201215451 - 4有化予式時’ §|揭示最能顯示發明特徵的化學式 盔 ««I、 六、發明說明： 【發明所屬之技術領域】201215451 - 4 when there is a formula § | reveals the chemical helmet that best shows the characteristics of the invention ««I, VI, invention description: [Technical field of invention]
本2技術可加熱式液珠裝置可廣泛使用於錄速溫控的檢驗設 又備專“其反應效率，確實達到即時定量檢測之目的。 【先前技術】 _ 隨著微機電技術（MEMS)及生醫技術的日新月異，近年來有 許多的技娜驗醫療照護的領域t，而祕騎n材也朝向微小 化個人化、奈米化、客制化及無線化的方向進行研發，最後還需能 達到節能省碳的綠色醫材。 微機電技術是能將生物晶片微小化的技術，因此配合生物醫學的 應用，衍生出生物微機電（Bio-MEMS)的技術領域，其目的在於開發 出微小化的可攜式之快速檢測儀器。生醫領域中，常需要溫度做為生 化的催化作用，例如PCR技術、RT-PCR、Digest等。 一種利用微機電製程技術完成微加熱器之先前技術[J〇urnal 〇f Thermal Sciences，46,580-588,2007]，其技術特徵在於利用金（Au)與 鈦（Ti)兩種金屬沈積在玻璃基材上，作為加熱的裝置，並給予一固定 之電壓可以產生相當的熱源。因此在功率（P)的換算上，可由 式表示： 式（1)中i為電流，R則為金屬的阻質。 PCR技術 3 201215451 聚合扭連鎖反應（polymerase chain reaction，PCR)是由 Kary Mullis 於1985年所發明’Mullis並因此獲得諾貝爾獎以及專利權[US 4,683，195] [US4,683,202]«PCR需要三步驟則是指⑴雙股分離 (denature):升溫到94 °C，打開DNA模版的雙股結構；（2)引子雜 交（annealing):降溫至50—65 °C，此時一對引子進入雙股DNA分 子中’分頭搜索與本身互補的鹼基序列並結合在此位置；（3)核酸合成 (extension):升溫至65 —75 °C，藉此活化聚合並結合在引子的3, 端，依著模版上的鹼基序列抓取周遭對應的dNTP連成新的核酸分子 鏈；兩個聚合酶以面對面的方向同時成長核酸分子鏈，直到雙雙走完 模版為止。然而傳統的PCR程序，因需要升降溫的控制，導致整個程 序的時間增加，以及非專一性的DNA反應’因此為能提升整體檢測的 反應時間，需要增加熱傳效率，及減少試劑體積。 傳統的PCR設備常需要利用快速的升降溫模組來對腔體内的金屬 平板加熱，並藉由熱板的溫度傳導給放置在金屬板的塑膠管，其體積 至少為15ul ’以三種不同的溫度65t，95χ：，75t進行循環。此時生 物訊號會以2"，進行訊號放大，當溫度循環一次時π值為1，當n到達 25至30次時，可以將很微量的生醫訊號放大。近年來配合螢光系統的 檢測’更可以達到即時定量（Real_time)檢測技術。 近年來，為能減少整體的反應時間，因此有兩段式的溫度進行pCR 的反應程序，其使用的方式為將Annealing/Extemi〇n，的溫度結合在一 起’而=enature的部分維持，因此整個反應的程序只需要兩個溫度點， 及可以完成PCR的反應程序。然而利用傳統的加熱系統仍需要有升降 溫的程序’因此仍會增加其反應的整體時間。 。除了在試劑的精進之外，利用微機電製程技術開發出來的微加熱 器應用於生醫的領域中，其具有快速升降溫及縮小試劑體積之優勢。 近年來由於PCR馳術已被開發，漸漸被應用至即時（Real_time) 檢測的領域巾’而目前使綠多的技術為光學式的檢測。在勞光即時 檢測系統中’其中又以SYBRGreen的檢測較常被使用而此勞 ^特性是會鎮埋於雙股的DNA中，因此#進行pCR的程序，及當 股的DNA數量越多時’其螢光強度會。—般傳騎制之設;， 201215451 其需要精準的光學元件，激發光源，如雷射，以及精密精確的光學鏡The 2 technology heatable liquid bead device can be widely used in the recording and temperature control inspection device and has the special "the reaction efficiency, and indeed achieves the purpose of real-time quantitative detection. [Prior Art] _ With micro-electromechanical technology (MEMS) and The technology of biomedical technology is changing with each passing day. In recent years, there have been many fields of medical care, and Mito is also developing in the direction of miniaturization, customization, customization and wirelessization. A green medical material that can achieve energy saving and carbon saving. Microelectromechanical technology is a technology that can miniaturize biochips. Therefore, in conjunction with biomedical applications, bio-MEMS technology has been developed with the aim of developing tiny Portable and rapid detection instruments. In the field of biomedical science, temperature is often required as a biochemical catalysis, such as PCR technology, RT-PCR, Digest, etc. A prior art that uses microelectromechanical process technology to complete microheaters [ J〇urnal 〇f Thermal Sciences, 46, 580-588, 2007], which is characterized in that gold (Au) and titanium (Ti) are deposited on a glass substrate as a heating device, and Giving a fixed voltage can produce a considerable heat source. Therefore, in terms of power (P) conversion, it can be expressed by the formula: i is the current in equation (1), and R is the resistivity of the metal. PCR technology 3 201215451 Polymerization twist-chain reaction (polymerase chain reaction, PCR) was invented by Kary Mullis in 1985 as 'Mullis and thus won the Nobel Prize and patent rights [US 4,683,195] [US 4,683,202] «PCR requires three steps to refer to (1) double-strand separation (denature): warming to 94 °C, open the double-strand structure of the DNA template; (2) primer hybridization (annealing): cooling to 50-65 °C, when a pair of primers enter the double-stranded DNA molecule's split search a base sequence complementary to itself and bound at this position; (3) nucleic acid synthesis (extension): heating up to 65-75 ° C, thereby activating polymerization and binding at the 3' end of the primer, depending on the base on the template The sequence captures the corresponding dNTPs into new nucleic acid molecular chains; the two polymerases simultaneously grow the nucleic acid molecular chains in a face-to-face direction until both pairs have finished the template. However, the traditional PCR procedure requires control of the temperature rise and fall. Whole program Increased, as well as non-specific DNA reactions', so it is necessary to increase the heat transfer efficiency and reduce the reagent volume in order to improve the reaction time of the overall detection. Traditional PCR equipment often needs to use a rapid temperature rise and fall module to the cavity. The metal plate is heated and transferred to the plastic tube placed on the metal plate by the temperature of the hot plate, and its volume is at least 15 ul 'cycle at three different temperatures of 65 t, 95 χ:, 75 t. At this time, the biosignal will be amplified by 2", and the π value will be 1 when the temperature is cycled once. When n reaches 25 to 30 times, a very small amount of biomedical signal can be amplified. In recent years, with the detection of fluorescent systems, real-time quantitative (Real_time) detection technology can be achieved. In recent years, in order to reduce the overall reaction time, there is a two-stage temperature reaction process for pCR, which is carried out by combining the temperatures of Annealing/Extemi〇n, and the part of the =enature is maintained, so The entire reaction procedure requires only two temperature points and a reaction procedure that can complete the PCR. However, the use of conventional heating systems still requires procedures for temperature rise and fall, thus still increasing the overall time of their reaction. . In addition to the refinement of reagents, micro-heaters developed using MEMS process technology are used in the field of biomedical applications, which have the advantages of rapid temperature rise and decrease of reagent volume. In recent years, since PCR has been developed, it has been gradually applied to the field of real-time detection, and currently the technology of green is optical detection. In the Luguang instant detection system, the detection of SYBRGreen is often used, and the characteristics of this work are buried in the double-stranded DNA. Therefore, the procedure of pCR is performed, and the more the amount of DNA in the stock is, the more 'The intensity of its fluorescence will be. General-style riding system;, 201215451 It requires precise optical components, excitation light sources, such as lasers, and precise and precise optical mirrors.
在1958年，Palecek發現DNA於電化學電極具有氧化還原的行為 反應。後續也開啟利用電化學進行DNA相關的檢測。一種利用電化學In 1958, Palecek discovered that DNA has a redox behavioral response at the electrochemical electrode. The subsequent use of electrochemistry for DNA-related detection is also initiated. Electrochemical utilization
方式來進行即時的PCR之先前技術[Biosensors and Bioelectronic、 2131-2136, 2009] ’其技術特徵在於利用高分子材料，如PDMS作為傳 輸微流體的流道，並使流體流經底部的三個不同加熱塊，來完成pCR 的反應程序。而為能即時偵測DNA的放大效果，將於試劑内部添加會 與DNA反應的試劑’如甲基蘭，此試劑會鑲埋至雙股螺旋的DNA中， 而當產生反應時，其電流訊號會降低。因此透過此方式來完成即時偵 測的PCR反應❶一種利用電化學檢測的裝置[us 7，135,294 ], [US7’393’644]其利用DNA的固定化技術’先使DNA固定於基板表面 之後，再給予DNA的試劑，並進行PCR的反應程序，最後量測其阻 抗（Impedance)訊號。 一般在進行DNA的量測有許多的方式，利用電極表面直接進行 DNA的sensor，但是表面若是以奈米化的表面進行檢測，由於奈米結 構的表面具有高細表面積，以及電極有直接制DNA雜力，因此 在奈米化的表面電極，將有助於£)]^八的感測。 本發明係糊微機電的製程方式，製作出提供生化程序所需之微 小加熱器，除了提供生化檢測時的熱能量之外，還於晶片表面提供檢 測的元件’做為生化反應時的即時檢測。經由晶月的設計，會驅使生 物分子往特定方向移動，然而將感·設計在特定域時，會使生 物分子在特定的區域被感測到。 在熱力學巾，自然職（FreeC_etiGn)的效應是目為溫度變化 密f變化所造成，因此當溫度錢猶（溫度不辦時）則會產生 此將導致液體内部的速度場變化。當液體内部受熱而 _ 生浮力，密度產生賊，糊液體加熱之後聯力改變導致 的八形成—流場，利用這樣的方式。也因此可以驅動液鱧 、刀子流過特疋之區域，如電極、光學檢測區。本發明之 作的微電極加熱，驅使液珠中間底部可以達到机，此時分子因受熱 201215451 進而產生浮力’驅使分子往上移動，而分子移動路徑會被液珠的外型 所侷限’因此到達液珠的頂部，會因幾何形狀而讓生物分子順勢往液· 珠周圍移動。生物分子到達底部之後又會因加熱的因素，在驅使生物 分子往上鑛，這樣的方式魏，可以完錢麵連鎖反應的献 循環。 ， 在加熱的過程中’液體的揮發將是一個重要議題，而礦物油常被 應用於PCR反應中避免揮發的一種液體，因此本發明之專利於裝置表 面製作具^驗體可轉放之設計，_ SU 8光贿合標準的黃光微 影製程技術（Photolithography)定義出兩種液體存放之區域，讓進行 PCR反應之試劑可以在加熱的過程巾經由礦物油的保護避免揮發。A prior art approach to performing real-time PCR [Biosensors and Bioelectronic, 2131-2136, 2009] 'Technical features are the use of polymeric materials such as PDMS as a flow path for transporting microfluidics and three different fluid flows through the bottom Heat the block to complete the pCR reaction procedure. In order to instantly detect the amplification effect of DNA, a reagent that reacts with DNA, such as methyl blue, is added to the reagent, and the reagent is embedded in the DNA of the double helix, and when the reaction is generated, the current signal is generated. Will decrease. Therefore, the PCR reaction for immediate detection is performed in this way, a device using electrochemical detection [us 7, 135, 294], [US 7 '393 '644] which uses DNA immobilization technology to fix the DNA on the surface of the substrate first. Then, the DNA reagent is administered, and the PCR reaction procedure is performed, and finally the impedance (Impedance) signal is measured. There are many ways to measure DNA, and the sensor of DNA is directly used on the surface of the electrode. However, if the surface is detected on a surface that is nanocrystalline, the surface of the nanostructure has a high surface area, and the electrode has direct DNA. Miscellaneous forces, therefore in the surface of the nanometerized electrode, will help with the sensing of £)]^. The invention relates to a micro-electromechanical process for producing a micro-heater required for providing a biochemical process, and in addition to providing thermal energy during biochemical detection, the component provided on the surface of the wafer is detected as a biochemical reaction. . Through the design of the crystal moon, the biomolecules are driven to move in a specific direction. However, when the sensor is designed in a specific domain, the biomolecule is sensed in a specific region. In the thermodynamics towel, the effect of FreeC_etiGn is caused by the change in temperature, so when the temperature is still (the temperature is not working), this will cause the velocity field inside the liquid to change. When the inside of the liquid is heated, the buoyancy is generated, the density is generated by the thief, and the eight-formed-flow field caused by the change of the joint force after the paste liquid is heated is utilized in this manner. It is therefore possible to drive the liquid helium and the area where the knife flows through the special area, such as the electrode and the optical detection area. The microelectrode of the invention is heated to drive the middle bottom of the liquid bead to reach the machine, and the molecule is heated by 201215451 to generate buoyancy to drive the molecules to move upward, and the molecular movement path is limited by the appearance of the liquid bead. The top of the bead will cause the biomolecule to move around the liquid and the bead due to the geometry. After the biomolecule reaches the bottom, it will drive the biomolecules to the ore due to the heating factor. In this way, Wei can complete the circulation of the chain reaction. 'Liquid volatilization will be an important issue during the heating process, and mineral oil is often used in a PCR reaction to avoid volatilization of a liquid. Therefore, the patent of the present invention is designed to be able to be transferred to the surface of the device. , _ SU 8 light bribe standard yellow photolithography technology (Photolithography) defines two areas of liquid storage, so that the reagents for PCR reactions can be protected from volatile oil in the heated process towel by mineral oil protection.
為能精痛的控制溫度到達-定的溫度，因此利用回鎖的電壓來監 控實際所產生的熱功率’根據式⑴，當製作出來的金屬阻值（R)= 能夠確定數值，因此經由所需的結果，來固定消耗功率（p)，因此時 時監控電流的變化⑴來做為生化實驗巾，溫度的精確控制。因此固 定冷卻基板之後來調整及監控加熱器的狀態來達到溫度調控的機制。 另一部份，此流場造成之擾動，會驅使待測物強制經過檢測器表面， 繼而將訊號擷取出來》透過軟體的控制，更可以有不同的溫度出現， 例如利用Plus的方式給予訊號。 為能自動化控制溫度及載入，可透過機構的設計，自動化載入晶 片及與探針卡進行精密接觸，並配合探針卡的精密固定，可以使的$ 試時的人為誤差及污染，因此晶片的訊號傳輸可以是利用探針卡的方 式、金屬導線的方式訊號傳出。 生物醫學常需要使用到精確溫度控制，微加熱器具有反應快降低 能量損耗的優勢。由於可以產生快速的溫度變化，因此^以用至 RT-PCR ( reverse transcription )’ Real-Time PCR 及 Digest 的領域中，聚 合酶連鎖反應被提出之後，已被大量應用生化、農業等相關產業。由 於傳統的PCR儀器是靠溫度來進行PCR的過程，因此除了生物樣本本 6 201215451 • 身的反應時間之外，儀器的溫度穩定度也將會增加PCR的反應過程的 時間。為能快速及微量體積的反應，目前提出一種微量體積，反應快 的裝置，以提供即時檢測的技術。 迎時檢測部分（Real-time detection ) 電化學原理 一般電化學的檢測試利用氧化還原的機制來進行樣本的檢測，本 發明經由溫度差的方式，驅使液體内的分子經由循環經過感測器之表 面’進行即時的生物樣本的監控。在感測器的部分還增加了奈米結椹 • 里丛提升接觸面積，不僅如此，可以透過表面的修飾，使具有專一性 的檢測。另外感測器表面之奈米結構，還可以使得液體分子的流動速 度發生變化’進而調控液體内部流場的速度。 登光檢測系搞. 本發明的即時檢測，一部份是利用光學式的檢測，因在反應時’ 液體會形成半圓的形狀，因此當有訊號光源產生時，液體會形成透鏡 的型態’將微弱的光源聚焦至感測器上面。 傳統的PCR常需要快速的升降溫，因此需要大功率的消耗，另外 也需要較多的生物試劑體積進行反應，另外進行榮光檢測時，螢光鏡 • 組將增加儀器系統的魔大體積，但是PCR技術成熟且被應用範圍相當 廣泛，且隨著能源概念提升，本發明將提出一種新穎的技術來針對傳 統儀器系統進行改善：-、降低儀器系統的功率消耗，達到省能源的 效益，二、減少生物試劑的體積：藉由新穎的微小化pcR晶片，來完 成微量體積的檢測，因為在許多的研究中，許多的生物樣本量少不易 取得且非常昂貴，因此儀微量的生物試舰本的使用，將是一個重 點’三、微小化系統：由於傳統的儀器系統是利用光學系統的檢測，In order to control the temperature to reach a constant temperature, the voltage of the lockback is used to monitor the actual generated thermal power. According to equation (1), when the metal resistance (R) = is determined, the value can be determined. The required result is to fix the power consumption (p), so the current change (1) is monitored as a biochemical test towel and the temperature is precisely controlled. Therefore, after fixing the substrate, the state of the heater is adjusted and monitored to achieve a temperature regulation mechanism. In another part, the disturbance caused by the flow field will force the object to be tested to pass through the surface of the detector, and then the signal will be taken out. Through the control of the software, different temperatures may occur, for example, using Plus to give signals. . In order to automatically control the temperature and loading, the design of the mechanism, the automatic loading of the wafer and the precise contact with the probe card, and the precise fixing of the probe card can make the human error and pollution of the test. The signal transmission of the chip can be signal transmission by means of a probe card or a metal wire. Biomedical science often requires precise temperature control, and micro-heaters have the advantage of fast response and reduced energy loss. Since rapid temperature changes can be generated, in the field of RT-PCR (reverse transcription)' Real-Time PCR and Digest, after the polymerase chain reaction has been proposed, biochemical, agricultural and other related industries have been widely used. Since traditional PCR instruments rely on temperature for PCR, the temperature stability of the instrument will increase the reaction time of the PCR process in addition to the reaction time of the biological sample. For rapid and micro-volume reactions, a micro-volume, fast-reacting device has been proposed to provide instant detection techniques. Real-time detection Electrochemical principle Generally, the electrochemical detection test uses a redox mechanism to detect a sample. The present invention drives a molecule in a liquid to pass through a sensor via a temperature difference. The surface 'is monitored for immediate biological samples. In the sensor part, the nano-crust is added. • The inner clump raises the contact area. Not only that, but the surface can be modified to make the detection specific. In addition, the nanostructure of the surface of the sensor can also change the flow velocity of the liquid molecules, thereby regulating the velocity of the internal flow field of the liquid. The light detection system of the present invention is partly based on the optical detection, because the liquid will form a semicircular shape during the reaction, so when a signal source is generated, the liquid will form a lens shape. Focus the weak light source on top of the sensor. Traditional PCR often requires rapid temperature rise and fall, so high power consumption is required, and more biological reagent volume is required for reaction. In addition, when the glory detection is performed, the fluoroscope group will increase the volume of the instrument system, but The PCR technology is mature and widely used, and with the improvement of the energy concept, the present invention will propose a novel technology to improve the traditional instrument system: - reduce the power consumption of the instrument system, and achieve energy-saving benefits, Reducing the volume of biological reagents: micro-volume detection is accomplished by novel miniaturized pcR wafers, because in many studies, many biological samples are less accessible and very expensive, so a small amount of biological test ship Use, will be a key 'three, miniaturized system: because the traditional instrument system is the use of optical system detection,
導致儀器系統的龐大，藉由微小化系統來達到節省材料避免資源 的效益。 '、M 是以，本案發明人鑑於上述習用即時定量pCR溫度控制裝置所衍 201215451 及該可加熱式液珠裝置於快速溫控的檢驗設備中之 PCR reverse pcr π 可以提高其反應效率’確實達到即時定量檢測之目 研發完成本件可加熱式液珠裝置。 【發明内容】 本發明為提供一可加熱式液珠裝置。 本發月之目的即在於利用該可力σ熱式液珠裝置，即時調控反麻過 程中溫度的變化。 ’ 本發明之[目的為期光學式檢測或電化學檢測來即時檢測該 可加熱式液珠裝置所完成的pCR反應。 本發明係以下面的實施例予以示範闡明，但本發明不受下述 例所限制。 【實施方式】 本發明可加熱式液珠裝置之具體詳細說明請參考圖一所示，反應裝 置100，其包含101第一液體、102第二液體、103加熱器' 1〇4保護 層、105對外導線、106基板、107冷卻裝置、108第一組感測元件、 1〇9第二組感測元件、110第三組感測元件、ηι第四組感測元件、ιΐ2 第一訊號源、113第二訊號源、114第一圓環層、115第二圓環層、116 第一循環線、117第二循環線、118第三循環線。 其中電極的製作方式可為利用微機電方式製作完成，為能使中間區 域加熱，因此加熱金屬103將採用白金的金屬，而對外導線1〇5則可 為金屬線（此例中是利用鋁作為導線）。利用電阻加熱的方式，當導線 金屬105通與一電壓或一電流時，加熱器103會因為電流或電壓之關 係而產生熱源，進而產生第一循環線116、第二循環線117、第三循環 線U8的循環軌跡。並透過感測區125之内部的第一組感測元件1〇8、 第二組感測元件109、第三組感測元件u〇、第四組感測元件lu，將 即時的反應訊號檢測出來。 201215451 以CR的技術來說’第一循環線116為pCR進行咖邱㈣及 :卿❿域’因此將感測區設計在此區，可以達到直接檢測的效果。 另需加溫度部分，可透過103產生特定溫度，進而產生第一循環線⑽、 第循裒線117、第二循環線us的軌跡並透過感測區125 β部的極 組進行即時的檢測。 圖二所顯示的溫度分佈圖是加熱器1〇3，透過112第一訊號源、ιΐ3 第二訊號源，並經由1〇5對外導線給予一 3伏特的電壓（3〇毫安培） 並穩定107冷卻裝置所得到之結果，此結果是利用紅外線熱像儀所量 測到的數據，其中在103的區域可以得到穩定的第一溫度3〇7(95<c)， 而107冷卻裝置則維持在第二溫度（6〇〇c)。 由圖二中可以得知，其升降溫的速度非常快，曲線3〇1為冷卻基板 107所提供之溫度，而302-305的曲線是給予不同電壓或電流至112及 113，並使其加熱元件1〇3產生不同的溫度點，利用這樣的方式，可以 在給予訊號之後得到直接的升溫曲線306，並且可以快速達到穩定的效 果305。 心 如圖四所示，反應裝置200，其包含ιοί第一液體、1〇2第二液體、 103加熱器、104保護層、1〇5對外導線、1〇6基板、107冷卻裝置、1〇8 第一組感測元件、109第二組感測元件、;11〇第三組感測元件、1U第 四組感測元件、112第一訊號源、113第二訊號源、114第一圓環層、 115第二圓環層、116第一循環線、117第二循環線、us第三循環線、 201第一光源、202第二光源、203第一阻撞層、204訊號接收器、205 支撐架。利用第一光源的照射在102的液體上面，生物分子會因放大 的結果，進而產生第一光源202 (榮光訊號），透過203將訊號分離出 來’而被訊號接收器204所偵測到’如圖四所示。為能控制ι〇2第二 液體内部的流速以便達到所需要的時間及反應，因此102第二液體將 添加不同比例之液體，其目的在於增加其黏滯係數，可以使得1〇2第 二液體的黏度不同’進而完成不同流速的控制。雷射的激發方式可為 側面激發螢光上方檢測，或為同軸光源激發並檢測。 實施例一、利用兩段式溫度操控來完成生化反應 由於微尺度的散熱具有升降溫快速的特性，因此本發明可加熱式液 201215451 珠裝置，其中之-項是·此特性完^ _兩段式的溫度控制 到PCR的放大效果。首先先使107冷卻裝置達到囊㈣的溫度點(幻， °C ) ’之後為能開始進行PCR的放大效果，因此利用1〇3加鱗極進行 加熱’使其達到denature的溫度（95t )，利用控斜間的方式來完成 PCR的放大機φ卜此方式可以避免升降溫過程賴祕。同時升降溫 及同時檢測，達到即時檢測的目的。 藉由103加熱電極控制，更可以達到三階段95〇c、65艽、7〇艽的溫 度控制。 實施例二、利用電化學機制來量測PCR的產物 利用電訊號來完成PCR產物的即時監測，透過微機電的製程技術來 製作出圖九所示的晶片示意圖，其包括1〇8第一組感測元件（工作電隹 極’ Work electrode)、109第二組感測元件（參考電極，reference electrode)、110第三組感測元件（計數電極，c〇unte丨.dectr〇de)、m 第四組感測元件（工作電極，Workelectrode)。此系統中，流體分子是 會因溫度場所影響進而驅使液體流動經過電極表面，進而被偵測到訊 號，量測值（可為電流值）增加或減少。因此本發明可加熱式液珠裝 置中之表面的電極，除了利用奈米結構來提升偵測靈敏度。還可使流 場可以產生變化。其設計的組合可以為對稱式或三明治式。 實施例三、利用光學的方式完成PCR產物的檢測 利用雷射激發液珠的PCR產物，並透夠過光學的檢測系統，可以來即 時檢測PCR的產物。其液體存在的狀態是以液珠的形式存在，因此當 螢光訊號產生之後，會被液體本身的聚焦特性，將螢光訊號傳送至檢 測器中，可為CCD、PMT，增強訊號的效果，如圖五所示利用側面光 源激發，在液體上方檢測之結果圖，從圖中可以看出，在兩分鐘之後， 其DNA已開始有反應，並在1〇分鐘之内完成反應的機制。 實施例四、微奈米化表面電極 本發明可加熱式液珠裝置中所使用的電極，也可採用微奈米化表面 電極(圖七）’圖六及圖七之感測區125内增加了第一組感測成長物 120、第二組感測成長物121、第三組感測成長物122、第四組感測成 長物123，該感測成長物不僅可以將116驅動過來的檢測物被感測區 201215451 内的電極檢測出來，還可以使流場的移動形成丨24的移動路徑，可以 同時改變流場(圖六）’及增加靈敏度。其中第一組感測成長物12〇、第 一組感測成長物121、第二組感測成長物122、第四組感測成長物123 更可以經過化學性、物理性的修飾來達到更專一性的反應。 為能確定感測區125的特性，利用電化學原理進行其特性之量測。 感測區125之内部的第一組感測元件1〇8、第三組感測元件11〇給予正 負電壓的切換，所測得之結果圖如圖八所示。 實施例五、RNA reverse transcription PCR€RT_PC^As a result, the instrument system is huge, and the miniaturization system can save the material and avoid the benefits of resources. ', M is, the inventor of the present invention in view of the above-mentioned instant quantitative pCR temperature control device derivative 201215451 and the heatable liquid bead device in the rapid temperature control inspection equipment PCR reverse pcr π can improve its reaction efficiency 'actually The purpose of real-time quantitative testing is to complete the heating-type bead device. SUMMARY OF THE INVENTION The present invention provides a heatable liquid droplet device. The purpose of this month is to use the force σ thermal liquid bead device to instantly control the temperature change during the anti-anatomy process. The present invention [is aimed at optical detection or electrochemical detection to immediately detect the pCR reaction performed by the heatable liquid droplet device. The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. [Embodiment] For a detailed detailed description of the heatable liquid bead apparatus of the present invention, as shown in FIG. 1, the reaction apparatus 100 includes 101 first liquid, 102 second liquid, 103 heater '1〇4 protective layer, 105 External conductor, 106 substrate, 107 cooling device, 108 first group of sensing elements, 1〇9 second group of sensing elements, 110 third group of sensing elements, ηι fourth group of sensing elements, ιΐ2 first signal source, 113 second signal source, 114 first annular layer, 115 second annular layer, 116 first circulating line, 117 second circulating line, 118 third circulating line. The electrode can be fabricated by using micro-electromechanical method. In order to heat the middle region, the heating metal 103 will be made of platinum metal, and the outer conductor 1〇5 can be metal wire (in this case, aluminum is used). wire). By means of resistance heating, when the wire metal 105 is connected to a voltage or a current, the heater 103 generates a heat source due to the relationship of current or voltage, thereby generating a first circulation line 116, a second circulation line 117, and a third cycle. The loop trajectory of line U8. And detecting the instantaneous reaction signal through the first group of sensing elements 1〇8, the second group of sensing elements 109, the third group of sensing elements u〇, and the fourth group of sensing elements lu inside the sensing area 125 come out. 201215451 In terms of the technology of CR, the first loop line 116 is the pCR for the Qiqiu (4) and the Qingyu domain. Therefore, the sensing area is designed in this area, and the direct detection effect can be achieved. In addition, a temperature portion is required to generate a specific temperature through 103, thereby generating a trajectory of the first circulation line (10), the circumscribing line 117, and the second circulation line us, and performing instantaneous detection through the pole group of the sensing portion 125 β. The temperature profile shown in Figure 2 is the heater 1〇3, through the first signal source 112, the ιΐ3 second signal source, and a voltage of 3 volts (3 mA) through the 1〇5 external conductor and stabilized 107 As a result of the cooling device, the result is data measured by an infrared camera, wherein a stable first temperature 3〇7 (95<c) is obtained in the region of 103, and the 107 cooling device is maintained at Second temperature (6〇〇c). As can be seen from Fig. 2, the temperature of the temperature rise and fall is very fast, the curve 3〇1 is the temperature provided by the cooling substrate 107, and the curve of 302-305 is to give different voltages or currents to 112 and 113 and heat it. Element 1 产生 3 produces different temperature points, in such a way that a direct temperature rise curve 306 can be obtained after the signal is given, and a stable effect 305 can be quickly achieved. As shown in FIG. 4, the reaction device 200 includes a first liquid, a second liquid, a 103 heater, a 104 protective layer, a 1 〇 5 external lead, a 1 〇 6 substrate, a 107 cooling device, and 1 〇. 8 first set of sensing elements, 109 second set of sensing elements, 11 〇 third set of sensing elements, 1U fourth set of sensing elements, 112 first signal source, 113 second signal source, 114 first circle Ring layer, 115 second ring layer, 116 first cycle line, 117 second cycle line, us third cycle line, 201 first light source, 202 second light source, 203 first blocking layer, 204 signal receiver, 205 support frame. By using the first light source to illuminate the liquid of 102, the biomolecule will generate a first light source 202 (Glory signal) due to the amplification result, and the signal will be separated by 203 and detected by the signal receiver 204. Figure 4 shows. In order to control the flow rate inside the second liquid of ι〇2 in order to achieve the required time and reaction, 102 the second liquid will add different proportions of liquid, the purpose of which is to increase the viscosity coefficient thereof, which can make 1〇2 second liquid The viscosity is different' and then the control of different flow rates is completed. The excitation of the laser can be detected by the side excitation fluorescence or by the coaxial source. Embodiment 1 The two-stage temperature control is used to complete the biochemical reaction. Since the micro-scale heat dissipation has the characteristics of rapid temperature rise and fall, the present invention can heat the liquid 201215451 bead device, wherein the item is · this characteristic is completed ^ _ two segments The temperature of the formula is controlled to the amplification effect of the PCR. First, let the 107 cooling device reach the temperature point of the capsule (4) (magic, °C)', and then start the PCR amplification effect, so use the 1〇3 scaled electrode to heat it to reach the denature temperature (95t). The method of controlling the tilting is used to complete the amplification machine φ of the PCR method, which can avoid the fading process of the temperature rise and fall. At the same time, the temperature is raised and lowered and detected at the same time to achieve the purpose of instant detection. With 103 heating electrode control, temperature control of three stages of 95〇c, 65艽, and 7〇艽 can be achieved. Embodiment 2: Using electrochemical mechanism to measure PCR products The electrical signals are used to complete the real-time monitoring of the PCR products, and the schematic diagram of the wafer shown in FIG. 9 is prepared through the microelectromechanical process technology, which includes the first group of 1〇8. Sensing element (Working electrode), 109 second sensing element (reference electrode), 110 third group of sensing elements (counting electrode, c〇unte丨.dectr〇de), m The fourth set of sensing elements (Work Electrode, Workelectrode). In this system, the fluid molecules are driven by the influence of the temperature field to drive the liquid to flow through the surface of the electrode, and then the signal is detected, and the measured value (which can be the current value) is increased or decreased. Therefore, the electrode on the surface of the heatable bead apparatus of the present invention is used in addition to the nanostructure to enhance the detection sensitivity. It also allows the flow field to change. The combination of designs can be either symmetrical or sandwich. Example 3: The detection of the PCR product by optical means The PCR product of the laser excitation bead and the optical detection system can be used to detect the PCR product immediately. The state in which the liquid exists is in the form of a liquid bead. Therefore, after the fluorescent signal is generated, the fluorescent signal is transmitted to the detector by the focusing characteristic of the liquid itself, which can be a CCD, a PMT, and an effect of enhancing the signal. As shown in Figure 5, the results of the detection above the liquid using the side light source excitation, it can be seen from the figure that after two minutes, the DNA has begun to react and complete the reaction mechanism within 1 minute. Embodiment 4: Micro-nanoated surface electrode The electrode used in the heatable liquid bead device of the present invention may also be increased by using a micro-nano surface electrode (Fig. 7) in the sensing region 125 of FIG. 6 and FIG. The first group of sensing growth objects 120, the second group of sensing growth objects 121, the third group of sensing growth objects 122, and the fourth group of sensing growth objects 123, the sensing growth objects can not only drive the detection of 116 The object is detected by the electrode in the sensing area 201215451, and the movement of the flow field can also form a moving path of the crucible 24, which can simultaneously change the flow field (Fig. 6)' and increase the sensitivity. The first group of sensing growth objects 12〇, the first group of sensing growth objects 121, the second group of sensing growth objects 122, and the fourth group of sensing growth objects 123 can be more chemically and physically modified to achieve more Specific response. In order to be able to determine the characteristics of the sensing region 125, the measurement of its characteristics is performed using an electrochemical principle. The first group of sensing elements 1〇8 and the third group of sensing elements 11〇 inside the sensing region 125 are switched between positive and negative voltages, and the measured result is shown in FIG. Example 5, RNA reverse transcription PCR € RT_PC^
反轉錄PCR之後_鮮rt_PCR，本拥可加赋液珠裝置也可 應用於在RT-PCR儀器令的溫控裝置，快速調控儀器中溫度的變化，以 提高RT-PCR反應效率。 實施例六、酵素分解作用(Enzyme Digeation)之應用 本發明可加熱式液珠裝置也可應用於生化反應中酵素分解作用 (Enzyme Digeation)所需要的溫度調控儀器，提高升降溫速度減少熱 耗損，以提高酵素的分解能力。 ”' 上列詳細說_針對本發明之可行實施例之具體說明，惟該實施 例並非用錄制本發明之柄翻’凡未雌本發明技藝精神所為之After reverse transcription PCR, fresh rt_PCR, this can be added to the temperature control device of the RT-PCR instrument to quickly adjust the temperature change in the instrument to improve the efficiency of the RT-PCR reaction. Example 6 Application of Enzyme Digeation The heatable liquid bead device of the present invention can also be applied to a temperature regulating instrument required for Enzyme Digeation in a biochemical reaction to increase the temperature rise and fall and reduce heat loss. To improve the ability of the enzyme to break down. The above is a detailed description of a possible embodiment of the present invention, but the embodiment is not a recording of the handle of the present invention.
施或變更’例如··該可加減液珠裝置應具有快速溫度控 制需求之設備，如㈣^量PCR、腿寒咖如pcR 之應料變化之等效性實施例，均聽含於本案之專利範 增 進上本Ϊ不但在方法型態上確屬創新，並能較習用物品 ^項功效，紅充分符合购性錢步性之法定發 ==㈣，_ #獅撕咖咖，以勵發 【圖式簡單說明】 圖一 圖二 、可加熱式液珠裝置結合電化學檢測裝 、加熱電極的溫度分佈區間曲線圖 置之即時檢測裝置示意圖 201215451 圖三、溫度曲線圖 圖四、可加熱式液珠裝置結合光學檢測裝置之即時檢測裝置示意圖 圖五、即時量測的訊號擷取曲線圖 圖六、奈米結構造成流場改變之示意圖 圖七、檢測電極表面奈米碳管示意圖 圖八、感測區測試結果曲線圖 圖九、晶片不意圖 【主要元件符號說明】 100聚合酶連鎖反應裝置 200聚合酶連鎖反應裝置 101第一液體 201第一光源 102第二液體 202第二光源 103加熱器 203第一阻擋層 104保護層 204訊號接收器 105對外導線 205支撐架 106基板 206螢光產生區 107冷卻裝置 301冷卻裝置107所提供之溫度 108第一組感測元件 302-305不同電壓或電流之溫度曲線 109第二組感測元件 306升溫速率 110第三組感測元件 307第一溫度 111第四組感測元件 308第二溫度 112第一訊號源 113第二訊號源 114第一圓環層 115第二圓環層 116第一循環線 117第二循環線 118第三循環線 120第一感測成長物 121第二感測成長物Application or change 'for example · The device capable of adding and subtracting liquid droplets should have fast temperature control requirements, such as (4) PCR, leg cold coffee, such as pcR, the equivalent of the embodiment of the changes, are included in the case The patent model is not only innovative in terms of method type, but also more effective than the use of items. Red is fully in line with the legality of purchasing money. == (4), _ #狮撕咖咖, to encourage [Simple diagram of the diagram] Figure 1 Figure 2. Schematic diagram of the instant detection device of the heatable liquid bead device combined with the electrochemical detection device and the temperature distribution interval of the heating electrode. 201215451 Figure 3. Temperature curve diagram 4. Heatable Schematic diagram of the instant detection device of the liquid bead device combined with the optical detection device. Figure 5. Schematic diagram of the instantaneous measurement signal. Figure 6. Schematic diagram of the flow field change caused by the nanostructure. Figure 7. Schematic diagram of the carbon nanotube on the surface of the detection electrode. Sensing area test result graph FIG. 9 , wafer not intended [main element symbol description] 100 polymerase chain reaction device 200 polymerase chain reaction device 101 first liquid 201 first light source 10 2 second liquid 202 second light source 103 heater 203 first barrier layer 104 protective layer 204 signal receiver 105 outer conductor 205 support frame 106 substrate 206 fluorescent generating region 107 cooling device 301 cooling device 107 provided by temperature 108 first Group sensing elements 302-305 different voltage or current temperature curves 109 second group of sensing elements 306 heating rate 110 third group of sensing elements 307 first temperature 111 fourth group of sensing elements 308 second temperature 112 first signal Source 113 second signal source 114 first annular layer 115 second annular layer 116 first circulation line 117 second circulation line 118 third circulation line 120 first sensing growth object 121 second sensing growth object
12 201215451 122第三感測成長物 123第四感測成長物 124第四循環線 125反應感測區12 201215451 122 Third Sensing Growth Object 123 Fourth Sensing Growth Product 124 Fourth Cycle Line 125 Reaction Sensing Area