白金、黃金、銀 '銅、鎳、 欽、石墨、奈米碳材或奈米金屬、或其他導電材質。 刖述之反應框體12可為一具黏性之Gene Frame® Spaeei^ABgene> 其中該反應框體12進一步可為一「田」字構型，便可形成四個反應槽 13，可供四個反應同時進行及偵測。 請參閱圖三A、B、C所示，為本創作之可溫控之微型生物分子反應晶 片1之導電基板11之另一態樣之剖面圖，與前述導電基版不同處係為： 該導電基版11係由導電塗層1111及基板1112所組成； 其中該導電塗層1111可以不同態樣塗覆於基板1112上，如圖三a所 示，係將導電塗層1111塗附於基版1112之任一表面上；如圖三B所示， 係將導電塗層1111塗附於兩基版1112中間；如圖三c所示，可將導電塗 層1111覆蓋整片基板1112。 前述之基板1112包含但不限於玻璃、石英或高分子材料，其中該高 分子材料係可為壓克力(PMMA) '聚碳酸酯(PC)或聚二甲矽氧烷(PDMS)等 適用材質》 前述之導電塗層1111係可為金屬氧化物半導體、導體、導電高分子或 奈米導體；S中該金屬氧化物半導體係可為銦錫氧化物(Indium Tin 0xide， ITO)銅鋅氧化物(in(jium zinc Oxide, IZO)、辞氧化物(Aluminum Zinc (^(^^(^、鎵鋅氧化物吻出贈—况如㈤⑺〜氣錫氧化物⑺·^Currently developing biochips can be roughly divided into three types: gene chip or DNA microarray, protein chip and Lab-on-a-chip. Gene chips are the fastest growing of all different types of biochips. Refers to the installation of thousands or tens of thousands of nucleic acid probes over a few square centimeters, providing a large number of genetic sequence related information through a single test. The functions of the laboratory wafers are designed according to different requirements, and different reactions are performed on the microcrystals. A biochemical experiment package that can be performed on a micro-laboratory wafer is currently known, including a polymerase chain lock with a chirp-enhancing function. Reaction (polymerase chain reaction, PCR), nucleic acid sequencing reaction, microfluidic manipulation, electrophoresis, mass spectrometry (MS), antigen-antibody binding, or general enzyme reaction. Polymerase chain reaction (PCR) has been widely used in the process of biomedical detection, and the increase in DNA replication can improve the sensitivity of biomedical detection. The traditional PCR device puts the sample in a plastic tube and puts it into a large-scale PCR device to generate a specific 4 M373489 - temperature 'time and frequency cycle, copying a large amount of DNA and performing subsequent tests to improve the discrimination of biomedical detection. . However, because the required sample size is large, it takes more than three hours; on the contrary, the micro-medical wafers manufactured by the micro-electromechanical process can improve the above shortcomings. In addition, 'there is often an instant quantitative PCR device (real_time quantitative pcR), used to detect the content of biomolecules (such as DNA) in real time. Different from traditional PCR devices, the real-time quantitative PCR device can detect the biological and molecular content in the reaction in addition to the PCR reaction. . When performing an immediate quantitative PCR reaction, the sample is placed in a capillary tube and placed in a large-scale real-time quantitative PCR device, and the device generates a cycle of a specific temperature, time, and number of times, by heating, heating, and cooling to promote the sample. The enzyme reacts to further replicate a large amount of DNA, and the sample towel contains a light-emitting substance f, so that the content of the fine biomolecule can be used. However, like the conventional PCR device, the instant uPCR device heats the molecular sample in the capillary by heat conduction in a conventional heating manner to react, so the device has high energy consumption, large volume, high cost, and no Disadvantages such as portability. In addition, a common biomolecule temperature-controlled culture device, such as a cell culture incubator; or a bio-degraded sub-culture monitoring device, such as a co-focus microscope combined with a small biomolecular incubator, is provided by a heater - Thermal energy is placed in the incubator to bring the incubator to a constant temperature, so that the biomolecules in the culture dish in the incubator can be grown and observed. However, similar to the conventional pcR device, such a biomolecular hybrid culture device, the crane is guided by the scale of the wire to provide the sample thermal energy, so the reaction temperature is inaccurate, the reaction time is time consuming, the device is large, and the micro operation is not easy. It can be seen that the above-mentioned conventional PCR device and temperature-controlled culture device still have many defects and difficulties, and it is not a good designer, and needs to be improved. IT indium tin oxide glass will be on a non-conducting glass substrate, dreaming - [S) M373489 layer of conductive gold > 1 material, which is made of indium tin oxide , ITO), hence the name ITO glass. Neodymium glass is one of the key components for the manufacture of LCD panels today, and its possible applications have become more common in recent years. Nonetheless, the application of biomolecular analysis has not been proposed so far. Therefore, in view of the shortcomings and difficulties arising from the above-mentioned conventional PCR devices and temperature-controlled incubators, the creators of this case have improved and innovated, and after years of painstaking research, they have finally successfully developed and developed a temperature control device. Micro biomolecule reaction wafers. [New content] The purpose of this creation is to provide a temperature-controllable micro biomolecular reaction wafer, which is simple in construction. Therefore, the method of preparing the wafer is extremely simple. The second purpose of this creation is to provide a temperature-controlled micro biomolecule reaction wafer which is disposable in design because of its simple process. Another object of the present invention is to provide a temperature-controlled micro biomolecular reaction wafer that can be combined with an optical detection system to simultaneously perform biomolecular content or biomolecular state at the time of biomolecular reaction. Detection. Another object of the present invention is to provide a temperature-controllable micro biomolecular reaction wafer, which is capable of accurately regulating temperature and performing various types of reactions requiring precise temperature control. The temperature-controllable micro biomolecular reaction wafer capable of achieving the above-mentioned creation includes at least: a conductive substrate having a reaction surface in direct contact with the biomolecule, and the conductive substrate has electrical conductivity and thermal conductivity; The body has a first surface and a second surface, wherein the first surface is an adhesive surface, and 6 M373489 adheres the adhesive surface of the reaction frame to the reaction surface of the conductive substrate to thereby form a loadable biomolecule The reaction tank of the sample. The aforementioned temperature-controllable microbiomolecular reaction wafer may further comprise a cover for covering the reaction tank. The aforementioned temperature-controllable micro biomolecule reaction wafer can be further combined with a power supply, a control unit and a temperature sensing unit for controlling the temperature of the wafer to a desired set value. The aforementioned temperature-controllable micro biomolecule reaction wafer can be further combined with an optical detection unit to detect the content of biomolecules in the reaction tank. The above-mentioned temperature-controllable micro biomolecule reaction wafer, wherein the reaction frame can further be in a "field" configuration, and four reaction tanks can be formed with the conductive substrate for four reactions to be simultaneously detected and detected. [Embodiment] This creation is based on the micro-small sub-reaction of the m-control. The advantages and characteristics of the citations will be further explained in the following detailed description and in conjunction with the schema. Kiss see figure -, figure - and figure aba The structural diagram and the perspective view of each component of the temperature-controllable biomolecule reaction wafer 1 of the present invention mainly include: - a conductive substrate u 'stored - a reverse surface m with a biomolecule directly twisted, and the conductive substrate 11 Electrically conductive and thermally conductive; - Reaction frame 12 'The frame 12 has a first surface 121 and a second surface 122, wherein the first surface 12! is an adhesive surface, which is the first of the reaction frame 12 The surface 12_ is on the reaction surface 111 of the conductive substrate M373489 11, thereby forming a reaction tank 3 capable of loading a sample of the biomolecule. The conductive substrate 11 is a conductor, and the conductor may be platinum, gold, silver 'copper. , nickel, chin, graphite, nano carbon or nano metal, or other conductive material. The reaction frame 12 described above may be a viscous Gene Frame® Spaeei^ABgene> wherein the reaction frame 12 is further For a "field" character configuration, it can be shaped Four reaction tank 13, for four simultaneous reaction and detection. Referring to FIG. 3A, B, and C, a cross-sectional view of another aspect of the conductive substrate 11 of the temperature-controllable microbiomolecular reaction wafer 1 of the present invention is different from the conductive substrate described above: The conductive substrate 11 is composed of a conductive coating 1111 and a substrate 1112; wherein the conductive coating 1111 can be applied to the substrate 1112 in different aspects, as shown in FIG. 3a, the conductive coating 1111 is coated on the substrate. On either surface of the plate 1112; as shown in FIG. 3B, a conductive coating 1111 is applied between the two substrates 1112; as shown in FIG. 3c, the conductive coating 1111 may cover the entire substrate 1112. The substrate 1112 includes, but is not limited to, glass, quartz or a polymer material, wherein the polymer material may be a suitable material such as acrylic (PMMA) polycarbonate (PC) or polydimethyl siloxane (PDMS). The conductive coating 1111 may be a metal oxide semiconductor, a conductor, a conductive polymer or a nanoconductor; in S, the metal oxide semiconductor may be an Indium Tin Oxide (ITO) copper zinc oxide. (in (jium zinc Oxide, IZO), the word oxide (Aluminum Zinc (^ (^^(^, gallium zinc oxide kiss out gift - condition as (5) (7) ~ gas tin oxide (7) · ^
Tin Oxide, FTO)或硒錫氧化物(Sn Selenium Oxide, SSO);其中該導電高分子 係可為聚苯胺(Polyaniline，PANI)、聚哒喀(Polypyrroie，ρργ)或聚(3,4·二氧乙 M373489 - 基塞吩）：聚苯乙浠續酸（[p〇ly(3,4-ethylenedioxythiophene)]:[poly(styrene sulfonate)]，PEDOT : PSS);其中該奈米導體係可為奈米碳材或奈米金屬。 前述之導電基板11之材料係具可透光特性或不可透光特性。 請參閱圖四所示，本創作之可溫控之微型生物分子反應晶片1平常備 用時’可將一防塵膜2黏貼於反應框體Π之第二表面122上’可遮蓋反應 槽13避免塵埃污染》其中該防塵膜2係為可撕貼之設計。 請參閱圖五所示，為本創作之可溫控之微型生物分子反應晶片1使用 • 狀態側視圖，將反應框體12黏貼於導電基板11之反應表面in上，以形成 一反應槽13。將生物分子4放置於反應槽13内，一蓋片3放置、黏貼於反 應框體12之第二表面122上，可遮蓋反應槽13，使反應槽成一密閉空間’ 除可避免不欲之污染外，更可避免溫度洩漏，藉此可讓反應槽13内之生物 分子反應於一穩定、恆定狀態下進行反應。其令該蓋片3可‘一蓋玻片、 玻璃片、塑谬片、或亦可為一導電基板、或任何適用形式之蓋片。 請同時參閱圖五及圖六所示，為本創作之使用狀態侧視圖及操作流程 # 圖；將可溫控之微型生物分子反應晶片1與電源供應器5進行連結，並將 該晶片1與溫度感測單元6進行連結’該溫度感測單元6及電源供應器5 皆連結至控制單元7;藉由電源供應器5提供一電源給晶片丨，使其產生熱 能以達到所欲之溫度，以促使生物分子4進行反應；並透過溫度感測單元6 偵測晶片1之溫度’並將偵測結果傳送至控制單元7，控制單元7會依所接 受的訊號進行判讀以得知溫度感測單元6所偵測之結果係用以判別溫度感 測單元ό產生之訊號是否與設定值相同、或高於設定值、或低於設定值。 若高於設定值，則該控制單元7傳送一訊號予電源供應器5，使電源供應器 M373489 5停止供電給晶片卜使晶片1自然降溫；若晶片！之溫度與設定值相同或 低於没定值’則雜制單元7傳送-城予電源供絲5，使電源供應器5 繼續供電給晶片1，藉此產生準破之固定或特定溫度變化。 接下來請同時參閱圖二、圖五、圖七及圖續示，與圖六不同處為本 創作之晶片1可進一步搭配一光學偵測系統8，用以即時偵測晶片丨上之生 物分子4之含量；首先將生物分子4樣品置於反應槽13内，並將蓋片3黏 貼於反應框體12上’以遮蓋賴反應槽丨3。上述電源供廳5、溫度 感測單元6及控制單元7，將反應槽13内之生物分子4進行加熱，促使其 進行反應。另外，光學檢測系統8至少包含：一光發射單元81，用以發射 一光源以照射至反應槽13内之生物分子4樣品；一光接收單元82，用以接 收光發射單元81所發射之光源並產生一訊號並傳送給分析單元83; 一分析 單元83，用以分析處理自光接收單元82傳來之訊號，藉此分析樣品中生物 分子13進行反應時之含量或狀態。 其中該光發射單元81係發射一光源以穿透透明之導電基板u上之反 應槽13内之生物分子4樣品，再由光接收單元82接收該光源； 其中該光發射單元81係發射一光源以照射至不透明之導電基板η上 之反應槽13内之生物分子4樣品，該光源再從該反應槽13内反射射出由 光接收單元82接收該光源。 前述之光發射單元81包含但不限於LED燈源、螢光燈源等適用於所 欲分析生物分子反應之光源 前述之光接收單元82包含但不限於光學濾鏡、或適用於接收前述光發 射單元81之光源接收器。Tin Oxide, FTO) or Sn Selenium Oxide (SSO); wherein the conductive polymer may be polyaniline (PANI), polypyrroie (ρργ) or poly (3, 4·2) Oxygen B M373489 - keetin): [p〇ly(3,4-ethylenedioxythiophene)]: [poly(styrene sulfonate)], PEDOT: PSS); wherein the nano-conducting system can be Nano carbon or nano metal. The material of the conductive substrate 11 described above has a light transmissive property or a non-transmissive property. Referring to FIG. 4, the temperature-controllable micro biomolecule reaction wafer 1 of the present invention can be attached to the second surface 122 of the reaction frame by a dust-proof film 2 to cover the reaction tank 13 to avoid dust. "Pollution" in which the dustproof film 2 is designed to be tearable. Referring to FIG. 5, the temperature-controlled micro biomolecule reaction wafer 1 of the present invention is bonded to the reaction surface in of the conductive substrate 11 by using a side view of the state to form a reaction tank 13. The biomolecule 4 is placed in the reaction tank 13, and a cover sheet 3 is placed and adhered to the second surface 122 of the reaction frame body 12, so that the reaction tank 13 can be covered, so that the reaction tank can be formed into a closed space, in addition to avoiding unwanted pollution. In addition, temperature leakage can be avoided, whereby the biomolecules in the reaction tank 13 can be reacted in a stable and constant state. It allows the cover sheet 3 to be a cover glass, a glass sheet, a plastic sheet, or a conductive substrate, or a cover sheet of any suitable form. Please also refer to FIG. 5 and FIG. 6 for the side view and operation flow of the creation state. The micro-biomolecular reaction wafer 1 with temperature control is connected with the power supply 5, and the wafer 1 is connected with The temperature sensing unit 6 is connected. The temperature sensing unit 6 and the power supply unit 5 are all connected to the control unit 7; a power supply is supplied to the wafer cassette by the power supply 5 to generate heat energy to reach a desired temperature. In order to cause the biomolecule 4 to react; and the temperature of the wafer 1 is detected by the temperature sensing unit 6 and the detection result is transmitted to the control unit 7, the control unit 7 performs the reading according to the received signal to know the temperature sensing. The result detected by the unit 6 is used to determine whether the signal generated by the temperature sensing unit is the same as the set value, or higher than the set value, or lower than the set value. If it is higher than the set value, the control unit 7 transmits a signal to the power supply 5, so that the power supply M373489 5 stops supplying power to the wafer to naturally cool the wafer 1; if the chip! If the temperature is the same as or lower than the set value, the hybrid unit 7 transmits the power supply to the power supply wire 5, so that the power supply 5 continues to supply power to the wafer 1, thereby generating a fixed or specific temperature change. Next, please refer to Figure 2, Figure 5, Figure 7 and the continuation of Figure 6. The wafer 1 which is different from Figure 6 can be further equipped with an optical detection system 8 for instantly detecting biomolecules on the wafer. The content of 4; first, the biomolecule 4 sample is placed in the reaction tank 13, and the cover sheet 3 is adhered to the reaction frame 12 to cover the reaction tank 3. The power supply chamber 5, the temperature sensing unit 6, and the control unit 7 heat the biomolecules 4 in the reaction tank 13 to cause the reaction. In addition, the optical detecting system 8 includes at least one light emitting unit 81 for emitting a light source to illuminate the sample of biomolecules 4 in the reaction tank 13 and a light receiving unit 82 for receiving the light source emitted by the light emitting unit 81. A signal is generated and transmitted to the analyzing unit 83. An analyzing unit 83 is configured to analyze the signal transmitted from the light receiving unit 82, thereby analyzing the content or state of the biomolecule 13 in the sample during the reaction. The light emitting unit 81 emits a light source to penetrate the sample of the biomolecule 4 in the reaction tank 13 on the transparent conductive substrate u, and then receives the light source by the light receiving unit 82; wherein the light emitting unit 81 emits a light source The sample of biomolecules 4 in the reaction vessel 13 on the opaque conductive substrate η is irradiated and reflected from the reaction vessel 13 to be received by the light receiving unit 82. The foregoing light emitting unit 81 includes, but is not limited to, an LED light source, a fluorescent light source, or the like, which is suitable for the light source to be analyzed. The light receiving unit 82 includes, but is not limited to, an optical filter, or is adapted to receive the aforementioned light emission. The light source receiver of unit 81.
TC 10 M373489 則述之分析單元83包含但不限於光譜儀(或二極體、光電倍增管等光電 檢測器）、電腦程式控制界面。 前述之生物分子4泛指一切具備生理、生化意義之化學分子、生物巨 分子及巨分子組裝而成之超分子構造，包含但不限於聚合形式之核酸(DNA 或RNA)、蛋白質、醣類及脂質等生物巨分子、巨分子組裝而成之病毒、細 菌、細胞、或非聚合物形式之有機分子與荷爾蒙、或其他適用之檢體。 - 當本創作之晶片1搭配前述光學偵測系統8時，於生物分子4中添加 • 螢光標記的探針’可與生物分子4進行雜合(hybridization)、或辨識生物分 子4並與之結合，藉此可藉由光學偵測系統8偵測反應槽13内生物分子4 進行反應時之含量或狀態。 前述光學偵測系統8包含但不限於吸收度檢測、螢光檢測、量子點檢 測、螢光共振能量轉移(fluorescence resonance energy transfer, FRET)檢測、 冷光檢測等放射光譜或吸收光譜檢測。 本創作之晶片1係可應用於PCR反應(Polymer Chain Reaction，聚合酶 _ 連鎖反應）、微型細胞培養器、微型發酵器、微型恆化器(micr〇 chem〇stat^、 微型細胞破碎器(micro cell disrupters)、微型薄膜分離器(micro membrane separators)及微型純化器(micro purifiers)等需要精確溫度控制條件之微型生 物反應器(micro bio-reactors) 〇 本創作之晶片1應用包含但不限於基因表現分析、蛋白質免疫分析、 藥物篩選'以細胞為主的分析，且該應用任擇地涉及到一種生物系統，較 佳為即時定量 PCR 裝置(real-time quantitative PCRp 本創作之晶片1之上述構造組件可被一體成型地微製造。 [S] 11 M373489 本創作係以下面的實施例予以示範闡明，但本創作不受下述實施例所 限制。 如上述之可溫控之微型生物分子反應晶片1，係可應用於各種需要精確 溫度控制之微型生物反應器上，例如：應用於DNA雜合(hybridization)反應 所須之溫控系統。以下將由一微型DNA雜合晶配合一光學檢測系統及溫 控系統用以檢測樣品中生物分子含量之實施例加以詳細介紹。 形成如前述圖一、圖二、圖五及圖八之晶片1，先將一具黏性之Gene Frame spacer (ABgene，UK)(反應框體I2)黏貼於ITO玻璃(導電基板11)之導 電表面上’並將一透明玻片（蓋片3)置放於該Gene Frame spacer上，形成可 裝載樣品之微量反應槽(如：20μ1之反應槽）。 請參照圖五、圖七之本創作使用狀態，生物分子4係以禽流感病毒 H5Nl(avian flu virus)做一示範說明’準備偵測禽流感病毒序列之特異性探 針，該特異性探針可與禽流感病毒H5N1序列互補。且該特異性探針上分 別帶有量子點 QD655(Quantum Dot 655，QD655，emission at 655nm)及 AlexaTC 10 M373489 The analysis unit 83 described above includes, but is not limited to, a spectrometer (or a photodetector such as a diode or a photomultiplier tube), and a computer program control interface. The aforementioned biomolecules 4 generally refer to all supramolecular structures assembled by chemical molecules, biological macromolecules and macromolecules having physiological and biochemical significance, including but not limited to polymeric forms of nucleic acids (DNA or RNA), proteins, sugars and Biological molecules such as lipids, macromolecules, viruses, bacteria, cells, or non-polymeric organic molecules and hormones, or other suitable samples. - When the wafer 1 of the present invention is paired with the optical detection system 8, the fluorescent probe labeled 'adds to the biomolecule 4' can be hybridized with the biomolecule 4, or the biomolecule 4 can be identified and In combination, the content or state of the biomolecule 4 in the reaction tank 13 during the reaction can be detected by the optical detection system 8. The optical detection system 8 includes, but is not limited to, absorption spectrum detection, fluorescence detection, quantum dot detection, fluorescence resonance energy transfer (FRET) detection, cold light detection, and the like, or emission spectrum detection. The wafer 1 of this creation can be applied to PCR reaction (Polymer Chain Reaction, polymerase_chain reaction), micro cell culture device, micro fermenter, micro-chemostat (micr〇chem〇stat^, micro cell breaker (micro) Micro bio-reactors requiring precise temperature control conditions such as cell disrupters, micro membrane separators, and micro purifiers. The wafer 1 application of the present invention includes, but is not limited to, genes. Performance analysis, protein immunoassay, drug screening 'cell-based analysis, and the application optionally involves a biological system, preferably a real-time quantitative PCRp. The assembly can be microfabricated in one piece. [S] 11 M373489 This creation is exemplified by the following examples, but the creation is not limited by the following examples. The temperature-controlled micro biomolecule reaction wafer as described above 1, can be applied to a variety of micro-bioreactors that require precise temperature control, for example: applied to DNA hybridization (hybridizatio n) Temperature control system required for the reaction. The following is a detailed description of an embodiment of a micro-DNA hybrid crystal combined with an optical detection system and a temperature control system for detecting the content of biomolecules in a sample. The formation is as shown in Figure 1 and Figure 2 above. , Figure 5 and Figure 8 of the wafer 1, first adhere a sticky Gene Frame spacer (ABgene, UK) (reaction frame I2) on the conductive surface of ITO glass (conductive substrate 11) 'and a transparent glass The sheet (cover sheet 3) is placed on the Gene Frame spacer to form a micro-reaction tank (for example, a reaction tank of 20 μl) capable of loading a sample. Referring to the creation state of Fig. 5 and Fig. 7, the biomolecule 4 is The avian flu virus H5N1 (avian flu virus) is used as a demonstration to describe the specific probe for the detection of the avian influenza virus sequence, which can be complementary to the avian influenza virus H5N1 sequence, and the specific probe is separately There are quantum dots QD655 (Quantum Dot 655, QD655, mission at 655nm) and Alexa
Flour 660 (emission at 690nm)之可激發(excitation — emissi〇n)出不同波長 之螢光物質，可藉由給予適當波長之光源用以激發之。 H5N1之探針(QD655 probe) : 3’端序列帶有qD655 5,-cagtggcgag(t/c)tccct-SH-QD655-3, H5N1 之探針(Alexa660 probe) : 5’端序列帶有 Aiexa66〇 -Alexa660-actggcaatcatggt(a/g)gC-3^ 將禽流感病毒麵1特探舰所冑球f、餘置於反應槽i3中 進行反應。當特異性探針與禽流感病毒進行雜合(hybridizati〇n)時給予一 激發光源(365咖間，其中帶有Q細之特異性探針便會產生榮光能量轉 12 -M373489 移(FRET)，此能量轉移之距離很短(約10-100 angstrom)，故若另一帶有Alexa Flour 660之特異性探針於此能量轉移距離内，便可接收由QD655所轉移之 能量，進而被激發出一紅光，便可由光接收單元82所接收(如：一連接光學 纖維(QP600-2-UV-VIS multimode fiber, Ocean Optics, USA)之 SMA 905 connector)。最後’藉由分析單元 83(如：以 UV-VIS spectrometer 分析 excitation spectrum ;以 16 bit CCD spectrophotometer 分析 emission spectrum)分析該螢 光之強度，用以判別該生物分子之濃度》 上述結合FRET技術及核普酸雜合(nucleic acid hybridization)之技術， 用以測試樣品中禽流感病毒H5N1之含量，生物分子4樣品分別為不同濃 度(0-50 nM)之目標序列(target sequence)，具有如下所示之序列；qD655探 針之濃度為50 nM ; Alexa Flour 660探針之濃度為ΐμΜ。 目標序列(target sequence):(為Η5Ν1之部分序列） 5^- aagatagacc agctaccatg attgccagtg ctagggaact tgccactgtt gaataaattg-3， 將上述生物分子4及反應所需之酵素、溶液置於反應槽i3中，給予〖το 玻璃一電壓以加熱之，促使反應槽13内之生物分子4進行雜合反應。結果 如圖九A-B所示，可觀察到隨著生物分子樣品濃度越高，於波長69〇聰時 所偵測之螢光強度(fluorescence intensity)越高(圖九A)，將圖九A之結果經 分析後’轉換如圖九B之柱狀圖，更明顯表示隨著生物分子樣品濃度越高， 其FRET值也越高。故，本創作之晶片丨經電熱反應後，確實可使反應槽 内之生物分子進行雜合反應，並可即時偵測其反應過程之狀態。 另，請參考圖五、圖七及圖十所示，本創作之晶片丨，可藉由控制其溫 [S] 13 M373489 度變化，以達成所欲之反應。結果如圖十所示，將晶片1經電熱反應，可 加熱生物分子4至65。0，使其變性(denature)。由於變性(denature)時，特異 性探針無法雜合(hybridization)至目標序列上，故僅會偵測到QD655之螢光 (於655nm有波峰）；當停止加熱後，反應槽溫度下降至25°C，同時，特異 性探針會與目標序列進行雜合並發生能量轉移(FRET)，故僅會偵測到Alexa Flour 660之螢光(於690 nm有波峰）。結果證實本創作之可溫控之微型生物 分子反應晶片1確實可精確溫控並進行所欲之生物分子反應。 本創作所提供之可溫控之微型生物分子反應晶片，與前述引證案及其 他習用技術相互比較時，更具有下列之優點： 本創作所提供之可溫控之微型生物分子反應晶片，其特點在於可直接 利用導電基版之電熱反應’藉由給予導電基板一電壓以產生所需之熱，以 使反應槽内之生物分子進行反應’故，大幅縮減所需之大型加熱裝置。 本創作提供之可溫控之微型生物分子反應晶片，其製備方式相當簡 便，僅需將反應框體黏附於導電基版上，便可得到本創作之晶片，故可大 幅降低製備之成本、提升其效率。 本創作提供之可溫控之微型生物分子反應晶片，其設計為可拋棄式設 計，故可避免樣品污染。 本創作提供之可溫控之微型生物分子反應晶片，除可提供精確之溫控 進行反應外，由於其簡便之設計，可搭配其他簡便適用之生物檢測裝置， 便於攜帶檢測。 本創作提供之可溫控之微型生物分子反應晶片，其中該反應框體可為 「田」字構型，故可形成四個反應槽，可供四個反應同時進行及偵測。 H373489 •上雜說縣針縣創狀-可竹_之綱，惟該實施例 並非用以關本創作之專利，凡未脫離本創作技藝精神所為之等效實 施或變更，例如•生物分子之種類、榮光標記物質等實施例，均應包含於 本案之專利範圍中。 綜上所述’本案不但在用途上確屬_，並能較㈣物品增進上述多 項功效，應已充分符合新雜及進步性之法補作專利要件爰依法提出 申請，.懇請貴局核准本件創作專獅請案，簡創作，至感德便。 【圖式簡單說明】 凊參閱以下有關本創作一較佳實施例之 圖，將可進-步瞭解本創作之技術内容及其目的功效及有其： 該實施例之附圖為： 圖-為本創作之可控之微型生物分子反應晶片之各元件結構圖； 圖二為本創作之可溫控之微型生物分子反應晶片之立體圖； 圖三Α為本創作之晶片的導電塗層塗附於基版之任一表面之示意 圖’圖三B為本創作之晶片的導電塗層塗附於兩基版中間之示意圖；圖三 C為本創作之晶片的導電塗層覆蓋整片基板之示意圖； 圖四為本創作之可溫控之微型生物分子反應晶片之平常備用狀態之 側視圖； 圖五為本創作之可溫控之微型生物分子反應晶片之使用狀態之側視 圖， 圖六為本創作之可溫控之微型生物分子反應晶片之操作流程圓； [Si 15 M373489 圖七為本創作之可溫控之微型生物分子反應晶片之另一態樣之操作 流程圖； 圖八為本創作之可溫控之微型生物分子反應晶片之另一態樣立體圖； 圖九A為檢測樣品中生物分子含量之螢光強度檢測光譜；圖九B為圖 九A之分析結果； 圖十為本創作之可溫控之微型生物分子反應晶片之可溫控性之結果 分析。 【主要元件符號說明】 1微型生物分子反應晶片 11導電基板 111反應表面 1111導電塗層 1112基板 12反應框體 121反應框體之第一表面 122反應框體之第二表面 13反應槽 2防塵臈 3蓋片 4生物分子 電源供應器 M373489 6溫度感測單元 7控制單元 8光學偵測系統 81光發射單元 82光接收單元 83分析單元Flour 660 (emission at 690nm) excites (emissi〇n) fluorescent materials of different wavelengths, which can be excited by giving a light source of appropriate wavelength. H5N1 probe (QD655 probe): 3' end sequence with qD655 5, -cagtggcgag(t/c)tccct-SH-QD655-3, H5N1 probe (Alexa660 probe): 5' end sequence with Aiexa66〇 -Alexa660-actggcaatcatggt(a/g)gC-3^ The avian influenza virus surface 1 special probe ship 胄 ball f, left in reaction tank i3 for reaction. When the specific probe is hybridized with the avian influenza virus (hybridizati〇n), an excitation light source is given (365 coffee room, in which a Q-specific probe is used to generate glory energy to 12-M373489 shift (FRET) The distance of this energy transfer is very short (about 10-100 angstrom), so if another probe with Alexa Flour 660 is within this energy transfer distance, the energy transferred by QD655 can be received and then excited. A red light can be received by the light receiving unit 82 (eg, an SMA 905 connector of a QP600-2-UV-VIS multimode fiber, Ocean Optics, USA). Finally, by the analyzing unit 83 (eg : Analysis of the excitation spectrum with a UV-VIS spectrometer; analysis of the intensity of the fluorescence by a 16 bit CCD spectrophotometer to determine the concentration of the biomolecule. The above-described combination of FRET technology and nucleic acid hybridization The technique is used to test the content of the avian influenza virus H5N1 in the sample, and the biomolecule 4 samples are respectively target sequences of different concentrations (0-50 nM), and have the sequence shown below; qD The concentration of the 655 probe is 50 nM; the concentration of the Alexa Flour 660 probe is ΐμΜ. Target sequence: (partial sequence of Η5Ν1) 5^- aagatagacc agctaccatg attgccagtg ctagggaact tgccactgtt gaataaattg-3, the above biomolecule 4 And the enzyme and solution required for the reaction are placed in the reaction tank i3, and a voltage of το glass is applied to heat, and the biomolecule 4 in the reaction tank 13 is caused to undergo a heterozygous reaction. The result is as shown in FIG. As the concentration of the biomolecule sample is higher, the fluorescence intensity detected at the wavelength of 69 〇 Cong is higher (Fig. 9A), and the result of Fig. 9A is analyzed and then converted as shown in Fig. 9B. The histogram, more obviously, shows that the higher the concentration of the biomolecule sample, the higher the FRET value. Therefore, the wafer of the present invention can be subjected to the hybrid reaction after the electrothermal reaction, and the biomolecule in the reaction tank can be heterozygous. It can instantly detect the state of the reaction process. In addition, please refer to Figure 5, Figure 7 and Figure 10. The wafer defect of this creation can be controlled by controlling its temperature [S] 13 M373489 degree. reaction. As a result, as shown in Fig. 10, the wafer 1 was subjected to an electrothermal reaction to heat the biomolecules 4 to 65.0 to denature them. Due to the denaturation, the specific probe cannot be hybridized to the target sequence, so only the fluorescence of QD655 (with peaks at 655 nm) is detected; when the heating is stopped, the temperature of the reaction tank drops to 25 At °C, at the same time, the specific probe will be mixed with the target sequence to generate energy transfer (FRET), so only the fluorescence of Alexa Flour 660 (with peaks at 690 nm) will be detected. As a result, it was confirmed that the temperature-controllable micro biomolecule reaction wafer 1 of the present invention can accurately temperature control and carry out the desired biomolecule reaction. The temperature-controlled micro biomolecule reaction chip provided by this creation has the following advantages when compared with the above cited documents and other conventional techniques: The temperature-controllable micro biomolecule reaction wafer provided by the creation, its characteristics The electrothermal reaction of the conductive substrate can be directly used to reduce the required large-scale heating device by giving a voltage to the conductive substrate to generate the required heat to react the biomolecules in the reaction cell. The temperature-controllable micro biomolecule reaction wafer provided by the present invention is prepared in a simple and convenient manner, and only needs to adhere the reaction frame to the conductive substrate to obtain the wafer of the present invention, thereby greatly reducing the cost and improvement of the preparation. Its efficiency. The temperature-controlled micro biomolecule reaction chip provided by this creation is designed to be disposable, so sample contamination can be avoided. The temperature-controlled micro biomolecule reaction chip provided by this creation can not only provide accurate temperature control for reaction, but also can be combined with other simple and suitable biological detection devices for easy carrying detection due to its simple design. The present invention provides a temperature-controlled micro biomolecule reaction wafer, wherein the reaction frame can be in the "field" configuration, so that four reaction tanks can be formed for simultaneous reaction and detection of four reactions. H373489 • The syllabus of the county of Xianxian County, which is not a patent for the creation of this article, which is equivalent to the implementation or alteration of the spirit of the creative technique, such as • the type of biomolecule Examples of glory marking substances, etc., should be included in the patent scope of this case. In summary, the case is not only in use, but also can enhance the above-mentioned multiple functions compared with (4) articles. It should be fully complied with the new and progressive laws to supplement the patent requirements and apply in accordance with the law. Please ask your bureau to approve the creation of this article. The lion is invited to the case, and the creation is simple. BRIEF DESCRIPTION OF THE DRAWINGS Referring to the following drawings relating to a preferred embodiment of the present invention, the technical content of the present invention and its purpose and effects can be further understood and the following: The drawings of the embodiment are: Figure 2 shows the structure of each component of the controllable micro biomolecule reaction wafer; Figure 2 is a perspective view of the temperature-controlled micro biomolecule reaction wafer of the creation; Figure III is a conductive coating of the wafer of the present invention. Schematic diagram of any surface of the base plate. FIG. 3B is a schematic view showing the conductive coating of the wafer of the present invention applied to the middle of the two substrates; FIG. 3C is a schematic view showing the conductive coating of the wafer of the present invention covering the entire substrate; Figure 4 is a side view of the normal standby state of the temperature-controllable micro biomolecule reaction wafer of the present invention; Figure 5 is a side view of the use state of the temperature-controllable micro biomolecule reaction wafer, Fig. 6 is a creation The operating cycle of the temperature-controllable micro biomolecule reaction wafer; [Si 15 M373489 Figure VII is a flow chart of another aspect of the temperature-controlled micro biomolecule reaction wafer of the present invention; Another perspective view of the temperature-controlled micro biomolecule reaction wafer of the present invention; FIG. 9A is a fluorescence intensity detection spectrum for detecting the biomolecule content in the sample; FIG. 9B is the analysis result of FIG. 9A; The result of temperature control of the temperature-controlled micro biomolecule reaction wafers of the creation. [Major component symbol description] 1 micro biomolecule reaction wafer 11 conductive substrate 111 reaction surface 1111 conductive coating 1112 substrate 12 reaction frame 121 reaction frame first surface 122 reaction frame second surface 13 reaction tank 2 dust 臈3 cover sheet 4 biomolecule power supply M373489 6 temperature sensing unit 7 control unit 8 optical detection system 81 light emitting unit 82 light receiving unit 83 analysis unit