TWI386253B - Heater-type tilting device - Google Patents
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Description
本發明技術可加熱式液珠裝置可廣泛使用於需快速溫控的檢驗設備中,例如即時定量PCR設備、RNA reverse transcription PCR(RT-PCR)設備等,提高其反應效率,確實達到即時定量檢測之目的。 The heating type liquid bead device of the invention can be widely used in an inspection device requiring rapid temperature control, such as an instant quantitative PCR device, an RNA reverse transcription PCR (RT-PCR) device, etc., to improve the reaction efficiency, and indeed achieve instant quantitative detection. The purpose.
隨著微機電技術(MEMS)及生醫技術的日新月異,近年來有許多的技術應用於醫療照護的領域中,而醫療照護器材也朝向微小化、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。 With the rapid development of micro-electromechanical technology (MEMS) and biomedical technology, many technologies have been used in the field of medical care in recent years, and medical care equipment is also becoming miniaturized, personalized, nano-customized, customized and wireless. In the direction of research and development, it is necessary to achieve green energy-saving materials that can save energy and save carbon.
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。 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 miniaturized portable rapid detection instruments. In the field of biomedicine, temperature is often required as a catalytic effect of biochemistry, such as PCR technology, RT-PCR, Digest, and the like.
一種利用微機電製程技術完成微加熱器之先前技術[Journal of 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(1)式表示:P=i 2 R………(1) A prior art technique for accomplishing microheaters using microelectromechanical process technology [Journal of Thermal Sciences, 46, 580-588, 2007], which is characterized in that gold (Au) and titanium (Ti) are deposited on a glass substrate. As a means of heating, and giving a fixed voltage can produce a comparable heat source. Therefore, in terms of power (P) conversion, it can be expressed by equation (1): P = i 2 R ... (1)
式(1)中i為電流,R則為金屬的阻質。 In the formula (1), i is a current, and R is a metal barrier.
PCR技術 PCR technology
聚合酶連鎖反應(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(1)雙股分離(denature):升溫到94℃,打開DNA模版的雙股結構;(2)引子雜交(annealing):降溫至50-65℃,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℃,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。 The polymerase chain reaction (PCR) was invented by Kary Mullis in 1985, and Mullis received the Nobel Prize and patent rights [US 4,683,195] [US 4,683,202]. PCR requires three steps to refer to (1) double stranding: heating to 94 ° C, open the double-strand structure of the DNA template; (2) primer hybridization (induction): cooling to 50-65 ° C, at this time a pair The primer enters the double-stranded DNA molecule, and searches for the base sequence complementary to itself and binds to this position; (3) nucleic acid synthesis (extension): the temperature is raised to 65-75 ° C, thereby activating the polymerization and binding to the 3 end of the primer, and grabbing the corresponding dNTPs according to the base sequence on the template to form a new nucleic acid molecular chain; two polymerases The nucleic acid molecular chain is simultaneously grown in a face-to-face direction until both models are finished. However, the traditional PCR procedure, due to the need to control the temperature rise and fall, leads to an increase in the time of the entire procedure, as well as a non-specific DNA reaction, so in order to improve the reaction time of the overall detection, it is necessary to increase the heat transfer efficiency and reduce the reagent volume.
傳統的PCR設備常需要利用快速的升降溫模組來對腔體內的金屬平板加熱,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65℃,95℃,75℃進行循環。此時生物訊號會以2n,進行訊號放大,當溫度循環一次時n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。 Conventional PCR equipment often needs to use a rapid temperature rise and fall module to heat the metal plate in the cavity, and the temperature of the hot plate is transferred to the plastic tube placed on the metal plate, which has a volume of at least 15 ul, at three different temperatures. The cycle was carried out at 65 ° C, 95 ° C, and 75 ° C. At this time, the biological signal will be amplified by 2 n , and the n value is 1 when the temperature is cycled once. When n reaches 25 to 30 times, a very small amount of the medical signal can be amplified. In recent years, with the detection of fluorescent systems, real-time detection technology can be achieved.
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/Extension,的溫度結合在一起,而Denature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。 In recent years, in order to reduce the overall reaction time, there is a two-stage temperature PCR reaction procedure, which is carried out by combining the temperatures of Annealing/Extension, while the part of Denature is maintained, so the entire reaction The program 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 the reaction.
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。 In addition to the refinement of reagents, micro-heaters developed using microelectromechanical process technology are used in the field of biomedical applications, which have the advantages of rapid temperature rise and decrease of reagent volume.
近年來由於PCR的技術已被開發,漸漸被應用至即時(Real-time)檢測的領域中,而目前使用較多的技術為光學式的檢測。在螢光即時檢測系統中,其中又以SYBR Green的檢測較常被使用,而此螢光染劑的特性是會鑲埋於雙股的DNA中,因此當進行PCR的程序,及當雙股的DNA數量越多時,其螢光強度會越強。一般傳統所使用之設備,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組。 In recent years, since the technology of PCR has been developed, it has gradually been applied to the field of real-time detection, and currently more used techniques are optical detection. In the fluorescent real-time detection system, the detection by SYBR Green is often used, and the characteristics of the fluorescent dye are embedded in the double-stranded DNA, so when the PCR process is performed, and when the double-strand The greater the amount of DNA, the stronger the fluorescence intensity. Equipment traditionally used requires precise optical components, excitation sources such as lasers, and precision optics.
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。一種利用電化學方式來進行即時的PCR之先前技術[Biosensors and Bioelectronics,24,2131-2136,2009],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,將於試劑內部添加會與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。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。 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. A prior art technique for performing real-time PCR using electrochemical methods [Biosensors and Bioelectronics, 24, 2131-2136, 2009], which is characterized by the use of a polymer material such as PDMS as a transmission The flow path of the microfluid is passed and the fluid is passed through three different heating blocks at the bottom 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 instant detection is completed in this way. A device utilizing electrochemical detection [US 7, 135, 294], [US 7,393,644] which uses DNA immobilization technology to immobilize DNA on a substrate surface, then to give DNA reagents, and to perform PCR reaction procedures, and finally to measure Its Impedance signal.
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接觸表面積,以及電極有直接量測DNA的能力,因此在奈米化的表面電極,將有助於DNA的感測。 There are many ways to perform DNA measurement, and the sensor of the DNA is directly used on the surface of the electrode, but if the surface is detected on a surface that is nanocrystalline, the surface of the nanostructure has a high contact surface area, and the electrode has direct measurement. The ability of DNA, therefore, on the surface electrode of nanocrystallization, will contribute to the sensing of DNA.
本發明係利用微機電的製程方式,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。經由晶片的設計,會驅使生物分子往特定方向移動,然而將感測器設計在特定的區域時,會使生物分子在特定的區域被感測到。 The invention utilizes the micro-electromechanical process to produce a tiny heater required for providing a biochemical process, in addition to providing thermal energy during biochemical detection, and also providing a detecting component on the surface of the wafer as an instant detection during biochemical reaction. . Via the design of the wafer, the biomolecule is driven to move in a specific direction. However, when the sensor is designed in a specific area, the biomolecule is sensed in a specific area.
在熱力學中,自然對流(Free Convection)的效應是因為溫度變化及密度變化所造成,因此當溫度有變化時(溫度不相等時)則會產生不穩定循環,因此將導致液體內部的速度場變化。當液體內部受熱而使微粒產生浮力,密度產生變化,利用液體加熱之後因浮力改變導致液體內部會自行形成一流場,利用這樣的方式。也因此可以驅動液體的分子流過特定之區域,如電極、光學檢測區。本發明之裝置利用製作的微電極加熱,驅使液珠中間底部可以達到95℃,此時分子因受熱進而產生浮力,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移動,利用這樣的方式循環,可以完成聚合酶連鎖反應的熱循環。 In thermodynamics, the effect of Free Convection is caused by temperature changes and density changes. Therefore, when the temperature changes (when the temperature is not equal), an unstable cycle will occur, which will result in a change in the velocity field inside the liquid. . When the inside of the liquid is heated to cause the particles to buoyant, the density changes, and after the liquid is heated, the inside of the liquid will form a first-class field due to the change of buoyancy, in such a manner. It is thus also possible to drive the molecules of the liquid through a specific area, such as an electrode, an optical detection zone. The device of the invention uses the prepared microelectrode to heat, and drives the bottom of the liquid bead to reach 95 ° C. At this time, the molecules generate buoyancy due to heat, which drives the molecules to move upward, and the molecular movement path is limited by the appearance of the liquid bead. Therefore, reaching the top of the bead will cause the biomolecule to move around the bead due to the geometry. After the biomolecule reaches the bottom, it will drive the biomolecule upwards due to the heating factor. By circulating in this way, the thermal cycle of the polymerase chain reaction can be completed.
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兩種液體可以存放之設計,利用SU-8光阻配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中經由礦物油的保護避免揮發。 During the heating process, the evaporation of liquid will be an important issue, and mineral oil is often A liquid used in a PCR reaction to avoid volatilization. Therefore, the patent of the present invention is designed to have two liquids that can be stored on the surface of the device, and two liquids are defined by using SU-8 photoresist in combination with standard yellow light photolithography (Photolithography). In the storage area, the reagents for the PCR reaction can be volatilized by the protection of mineral oil during the heating process.
為能精確的控制溫度到達一定的溫度,因此利用回饋的電壓來監控實際所產生的熱功率,根據式( ),當製作出來的金屬阻值(R)是能夠確定數值,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(i)來做為生化實驗中,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。 In order to accurately control the temperature to reach a certain temperature, the voltage of the feedback is used to monitor the actual generated thermal power. According to the formula ( ), when the metal resistance (R) is determined, the value can be determined. As a result, the power consumption (P) is fixed, so that the current change (i) is constantly monitored as a precise control of the temperature in the biochemical experiment. Therefore, after the substrate is fixedly cooled, the state of the heater is adjusted and monitored to achieve a temperature regulation mechanism. In another part, the disturbance caused by this 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, it is possible to have different temperatures, for example, using Plus to give signals.
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,可以使的測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。 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 during the test, so the wafer The signal transmission can be transmitted by means of a probe card or a metal wire.
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,因此可以用至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。 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, it can be used in the fields of RT-PCR (reverse transcription), Real-Time PCR and Digest. After the polymerase chain reaction has been proposed, it has been widely used in biochemical, agricultural and other related industries. Since the conventional PCR instrument performs the PCR process by temperature, 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 itself. In order to respond quickly and in a small volume, a device with a small volume and fast response has been proposed to provide an instant detection technique.
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,驅使液體內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構用以提升接觸面積,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,進而調控液體內部流場的速度。 Generally, the electrochemical detection test utilizes the mechanism of redox to perform sample detection. The present invention drives the molecules in the liquid to circulate through the surface of the sensor via a temperature difference to perform real-time monitoring of the biological sample. In the sensor part, a nano structure is added to increase the contact area, and not only the surface modification can be made to have a specific detection. 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.
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。 In the instant detection of the present invention, part of the detection is by optical detection, because the liquid will form a semicircular shape during the reaction, so when a signal source is generated, the liquid forms a lens shape, and the weak light source is focused to Above the sensor.
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,另外進行螢光檢測時,螢光鏡組將增加儀器系統的龐大體積,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:一、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,因此利用微量的生物試劑樣本的使用,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,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浪費的效益。 Traditional PCR often requires rapid temperature rise and fall, so it requires high power consumption. In addition, more biological reagent volume is required for reaction. In addition, when fluorescent detection is performed, the fluoroscope group will increase the bulk of the instrument system, but PCR The 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: First, reduce the power consumption of the instrument system, achieve energy-saving benefits, and second, reduce 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 the use of micro-reagent samples is used. It will be a key point. Third, the miniaturization system: Since the traditional instrument system utilizes the detection of the optical system, the instrument system is huge, and the miniaturization system can save the material and avoid the waste of resources.
是以,本案發明人鑑於上述習用即時定量PCR溫度控制裝置所衍生的各項缺點,以及該可加熱式液珠裝置於快速溫控的檢驗設備中之廣泛應用,例如即時定量PCR設備、RNA reverse transcription PCR(RT-PCR)設備等,可以提高其反應效率,確實達到即時定量檢測之目的,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可加熱式液珠裝置。 Therefore, the inventors of the present invention have various disadvantages derived from the above-mentioned instant quantitative PCR temperature control device, and the wide application of the heatable liquid bead device in a rapid temperature control inspection device, such as an instant quantitative PCR device, RNA reverse Transcription PCR (RT-PCR) equipment, etc., can improve the reaction efficiency, and indeed achieve the purpose of real-time quantitative detection. It is improved and innovated by 亟思, and after years of painstaking research, finally successfully developed this piece of heatable liquid bead device. .
本發明為提供一可加熱式液珠裝置。 The present invention provides a heatable bead apparatus.
本發明之目的即在於利用該可加熱式液珠裝置,即時調控反應過程中溫度的變化。 The object of the present invention is to use the heatable liquid bead device to instantly control the temperature change during the reaction.
本發明之另一目的為利用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。 Another object of the present invention is to use an optical detection or electrochemical detection to instantly detect a PCR reaction performed by the heatable liquid droplet device.
本發明係以下面的實施例予以示範闡明,但本發明不受下述實施例所限制。 The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.
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、104保護層、105對外導線、106基板、107冷卻裝置、108第一組感測元件、109第二組感測元件、110第三組感測元件、111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、118第三循環線。 For a detailed description of the heatable bead device of the present invention, please refer to FIG. 1 , a reaction device 100 including 101 first liquid, 102 second liquid, 103 heater, 104 protective layer, 105 external lead, 106 substrate, 107 cooling device, 108 first group of sensing elements, 109 second group of sensing elements, 110 third group of sensing elements, 111 fourth group of sensing elements, 112 first signal source, 113 second signal source, 114 A ring layer, 115 second ring layer, 116 first cycle line, 117 second cycle line, 118 third cycle line.
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105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118的循環軌跡。並透過感測區125之內部的第一組感測元件108、第二組感測元件109、第三組感測元件110、第四組感測元件111,將即時的反應訊號檢測出來。 The electrode can be fabricated by using a micro-electromechanical method. In order to heat the intermediate portion, the heating metal 103 will be made of platinum metal, and the outer conductor 105 can be a metal wire (in this case, aluminum is used as the 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 circular trajectory of line 118. And the instant response signal is detected through the first group of sensing elements 108, the second group of sensing elements 109, the third group of sensing elements 110, and the fourth group of sensing elements 111 inside the sensing area 125.
以PCR的技術來說,第一循環線116為PCR進行annealing及extension區域,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116、第二循環線117、第三循環線118的軌跡並透過感測區125內部的電極組進行即時的檢測。 In the technique of PCR, the first loop line 116 performs the annealing and extension regions for the PCR, so that the sensing region is designed in this region, and the direct detection effect can be achieved. In addition, a temperature portion is required to generate a specific temperature through the 103, thereby generating a trajectory of the first circulation line 116, the second circulation line 117, and the third circulation line 118, and performing an instantaneous detection through the electrode group inside the sensing region 125.
圖二所顯示的溫度分佈圖是加熱器103,透過112第一訊號源、113 第二訊號源,並經由105對外導線給予一3伏特的電壓(30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307(95℃),而107冷卻裝置則維持在第二溫度(60℃)。 The temperature profile shown in Figure 2 is the heater 103, through the first signal source 112, 113 The second signal source, and a voltage of 3 volts (30 mA) is applied to the external conductor via 105, and the result obtained by the 107 cooling device is stabilized. The result is data measured by an infrared thermal imager, wherein at 103 The zone can be stabilized at a first temperature of 307 (95 ° C) while the 107 cooling device is maintained at a second temperature (60 ° C).
由圖三中可以得知,其升降溫的速度非常快,曲線301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103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。 As can be seen from FIG. 3, the temperature of the temperature rise and fall is very fast, the curve 301 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 to make the heating element 103 Different temperature points are generated, 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.
如圖四所示,反應裝置200,其包含101第一液體、102第二液體、103加熱器、104保護層、105對外導線、106基板、107冷卻裝置、108第一組感測元件、109第二組感測元件、110第三組感測元件、111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、118第三循環線、201第一光源、202第二光源、203第一阻擋層、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(螢光訊號),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102第二液體內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102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。 As shown in FIG. 4, the reaction device 200 includes 101 first liquid, 102 second liquid, 103 heater, 104 protective layer, 105 outer lead, 106 substrate, 107 cooling device, 108 first set of sensing elements, 109 a second set of sensing elements, 110 a third set of sensing elements, 111 a fourth set of sensing elements, 112 a first signal source, 113 a second signal source, 114 a first ring layer, 115 a second ring layer, 116 First cycle line, 117 second cycle line, 118 third cycle line, 201 first light source, 202 second light source, 203 first barrier layer, 204 signal receiver, 205 support frame. When the first light source is irradiated on the liquid of 102, the biomolecule generates a first light source 202 (fluorescent signal) due to the amplification result, and the signal is separated by the 203, and is detected by the signal receiver 204. As shown in Figure 4. In order to control the flow rate inside the second liquid 102 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, so that the viscosity of the second liquid of 102 can be different. In turn, the control of different flow rates is completed. The excitation of the laser can be detected above the side-excited fluorescence or excited and detected by the coaxial source.
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珠裝置,其中之一項是利用此特性完成。利用兩段式的溫度控制來達到PCR的放大效果。首先先使107冷卻裝置達到annealing的溫度點(65℃),之後為能開始進行PCR的放大效果,因此利用103加熱電極進行加熱,使其達到denature的溫度(95℃),利用控制時間的方式來完成PCR的放大機制。此方式可以避免升降溫過程的熱損耗。同時升降溫及同時檢測,達到即時檢測的目的。 Since the micro-scale heat dissipation has the characteristics of rapid temperature rise and fall, the heatable liquid droplet device of the present invention, one of which is completed by utilizing this characteristic. A two-stage temperature control is used to achieve amplification of the PCR. First, the 107 cooling device is first brought to the annealing temperature point (65 ° C), and then the amplification effect of the PCR can be started. Therefore, the heating electrode of 103 is used to heat the temperature to reach the denature temperature (95 ° C), and the time is controlled. To complete the amplification mechanism of PCR. This method can avoid heat loss during the temperature rise and fall process. At the same time, the temperature is raised and lowered and detected at the same time to achieve the purpose of instant detection.
藉由103加熱電極控制,更可以達到三階段95℃、65℃、70℃的溫度控制。 With 103 heating electrode control, temperature control of three stages of 95 ° C, 65 ° C, and 70 ° C can be achieved.
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108第一組感測元件(工作電極,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,Counter electrode)、111第四組感測元件(工作電極,Work electrode)。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。 Using the electrical signal to complete the real-time monitoring of the PCR product, the schematic diagram of the wafer shown in FIG. 9 is fabricated through the microelectromechanical process technology, which includes 108 first set of sensing elements (Work electrodes) and 109 second senses. Measuring element (reference electrode), 110 third group of sensing elements (counter electrode), 111 fourth group of sensing elements (Working Electrode). In this system, the fluid molecules will drive the liquid to flow through the surface of the electrode due to the influence of the temperature field, 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 device of the present invention uses a nanostructure to enhance the detection sensitivity. It is also possible to make the flow field changeable. The combination of designs can be either symmetrical or sandwich.
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10分鐘之內完成反應的機制。 By using a laser to excite the PCR product of the bead and through the optical detection system, the PCR product can be detected 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, as can be seen from the figure, after two minutes, the DNA has begun to react and complete the reaction mechanism within 10 minutes.
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內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125內的電極檢測出來,還可以使流場的移動形成124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更可以經過化學性、物理性的修飾來達到更專一性的反應。 The electrode used in the heatable bead device of the present invention may also be a micro-nano surface electrode (Fig. 7). The first group of sensing growth objects 120 are added to the sensing area 125 of FIG. 6 and FIG. The second group senses the growth object 121, the third group of sensing growth objects 122, and the fourth group of sensing growth objects 123. The sensing growth object can not only detect the 116-driven detection object by the electrode in the sensing area 125. When it comes out, it can also make the movement of the flow field form a moving path of 124, which can change the flow field at the same time (Fig. 6) and increase the sensitivity. 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 can be more chemically and physically modified to achieve a more specific one. Sexual reaction.
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之內部的第一組感測元件108、第三組感測元件110給予正 負電壓的切換,所測得之結果圖如圖八所示。 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 set of sensing elements 108 and the third set of sensing elements 110 inside the sensing region 125 are given positive The negative voltage is switched, and the measured result is shown in Figure 8.
反轉錄PCR之後繼續接著RT-PCR,本發明可加熱式液珠裝置也可應用於在RT-PCR儀器中的溫控裝置,快速調控儀器中溫度的變化,以提高RT-PCR反應效率。 Following reverse transcription PCR, followed by RT-PCR, the heatable bead device of the present invention can also be applied to a temperature control device in an RT-PCR instrument to rapidly regulate temperature changes in the instrument to improve the efficiency of the RT-PCR reaction.
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。 The heatable liquid bead device of the invention can also be applied to the temperature regulating instrument required for Enzyme Digeation in the biochemical reaction, thereby improving the temperature rise and fall, reducing the heat loss, and improving the decomposition ability of the enzyme.
上列詳細說明係針對本發明之可行實施例之具體說明,惟該實施例並非用以限制本發明之專利範圍,凡未脫離本發明技藝精神所為之等效實施或變更,例如:該可加熱式液珠裝置應用於具有快速溫度控制需求之設備,如即時定量PCR、RNA reverse transcription PCR(RT-PCR)等之應用等變化之等效性實施例,均應包含於本案之專利範圍中。 The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention. Equivalent embodiments of the liquid bead device applied to devices with rapid temperature control requirements, such as real-time quantitative PCR, RNA reverse transcription PCR (RT-PCR), etc., are all included in the patent scope of the present application.
綜上所述,本案不但在方法型態上確屬創新,並能較習用物品增進上述多項功效,應已充分符合新穎性及進步性之法定發明專利要件,爰依法提出申請,懇請 貴局核准本件發明專利申請案,以勵發明,至感德便。 In summary, this case is not only innovative in terms of method and form, but also can enhance the above-mentioned multiple functions compared with conventional articles. It should fully comply with the statutory invention patent requirements of novelty and progressiveness, and apply for it according to law. This invention patent application, in order to invent invention, to the sense of virtue.
100‧‧‧聚合酶連鎖反應裝置 100‧‧‧ polymerase chain reaction device
101‧‧‧第一液體 101‧‧‧First liquid
102‧‧‧第二液體 102‧‧‧Second liquid
103‧‧‧加熱器 103‧‧‧heater
104‧‧‧保護層 104‧‧‧Protective layer
105‧‧‧對外導線 105‧‧‧External wire
106‧‧‧基板 106‧‧‧Substrate
107‧‧‧冷卻基板 107‧‧‧Cooling the substrate
108‧‧‧第一組感測元件 108‧‧‧First set of sensing elements
109‧‧‧第二組感測元件 109‧‧‧Second set of sensing elements
110‧‧‧第三組感測元件 110‧‧‧The third set of sensing elements
111‧‧‧第四組感測元件 111‧‧‧Fourth set of sensing elements
112‧‧‧第一訊號源 112‧‧‧first signal source
113‧‧‧第二訊號源 113‧‧‧Second signal source
114‧‧‧第一圓環層 114‧‧‧First ring layer
115‧‧‧第二圓環層 115‧‧‧second ring layer
116‧‧‧第一循環線 116‧‧‧First cycle line
117‧‧‧第二循環線 117‧‧‧second cycle line
118‧‧‧第三循環線 118‧‧‧ third cycle line
120‧‧‧第一感測成長物 120‧‧‧First Sensing Growth
121‧‧‧第二感測成長物 121‧‧‧Second Sensing Growth
122‧‧‧第三感測成長物 122‧‧‧ Third Sensing Growth
123‧‧‧第四感測成長物 123‧‧‧ Fourth Sensing Growth
124‧‧‧第四循環線 124‧‧‧ fourth cycle line
125‧‧‧反應感測區 125‧‧‧Response sensing area
200‧‧‧聚合酶連鎖反應裝置 200‧‧‧ polymerase chain reaction device
201‧‧‧第一光源 201‧‧‧First light source
202‧‧‧第二光源 202‧‧‧second light source
203‧‧‧第一阻擋層 203‧‧‧ first barrier
204‧‧‧訊號接收器 204‧‧‧Signal Receiver
205‧‧‧支撐架 205‧‧‧Support frame
206‧‧‧螢光產生區 206‧‧‧Fluorescent area
301‧‧‧冷卻裝置107所提供之溫度 301‧‧‧The temperature provided by the cooling device 107
302-305‧‧‧不同電壓或電流之溫度曲線 302-305‧‧‧ Temperature curves for different voltages or currents
306‧‧‧升溫速率 306‧‧‧ Heating rate
307‧‧‧第一溫度 307‧‧‧First temperature
308‧‧‧第二溫度 308‧‧‧second temperature
圖一、可加熱式液珠裝置結合電化學檢測裝置之即時檢測裝置示意圖 Figure 1. Schematic diagram of the instant detection device of the heatable liquid bead device combined with the electrochemical detection device
圖二、加熱電極的溫度分佈區間曲線圖 Figure 2. Temperature distribution interval curve of the heating electrode
圖三、溫度曲線圖 Figure 3, temperature curve
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 Figure 4. Schematic diagram of the instant detection device of the heatable liquid bead device combined with the optical detection device
圖五、即時量測的訊號擷取曲線圖 Figure 5. Signal measurement curve for real-time measurement
圖六、奈米結構造成流場改變之示意圖 Figure 6. Schematic diagram of the flow field change caused by the nanostructure
圖七、檢測電極表面奈米碳管示意圖 Figure 7. Schematic diagram of the carbon nanotubes on the surface of the test electrode
圖八、感測區測試結果曲線圖 Figure 8. Curve of test results in the sensing area
圖九、晶片示意圖 Figure 9. Schematic diagram of the wafer
100‧‧‧聚合酶連鎖反應裝置 100‧‧‧ polymerase chain reaction device
101‧‧‧第一液體 101‧‧‧First liquid
102‧‧‧第二液體 102‧‧‧Second liquid
103‧‧‧加熱器 103‧‧‧heater
104‧‧‧保護層 104‧‧‧Protective layer
105‧‧‧對外導線 105‧‧‧External wire
106‧‧‧基板 106‧‧‧Substrate
107‧‧‧冷卻基板 107‧‧‧Cooling the substrate
108‧‧‧第一組感測元件 108‧‧‧First set of sensing elements
109‧‧‧第二組感測元件 109‧‧‧Second set of sensing elements
110‧‧‧第三組感測元件 110‧‧‧The third set of sensing elements
111‧‧‧第四組感測元件 111‧‧‧Fourth set of sensing elements
112‧‧‧第一訊號源 112‧‧‧first signal source
113‧‧‧第二訊號源 113‧‧‧Second signal source
114‧‧‧第一圓環層 114‧‧‧First ring layer
115‧‧‧第二圓環層 115‧‧‧second ring layer
116‧‧‧第一循環線 116‧‧‧First cycle line
117‧‧‧第二循環線 117‧‧‧second cycle line
118‧‧‧第三循環線 118‧‧‧ third cycle line
125‧‧‧反應感測區 125‧‧‧Response sensing area
200‧‧‧聚合酶連鎖反應裝置 200‧‧‧ polymerase chain reaction device
201‧‧‧第一光源 201‧‧‧First light source
202‧‧‧第二光源 202‧‧‧second light source
203‧‧‧第一阻擋層 203‧‧‧ first barrier
204‧‧‧訊號接收器 204‧‧‧Signal Receiver
205‧‧‧支撐架 205‧‧‧Support frame
206‧‧‧螢光產生區 206‧‧‧Fluorescent area
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