WO2002090912A1 - Methods for measuring and controlling the temperature of liquid phase in micro passage of microchip, device for the methods, and microchip - Google Patents

Methods for measuring and controlling the temperature of liquid phase in micro passage of microchip, device for the methods, and microchip Download PDF

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Publication number
WO2002090912A1
WO2002090912A1 PCT/JP2001/010450 JP0110450W WO02090912A1 WO 2002090912 A1 WO2002090912 A1 WO 2002090912A1 JP 0110450 W JP0110450 W JP 0110450W WO 02090912 A1 WO02090912 A1 WO 02090912A1
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Prior art keywords
temperature
microchip
liquid phase
microchannel
measuring
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PCT/JP2001/010450
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French (fr)
Japanese (ja)
Inventor
Takehiko Kitamori
Maxim Slyadnev
Manabu Tokeshi
Akihide Hibara
Yukio Kin
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Kanagawa Academy Of Science And Technology
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Priority to JP2002588124A priority Critical patent/JP4166091B2/en
Publication of WO2002090912A1 publication Critical patent/WO2002090912A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
    • G01K11/00—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00
    • G01K11/20—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00 using thermoluminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
    • G01K7/00—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements
    • G01K7/42—Circuits effecting compensation of thermal inertia; Circuits for predicting the stationary value of a temperature
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1861—Means for temperature control using radiation
    • B01L2300/1872—Infrared light

Definitions

  • the invention of this application relates to a method for measuring and controlling the temperature of a liquid phase in a microchip microchannel, an apparatus therefor, and a microchip.
  • microchips that use microchannels (microchannels) formed on a substrate such as glass as a chemical reaction field have been known. Chemical synthesis using such microchips as integrated chemical devices has been known. The precise analysis of trace components has been studied from various viewpoints and many proposals have been made.
  • Integrating chemical processes on a chip eliminates the need to handle analytes and reagents during analysis or synthesis, and allows fluid control, sample processing, multi-step reactions, temperature control, separation, and separation on a single device. Because detection will be possible, significant future development is expected in the fields of analytical chemistry, biochemistry, and medical science. One of the elements needed for consideration in this area is knowledge of chemistry in microspace. Compared to macroscale systems, integrated chemical systems have several advantages that derive as a result of basic scale principles. Reducing reagent consumption and disposal is directly provided by the small size. Also control of mixing and heat The benefits to speed and efficiency come from the mass and heat transfer driven by diffusion.
  • microdevices for chemical reactions include, for example, the previously reported polymerase chain reaction (PCR) amplification of DNA fragments, precolumn and postcolumn derivatization, flow injection analysis, and organic synthesis. It has been proved by application to such as.
  • the inventors of the present application have already proposed a microchip capable of performing a chelate reaction, an enzymatic reaction, a liquid-liquid extraction, and a peas-filled immunoassay.
  • the temperature of the liquid phase such as the solution in the microchip microchannel must be increased. It is necessary to accurately measure the temperature and control the temperature of the liquid phase reaction in the microchannel.
  • the temperature in the past, such temperature measurement has hardly been studied, and the temperature must be appropriately adjusted. In fact, it has not been realized to control the reaction temperature, in particular, to optimize the heating means required for the chemical reaction. Therefore, by controlling the reaction temperature, the reaction rate and the reaction selectivity are improved. There were great restrictions on this. However, there was a reason for this situation. This is because the heat capacity of the liquid phase in the microchip microchannel was extremely small, and it was considered extremely difficult to accurately measure the liquid phase temperature in the microchannel.
  • the thermal time constant can be reduced due to the small heat capacity in the microchannel and the high thermal conductivity of the device substrate.
  • Miniaturized reactors using electrothermal Joule heating for rapid thermal cycling have already been reported, with 30 / s The heating rate and cooling rate of 4 / s have been revealed.
  • the heating / cooling speed of the depiice by Joule heating block by electric heating was limited by the absolute heat capacity of the block itself.
  • local heating limits the lateral resolution due to the thermal conductivity of the chip material, making it difficult to manufacture devices with multiple independent heating points. Even with integrated heaters on the chip, these devices still required complex manufacturing processes and limited the flexibility to easily change the microchip design.
  • the solution temperature can be directly monitored by measuring the electric resistance.
  • a volume of about nanoliter can be locally localized with sufficient accuracy.
  • the invention of the present application solves the above-mentioned problems, and can accurately measure the liquidus temperature in the microchannel of the microchip.
  • the task is to provide new technical means that enable a great degree of temperature control. Disclosure of the invention
  • This application provides the invention as follows to solve the above problems.
  • a method for measuring the temperature of the liquid phase in a microchip microchannel wherein the liquid phase temperature in the microchip microchannel is measured in a non-contact manner by detecting the emission intensity from a fluorescent substance.
  • a method for measuring the temperature of a liquid phase in a microchip microchannel wherein the liquid phase temperature in the microchip microchannel is measured by a temperature detecting means in contact with the liquid phase in the microchannel.
  • thermocouple The method for measuring the temperature of a liquid phase in a microchip microchannel according to the above item 3 or 4, wherein the temperature detecting means is a thermocouple.
  • thermocouple inserted and arranged in a fine groove formed on the side of the fine channel.
  • a method for controlling the temperature of the liquid phase in a microchip microchip microchannel wherein the liquid phase in the microchip microchannel is heated by irradiating an infrared (IR) laser to the liquid phase in the microchip microchannel.
  • IR infrared
  • a temperature measuring device for a liquid phase in a microchip microchannel 8. Equipped with a microchip placement part with a microchannel, a detection unit for the emission intensity from the fluorescent substance in the microchannel, and a calculation unit that calculates the temperature of the liquid phase in the microchannel from the emission intensity.
  • a temperature measuring device for a liquid phase in a microchip microchannel 8. Equipped with a microchip placement part with a microchannel, a detection unit for the emission intensity from the fluorescent substance in the microchannel, and a calculation unit that calculates the temperature of the liquid phase in the microchannel from the emission intensity.
  • Temperature measurement of the liquid phase in the microchip microchannel characterized by having a temperature detection means that comes into contact with the liquid phase in the microchip microchannel, along with the microchip placement part having the microchannel. apparatus.
  • a microchip thermal reaction device wherein the temperature measuring device according to 8 or 9 is provided together with a heating means for a liquid phase in a microchip microchannel.
  • thermo reactor according to the item 10 wherein the heating means is an infrared laser irradiation means.
  • a microchip characterized by being provided with a temperature detection means for contacting a liquid phase in the microchannel together with the microchannel.
  • thermocouple 13 The microchip as described in 12 above, wherein the temperature detecting means is a thermocouple.
  • FIG. 1 is a configuration diagram exemplifying a system for measuring temperature by non-contact fluorescence intensity.
  • FIG. 2 is a diagram showing an example of temperature measurement using a thermocouple.
  • FIG. 3 is a diagram illustrating irradiation of an infrared laser at a plurality of points.
  • FIG. 4 is a diagram illustrating an example of a device configuration according to the embodiment.
  • FIG. 5 is a diagram illustrating a calibration curve of temperature measurement based on fluorescence intensity.
  • FIG. 6 is a temperature distribution diagram at each flow rate of the solution due to IR laser irradiation.
  • FIG. 7 is a diagram illustrating rapid cycling of the temperature measured by the fluorescence quench.
  • FIG. 8 is a diagram exemplifying a time transition of the IR irradiation between nos obtained experimentally.
  • FIG. 9 is a diagram exemplifying a result of reaction control on a chip by IR laser heating.
  • FIG. 10 is a diagram showing a normalized FFT spectrum obtained from the TLM signal.
  • FIG. 11 is a diagram illustrating a profile of fluorescence intensity detection in a PCR cycle.
  • FIG. 12 is a diagram exemplifying the state of DNA sampling as temperature detection by thermocouple means.
  • a method for measuring a liquidus temperature in a microchannel of a microchip is as follows.
  • the liquid phase in the microchannel of the microchip is As a method of measuring temperature
  • ⁇ C> Heating method by irradiating the liquid phase in the channel with infrared (IR) laser
  • the principle of the non-contact optical temperature measurement method ⁇ A> is based on the principle of detecting the emission intensity of a fluorescent substance present in the liquid phase.
  • the fluorescent substance various kinds of fluorescent substances which do not participate in the liquid phase reaction in the microchannel or are not essential even if they participate may be used.
  • rhodamine dye substance Rhodamine dye substance
  • Such a method of measuring the temperature of the liquid phase in the fine channel by detecting the emission intensity of the fluorescent substance is extremely useful for temperature measurement in a small space where the heat capacity is extremely small. Since it is non-contact, it does not affect the heating or cooling of the liquid phase, accurately measures the temperature, and appropriately controls the heating or cooling conditions of the liquid phase according to the measured temperature. It is possible to do.
  • FIG. 1 illustrates a system for measuring the temperature from the detection of the fluorescence intensity in a non-contact manner.
  • the temperature of the liquid phase in the microchannel (2) formed in the microchip (1) can be measured as fluorescence intensity detection.
  • the liquid phase heating means As an example, an IR (infrared) laser irradiation means is exemplified.
  • the system includes a microchip (1) having a microchannel (2), a fluorescence intensity detection unit, and a calculation unit for calculating a temperature from the emission intensity. Also, by providing an IR (infrared) laser heating means as in the example in Fig. 1, it is possible to configure a thermal reaction device that can control temperature conditions with high accuracy. For the detection of the reaction, a means of a thermal lens microscope established by the inventors of the present application and the like can be employed.
  • a temperature detecting means that comes into contact with the liquid phase in the microchannel of the microchip is used. Since the fine channels are fine, for example, with a depth of 500 m or less and a width of 300 ⁇ m or less, they may have a thermal effect on the liquid phase in such fine channels. Temperature sensing means are used so as to have little or no practical effect on the required liquid phase flow.
  • thermocouple can be suitably used.
  • Fig. 2 shows that a thermocouple is used as a temperature detecting means and inserted into a groove (3) formed on the side of the microchannel (2) so that the thermocouple contacts the liquid phase.
  • the example shown in FIG. The photo shows an enlarged example of the contact part (4).
  • the groove (3) can be formed by a drill or the like, and a K-type thermocouple or the like can be used.
  • the effect of direct contact with the liquid phase can be substantially negligible. This has also been confirmed in the PCR of the DNA fragment.
  • a microchip with a built-in temperature detecting means is valuable in the configuration of the microchip itself.
  • the method and apparatus of the present invention in which the liquid phase is heated using the photothermal effect of an infrared laser (IR laser), can rapidly and locally control an enzymatic reaction on a microchip under flow conditions.
  • IR laser infrared laser
  • Non-contact The dynamic change and three-dimensional distribution of the temperature are measured by using the tactile spectral temperature detection method, and the results are compared with the results of numerical simulation analysis.
  • a special feature of the heating method using an infrared laser is that the size of the object to be heated is extremely small, only 5 nL, as compared with the existing system using an electric heating chip.
  • a rapid heating rate can be realized by directly heating a sample having an extremely small heat capacity, and by using a glass substrate, heat is efficiently removed by transferring heat to the glass substrate. Cooling can be performed quickly. Temperature level reproducibility is ensured by keeping the stay time less than 0.5 seconds.
  • the enzymatic reaction on the chip has been successfully controlled with a time resolution of, for example, 0.6 seconds by means of periodic heating using photothermal heat with an IR laser. Since the IR diode laser is compact, it is suitable for a design with a smaller system.
  • heating with an infrared (IR) laser has the special feature that multipoint heating can be controlled as appropriate, and can be controlled as a rapid heating and cooling cycle along a fine channel. are doing.
  • IR infrared
  • FIGS. 1 and 2 a minute channel having a Y-shaped junction was formed by a known etching method.
  • the substrate is glass.
  • the microchannel is 250 zm wide and 100 m deep.
  • the length of the reaction channel behind the Y-shaped junction is 4.0 cm.
  • a side channel was provided by a diamond drill in a direction perpendicular to the channel etched from the end of the bonded chip in the same manner as in FIG. The side channel was cut until it merged with the etched channel. The intersection was located 2 cm downstream from the entrance.
  • the overall width of the etched microchannel was 250 m, the depth was 100 ⁇ m, and the diameter of the side channel was 500 m.
  • 4-Antiaminopyrine (4-1 AAP) and N-ethyl N- (2-hydroxy-3-sulfopropyl) -1m-toluidine (TOOS) for biochemistry were purchased from Dojin Research Institute (Kumamoto City).
  • . Radish peroxidase (HRP, EC 1.1.1.1.7, specific activity 200 units / mg) and 35% hydrogen peroxide solution (for electronic industry) were purchased from Wako Pure Chemical (Osaka). All reagents were used without any particular purification. All water used was ultra-pure water from a water purification system (TW-600 RU, Nomura Microscience, Kanagawa).
  • Substrate stock solution (2 X 1 0 ⁇ 3 M , 4 one AAP and TOOS; 1 0'' ⁇ ⁇ 2 0 2 ) was prepared by phosphate buffer solution (PB S, ⁇ 7. 4) . These were stored at 10 in a refrigerator and diluted appropriately with PBS before use. HRP solutions (20 units ZmL) were prepared daily from the solid enzyme, monitored for absorbance at 403 nm, assayed for concentration, and further diluted as needed. 4 one AAP and TOO S concentration ranges of the working fluid, 2 X 10- 4 M, H ? 1 to 5 units / 1! 1, H 2 0 2 was 1 ⁇ 5 X 1 0- 5 M. Equal volumes (each LML) mixed solution of HRP and 4-AAP immediately before use and (solution 1) was equal volume mixed liquid of TOO S and H 2 0 2 (solution 2) was prepared.
  • PB S phosphate buffer solution
  • One beam of the IR laser was converged by a lens to form a beam with a waist diameter of 150M.
  • the output of the IR laser was controlled by a computer equipped with a digital-to-analog conversion board (DAC) (PCI-6035, National Instruments, Austin, TX).
  • DAC digital-to-analog conversion board
  • the heating point was set at 1.5 cm downstream from the Y-shaped junction.
  • the reaction medium was heated in a flow system. 4-AAP -T00S-H 2 0 2 -HRP reaction product was continuously transported to the detection point of the thermal lens microscope provided 5 dragon downstream further (TLM).
  • ADC analog-to-digital conversion
  • the ambient temperature of the microchip is controlled by a Peltier temperature stage equipped with a controller (temperature range-25 to +99, accuracy and stability ⁇ 0.1; PE-60, Linkam Scientific Instruments, Tadworth, UK). Held. As shown in Fig. 4, a chip with side channels was placed on the stage. The steady-state temperature on the chip was monitored by two calibrated thermocouples (Chromel-Alumel type, wire tip diameter 25 I; Anbe Soldering Technologies Ltd., Yokohama). One thermocouple was placed above the Peltier stage and the other was placed inside a 500 m side channel and sealed with epoxy adhesive. The thermocouple tip protruded ⁇ 100 m into the main channel.
  • the tip was 1 cm upstream from the IR irradiation point to break up the local heating effect. Typically, the temperature difference between the two sensors did not exceed 0.2.
  • a microchip with a Peltier stage can handle liquids without disturbing the position of the microchip with respect to the IR irradiation optical system. , I installed it stably. To obtain stable thermal contact
  • the microchip's park temperature was constantly monitored with two thermocouples. Temperature change and spatial temperature distribution in the microchannel were monitored by fluorescence quenching.
  • the samples were used rhodamine 3 B dye (Rh- 3Bb, Exciton, Inc. (Di tons, Ohio), manufactured by) an aqueous solution of (10- 5 M).
  • An Ar + ion laser (514.5 ni, cut at 1025 Hz) was used to excite the fluorescence. As shown in Fig.
  • the time constant of the lock-in amplifier was changed in the range of 12 to 400 ms.
  • the fluorescence signal was digitized at the ADC pod, as in the case of TLM detection, and then sent to a computer. Fluorescent images were captured on a PC equipped with a video frame grabber pod.
  • thermocouple readings showed that the solution temperature did not change over the flow rate range studied (0-5 cm / s).
  • the solution temperature was in equilibrium with the surrounding substrate temperature, as the intrinsic diffusion time through a 100 m water-filled microchannel was only 70 ms. Since the solution residence time is much longer than the intrinsic time (0.5 s even at the fastest linear velocity of 5 cm / s studied), such an equilibrium state was established even under flowing conditions.
  • the normalized calibration curves generated on different days were very stable as shown in Figure 3, confirming an RSD of 3%.
  • the accuracy of the temperature measurement by the fluorescence quenching method was ⁇ 0.5.
  • the calibration curve was independent of solution flow rate over the range studied.
  • FIG. 2 The fluorescence microscope image clearly showed that the intensity was locally reduced when the channel was irradiated with the IR laser.
  • Figure 6 shows the temperature distribution image when the flow rate of the solution was changed. This image is a top view of a small channel, 1 orchid long and 250 m wide. The boundary between the top and bottom of each image corresponds to the interface between water and glass. The microchip was held at a constant temperature by a Peltier stage, and the IR laser heated only a certain part of the channel. The original intensity image was normalized with the image obtained in step 10, and the fluorescence intensity ratio obtained for each pixel was converted to its temperature using the calibration curve in FIG.
  • the images obtained showed that the spatial temperature distribution along the channel clearly reflected the effect of the solution flow rate.
  • the localization of temperature was closely related to the IR irradiation volume for low flow rates, and the higher the flow rate, the more the local area spread and moved downstream.
  • the images show that the spatial distribution along the microchannel is not uniform. There were two reasons for this: the IR intensity at the focal plane of the microscope objective had a spatial Gaussian distribution, and the heat transfer to the substrate parc at the water-glass interface was effective. . Overall, the temperature near the water-glass interface was 40-50% of the maximum temperature in the center of the IR beam for the range of flow rates studied.
  • the intermediate level T 2 was adjusted by changing the output of the IR laser (10, 20, 30, 40, 60, 70, 8 OmW).
  • the observed temperature reproducibility between cycles arises from the reproducibility inherent in the heat dissipation path due to diffusion, and the latter reproducibility depends on the high thermal conductivity of the glass substrate maintained at a constant temperature by the Peltier stage. Brought. Both the temperature rise and the cooling reached the equilibrium temperature. The problem of excess and deficiency has been completely eliminated. As a result, this system can produce thermal cycles with sufficient resolution and residence times of less than 1 s.
  • Figure 8b shows the experimentally determined time course of IR irradiation for 1 s.
  • the transition of irradiation for 1 s calculated is also given.
  • the heating rate was 67 / s and the cooling rate was 53 / s.
  • the temperature difference ⁇ ⁇ 27 ⁇ : was divided by the time required to decrease (0.51 s).
  • the heating / cooling rate of the center of the IR laser beam observed in the experiment was independent of the initial temperature and flow velocity within the range of the studied conditions, and was in good agreement with the results of numerical analysis.
  • Direct heating of the electrolyte in the microchannels by electroheating could raise both the heating rate and the cooling rate up to a maximum of 20, but the volume to be heated was still a number.
  • Further reducing the directly heated volume to about ⁇ 150 nL provided heating and cooling rates of 60 / s and 20 / s, respectively.
  • Rapid NoboriAtsushihiya ⁇ degree is greater in the heated the volume 5 nL an even and small and thermal capacity 2 X 10- 5 J / K Ikoto and small surface to volume ratio of the channel is 280 cm- 1, It was achieved by doing so.
  • the transition from the starting temperature to the higher temperature, and vice versa, takes less than 1 s to achieve, enabling rapid temperature control on the chip. It should be noted that rapid cooling is possible by using natural heat transfer to the glass substrate while maintaining the entire chip at a constant temperature, without using a blower or a system for sending compressed air repeatedly. Deserve.
  • Figure 9 shows typical results for controlling the reaction on the chip by IR laser heating under flow conditions.
  • the IR laser was operated regularly.
  • the laser was turned on and off with a 50% duty cycle for the indicated time.
  • the time course showed that the TLM signal followed a regular pattern related to that of IR irradiation.
  • the TLM signal was subjected to a fast Fourier transform (FFT) to reveal rapid control data of the reaction with an IR irradiation time shorter than 1 s.
  • FFT fast Fourier transform
  • Figure 10 shows the normalized FFT spectrum obtained from the TLM signal. This spectrum showed a sharp characteristic frequency exceeding the noise level. For I s and 0.6 s, some are probably due to the increased noise contribution at that frequency. Harmonics were observed. The characteristic frequency was consistent with the IR irradiation time used. This confirms that the reaction rate is externally controlled by IR irradiation.
  • FIG. 11 exemplifies the profile of fluorescence intensity detection of mouth-damine-13B in the case of 45 cycles of PCR in the same manner as in Example 1.
  • the cycle is
  • I R 1 2 OmW, 9 5-2 s
  • I R OmW, 6 8— 6 s
  • the temperature change of DNA melting was observed using a temperature detecting means by a thermocouple provided in the microchip illustrated in FIGS. 2 and 4.
  • the PCR amplified DNA fragment labeled with the ds-DNA specific dye S YBER-Greenl was introduced into the microchannel, and heated to 60 to 98 by external heating means.
  • the heating rate was 0.2 / s.
  • Figure 12 shows the results of DNA melting.
  • the solid line shows the case of the 500 bp ⁇ -phage DNA fragment, and the broken line shows the case of the NTC (negative template control) sample.
  • thermocouple of embedded type as a temperature detecting means was effective.
  • a new technical means that can accurately measure the liquidus temperature in a microchannel of a microchip and can quickly and appropriately control the temperature. Can be provided.

Abstract

Novel technical means capable of measuring and controlling the temperature of a liquid phase in a micro passage of a microchip highly accurately by measuring the temperature of the liquid phase in the micro passage of the microchip in a noncontact manner by measuring the luminous intensity of light from a fluorescent substance or by measuring it by means of a sensing means buried in the microchip, and by heating the liquid phase by irradiating it with infrared laser beam.

Description

明 細 書 マイクロチップ微細流路内液相の温度の測定方法と制御方法、  Description Method for measuring and controlling the temperature of the liquid phase in the microchip microchannel,
そのための装置並びにマイクロチップ 技術分野  Equipment and microchip for that purpose
この出願の発明は、 マイクロチップ微細流路内液相の温度の測定方 法と制御方法、 そのための装置並びにマイクロチップに関するもので The invention of this application relates to a method for measuring and controlling the temperature of a liquid phase in a microchip microchannel, an apparatus therefor, and a microchip.
¾) ¾ o 背景技術 ¾) ¾ o Background technology
従来より、 ガラス等の基板に形成した微細流路 （マイクロチャンネ ル） を化学反応場として利用するマイクロチップが知られており、 こ のような集積化した化学デバイスとしてのマイクロチップによって、 化学合成や微量成分の精密分析を行うことが様々な観点から検討され 、 数多くの提案がなされてきている。  Conventionally, microchips that use microchannels (microchannels) formed on a substrate such as glass as a chemical reaction field have been known. Chemical synthesis using such microchips as integrated chemical devices has been known. The precise analysis of trace components has been studied from various viewpoints and many proposals have been made.
チップ上に化学過程を統合すると、 分析または合成を行う途中で分 析試料や試薬を取り扱う必要がなく、 単一のデバイス上で流体の制御 、 試料の処理、 多段階反応、 温度制御、 分離および検出を行うことが 可能になるため、 分析化学、 生化学および医療科学等の分野で将来の 大きな発展が期待される。 この分野での検討のために必要とされる要 素の一つは、 微小空間での化学に関する知識にある。 マクロスケール の系と比べて、 集積化した化学系は、 基本的な規模の原理の結果とし て派生するいくつかの利点を持っている。 試薬の消費および廃棄の削 減は小型によって直接的にもたらされる。 また、 混合および熱の制御 の速度および効率に対する利益は拡散によって駆動される質量および 熱の移動から生まれる。 化学反応のための微小デバイスのスケールメ リッ トは、 たとえば、 すでに報告されている D N Aフラグメントのポ リメラ一ゼ連鎖反応 （P C R ) 増幅法、 プレカラムおよびポストカラ ム誘導体化、 フローインジェクション分析法、 および有機合成などへ の応用によって証明されている。 そして、 この出願の発明者らによつ て、 キレート反応や、 酵.素反応、 液液抽出およびピーズ充填免疫アツ セィを行うことができるマイクロチップがすでに提案されている。 Integrating chemical processes on a chip eliminates the need to handle analytes and reagents during analysis or synthesis, and allows fluid control, sample processing, multi-step reactions, temperature control, separation, and separation on a single device. Because detection will be possible, significant future development is expected in the fields of analytical chemistry, biochemistry, and medical science. One of the elements needed for consideration in this area is knowledge of chemistry in microspace. Compared to macroscale systems, integrated chemical systems have several advantages that derive as a result of basic scale principles. Reducing reagent consumption and disposal is directly provided by the small size. Also control of mixing and heat The benefits to speed and efficiency come from the mass and heat transfer driven by diffusion. The scale benefits of microdevices for chemical reactions include, for example, the previously reported polymerase chain reaction (PCR) amplification of DNA fragments, precolumn and postcolumn derivatization, flow injection analysis, and organic synthesis. It has been proved by application to such as. The inventors of the present application have already proposed a microchip capable of performing a chelate reaction, an enzymatic reaction, a liquid-liquid extraction, and a peas-filled immunoassay.
しかしながら、 たとえば P C Rや、 生物化学的あるいは合成化学的 反応等の各種の応用分野にこのようなマイクロチップの利用を拡大す るためには、 マイクロチップ微細流路内の溶液等の液相の温度を正確. に測定し、 微細流路内の液相反応の温度を制御することが必要である が、 従来では、 このような温度測定についてはほとんど検討されてお らず、 また、 適切に温度を制御すること、 特に化学反応に必要とされ る加熱手段の最適化は実現されていないのが実情であって、 このため 、 反応温度を制御することによって反応速度や反応選択性を向上させ ることには大きな制約があった。 ただ、 このような状況については理 由があった。 それと言うのも、 マイクロチップ微細流路内の液相の熱 容量は極めて小さいため、 微細流路内の液相温度を正確に測定するこ とは極めて難しいと考えられていたからである。  However, in order to expand the use of such microchips in various application fields such as PCR and biochemical or synthetic chemical reactions, the temperature of the liquid phase such as the solution in the microchip microchannel must be increased. It is necessary to accurately measure the temperature and control the temperature of the liquid phase reaction in the microchannel. However, in the past, such temperature measurement has hardly been studied, and the temperature must be appropriately adjusted. In fact, it has not been realized to control the reaction temperature, in particular, to optimize the heating means required for the chemical reaction. Therefore, by controlling the reaction temperature, the reaction rate and the reaction selectivity are improved. There were great restrictions on this. However, there was a reason for this situation. This is because the heat capacity of the liquid phase in the microchip microchannel was extremely small, and it was considered extremely difficult to accurately measure the liquid phase temperature in the microchannel.
そして、 チップ上で反応混合物の熱処理を行うと、 微小チャンネル 内の熱容量が小さくデバイス基板の熱伝導性が高いために熱時間定数 を小さくすることができるため、 多くの応用分野でチップ上での反応 混合物の熱処理が要望されている。 迅速熱サイクルに電熱ジュール加 熱を使用する小型化した反応器はすでに報告されており、 3 0で/ s の昇温速度と 4 / sの冷却速度が明らかにされている。 しかしなが ら、 電熱によるジュール加熱プロックによるデパイスの昇温/冷却速 度にはブロック自体の絶対熱容量による限界があった。 その上、 外部 的に接触させるデバイスの場合、 局部的加熱は、 チップ材料の熱伝導 性によって横方向の分解能に限界があり、 独立した多数の加熱点を持 つデバイスは製作困難である。 チップ上に集積したヒータの場合でも 、 やはりこれらのデバイスは複雑な製造工程が必要であり、 マイクロ チップの設計を簡単に変更する柔軟性に制約があった。 When the heat treatment of the reaction mixture is performed on the chip, the thermal time constant can be reduced due to the small heat capacity in the microchannel and the high thermal conductivity of the device substrate. There is a need for heat treatment of the reaction mixture. Miniaturized reactors using electrothermal Joule heating for rapid thermal cycling have already been reported, with 30 / s The heating rate and cooling rate of 4 / s have been revealed. However, the heating / cooling speed of the depiice by Joule heating block by electric heating was limited by the absolute heat capacity of the block itself. In addition, in the case of externally contacted devices, local heating limits the lateral resolution due to the thermal conductivity of the chip material, making it difficult to manufacture devices with multiple independent heating points. Even with integrated heaters on the chip, these devices still required complex manufacturing processes and limited the flexibility to easily change the microchip design.
加熱デバイスの熱容量が大きいという問題を解決し、 試料への熱伝 導を容易にするため、 電解質の抵抗を利用して溶媒を直接加熱するこ とが提案されてもいる。 これによつて最高 2 0でまでの昇温冷却速度 が達成できた。 しかし、 この方法によれば、 電気抵抗を測定すること によって、 溶液温度を直接モニターすることができるが、 特に電気伝 導率が変化する試料の場合、 ナノリットル程度の体積を十分な精度で 局部的に制御しながら加熱するには制約がある。  In order to solve the problem of large heat capacity of the heating device and to facilitate heat transfer to the sample, it has been proposed to directly heat the solvent using the resistance of the electrolyte. As a result, a heating / cooling rate of up to 20 was achieved. However, according to this method, the solution temperature can be directly monitored by measuring the electric resistance. Particularly, in the case of a sample in which the electric conductivity changes, a volume of about nanoliter can be locally localized with sufficient accuracy. There is a restriction in heating while controlling the temperature.
一方、 赤外線 （ I R ) による非接触加熱法を利用してマイクロリツ トルまたはそれより 1桁小さい量の試料でゲノム D N Aを P C R増幅 する方法が Oda ら、 および Humer と Landers によって発表されている。 それによれば、 I R照射源タングステンランプが、 そして冷却を行う にはゲートを設けた圧縮空気が使用されている。 この方法では 6 5で / sの昇温速度と 2 0 T / s の冷却速度が実現した。 しかし、 この熱 サイクルシステムはマイクロチップとの整合性が取られていないため 、 いくつかの操作は手動で行われた。 その上、 流速の制御が行われな かった。 また、 使用されたタングステンランプは非干渉性の非点光源 であるために焦点が比較的大きく、 断面積の小さいマイクロチップの チヤンネルに適用するには、 こうした加熱デバイスとしての効率には 制限がある。 On the other hand, Oda et al., And Humer and Landers have published a method of PCR-amplifying genomic DNA using microliters or an order of magnitude smaller using non-contact heating by infrared (IR) heating. According to the report, a tungsten lamp for IR irradiation is used, and gated compressed air is used for cooling. With this method, a heating rate of 65 / s and a cooling rate of 20 T / s were realized. However, some operations were performed manually because the heat cycle system was not compatible with the microchip. In addition, there was no control over the flow velocity. Also, since the tungsten lamp used is a non-coherent, non-point light source, it has a relatively large focal point and a microchip with a small cross-sectional area. There are limitations on the efficiency of such heating devices for channel applications.
そこで、 この出願の発明は、 以上のような問題点を解消し、 マイクロ チップの微細流路内の液相温度を精度よく測定することができ、 また 、 実際的に適切に、 しかも簡便に自由度の大きい温度制御を可能とす る、 新しい技術手段を提供することを課題としている。 発明の開示  Therefore, the invention of the present application solves the above-mentioned problems, and can accurately measure the liquidus temperature in the microchannel of the microchip. The task is to provide new technical means that enable a great degree of temperature control. Disclosure of the invention
この出願は、 上記の課題を解決するものとして、 以下のとおり発明 を提供する。  This application provides the invention as follows to solve the above problems.
1 . マイクロチップ微細流路内の液相温度を蛍光物質からの発光強 度の検知によって非接触で測定することを特徴とするマイクロチップ 微細流路内液相の温度測定方法。  1. A method for measuring the temperature of the liquid phase in a microchip microchannel, wherein the liquid phase temperature in the microchip microchannel is measured in a non-contact manner by detecting the emission intensity from a fluorescent substance.
2 . 蛍光物質がローダミン色素物質であることを特徴とする前記 1 のマイクロチップ微細流路内液相の温度測定方法。  2. The method for measuring the temperature of a liquid phase in a microchip microchannel according to the above item 1, wherein the fluorescent substance is a rhodamine dye substance.
3 . マイクロチップ微細流路内の液相温度を、 微細流路内の液相に 接触させた温度検知手段により測定することを特徴とするマイクロチ ップ微細流路内液相の温度測定方法。  3. A method for measuring the temperature of a liquid phase in a microchip microchannel, wherein the liquid phase temperature in the microchip microchannel is measured by a temperature detecting means in contact with the liquid phase in the microchannel.
4 . 温度検知手段はその一部または全部がマイクロチップに埋設も しくは載置されていることを特徵とする前記 3のマイクロチップ流路 内液相の温度測定方法。 ， 4. The method for measuring the temperature of a liquid phase in a microchip flow path according to the above 3, wherein the temperature detecting means is partially or entirely embedded or mounted on the microchip. ,
5 . 温度検知手段が熱電対であることを特徵とする前記 3または 4 のマイクロチップ微細流路内液相の温度測定方法。 5. The method for measuring the temperature of a liquid phase in a microchip microchannel according to the above item 3 or 4, wherein the temperature detecting means is a thermocouple.
6 . 微細流路に対してその側部に形成した微細溝に挿入配置した熱 電対により温度を測定することを特徴とする前記 5のマイクロチップ 微細流路内液相の温度測定方法。 6. The microchip according to the above-mentioned 5, wherein the temperature is measured by a thermocouple inserted and arranged in a fine groove formed on the side of the fine channel. A method for measuring the temperature of a liquid phase in a fine channel.
7 . マイクロチップ微細流路内の液相を赤外線 （ I R ) レーザーの 照射によって加熱して液相温度を制御することを特徴とするマイク口 チップ微細流路内液相の温度制御方法。  7. A method for controlling the temperature of the liquid phase in a microchip microchip microchannel, wherein the liquid phase in the microchip microchannel is heated by irradiating an infrared (IR) laser to the liquid phase in the microchip microchannel.
8 . 微細流路を有するマイクロチップの配置部とともに、 微細流路 内の蛍光物質からの発光強度の検知部と、 この発光強度から微細流路 内液相の温度を算出する演算部とを備えていることを特徴とするマイ クロチップ微細流路内液相の温度測定装置。  8. Equipped with a microchip placement part with a microchannel, a detection unit for the emission intensity from the fluorescent substance in the microchannel, and a calculation unit that calculates the temperature of the liquid phase in the microchannel from the emission intensity. A temperature measuring device for a liquid phase in a microchip microchannel.
9 . 微細流路を有するマイクロチップ配置部とともに、 マイクロチ ップ微細流路内の液相に接触する温度検知手段を備えていることを特 徴とするマイクロチップ微細流路内液相の温度測定装置。  9. Temperature measurement of the liquid phase in the microchip microchannel characterized by having a temperature detection means that comes into contact with the liquid phase in the microchip microchannel, along with the microchip placement part having the microchannel. apparatus.
1 0 . 前記 8または 9の温度測定装置が、 マイクロチップ微細流路 内液相の加熱手段とともに備えられていることを特徴とするマイクロ チップ熱反応装置。  10. A microchip thermal reaction device, wherein the temperature measuring device according to 8 or 9 is provided together with a heating means for a liquid phase in a microchip microchannel.
1 1 . 加熱手段が赤外線レーザ一照射手段であることを特徴とする 前記 1 0の熱反応装置。  11. The thermal reactor according to the item 10, wherein the heating means is an infrared laser irradiation means.
1 2 . 微細流路とともに、 微細流路内の液相に接触する温度検知手 段が備えられていることを特徴とするマイクロチップ。  1 2. A microchip characterized by being provided with a temperature detection means for contacting a liquid phase in the microchannel together with the microchannel.
1 3 . 温度検知手段が熱電対であることを特徵とする前記 1 2のマ ィク Πチップ。 図面の簡単な説明  13. The microchip as described in 12 above, wherein the temperature detecting means is a thermocouple. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 非接触での蛍光強度による温度測定のシステムを例示した 構成図である。  FIG. 1 is a configuration diagram exemplifying a system for measuring temperature by non-contact fluorescence intensity.
図 2は、 熱電対による温度測定の例を示した図である。 図 3は、 赤外線レーザーの複数点での照射を例示した図である。 FIG. 2 is a diagram showing an example of temperature measurement using a thermocouple. FIG. 3 is a diagram illustrating irradiation of an infrared laser at a plurality of points.
図 4は、 実施例における装置構成例を示した図である。  FIG. 4 is a diagram illustrating an example of a device configuration according to the embodiment.
図 5は、 蛍光強度による温度測定の較正曲線を例示した図である。 図 6は、 I Rレーザー照射にともなう溶液の流速毎の温度分布図で ある。  FIG. 5 is a diagram illustrating a calibration curve of temperature measurement based on fluorescence intensity. FIG. 6 is a temperature distribution diagram at each flow rate of the solution due to IR laser irradiation.
図 7は、 蛍光クェンチによって測定した温度の迅速サイクリングを 例示した図である。  FIG. 7 is a diagram illustrating rapid cycling of the temperature measured by the fluorescence quench.
図 8は、 実験的に求めたノ s間の I R照射の時間推移を例示した図 である。  FIG. 8 is a diagram exemplifying a time transition of the IR irradiation between nos obtained experimentally.
図 9は、 I Rレーザー加熱によるチップ上の反応制御の結果を例示 した図である。  FIG. 9 is a diagram exemplifying a result of reaction control on a chip by IR laser heating.
図 1 0は、 T L M信号から得られた規格化 F F Tスぺクトルを示し た図である。  FIG. 10 is a diagram showing a normalized FFT spectrum obtained from the TLM signal.
図 1 1は、 P C Rサイクルの蛍光強度検知のプロファイルを例示し た図である。  FIG. 11 is a diagram illustrating a profile of fluorescence intensity detection in a PCR cycle.
図 1 2は、 D N A me l t ingの状態を熱電対手段による温度検知として 例示した図である。 発明を実施するための最良の形態  FIG. 12 is a diagram exemplifying the state of DNA sampling as temperature detection by thermocouple means. BEST MODE FOR CARRYING OUT THE INVENTION
まず、 この出願の発明では、 マイクロチップの微細流路内の液相温 度の測定方法として、  First, in the invention of this application, a method for measuring a liquidus temperature in a microchannel of a microchip is as follows.
< A>非接触で光学的に液相温度を測定する方法  <A> Non-contact method for optically measuring liquidus temperature
< B >液相との直接的接触により液相温度を測定する方法  <B> Method of measuring liquid phase temperature by direct contact with liquid phase
の 2種の方法を提供する。 The two methods are provided.
そして、 この出願の発明では、 マイクロチップの微細流路内の液相 温度の測定方法として、 And, in the invention of this application, the liquid phase in the microchannel of the microchip is As a method of measuring temperature,
< C >流路内液相に赤外線 （ I R ) レーザーを照射して加熱する方 法  <C> Heating method by irradiating the liquid phase in the channel with infrared (IR) laser
を提供する。 I will provide a.
まず < A >の非接触光学式の温度測定法では、 液相内に存在させた 蛍光物質からの発光強度の検知をその原理としている。 蛍光物質とし ては、 微細流路での液相反応に関与しない、 もしくは関与しても本質 的ではない蛍光性物質の各種のものが使用されてよく、 たとえばロー ダミン色素物質（Rhodamine dye) がある。 このような蛍光物質の発光強 度の検知による微細流路内液相の温度測定方法は、 微小空間で、 熱容 量が極めて小さい状況の温度測定にとって極めて有用である。 非接触 であることから、 液相の加熱あるいは冷却に影響を及ぼすことはなく 、 精度よく温度を測定し、 この測定された温度に応じて液相の加熱あ るいは冷却の条件操作を的確に行うことが可能になる。  First, the principle of the non-contact optical temperature measurement method <A> is based on the principle of detecting the emission intensity of a fluorescent substance present in the liquid phase. As the fluorescent substance, various kinds of fluorescent substances which do not participate in the liquid phase reaction in the microchannel or are not essential even if they participate may be used. For example, rhodamine dye substance (Rhodamine dye substance) may be used. is there. Such a method of measuring the temperature of the liquid phase in the fine channel by detecting the emission intensity of the fluorescent substance is extremely useful for temperature measurement in a small space where the heat capacity is extremely small. Since it is non-contact, it does not affect the heating or cooling of the liquid phase, accurately measures the temperature, and appropriately controls the heating or cooling conditions of the liquid phase according to the measured temperature. It is possible to do.
図 1は、 このような非接触で蛍光強度の検出から温度測定するため のシステムを例示したものである。 この例においては、 マイクロチッ プ （ 1 ) に形成された微細流路 （2 ) における液相の温度を、 蛍光強 度の検出として測定可能としており、 また、 この例では、 液相の加熱 手段として、 I R (赤外線） レーザーの照射手段を例示してもいる。  FIG. 1 illustrates a system for measuring the temperature from the detection of the fluorescence intensity in a non-contact manner. In this example, the temperature of the liquid phase in the microchannel (2) formed in the microchip (1) can be measured as fluorescence intensity detection. In this example, the liquid phase heating means As an example, an IR (infrared) laser irradiation means is exemplified.
システム （装置） としては、 微細流路 （ 2 ) を有するマイクロチッ プ （ 1 ) の配置部とともに、 蛍光強度の検知部と、 この発光強度から 温度を算出する演算部とを備えている。 また、 図 1の例のように I R (赤外線） レーザー加熱手段を備えることによって、 温度条件を精度 良くコントロールすることのできる熱反応装置を構成することもでき る。 反応の検出には、 この出願の発明者らが確立した熱レ ズ顕微鏡の 手段等を採用することができる。 The system (equipment) includes a microchip (1) having a microchannel (2), a fluorescence intensity detection unit, and a calculation unit for calculating a temperature from the emission intensity. Also, by providing an IR (infrared) laser heating means as in the example in Fig. 1, it is possible to configure a thermal reaction device that can control temperature conditions with high accuracy. For the detection of the reaction, a means of a thermal lens microscope established by the inventors of the present application and the like can be employed.
< B >の接触方式による温度測定では、 マイクロチップの微細流路 内の液相に接触する温度検知手段が用いられる。 微細流路は、 たとえ ば深さが 5 0 0 m以下、 幅 3 0 0 以下と微細であることから、 このような微細流路内での液相に対して熱的な影響を及ぼすことがな く、 また必要とされる液相の流れに実際的な影響をほとんど与えるこ とがないように、 温度検知手段が使用される。  In the temperature measurement by the contact method <B>, a temperature detecting means that comes into contact with the liquid phase in the microchannel of the microchip is used. Since the fine channels are fine, for example, with a depth of 500 m or less and a width of 300 μm or less, they may have a thermal effect on the liquid phase in such fine channels. Temperature sensing means are used so as to have little or no practical effect on the required liquid phase flow.
このような温度検知手段としては各種の温度センサーが考慮される が、 この出願の発明においては、 その一部または全部がマイクロチッ プに埋設もしくは載置されるもの、 特に、 より具体的には、 熱電対を 好適に使用することができる。  Various temperature sensors are considered as such a temperature detecting means. In the invention of this application, a part or the whole of the temperature sensor is embedded or placed in a microchip, and more specifically, A thermocouple can be suitably used.
たとえば図 2は、 熱電対を温度検知手段とし、 微細流路 （ 2 ) に対 してその側部に形成した溝 （ 3 ) にこれを挿入配置して、 熱電対が液 相に接触するようにした例を示している。 写真は、 接触部 （4 ) を拡 大して例示したものである。 実際にも、 溝 （3 ) についてはドリル等 により形成することができ、 K—型熱電対等を使用することができる 。 図 2の例においては、 液相との直接接触による影響は実質的にほと んどないものとすることができる。 このことは、 D N A断片の P C R においても確認されている。  For example, Fig. 2 shows that a thermocouple is used as a temperature detecting means and inserted into a groove (3) formed on the side of the microchannel (2) so that the thermocouple contacts the liquid phase. The example shown in FIG. The photo shows an enlarged example of the contact part (4). Actually, the groove (3) can be formed by a drill or the like, and a K-type thermocouple or the like can be used. In the example of FIG. 2, the effect of direct contact with the liquid phase can be substantially negligible. This has also been confirmed in the PCR of the DNA fragment.
図 2に例示のように、 温度検知手段が組込まれたマイクロチップは 、 マイクロチップの構成そのものにおいて価値のあるものとなる。 赤外線レーザー （ I Rレーザー） の光熱効果を利用して液相を加熱 するこの出願の発明の方法と装置では、 流通条件下でマイクロチップ 上の酵素反応を迅速かつ局部的に制御することができる。 前記の非接 触での分光温度検知法を利用して、 温度の動的変化と立体的分布を測 定し、 その結果を数値シミュレーション分析の結果と比較すると、 た とえば、 この出願の発明の方法と装置では、 従来のシステムより 3 0 倍、 小型化した電熱熱サイクルより 3〜 6倍速い、 それぞれ 6 7で7 sおよび 5 3 t: s という超高速での昇温、 冷却速度で操作すること ができる。 赤外線レーザーを使う加熱法の特徵は、 電熱加熱によるチ ップを使つた既存のシステムと比べて、 加熱対象の大きさが著しく小 さく、 わずか 5 n Lであるという点にある。 熱容量が極めて小さい試 料を直接加熱することで迅速な加熱速度を実現することができ、 また 、 ガラス基板を使用することで、 これに熱を移すことで熱の除去を効 率的に行い、 冷却を迅速に行うことができる。 温度レベルの再現性は 、 滞在時間を 0 . 5秒より短くすることで確保される。 実際、 チップ 上の酵素反応は、 I Rレーザーによる光熱による定期的な加熱によつ て、 たとえば 0 . 6秒の時間分解能で制御することに成功している。 I Rダイォードレーザ一はコンパクトであるため、 システムを小型化 したデザィンに好適である。 As illustrated in FIG. 2, a microchip with a built-in temperature detecting means is valuable in the configuration of the microchip itself. The method and apparatus of the present invention, in which the liquid phase is heated using the photothermal effect of an infrared laser (IR laser), can rapidly and locally control an enzymatic reaction on a microchip under flow conditions. Non-contact The dynamic change and three-dimensional distribution of the temperature are measured by using the tactile spectral temperature detection method, and the results are compared with the results of numerical simulation analysis. Can operate at an ultra-high speed of 7s and 53t: s, respectively, 30 times faster than the conventional system and 3-6 times faster than the miniaturized electrothermal cycle. it can. A special feature of the heating method using an infrared laser is that the size of the object to be heated is extremely small, only 5 nL, as compared with the existing system using an electric heating chip. A rapid heating rate can be realized by directly heating a sample having an extremely small heat capacity, and by using a glass substrate, heat is efficiently removed by transferring heat to the glass substrate. Cooling can be performed quickly. Temperature level reproducibility is ensured by keeping the stay time less than 0.5 seconds. In fact, the enzymatic reaction on the chip has been successfully controlled with a time resolution of, for example, 0.6 seconds by means of periodic heating using photothermal heat with an IR laser. Since the IR diode laser is compact, it is suitable for a design with a smaller system.
赤外線 （ I R ) レーザーによる加熱は、 たとえば図 3に例示したよ うに、 微細流路に沿って、 多点加熱が適宜に、 しかも迅速な加熱、 冷 却のサイクルとして制御可能であるという特徵も有している。  As shown in Fig. 3, for example, heating with an infrared (IR) laser has the special feature that multipoint heating can be controlled as appropriate, and can be controlled as a rapid heating and cooling cycle along a fine channel. are doing.
そこで以下に実施例を示し、 さらに詳しくこの出願の発明について 説明する。 実 施 例  Then, an example is shown below and the invention of this application will be described in more detail. Example
実施例 1 Example 1
( A ) マイクロチップ 図 1および図 2に例示したように Y形接合点を有する微小チャンネ ルを公知のエッチング法によって作成した。 基板はガラスである。 微 小チヤンネルは幅が 2 50 zmで、 深さが 100 mである。 Y形接 合点からうしろの反応チヤンネルの長さは 4. 0 c mである。 (A) Microchip As illustrated in FIGS. 1 and 2, a minute channel having a Y-shaped junction was formed by a known etching method. The substrate is glass. The microchannel is 250 zm wide and 100 m deep. The length of the reaction channel behind the Y-shaped junction is 4.0 cm.
温度測定のために、 接合したチップの端からエッチングしたチャン ネルに対して直角方向に、 ダイヤモンドドリルでサイ ドチャンネルを 図 2の場合と同様に設けた。 サイ ドチャンネルは、 エッチングしたチ ャンネルと合流するところまで切削した。 交差点は入り口から 2 c m 下流にあるようにした。 全体としてエッチングした微小チャンネルの 幅は 250 m, 深さは 1 00 ^m、 そしてサイ ドチヤンネルの直径 は 500 mとした。  For temperature measurement, a side channel was provided by a diamond drill in a direction perpendicular to the channel etched from the end of the bonded chip in the same manner as in FIG. The side channel was cut until it merged with the etched channel. The intersection was located 2 cm downstream from the entrance. The overall width of the etched microchannel was 250 m, the depth was 100 ^ m, and the diameter of the side channel was 500 m.
(B) チップ上の化学反応  (B) Chemical reaction on the chip
生化学用の 4一アンチアミノピリン （4一 AAP) および N—ェチ ルー N— ( 2—ヒドロキシー 3—スルフォプロピル） 一m—トルイジ ン （TOOS) は同仁研究所 （熊本市） から購入した。 大根ペルォキ シダ一ゼ （HRP， EC 1. 1 1. 1. 7、 比活性度 200単位/ m g) および 35 %過酸化水素水 （電子工業用） は、 和光純薬 （大阪） から購入した。 すべての試薬は特に精製を行わずに使用した。 水は全 実験を通してすべて水精製システム （TW— 6 00 RU、. 野村ミクロ サイエンス、 神奈川） から得た超純粋水を使用した。 基質原液 （2 X 1 0 ~3M, 4一 AAPおよび TOOS ； 1 0''Μ Η202) はリン酸塩緩 衝液 （PB S, ρΗ 7. 4) によって調製した。 これらは冷蔵庫中、 1 0でで保存し、 実験前に適宜 PB Sで希釈して使用した。 HRP溶 液 （20単位 ZmL) は固体の酵素から毎日調製し、 403 nmで吸 光度をモニターして濃度を検定し、 必要に応じてさらに希釈した。 4一 AAPおよび TOO S作業用液の濃度範囲は、 2 X 10-4M、 H ？は1〜5単位/1!1 、 H202は 1〜5 X 1 0— 5Mとした。 使用直前 に HRPと 4—AAPとの等体積 （各 lmL) 混合液 （溶液 1) と、 TOO Sと H202との等体積混合液 （溶液 2) を調製した。 4-Antiaminopyrine (4-1 AAP) and N-ethyl N- (2-hydroxy-3-sulfopropyl) -1m-toluidine (TOOS) for biochemistry were purchased from Dojin Research Institute (Kumamoto City). . Radish peroxidase (HRP, EC 1.1.1.1.7, specific activity 200 units / mg) and 35% hydrogen peroxide solution (for electronic industry) were purchased from Wako Pure Chemical (Osaka). All reagents were used without any particular purification. All water used was ultra-pure water from a water purification system (TW-600 RU, Nomura Microscience, Kanagawa). Substrate stock solution (2 X 1 0 ~ 3 M , 4 one AAP and TOOS; 1 0''Μ Η 2 0 2 ) was prepared by phosphate buffer solution (PB S, ρΗ 7. 4) . These were stored at 10 in a refrigerator and diluted appropriately with PBS before use. HRP solutions (20 units ZmL) were prepared daily from the solid enzyme, monitored for absorbance at 403 nm, assayed for concentration, and further diluted as needed. 4 one AAP and TOO S concentration ranges of the working fluid, 2 X 10- 4 M, H ? 1 to 5 units / 1! 1, H 2 0 2 was 1~5 X 1 0- 5 M. Equal volumes (each LML) mixed solution of HRP and 4-AAP immediately before use and (solution 1) was equal volume mixed liquid of TOO S and H 2 0 2 (solution 2) was prepared.
液体試料の流量調節 （0. 1〜 10 1 分） は、 2本のシリンジ (lmL， 1 7 1 0 TLL, ゾヽミルトン社製、 Reno, N V) を備えた マイクロシリンジポンプ （KDS 200, KD Scientific社製， ポ ストン， マサチューセッツ州） で行った。 既に既報に書いたように、 シリンジに溶液 1および溶液 2を入れ、 溶融シリカ製毛細管と PTF Eコネクターを通して、 チップ上の Y形接合点上流の 2つの入り口か ら各溶液をマイクロチップに注入した。  To adjust the flow rate of the liquid sample (0.1 to 101 min), use a micro-syringe pump (KDS 200, KD) equipped with two syringes (1 mL, 1710 TLL, manufactured by Zodimil, Reno, NV). Scientific, Poston, Mass.). As described in the previous report, solution 1 and solution 2 were placed in a syringe, and each solution was injected into the microchip through two inlets upstream of the Y-shaped junction on the chip through a fused silica capillary and a PTFE connector. .
(C) 赤外線（IR)レーザーによる加熱  (C) Infrared (IR) laser heating
微小チャンネルの局所の温度を上げるには、 ダイオードレーザ一 （ λ c = 1472 nm, 150 mW; AF4A212P1, ANRITSU ， 神奈川） を使用して溶 媒を加熱した。 IRレーザ一ビームはレンズで収束させ、 ウェスト直径 が 150 Mのビームとした。 To raise the temperature of the micro-channel local, diode lasers one (λ c = 1472 nm, 150 mW; AF4A212P1, ANRITSU, Kanagawa) was heated Solvent using. One beam of the IR laser was converged by a lens to form a beam with a waist diameter of 150M.
プログラム化した加熱実験では、 デジタルからアナログに変換する 変換ボード （DAC ) を搭載したコンピュータで IRレーザーの出力を制御 した （PCI-6035, National Instruments, オースチン， テキサス州）。 加熱点は Y形接合点から下流に向かって 1.5cmの所に定めた。 反応媒 体は流通系で加熱した。 4-AAP -T00S-H202 -HRP 反応生成物は、 さら に 5 龍下流に設けた熱レンズ顕微鏡 （TLM ) の検出点に連続的に輸送 した。 In the programmed heating experiment, the output of the IR laser was controlled by a computer equipped with a digital-to-analog conversion board (DAC) (PCI-6035, National Instruments, Austin, TX). The heating point was set at 1.5 cm downstream from the Y-shaped junction. The reaction medium was heated in a flow system. 4-AAP -T00S-H 2 0 2 -HRP reaction product was continuously transported to the detection point of the thermal lens microscope provided 5 dragon downstream further (TLM).
(D) 熱レンズ顕微鏡（TLM) による検出  (D) Detection by thermal lens microscope (TLM)
着色反応生成物は、 発明者らによってその方法が確立された TLMで検 出した。 簡単に述べると、 514.5 ni, 200 mWで運転し、 1025 Hz で機械 的に切られる Ar+イオンレーザ一 （SHG-95, Lexel Laser Inc, フリー モント， カリフォルニア州） を励起レーザーとして使用した。 反応物質 は 515 nm付近に弱い吸収しか持たないが、 生成物はこの領域に強い吸 収を示す （Amax = 555 ， ε =3.2 x 104 Μ— 1 cm—1)。 プローブ用レ一 ザ一には He— Neレーザー （632.8 n , 5m ; Melles Griot,カールズパッ ド， カリフォルニア州） を使用した。 TLM信号はアナログ—デジタル変 換 （ADC ) ボードを使用し、 サンプリング時間 20ミリ秒でデジタル化し た。 データの取り込みと処理を行うプログラムは、 LabView ソフトゥェ ァ ，パッケージ （National Instrument ) を使って書いた。 The colored reaction product is analyzed by TLM, the method of which has been established by the inventors. Issued. Briefly, an Ar + ion laser (SHG-95, Lexel Laser Inc, Fremont, CA) operated at 514.5 ni, 200 mW, and mechanically cut at 1025 Hz was used as the excitation laser. The reactant has only a weak absorption around 515 nm, but the product shows a strong absorption in this region (A max = 555, ε = 3.2 x 104 cm- 1 cm- 1 ). A He—Ne laser (632.8 n, 5 m; Melles Griot, Carlspad, Calif.) Was used for the probe laser. The TLM signal was digitized using an analog-to-digital conversion (ADC) board with a sampling time of 20 ms. The data acquisition and processing program was written using LabView software and a package (National Instrument).
(E) 微小チャンネル内の温度測定  (E) Temperature measurement in a small channel
マイクロチップの環境温度は、 コントローラ （温度範囲— 25〜+99 で 、 精度お よ び安定性 ± 0.1 で ； PE-60 ， Linkam Scientific Instruments, Tadworth, U. K. ) を備えたペルチェ温度ステージによつ て保持した。 図 4に例示したように、 サイ ドチャンネルを持つチップ をステージ上に置いた。 チップ上の定常状態の温度は、 較正した 2本 の熱電対 （クロメル—アルメルタイプ、 導線先端直径 25 I ；安部実 装技術研究所 （Anbe Soldering Technologies Ltd. ) , 横浜） でモニタ した。 熱電対の 1本はペルチェステージの上部に、 残り 1本は 500 m のサイ ドチャンネルの内側に設置し、 エポキシ接着剤でシールした。 熱電対の先端は幹線チャンネル内に〜 100 m突き出した。 局部的な光 熱加熱効果を裂けるため、 先端は IR照射点から 1 cm上流にあるように した。 典型的には、 両センサの温度差が 0.2 でを超えないようにした。 ペルチェステージを備えたマイクロチップは、 IR照射光学系に対する マイクロチップの位置が乱れを与えないで液体の取扱いができるよう 、 しっかり安定させて取り付けた。 安定した熱接触が得られるようにThe ambient temperature of the microchip is controlled by a Peltier temperature stage equipped with a controller (temperature range-25 to +99, accuracy and stability ± 0.1; PE-60, Linkam Scientific Instruments, Tadworth, UK). Held. As shown in Fig. 4, a chip with side channels was placed on the stage. The steady-state temperature on the chip was monitored by two calibrated thermocouples (Chromel-Alumel type, wire tip diameter 25 I; Anbe Soldering Technologies Ltd., Yokohama). One thermocouple was placed above the Peltier stage and the other was placed inside a 500 m side channel and sealed with epoxy adhesive. The thermocouple tip protruded ~ 100 m into the main channel. The tip was 1 cm upstream from the IR irradiation point to break up the local heating effect. Typically, the temperature difference between the two sensors did not exceed 0.2. A microchip with a Peltier stage can handle liquids without disturbing the position of the microchip with respect to the IR irradiation optical system. , I installed it stably. To obtain stable thermal contact
、 2つの熱電対でマイクロチップのパルク温度を絶えずモニタした。 微小チャンネル内の温度変化と空間的な温度分布は蛍光クェンチ法 によってモニタした。 試料にはローダミン 3 B染料 （Rh- 3Bb、 Exciton 社 （ディ トン， オハイオ州） 製， ） の水溶液 （10— 5 M) を使用した。 蛍 光の励起には Ar+イオンレーザ一 （514.5 ni, 1025 Hz で切る） を使用 した。 図 4 に示すように、 ショー トカッ トフィル夕 （ λ 50¾=570 nm, Melles Griot) とバンドパス ' フィルタ （ λ c=595 ± 10nm, Mel les Gropt ) で濾光した蛍光は、 ロックイ ンアンプ （NF Electronic Instruments 、 横浜） と CCD ビデオカメラ （KY-F55B ， 日本ビクター株 式会社 （Victor Co. of Japan Ltd.) ， 横浜； 分解能 480 X360 画素 ) に接続したシリコン ' ホトダイオード （ET-2010, Electrooptics Technology, トラパーズシティ， ミシガン州） でモニタした。 ロックィ ンアンプの時定数は 12〜400 msの範囲で変えた。 蛍光信号は、 TLM検出 の場合と同じように ADC ポードでデジタル化しそれからコンピュータに 送った。 蛍光画像は、 ビデオ · フレームグラバー ·ポ一ドを搭載した PCで捕捉した。 The microchip's park temperature was constantly monitored with two thermocouples. Temperature change and spatial temperature distribution in the microchannel were monitored by fluorescence quenching. The samples were used rhodamine 3 B dye (Rh- 3Bb, Exciton, Inc. (Di tons, Ohio), manufactured by) an aqueous solution of (10- 5 M). An Ar + ion laser (514.5 ni, cut at 1025 Hz) was used to excite the fluorescence. As shown in Fig. 4, the fluorescence filtered by the short cut filter (λ 50¾ = 570 nm, Melles Griot) and the bandpass filter (λ c = 595 ± 10 nm, Mel les Gropt) is converted to the lock-in amplifier (NF Electronic Instruments, Yokohama) and a CCD video camera (KY-F55B, Victor Co. of Japan Ltd., Yokohama; resolution: 480 x 360 pixels) Silicon 'photodiode (ET-2010, Electrooptics Technology, (Pars City, Michigan). The time constant of the lock-in amplifier was changed in the range of 12 to 400 ms. The fluorescence signal was digitized at the ADC pod, as in the case of TLM detection, and then sent to a computer. Fluorescent images were captured on a PC equipped with a video frame grabber pod.
(F) 結果と考察  (F) Results and discussion
① まず、 Rh-3B 水溶液の螢光クェンチによって、 微細流路 （チャン ネル） 内の水が、 IRによって局所的に加熱されることを確認した。 図 4に示す装置で測定した結果、 Rh-3B の蛍光強度は溶液温度が高くなる につれて大きく減少することがわかった。 図 5は、 ペルチェステージ でマイクロチップのバルク環境温度を制御して得られた較正曲線を示 す。 この曲線が Ar+イオンレーザーの励起強度に左右されないよう、 蛍 光強度を 10でにおける強度に規格化した。 測定前にチップの温度を 3 分間平衡化させ、 それからサイドチャンネルに挿入した熱電対の読み の平均値を溶液温度として使用した。 このようにして熱電対の読みを モニターした結果、 溶液の温度は、 検討した流速範囲 （0〜5 cm/s ) で変化しないことがわかった。 水を満たした 100 m の微小チャンネル を通過する固有拡散時間はわずか 70 ms に過ぎないため、 溶液温度は周 囲の基板温度と平衡状態にあった。 固有時間に比べて溶液滞在時間は はるかに長いため （検討した最も速い線速度 5 cm/s でも 0. 5 s )、 流 通条件でさえこのような平衡状態が成立した。 (1) First, it was confirmed that the water in the microchannel was locally heated by IR due to the fluorescence quenching of the Rh-3B aqueous solution. As a result of measurement using the apparatus shown in Fig. 4, it was found that the fluorescence intensity of Rh-3B greatly decreased as the solution temperature increased. Figure 5 shows the calibration curve obtained by controlling the microchip bulk ambient temperature at the Peltier stage. The fluorescence intensity was normalized to an intensity at 10 so that this curve was not affected by the excitation intensity of the Ar + ion laser. Set the tip temperature to 3 before measurement. Equilibrate for one minute and then use the average reading of the thermocouple inserted in the side channel as the solution temperature. Monitoring the thermocouple readings in this manner showed that the solution temperature did not change over the flow rate range studied (0-5 cm / s). The solution temperature was in equilibrium with the surrounding substrate temperature, as the intrinsic diffusion time through a 100 m water-filled microchannel was only 70 ms. Since the solution residence time is much longer than the intrinsic time (0.5 s even at the fastest linear velocity of 5 cm / s studied), such an equilibrium state was established even under flowing conditions.
感度は温度の上昇と共に低下するものの、 検討した温度範囲 （10〜 90¾ ) で観察された強度の低下は 5倍を超えた。 蛍光強度が低下する 原因は、 染料分子の励起状態での寿命が短くなるためではないかと思 われる。 事実、 図 5に示した蛍光クェンチの結果は、 公知の文献に記 載されている、 よく似た蛍光性分子 Rh- Bの、 より狭い温度範囲 14〜63 でにおける水溶液データと定量的に一致した。  Although the sensitivity decreased with increasing temperature, the decrease in intensity observed over the temperature range studied (10-90¾) was more than 5-fold. The cause of the decrease in fluorescence intensity is probably due to the shortened life of the dye molecules in the excited state. In fact, the results of the fluorescence quench shown in Figure 5 are in quantitative agreement with the aqueous solution data of a similar fluorescent molecule, Rh-B, in a narrower temperature range of 14 to 63, as described in the known literature. did.
異なる日に作成した規格化した較正曲線は図 3に示したとおり非常 に安定しており、 RSD は 3 % であることが確認された。 蛍光クェンチ法 による温度測定の精度は ± 0. 5 でであった。 較正曲線は、 検討した範囲 で溶液の流速には無関係であった。  The normalized calibration curves generated on different days were very stable as shown in Figure 3, confirming an RSD of 3%. The accuracy of the temperature measurement by the fluorescence quenching method was ± 0.5. The calibration curve was independent of solution flow rate over the range studied.
IR出力が一定の条件で温度上昇 Δ Tの初期溶液温度 Tへの依存性を 調べた結果によれば、 Δ Τは、 比例定数 Δ Τ / T =—0. 07での関係によ つて、 Τが高くなるにつれて低下した。 このことは、 1470 nm における 水のモル吸光係数が温度の上昇と共に低下するという公知の報告と一 致した。 水のこうした特性のために、 初期温度が異なると、 Δ Τ対 IR レーザー出力の依存性が、 高い IR出力において、 わずかに直線から外 れた。 しかし、 いずれにしても所与の初期温度と IRレーザ一出力にお いて、 Δ Τの再現性はかなり良好であった （RSD = 4%)。 According to the results of examining the dependence of the temperature rise ΔT on the initial solution temperature T under the condition that the IR output is constant, Δ Τ is expressed by the relation of the proportionality constant Δ Τ / T = -0.07 It decreased as Τ increased. This is consistent with known reports that the molar extinction coefficient of water at 1470 nm decreases with increasing temperature. Due to these properties of water, at different initial temperatures, the dependence of ΔΤ vs. IR laser power deviated slightly at high IR powers. However, in any case, given the initial temperature and the IR laser power The reproducibility of ΔΤ was fairly good (RSD = 4%).
② 蛍光顕微鏡画像を見ると、 IRレーザーをチャンネルに照射した ときに局部的に強度が低下していることがはっきりと認められた。 溶 液の流速を変えたときの温度分布画像を図 6に示す。 この画像は長さ 1 蘭 、 幅 250 m の微小チャンネルを上から見たものである。 各画像 の頂部と底部の境が、 水とガラスの界面に相当する。 マイクロチップ はペルチェステージによって一定の温度に保たれ、 IRレーザーはチヤ ンネルの所定部分のみを加熱した。 もとの強度画像は、 10でで得られ た画像で規格化され、 各画素に対して得られた蛍光強度比は図 5の較 正曲線を使ってその温度に変換した。  ② The fluorescence microscope image clearly showed that the intensity was locally reduced when the channel was irradiated with the IR laser. Figure 6 shows the temperature distribution image when the flow rate of the solution was changed. This image is a top view of a small channel, 1 orchid long and 250 m wide. The boundary between the top and bottom of each image corresponds to the interface between water and glass. The microchip was held at a constant temperature by a Peltier stage, and the IR laser heated only a certain part of the channel. The original intensity image was normalized with the image obtained in step 10, and the fluorescence intensity ratio obtained for each pixel was converted to its temperature using the calibration curve in FIG.
得られた画像は、 チャンネルに沿う空間温度分布が溶液の流速の影 響を、 明確に反映していることを示した。 温度の局部化は低流速に対 して IR照射体積と密接に関係し、 流速が速くなるほど局部は広がり下 流側に移動した。 微小チャンネルに沿った空間分布が均一でないこと は画像からわかる。 これには 2つの理由があった ：顕微鏡対物レンズ の焦点面での IR強度は空間ガウス分布をとること、 水一ガラス界面に おける基板パルクへの熱移動は効果的に行われること、 である。 全体 として、 検討した流速範囲に対して、 水一ガラス界面近くの温度は IR ビーム中心部の最高温度の 40〜 50% であった。  The images obtained showed that the spatial temperature distribution along the channel clearly reflected the effect of the solution flow rate. The localization of temperature was closely related to the IR irradiation volume for low flow rates, and the higher the flow rate, the more the local area spread and moved downstream. The images show that the spatial distribution along the microchannel is not uniform. There were two reasons for this: the IR intensity at the focal plane of the microscope objective had a spatial Gaussian distribution, and the heat transfer to the substrate parc at the water-glass interface was effective. . Overall, the temperature near the water-glass interface was 40-50% of the maximum temperature in the center of the IR beam for the range of flow rates studied.
この観察は、 数値シミュレーションの結果と一致した。 事実、 画像 から、 温度分布をチャンネルに沿って詳しく調べてみると、 計算され た依存性との間に相関性のあることがわかった。 数値シミュレ一ショ ンにおいて、 ただ 1つのパラメータ、 熱源の出力密度を合わせること により、 実験データと計算データとが定量的に一致した。 このパラメ 一夕の調節は、 界面での IR照射の散乱と反射、 およびチップの真の 3 D形状を考慮するためのものである。 This observation was consistent with the results of the numerical simulation. In fact, the images showed that a closer examination of the temperature distribution along the channel showed a correlation with the calculated dependencies. By combining only one parameter, the power density of the heat source, in the numerical simulation, the experimental data and the calculated data were quantitatively matched. The adjustment of this parameter overnight is due to the scattering and reflection of IR radiation at the interface and the true 3 This is for considering the D shape.
上述の一致によって、 ガラス基板への移動が主要な熱の散逸経路で あることを確認することが可能となった。 すなわち、 低流速における 拡散による熱移動と、 高流速で水が流れる場合は、 さらに対流による 熱の除去が加わり、 この熱移動は促進されることである。  The above agreement made it possible to confirm that transfer to the glass substrate was a major heat dissipation path. In other words, heat transfer by diffusion at low flow velocity and removal of heat by convection when water flows at high flow velocity are added, and this heat transfer is promoted.
③ 温度の動的な変化特性を実験的に得るために、 ロックインアン プによる検知スギ一ムを使用し、 IRビーム中心部の単一スポッ ト測定 に蛍光クェンチ法を使用した。 蛍光クェンチ法によって測定した温度 の迅速サイク リ ングを図 7 に示す。 10 cyc l es. 3. 5s/cyc l e, dwe l l t iie : l. 18s per s tep.である。 IRレーザ一出力をプログラミングして 3 種類のレベルサイクルの各サイクルを発生させた。 最初のレベル は IRレーザーのゼロ出力 （ O mW) に相当し、 最大レベル T 3は IRレーザ —の最大出力 （ 1 0 O mW) に相当する。 それに対して中間レベル T 2 は IRレーザーの出力を変えること（10, 20, 30, 40, 60, 70， 8 OmW)によって 調節した。 観測されたサイクル間の温度再現性は、 拡散による熱散逸 経路に固有の再現性から生じ、 後者の再現性はペルチェステージによ つて一定温度に保たれたガラス基板の熱伝導性の高さによってもたら された。 昇温も冷却も平衡温度に到達した。 過不足の問題が完全に排 除された。 結果として、 このシステムは、 十分な分解能で滞在時間が 1 sより短い温度サイクルを生み出すことができる。 (3) In order to experimentally obtain the dynamic change characteristics of temperature, a detection scheme using a lock-in amplifier was used, and a fluorescence quench method was used for single spot measurement at the center of the IR beam. Figure 7 shows the rapid cycling of the temperature measured by the fluorescence quenching method. 10 cyc les. 3.5 s / cyc le, dwe llt iie: l. 18 s per s tep. One IR laser output was programmed to generate each of the three level cycles. The first level corresponds to the zero power (O mW) of the IR laser, and the maximum level T 3 corresponds to the maximum power (10 O mW) of the IR laser. On the other hand, the intermediate level T 2 was adjusted by changing the output of the IR laser (10, 20, 30, 40, 60, 70, 8 OmW). The observed temperature reproducibility between cycles arises from the reproducibility inherent in the heat dissipation path due to diffusion, and the latter reproducibility depends on the high thermal conductivity of the glass substrate maintained at a constant temperature by the Peltier stage. Brought. Both the temperature rise and the cooling reached the equilibrium temperature. The problem of excess and deficiency has been completely eliminated. As a result, this system can produce thermal cycles with sufficient resolution and residence times of less than 1 s.
温度の上昇下降速度を詳細に検査するため、 図 8 bは、 実験的に求 めた 1 s間の IR照射の時間推移を示す。 比較のため計算で求めた 1 s 間の照射の推移もあげてある。 図から直線近似で得られた実験による 昇温速度は 67で/ s、 冷却速度は 53で/ sであった。 昇温に対しては 100 レベルまで上がるに要する時間 （0. 4s)、 冷却に対しては レベルまで 下がるに要する時間 （0. 51 s ) で温度差 Δ Τ = 27Χ:を割った。 実験によ つて観測された IRレーザービーム中心部の昇温冷却速度は、 検討した 条件の範囲内では初期温度および流速には無関係であり、 数値解析の 結果と良く一致した。 To examine the temperature rise and fall rates in detail, Figure 8b shows the experimentally determined time course of IR irradiation for 1 s. For comparison, the transition of irradiation for 1 s calculated is also given. In the experiment obtained by linear approximation from the figure, the heating rate was 67 / s and the cooling rate was 53 / s. Time required to raise to 100 level for heating (0.4s), level for cooling The temperature difference Δ Δ = 27Χ: was divided by the time required to decrease (0.51 s). The heating / cooling rate of the center of the IR laser beam observed in the experiment was independent of the initial temperature and flow velocity within the range of the studied conditions, and was in good agreement with the results of numerical analysis.
この出願の発明の非接触 IRレーザー加熱法によると、 観測された極 めて迅速な昇温、 冷却速度は、 他の加熱デバイスに対して報告されて いるものをしのいでいる。 ジュール効果に基づく接触式電熱ヒ一夕を 備えたマイクロチップの場合、 30 Vsおよび 4 で/ sのそれぞれ昇温速度 および冷却速度が実現された。 微小チャンネル内の電解質を電熱で直 接加熱する方法によって、 昇温速度も冷却速度も共に最高 20 まで上 げることができたが、 加熱される体積は、 依然として数 であった。 直接加熱される体積をさらに〜 150nL 程度まで減らすと、 60で/ sおよび 20で/ sのそれぞれ昇温速度および冷却速度が得られた。 迅速な昇温冷 却速度は、 加熱される体積が 5 nLであって熱容量が 2 X 10—5J/K と小さ いこと、 そして微小チャンネルの表面積対体積の比が 280 cm—1で大きい ことによって実現されたものである。 開始温度から高い温度への遷移 も、 また逆の遷移もその実現に 1 s もかからないため、 チップ上の迅 速な温度制御が可能になる。 送風機や圧縮空気を送るシステムを反復 的に使用しなくても、 むしろチップ全体は一定の温度に保ったままガ ラス基板への自然な熱移動によって、 迅速な冷却が可能であることは 注目に値する。 According to the non-contact IR laser heating method of the invention of this application, the extremely rapid heating and cooling rates observed surpass those reported for other heating devices. For microchips equipped with a contact-type electrothermal heater based on the Joule effect, heating and cooling rates of 30 Vs and 4 / s, respectively, were achieved. Direct heating of the electrolyte in the microchannels by electroheating could raise both the heating rate and the cooling rate up to a maximum of 20, but the volume to be heated was still a number. Further reducing the directly heated volume to about ~ 150 nL provided heating and cooling rates of 60 / s and 20 / s, respectively. Rapid NoboriAtsushihiya却速degree is greater in the heated the volume 5 nL an even and small and thermal capacity 2 X 10- 5 J / K Ikoto and small surface to volume ratio of the channel is 280 cm- 1, It was achieved by doing so. The transition from the starting temperature to the higher temperature, and vice versa, takes less than 1 s to achieve, enabling rapid temperature control on the chip. It should be noted that rapid cooling is possible by using natural heat transfer to the glass substrate while maintaining the entire chip at a constant temperature, without using a blower or a system for sending compressed air repeatedly. Deserve.
④ 前記のチップ上での化学反応では、 微小チャンネル内を流れる溶 液の体積がきわめて小さい、 IRレーザー加熱デバイスを、 流通条件下 での化学反応の迅速な温度制御に応用した。 微小チャンネル内の IRレ 一ザ—ビームの位置は、 Rh-3B 溶液の蛍光を見ながら調節した。 シリン ジ溶液を変えても IRレーザーの位置に支障は発生しなかった。 加熱の 効率は、 流す Rh-3B 溶液を逐次取り替えることによって検査した。 した がって、 8 : 2 水—エタノール混合物で 3回 （ l mL)、 それから純水 （ 1 mL) でマイクロチップを洗浄したのち、 チップ中で酵素反応を行った 酵素反応の速度は温度が高くなるにつれて速くなるため、 ペルチェ ステージを 10 に冷却して初期速度を低く保った。 反応条件は、 最適 となるように酵素濃度と流速によって調節し、 IRレーザー加熱中に発 生する反応生成物プラグのきれいな TLM検出を確保した。 要点は、 局部 加熱に対する感度を十分維持しながら、 IRを照射しない時は、 生成物 の蓄積のパックグラウンドをできるだけ小さくすることであった。 最 適条件として、 HRP 濃度を 2. 5 単位/ mL (溶液 1 )、 H 20 2濃度を 10—5 M (溶液 2 )、 流速を 5 17分 （線速度 6. 7 mm/s) とした。 予備実験から 、 酵素溶液の原液に IRを連続照射したあとでも HRP 活性度は変わらない ことが確認された。 で は In the above-mentioned chemical reaction on the chip, an IR laser heating device, in which the volume of the solution flowing in the microchannel is extremely small, was applied to rapid temperature control of the chemical reaction under flow conditions. IR Les monodentate within microchannel - position of the beam, R h - was adjusted while observing the fluorescence of 3B solution. Shirin Changing the solution did not affect the position of the IR laser. Heating efficiency was checked by successively changing the flowing Rh-3B solution. Therefore, the microchip was washed three times with an 8: 2 water-ethanol mixture (1 mL) and then with pure water (1 mL), and the enzymatic reaction was performed in the chip. The higher the speed, the faster the speed, so the Peltier stage was cooled to 10 to keep the initial speed low. Reaction conditions were adjusted for optimal enzyme concentration and flow rate to ensure clean TLM detection of reaction product plugs generated during IR laser heating. The point was to keep the background of product accumulation as low as possible when not irradiating while maintaining sufficient sensitivity to local heating. As optimal conditions, the HRP concentration 2.5 Units / mL (solution 1), H 2 0 2 concentration 10- 5 M (solution 2), a flow rate of 5 17 minutes (the linear velocity 6. 7 mm / s) did. From preliminary experiments, it was confirmed that the HRP activity did not change even after continuous irradiation of the stock solution of the enzyme solution with IR.
流通条件下で行った IRレーザ一加熱によるチップ上の反応の制御に 対する代表的な結果を図 9に示す。 この実験中 IRレーザーは定期的に 運転した。 レーザ一は、 指示された時間に対して 50% のデューティサイ クルでオン ·オフした。 時間的な推移から、 TLM信号は IR照射のそれと 関連した規則的なパターンに従うことがわかった。  Figure 9 shows typical results for controlling the reaction on the chip by IR laser heating under flow conditions. During this experiment, the IR laser was operated regularly. The laser was turned on and off with a 50% duty cycle for the indicated time. The time course showed that the TLM signal followed a regular pattern related to that of IR irradiation.
1 sより短い IR照射時間で反応の迅速な制御データを明らかにする ため、 TLM信号を高速でフーリエ変換 （FFT ) した。 TLM 信号から得ら れる規格化 FFT スぺクトルを図 1 0に示す。 このスぺクトルは雑音レべ ルを超えたシャープな特徴的な周波数を示した。 I s および 0. 6 s の場 合、 おそらくその周波数で雑音の寄与が増すためと思われるいくつか の高調波が観測された。 特徴的な周波数は、 使用される IR照射時間と 一致した。 このことは、 反応速度が IR照射によって外部から制御され ることを裏づけている。 The TLM signal was subjected to a fast Fourier transform (FFT) to reveal rapid control data of the reaction with an IR irradiation time shorter than 1 s. Figure 10 shows the normalized FFT spectrum obtained from the TLM signal. This spectrum showed a sharp characteristic frequency exceeding the noise level. For I s and 0.6 s, some are probably due to the increased noise contribution at that frequency. Harmonics were observed. The characteristic frequency was consistent with the IR irradiation time used. This confirms that the reaction rate is externally controlled by IR irradiation.
実施例 2 Example 2
図 1 1は、 実施例 1 と同様にして、 45サイクル P C Rの場合の口 —ダミン一 3 Bの蛍光強度検知のプロファイルを例示したものである 。 サイクルは、  FIG. 11 exemplifies the profile of fluorescence intensity detection of mouth-damine-13B in the case of 45 cycles of PCR in the same manner as in Example 1. The cycle is
I R= 1 2 OmW, 9 5 - 2 s  I R = 1 2 OmW, 9 5-2 s
I R= OmW, 6 8で— 6 s  I R = OmW, 6 8— 6 s
の加熱、 冷却のサイクルを示したものである。 This shows the cycle of heating and cooling of the steel.
実施例 3 Example 3
図 2および図 4に例示したマイクロチップ内に設けた熱電対による 温度検知手段を用いて、 DNA meltingの温度変化を観察した。  The temperature change of DNA melting was observed using a temperature detecting means by a thermocouple provided in the microchip illustrated in FIGS. 2 and 4.
d s一 DNA特異染料 S YBER-Greenlで標識された P CR増 幅後の DNA断片を微小チャンネル内に導入し、 外部加熱手段によつ て 60から 9 8でに加熱した。 昇温速度は 0. 2で/ sとした。  The PCR amplified DNA fragment labeled with the ds-DNA specific dye S YBER-Greenl was introduced into the microchannel, and heated to 60 to 98 by external heating means. The heating rate was 0.2 / s.
A r+イオンレーザー （4 8 8 nm、 1 0 0 mW) を蛍光励起源とし て用いた。  An Ar + ion laser (488 nm, 100 mW) was used as a fluorescence excitation source.
図 1 2は、 DNA meltingの結果を示したものである。 実線は 5 00 b pの λ—ファージ D NA断片の場合を、 破線は N T C (negative template control) 試料の場合を示している。  Figure 12 shows the results of DNA melting. The solid line shows the case of the 500 bp λ-phage DNA fragment, and the broken line shows the case of the NTC (negative template control) sample.
温度検知手段としてのチップ埋込み型の熱電対が有効であることが 確認された。 産業上の利用可能性 It was confirmed that a thermocouple of embedded type as a temperature detecting means was effective. Industrial applicability
以上詳しく説明したとおり、 この出願の発明によって、 マイクロチ ップの微細流路内の液相温度を精度よく測定することができ、 また迅 速に、 かつ適切に温度制御することのできる新しい技術手段を提供す ることができる。  As described in detail above, according to the invention of this application, a new technical means that can accurately measure the liquidus temperature in a microchannel of a microchip and can quickly and appropriately control the temperature. Can be provided.

Claims

請求の範囲 The scope of the claims
1 . マイクロチップ微細流路内の液相温度を蛍光物質からの発光強 度の検知によって非接触で測定することを特徴とするマイクロチップ 微細流路内液相の温度測定方法。 1. A method for measuring the temperature of the liquid phase in a microchip microchannel, wherein the liquid phase temperature in the microchip microchannel is measured in a non-contact manner by detecting the emission intensity from a fluorescent substance.
2 . 蛍光物質がローダミン色素物質であることを特徴とする請求項 1のマイクロチップ微細流路内液相の温度測定方法。  2. The method according to claim 1, wherein the fluorescent substance is a rhodamine dye substance.
3 . マイクロチップ微細流路内の液相温度を、 微細流路内の液相に 接触させた温度検知手段により測定することを特徴とするマイクロチ ップ微細流路内液相の温度測定方法。 3. A method for measuring the temperature of a liquid phase in a microchip microchannel, wherein the liquid phase temperature in the microchip microchannel is measured by a temperature detecting means in contact with the liquid phase in the microchannel.
4 . 温度検知手段はその一部または全部がマイクロチップに埋設も しくは載置されていることを特徵とする請求項 3のマイクロチップ流 路内液相の温度測定方法。  4. The method for measuring the temperature of a liquid phase in a microchip channel according to claim 3, wherein a part or all of the temperature detecting means is embedded or mounted on the microchip.
5 . 温度検知手段が熱電対であることを特徴とする請求項 3または 4のマイクロチップ微細流路内液相の温度測定方法。  5. The method for measuring the temperature of a liquid phase in a microchip microchannel according to claim 3, wherein the temperature detecting means is a thermocouple.
6 . 微細流路に対してその側部に形成した微細溝に挿入配置した熱 電対により温度を測定することを特徵とする請求項 5のマイクロチッ プ微細流路内液相の温度測定方法。  6. The method for measuring the temperature of a liquid phase in a microchip microchannel according to claim 5, wherein the temperature is measured by a thermocouple inserted into a microgroove formed on a side of the microchannel. .
7 . マイクロチップ微細流路内の液相を赤外線 （ I R ) レーザ一の 照射によって加熱して液相温度を制御することを特徴とするマイク口 チップ微細流路内液相の温度制御方法。  7. A method for controlling the temperature of a liquid phase in a microchip microchip microchannel, wherein the liquid phase temperature in the microchip microchannel is controlled by heating the liquid phase in the microchip microchannel by irradiation of an infrared (IR) laser.
8 . 微細流路を有するマイクロチップの配置部とともに、 微細流路 内の蛍光物質からの発光強度の検知部と、 この発光強度から微細流路 内液相の温度を算出する演算部とを備えていることを特徴とするマイ クロチップ微細流路内液相の温度測定装置。 8. Equipped with a microchip placement part with a microchannel, a detection unit for the emission intensity from the fluorescent substance in the microchannel, and a calculation unit that calculates the temperature of the liquid phase in the microchannel from the emission intensity. A temperature measuring device for a liquid phase in a microchip microchannel.
9 . 微細流路を有するマイクロチップ配置部とともに、 マイクロチ ップ微細流路内の液相に接触する温度検知手段を備えていることを特 徵とするマイクロチップ微細流路内液相の温度測定装置。 9. Temperature measurement of the liquid phase in the microchip microchannel, characterized by comprising a temperature detection means that comes into contact with the liquid phase in the microchip microchannel, in addition to the microchip disposition section having the microchannel. apparatus.
1 0 . 請求項 8または 9の温度測定装置が、 マイクロチップ微細流路 内液相の加熱手段とともに備えられていることを特徴とするマイク口 チップ熱反応装置。  10. A microphone-tip chip thermal reaction device, wherein the temperature measurement device according to claim 8 or 9 is provided together with a heating means for a liquid phase in a microchip microchannel.
1 1 . 加熱手段が赤外線レーザー照射手段であることを特徴とする請 求項 1 0の熱反応装置。  11. The thermal reactor according to claim 10, wherein the heating means is an infrared laser irradiation means.
1 2 . 微細流路とともに、 微細流路内の液相に接触する温度検知手段 が備えられていることを特徴とするマイクロチップ。  1 2. A microchip characterized by being provided with a temperature detecting means for contacting a liquid phase in the fine channel together with the fine channel.
1 3 . 温度検知手段が熱電対であることを特徴とする請求項 1 2のマ イク口チップ。  13. The micro mouth tip according to claim 12, wherein the temperature detecting means is a thermocouple.
PCT/JP2001/010450 2001-05-07 2001-11-29 Methods for measuring and controlling the temperature of liquid phase in micro passage of microchip, device for the methods, and microchip WO2002090912A1 (en)

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