WO2002090912A1

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

Info

Publication number: WO2002090912A1
Application number: PCT/JP2001/010450
Authority: WO
Inventors: Takehiko Kitamori; Maxim Slyadnev; Manabu Tokeshi; Akihide Hibara; Yukio Kin
Original assignee: Kanagawa Academy Of Science And Technology
Priority date: 2001-05-07
Filing date: 2001-11-29
Publication date: 2002-11-14
Also published as: JP4166091B2; JPWO2002090912A1

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

Description Method for measuring and controlling the temperature of the liquid phase in the microchip microchannel,

Equipment and microchip for that purpose

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 Background technology

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.

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.

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.

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.

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.

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.

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

This application provides the invention as follows to solve the above problems.

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. 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. 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. 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. 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. 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. 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. 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. 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.

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.

11. The thermal reactor according to the item 10, wherein the heating means is an infrared laser irradiation means.

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.

13. The microchip as described in 12 above, wherein the temperature detecting means is a thermocouple. BRIEF DESCRIPTION OF THE FIGURES

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. BEST MODE FOR CARRYING OUT THE INVENTION

First, in the invention of this application, a method for measuring a liquidus temperature in a microchannel of a microchip is as follows.

<A> Non-contact method for optically measuring liquidus temperature

<B> Method of measuring liquid phase temperature by direct contact with liquid phase

The two methods are provided.

And, in the invention of this application, 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

I will provide a.

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.

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.

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.

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.

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.

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.

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.

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.

Then, an example is shown below and the invention of this application will be described in more detail. Example

Example 1

(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.

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) Chemical reaction on the chip

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.

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) Infrared (IR) laser heating

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.

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) Detection by thermal lens microscope (TLM)

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) Temperature measurement in a small channel

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. 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) Results and discussion

(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.

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.

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.

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 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.

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.

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) 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.

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.

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.

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.

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.

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.

Example 2

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

This shows the cycle of heating and cooling of the steel.

Example 3

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.

An Ar + ion laser (488 nm, 100 mW) was used as a fluorescence excitation source.

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.

It was confirmed that a thermocouple of embedded type as a temperature detecting means was effective. Industrial applicability

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

The scope of the claims

2. The method according to claim 1, wherein the fluorescent substance is a rhodamine dye substance.

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. 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. 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. 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.

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.

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.

11. The thermal reactor according to claim 10, wherein the heating means is an infrared laser irradiation means.

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.

13. The micro mouth tip according to claim 12, wherein the temperature detecting means is a thermocouple.