JP6631867B2 - Method for measuring temperature of liquid in microchannel - Google Patents

Description

  The present disclosure relates to a method for measuring the temperature of a liquid in a microchannel. More specifically, the present invention relates to a thermal reactor in a microchannel chip, particularly to a temperature measuring method in a polymerase chain reaction (PCR) step used in a gene amplification step.

  For measuring the temperature of a liquid in a microchannel, there is disclosed a method of arranging a metal thin film serving as a thermocouple in the channel to measure the temperature (for example, see Patent Document 1).

  Further, as a method for measuring the temperature of a fluid in a non-contact manner, a measurement method utilizing the temperature dependence of the fluorescence intensity of a fluid containing a fluorescent dye has been disclosed (for example, see Non-Patent Document 1). In this measurement method, the molecular beacon-type molecular probe changes its binding state in the molecule due to a change in temperature, so that the fluorescence intensity changes. In a proton exchange type molecular probe, the fluorescent dye undergoes proton exchange due to a temperature change, and its quantum efficiency changes, so that the fluorescence intensity changes. In this measuring method, the temperature of the fluid can be estimated by measuring the fluorescence intensity.

JP 2006-130599 A

T. Barilero, T.W. Le Saux, C.I. Gosse, and L.M. Julien, "Fluorescent Thermometers for Dual-Emission-Wavelength Measurements: Molecular Engineering and Application to Thermal Imaging in the Future. Chem. 2009, 81, 7988-8000

  However, the method of arranging a metal thin film in a microchannel as described above has a problem that a manufacturing process is complicated and a special manufacturing apparatus is required.

  In addition, as described above, the temperature measurement method using the emission intensity of the fluorescent dye requires expensive optical equipment including an excitation light having a specific wavelength (for example, laser light) and a highly sensitive image sensor for detecting fluorescence. In addition, there is a problem that the device configuration is complicated.

  An object of the present disclosure is to provide a method that can easily measure the temperature of a liquid in a microchannel of a microchannel chip without going through a complicated manufacturing process and without introducing expensive optical equipment. And

The method for measuring the temperature of the liquid in the microchannel according to the present disclosure includes receiving light from a liquid containing cresol red in the microchannel,
Measuring the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less for the received light;
Calculating the temperature of the liquid in the microchannel based on the measured light intensity;
including.

  According to the method for measuring the temperature of a liquid in a microchannel according to the present disclosure, the temperature of the liquid in the microchannel can be easily measured without going through complicated manufacturing steps and without introducing expensive optical equipment. Can be measured.

FIG. 1 is a schematic diagram illustrating a configuration of a temperature measurement system including a device for measuring a temperature of a liquid in a microchannel according to a first embodiment. It is a top view which shows an example of a microchannel chip. FIG. 2B is a cross-sectional view illustrating a cross-sectional structure viewed from the AA direction in FIG. 2A. It is another example of the temperature reference in which the absorbance and the temperature were associated. It is another example of a temperature reference in which light intensity and temperature are associated. FIG. 3 is a block diagram illustrating an example of a physical configuration of a liquid temperature calculation unit. 6 is a graph showing the relationship between the wavelength of light transmitted through a liquid to which cresol red as a dye material is added and the absorptance. 5 is a graph showing the relationship between the wavelength of light transmitted through a liquid to which xenon cyanol, which is a dye material, and the absorptance are shown. 4 is a graph showing the relationship between the wavelength of light transmitted through a liquid to which bromophenol blue as a dye material is added and the absorptance. 5 is a graph showing a relationship between an absorption rate and a wavelength of light transmitted through a liquid to which orange G as a coloring material is added. 4 is a graph showing the relationship between the absorptance of light of a predetermined wavelength and the temperature for each liquid to which four types of dye materials are added in Example 1. 5 is an example of a flowchart of a temperature measuring method for outputting a temperature using the measured light intensity and a temperature reference. It is an example of the flowchart of the temperature measurement method of outputting a temperature using the calculated absorbance and the temperature reference. FIG. 7 is a schematic diagram illustrating a configuration of a temperature measurement system including a device for measuring a temperature of a liquid in a microchannel according to a second embodiment. 9 is a grayscale drawing of an image obtained by imaging a liquid containing cresol red in a microchannel with a solid-state imaging device of a temperature measuring device according to Embodiment 2 and passing through a red filter at 25 ° C. 10 is a grayscale drawing of an image obtained by imaging a liquid containing cresol red in a microchannel with a solid-state imaging device of the temperature measurement device according to Embodiment 2 and passing through a red filter at 60 ° C. 9 is a grayscale drawing of an image obtained by imaging a liquid containing cresol red in a microchannel with a solid-state imaging device of a temperature measuring device according to Embodiment 2 and passing through a red filter at 68 ° C. 10 is a grayscale drawing of an image obtained by imaging a liquid containing cresol red in a microchannel with a solid-state imaging device of a temperature measurement device according to Embodiment 2 and passing through a red filter at 98 ° C. 9 is a graph showing a relationship between a detection rate and a temperature in the temperature measuring method according to the second embodiment. 9 is a gray scale drawing of an electrophoresis photograph of an amplification product subjected to PCR using the temperature measurement method of Example 3 and an amplification product of Comparative Example.

The method for measuring the temperature of the liquid in the microchannel according to the first aspect includes the steps of receiving light from a liquid containing cresol red in the microchannel,
Measuring the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less for the received light;
Calculating the temperature of the liquid in the microchannel based on the measured light intensity;
including.
With the above configuration, the temperature of the liquid in the microchannel in the microchannel chip can be accurately measured without going through complicated manufacturing steps and without introducing expensive optical equipment. This makes it possible to accurately control the temperature of the polymerase chain reaction (PCR) in which the heating step and the cooling step are repeated.

  In the method for measuring the temperature of a liquid in a microchannel according to a second aspect, in the first aspect, in the step of calculating the temperature of the liquid, the absorbance is calculated from the measured light intensity, and the calculated absorbance is used. May be used to calculate the temperature of the liquid in the microchannel.

In the method for measuring the temperature of a liquid in a microchannel according to a third aspect, in the first aspect, the step of measuring the intensity of the light of the at least one wavelength may be performed in a wavelength range of 340 nm or more and less than 470 nm. Measure the light intensity of the wavelength above the point,
In the step of calculating the temperature of the liquid in the microchannel, the temperature of the liquid in the microchannel is determined based on the sum or average of the light intensities of the two or more wavelengths in the wavelength range of 340 nm or more and less than 470 nm. It may be calculated.

In the method for measuring the temperature of a liquid in a microchannel according to a fourth aspect, in the first aspect, the step of measuring the intensity of the light of at least one wavelength may be performed in a wavelength range of more than 470 nm and not more than 640 nm. Measure the light intensity at two or more wavelengths,
In the step of calculating the temperature of the liquid in the microchannel, the temperature of the liquid in the microchannel is based on the sum or average of the light intensities of the two or more wavelengths in a wavelength range exceeding 470 nm and not more than 640 nm. The temperature may be calculated.

According to a fifth aspect of the present invention, in the method for measuring the temperature of a liquid in a micro flow channel according to the first aspect, the step of measuring the intensity of the light having the at least one wavelength may include one of a wavelength range of 340 nm or more and less than 470 nm. Measuring the light intensity of the point wavelength and the light intensity of one point wavelength in the wavelength range exceeding 470 nm and not more than 640 nm,
In the step of calculating the temperature of the liquid in the microchannel, the light intensity of the one wavelength in the wavelength range of 340 nm or more and less than 470 nm and the light intensity of one wavelength in the wavelength range exceeding 470 nm and 640 nm or less are used. The temperature of the liquid in the microchannel may be calculated based on a difference between the light intensity and the light intensity.

  In the method for measuring the temperature of a liquid in a micro flow channel according to a sixth aspect, in the second aspect, in the step of calculating the temperature of the liquid in the micro flow channel, a numerical value based on the calculated absorbance is stored in advance. The temperature of the liquid in the microchannel may be calculated based on a temperature reference indicating the relationship between the absorbance and the temperature.

  The method for measuring the temperature of a liquid in a microchannel according to a seventh aspect is the method for measuring a temperature of a liquid in a microchannel according to the sixth aspect, further comprising: a temperature reference indicating the relationship between the absorbance and the temperature; The method may include calibrating a temperature reference indicating a relationship between the absorbance and the temperature based on the temperature of the liquid in the micro flow channel.

The method for measuring the temperature of a liquid in a microchannel according to an eighth aspect is the method of any one of the first, second, fourth, sixth, and seventh aspects, wherein the intensity of light having at least one wavelength is determined. The measuring may include measuring the intensity of the received light that has passed through a red filter that does not transmit at least light in a wavelength range of 470 nm or less.
With the above configuration, in particular, it is possible to obtain only the signal intensity of R (red wavelength region), and it is possible to remove the influence of the background signal from another color gamut, especially G (green wavelength region). Thereby, a high temperature measurement range and a high measurement accuracy can be obtained.

  A method for detecting a temperature change of a liquid in a micro flow channel according to a ninth aspect includes performing the method for measuring a temperature of a liquid in the micro flow channel according to the first aspect twice, and performing the first time measurement of the temperature in the micro flow channel. And the temperature of the liquid in the micro flow channel for the second time are calculated, and the temperature change of the liquid in the micro flow channel is detected.

A method for detecting a change in temperature of a liquid in a microchannel according to a tenth aspect includes receiving light from a liquid containing cresol red in the microchannel,
Obtaining a first light intensity by measuring the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm to 640 nm for the received light;
Receiving the light, and repeating the step of measuring the intensity of the light of the at least one wavelength, obtaining a second light intensity,
Comparing the first light intensity with the second light intensity and detecting a temperature change of the liquid in the microchannel from a change in the light intensity;
including.

The microchannel chip according to the eleventh aspect is a microchannel chip having a microchannel for holding a liquid,
At least one surface is made of a material that transmits light of at least one wavelength except for a wavelength of 470 nm in a wavelength range of 340 nm or more and 640 nm or less, and includes a microchannel including a channel that can be heated or cooled;
A cresol red carrying unit carrying cresol red to be supplied to the liquid in the microchannel,
Having.

  The microchannel chip according to a twelfth aspect is the microchannel chip according to the eleventh aspect, wherein the cresol red carrying portion is arranged such that cresol red is supplied to the liquid flowing through the microchannel in a constant amount per unit time. It may be.

A temperature measuring device for liquid in the microchannel according to a thirteenth aspect, a light receiving unit that receives light from the liquid containing cresol red in the microchannel,
A light intensity measuring unit that measures the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less for the received light;
A liquid temperature calculation unit that calculates the temperature of the liquid in the microchannel based on the measured light intensity,
including.

  An apparatus for measuring the temperature of a liquid in a microchannel according to a fourteenth aspect is the apparatus for measuring a liquid temperature in the thirteenth aspect, wherein the liquid temperature calculation unit calculates an absorbance from the measured light intensity, based on the calculated absorbance. The temperature of the liquid in the microchannel may be calculated.

  In the temperature measuring device for a liquid in a microchannel according to a fifteenth aspect, in the fourteenth aspect, the liquid temperature calculator indicates a relationship between the absorbance and the temperature stored in advance for the calculated absorbance. The temperature of the liquid in the microchannel may be calculated based on a temperature reference.

The apparatus for measuring the temperature of a liquid in a microchannel according to a sixteenth aspect is the apparatus according to any one of the thirteenth to fifteenth aspects, wherein the light intensity measurement unit includes a spectroscope for wavelength-separating the received light. May be.
With the above configuration, a high temperature measurement range and a high measurement accuracy can be obtained.

In the temperature measuring device for a liquid in a microchannel according to a seventeenth aspect, in any one of the thirteenth to sixteenth aspects, the light intensity measuring unit may include a solid-state imaging device.
With the above configuration, the temperature of the liquid in the microchannel can be measured with a small and simple setup.

An apparatus for measuring the temperature of a liquid in a microchannel according to an eighteenth aspect is the apparatus according to the seventeenth aspect, wherein the light intensity measuring unit is configured to transmit at least a red light that does not transmit light in a wavelength range of 470 nm or less for the received light. It may further include a filter.
With the above configuration, in particular, it is possible to obtain only the signal intensity of R (red wavelength region), and it is possible to remove the influence of the background signal from another color gamut, especially G (green wavelength region). Thereby, a high temperature measurement range and a high measurement accuracy can be obtained.

A liquid temperature measuring method according to a nineteenth aspect, receiving light from a liquid containing cresol red,
For the received light, measuring the intensity of light of at least one wavelength excluding light of a wavelength of 470 nm in a wavelength range of 340 nm or more and 640 nm or less;
Calculating the temperature of the liquid based on the measured light intensity,
including.

  In the liquid temperature measuring method according to a twentieth aspect, in the nineteenth aspect, in the step of calculating the temperature of the liquid, an absorbance is calculated from the measured light intensity, and the liquid is measured based on the calculated absorbance. May be calculated.

  Hereinafter, a device for measuring the temperature of a liquid in a microchannel and a method for measuring the temperature of a liquid in a microchannel according to an embodiment of the present disclosure will be described with reference to the accompanying drawings. In the drawings, substantially the same members are denoted by the same reference numerals.

(Embodiment 1)
<Temperature measurement system>
FIG. 1 is a schematic diagram showing a configuration of a temperature measurement system 100 including a device 10 for measuring the temperature of a liquid in a microchannel according to the first embodiment. The temperature measurement system 100 includes a device 10 for measuring the temperature of a liquid in a microchannel, a microchannel chip 11, and a light source 13. The temperature measurement system 100 receives light from a liquid containing cresol red in the microchannel 12 of the microchannel chip 11 of the microchannel chip 11 and has at least one wavelength except for a wavelength of 470 nm in a wavelength range of 340 nm or more and 640 nm or less. The light intensity is measured, the absorbance is calculated from the measured light intensity, and the temperature of the liquid is calculated based on the calculated absorbance.
According to the temperature measurement system 100, the temperature of the liquid in the microchannel in the microchannel chip can be accurately measured without passing through complicated manufacturing steps and without introducing expensive optical equipment.

<Micro channel chip>
FIG. 2A is a plan view of an example of the microchannel chip 11, and FIG. 2B is a cross-sectional view showing a cross-sectional structure as viewed from the AA direction in FIG. 2A. The microchannel chip 11 includes at least a microchannel 12 for holding a liquid. The microchannel chip 11 shown in the plan view of FIG. 2A has an upper right inlet 19a, a lower left outlet 19b, and a microchannel 12 connecting the inlet 19a and the outlet 19b. In addition, the inlet 19a and the outlet 19b may be reversed. Further, there may be a plurality of entrances and a plurality of exits.

  The surface of the microchannel chip 11 has a channel 12 having a width equal to or less than a predetermined width. The width of the flow channel 12 is, for example, 1 μm or more and 1 mm or less. The depth of the flow channel 12 is, for example, 1 μm or more and 1 mm or less. The channel 12 is composed of at least a side surface and a bottom surface. The flow channel 12 has a concave shape when viewed from a cross section of the micro flow channel chip 11 perpendicular to the traveling direction of the flow channel 12. Note that a clear boundary is not necessarily formed between the side surface and the bottom surface of the flow channel 12. Further, as shown in FIG. 2B, the flow channel 12 may have an upper surface. Further, it is desirable that the side surface and the bottom surface of the flow channel 12 are made of a material having a predetermined or higher translucency with respect to light output from the light source 13 described later. In particular, it is desirable to have a predetermined or higher transmittance for light in a wavelength range of 340 nm to 640 nm. The material of the flow channel 12 is, for example, glass or plastic.

In the flow path 12, a liquid to be detected is arranged. Specifically, for example, the liquid is introduced from the inlet 19a, and is drawn out from the lower left outlet 19b via the meandering micro flow channel 12. In this case, the liquid flows in the micro flow channel 12. After the introduction of the liquid, the inlet 19a and the outlet 19b may be sealed to hold the liquid in the microchannel 12.
Further, cresol red as a coloring material is disposed in the flow channel 12. That is, the liquid and cresol red flow through the flow channel 12. Specifically, cresol red may be added to the liquid in advance before being introduced into the microchannel chip 11, and a liquid containing cresol red may be introduced into the microchannel chip 11. Alternatively, for example, as shown in FIG. 2A, a cresol red holding portion 30 that holds cresol red may be provided in front of the meandering flow channel 12 of the micro flow channel chip 11. The cresol red carrying section 30 may supply cresol red to the liquid introduced into the inlet 19a so that the liquid containing cresol red flows through the microchannel 12. Further, cresol red may be arranged in the cresol red carrying section 30 so that cresol red is supplied at a constant rate per unit time. As a means for supplying a fixed amount of cresol red per unit time, a minute flow path containing a cresol red solution may be provided as the cresol red holding unit 30 and may be joined to the micro flow path 12 by a fixed amount. Alternatively, cresol red may be wrapped in a film that dissolves in a fixed amount in a liquid and exposed to the liquid. The configuration of the cresol red carrier 30 is not limited to the above example.

In addition, cresol red shows red to PH lower than the first discoloration range of pH 0.2 to 1.8, and shows yellow to PH higher than the first discoloration range. In addition, cresol red shows yellow at a pH lower than the second color change range of pH 7.2 to 8.8, and shows red at a pH higher than the second color change range.
Therefore, the liquid to be measured is preferably a liquid that does not cause a pH change before and after the step of changing the temperature. Further, it is more preferable that the liquid itself is a buffer or that a buffer is added to the liquid.

  Note that the liquid is preferably a material having a predetermined or higher translucency with respect to light output from the light source 13 described later. Examples of the liquid retained in the micro flow channel 12 of the micro flow channel chip 11 include a biological sample containing DNA, RNA, and fragments thereof, and a buffer aqueous solution containing potassium chloride, tris-hydrochloric acid, or tricine potassium hydroxide. is there.

  The microchannel chip 11 has a temperature controller 17 for heating or cooling the liquid in the channel 12 to adjust the temperature. As the temperature adjusting device 17, a known temperature adjusting device is used to adjust the temperature of the flow channel 12 of the microchannel chip 11 to a predetermined temperature. For example, a hot plate or a Peltier device may be used as the temperature adjusting device 17. Furthermore, a temperature may be adjusted by forming a metal layer on the bottom surface of the microchannel chip 21 and applying a current to the metal layer to heat the channel 12.

<Light source>
The light source 13 outputs light toward the flow path 12. More precisely, light is output from the light source 13 toward the fluid flowing through the flow channel 12. The light source 13 is arranged so as to face the surface of the microchannel chip 11 having the channel 12. For example, the light source 13 may be arranged so as to be provided on the microchannel chip 11. The light source 13 outputs light having a predetermined wavelength range. The predetermined wavelength range is a wavelength range from 340 nm to 640 nm and excluding a wavelength of 470 nm. A desirable predetermined wavelength range is a wavelength range larger than 470 nm and 640 nm or less. Further, the predetermined wavelength range may be 380 nm or more and 640 nm or less, and may be a wavelength range excluding the wavelength of 470 nm. This is because the light source 13 outputs light in the visible light wavelength range of 380 nm or more, so that the user can check the light output from the light source 13 with his eyes.
As the light source 13, for example, a xenon lamp, a halogen lamp, or white LED illumination can be used. In addition, the light source 13 may be natural light such as sunlight or lighting in a building. In the case of lighting in the sun or in a building, the channel 12 may be arranged to receive light.

<Liquid temperature measurement device>
The liquid temperature measurement device 10 includes at least a spectroscope 15, a detector 16, and a liquid temperature calculator 18.
The spectroscope 15 receives the light transmitted through the micro flow channel 12. As described above, the liquid containing cresol red is held in the micro flow channel 12. That is, the light received by the light source spectroscope 15 is light that has passed through the liquid containing cresol red in the microchannel 12. The spectroscope 15 is arranged to face the light source 13 with the microchannel chip 11 interposed therebetween, for example. In the spectroscope 15, the received light is wavelength-separated at least in a wavelength range of 340 nm to 640 nm. As the spectroscope 15, for example, a spectroscope using a prism, a diffraction grating, a filter, or the like, an interference spectroscope, or the like can be used.
The detector 16 measures the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm to 640 nm for the light received by the spectroscope 15. Any detector can be used as long as it can detect the intensity of light after wavelength separation. As the detector 16, for example, a CCD array, a CMOS array, a photomultiplier, or the like can be used. Note that the spectroscope 15 and the detector 16 may be integrally configured as a spectrophotometer. Further, the spectroscope 15 and the detector 16 may be configured as a solid-state imaging device as described in the second embodiment.

The liquid temperature calculator 18 calculates the absorbance from the measured light intensity. The liquid temperature calculator 18 receives the output intensity of the light output from the light source 13 and calculates the absorbance from the ratio of the measured light intensity to the light output intensity. The liquid temperature calculation unit 18 outputs the temperature of the liquid in the micro flow channel 12 based on the calculated absorbance and the temperature reference 36 held in advance. The temperature reference 36 is a rule for determining the temperature based on the measured light intensity. An example of the temperature reference 36 is (temperature) = a (coefficient) * X (temperature measured by the spectroscope 15) + (room temperature).
FIG. 3 shows another example of the temperature reference 37 held in advance. The temperature reference 37 is a table in which the absorbance and the temperature are associated with each other. The liquid temperature calculation unit 18 outputs a temperature corresponding to the calculated absorbance with reference to the temperature reference 37. If there is no calculated absorbance value in the absorbance value included in the temperature reference 37, the liquid temperature calculation unit 18 determines a value closest to the calculated absorbance among the absorbance values included in the temperature reference 37. The output has the temperature. Alternatively, the liquid temperature calculator 18 does not output the temperature when the calculated absorbance value is not included in the absorbance value included in the temperature reference 37.
FIG. 4 shows another example of the temperature reference 38 held in advance. The temperature reference 38 is a table in which the measured light intensity is associated with the temperature. The liquid temperature calculator 18 receives the output intensity of the light output from the light source 13 and outputs a temperature corresponding to the measured light intensity with reference to the temperature reference 38. The temperature reference 38 includes a value previously obtained as the light intensity based on the intensity of the light output from the light source 13 and the like. That is, the light intensities included in the temperature reference shown in FIG. 4 are described as A, B, and C, but A, B, and C included in FIG. Corresponding to the value of
When there is no measured light intensity value in the light intensity value included in the temperature reference 38, the liquid temperature calculation unit 18 determines that the measured light intensity is the highest among the light intensity values included in the temperature reference 38. Output a temperature with a close value. Alternatively, the liquid temperature calculation unit 18 does not output the temperature when the measured light intensity value is not included in the light intensity value included in the temperature reference 38.

The liquid temperature calculator 18 is configured by, for example, a computer shown in FIG.
FIG. 5 is a block diagram illustrating an example of a physical configuration of the liquid temperature calculation unit 18. The liquid temperature calculator 18 includes a CPU 31, a memory 32, an input / output unit 33, a display device 34, a storage device 35, and an interface 37. Further, the storage device 35 stores a temperature reference 36 indicating the relationship between the absorbance and the temperature used when calculating the temperature of the liquid. For example, the liquid temperature calculation unit 18 calculates the temperature of the liquid using the calculated absorbance and a temperature reference 36 indicating a relationship between the absorbance and the temperature held in advance. The temperature reference 36 is information in which the absorbance at a predetermined wavelength (573 nm) and the temperature are associated with each other with respect to a liquid containing a predetermined concentration of cresol red, as shown in FIG.

  The present inventor has found that not only the predetermined wavelength (573 nm) shown in FIG. 10 but also the relationship of the absorptance to the wavelength of light transmitted through the liquid containing cresol red shown in FIG. The present invention was found for the first time and completed the present invention of calculating the temperature of the liquid using the temperature change of the absorption rate.

  FIG. 6 is a graph showing the relationship between the wavelength of light transmitted through a liquid to which cresol red as a coloring material is added and the absorptance. FIG. 6 shows the relationship between the wavelength and the absorptance at each of 25 ° C., 55 ° C., 70 ° C., and 95 ° C. As shown in FIG. 6, the liquid containing cresol red has at least four peaks in the wavelength range from 200 nm to 800 nm. Peaks having a remarkable relationship between the absorptance and the temperature are a peak at about 430 nm and a peak at about 570 nm. In addition, the light absorptivity in the wavelength range from 340 nm to 640 nm including these peaks changes significantly in the temperature range from 20 ° C to 100 ° C. Furthermore, in the wavelength range of 340 nm or more and 470 nm or less, there is a positive correlation that the absorption rate (similarly, the absorbance) increases as the temperature rises. It has a negative correlation with decreasing absorption. At 470 nm, the absorptance is almost constant regardless of the temperature.

  The liquid temperature calculation unit 18 is not limited to the predetermined wavelength (573 nm) shown in FIG. 10, but may obtain the absorbance for the intensity of light of another wavelength and calculate the temperature of the liquid based on the obtained absorbance. For example, the light intensity of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less is measured, the absorbance is calculated from the measured light intensity, and the temperature of the liquid is calculated based on the calculated absorbance. May be. Note that the temperature of the liquid may be calculated based on the measured light intensity without calculating the absorbance.

Further, the following three cases are used as cases where light intensities of a plurality of wavelengths are used in the wavelength range of 340 nm to 640 nm.
a) About two or more wavelengths in the wavelength range of 340 nm or more and less than 470 nm The liquid temperature calculation unit 18 measures the light intensity of two or more wavelengths in the wavelength range of 340 nm or more and less than 470 nm, and The respective absorbances may be calculated from the light intensities of the wavelengths, and the temperature of the liquid may be calculated based on the sum or average of the calculated absorbances.
As described above, in the wavelength range of 340 nm or more and less than 470 nm, there is a positive correlation that the absorptance (similarly, the absorbance) increases as the temperature rises. Thus, the sum or average of the absorbances at two or more wavelengths in the above wavelength range also has a positive correlation with the temperature, so that the temperature can be calculated by the calculated sum or average.
b) About two or more wavelengths in the wavelength range exceeding 470 nm and 640 nm or less The liquid temperature calculation unit 18 measures the light intensity at two or more wavelengths in the wavelength range exceeding 470 nm and 640 nm or less. The respective absorbances may be calculated from the light intensities at two or more wavelengths, and the temperature of the liquid may be calculated based on the sum or average of the calculated absorbances.
As described above, in the wavelength range exceeding 470 nm and equal to or less than 640 nm, there is a negative correlation that the absorptance decreases as the temperature increases. Similarly, the sum or average of the absorbances at two or more wavelengths in the above wavelength range also has a negative correlation with the temperature, so that the temperature can be calculated from the calculated sum or average.
c) About one wavelength on the short wavelength side (340 nm or more) and one wavelength on the long wavelength side (640 nm or less) with 470 nm as a boundary The liquid temperature calculation unit 18 calculates the wavelength range from 340 nm to less than 470 nm. And the light intensity of one wavelength in the wavelength range exceeding 470 nm and 640 nm or less is measured, and the light intensity of one wavelength in the wavelength range of 340 nm or more and less than 470 nm is measured. The liquid temperature may be calculated based on the value obtained by adding 1 to the difference between the absorbance calculated from the above and the absorbance calculated from the light intensity of one wavelength in the wavelength range exceeding 470 nm and 640 nm or less. .
In each of the above cases, the absorbance is calculated from the measured light intensity, but the temperature of the liquid may be calculated based on the measured light intensity without calculating the absorbance. In the case of directly using the measured non-standardized light intensity, in the case of c), the light intensity of one wavelength in the wavelength range of 340 nm or more and less than 470 nm and the light intensity of one wavelength in the wavelength range exceeding 470 nm and 640 nm or less are used. The difference (including positive and negative) or the ratio with the absorbance calculated from the light intensity at one wavelength may be used as it is.

  As described above, the tendency of the temperature change of the absorptance (similarly, the absorbance) differs at the boundary of 470 nm. In other words, if the absorbance on the short wavelength side and the absorbance on the long wavelength side are added as they are at the boundary of 470 nm, the sign of the variation with respect to the temperature is reduced. Therefore, by taking the difference between them, the amount of change with respect to temperature is included with the same sign. Further, the difference between the two values may be positive or negative depending on the magnitude relationship between the respective values. Therefore, since the value of the absorbance at 573 nm at 25 ° C. is standardized as 1, the value calculated by adding 1 to the difference between the two and making the entire numerical value a positive value regardless of the magnitude relationship between the two, A simple correlation with temperature can be obtained. The value to be added is not limited to 1 if the whole numerical value can be made a positive value.

  The liquid temperature calculation unit 18 may calibrate the temperature reference 36 based on the numerical value based on the calculated absorbance and the temperature of the liquid separately measured. In the actual temperature measurement system 100, the relationship between the absorbance and the temperature may deviate from the temperature reference 36 stored in advance. By performing the temperature reference calibration as described above, more accurate temperature measurement can be performed.

<Temperature measurement method>
Hereinafter, FIG. 11 shows an example of a temperature measurement method of the temperature measurement system 100 that outputs a temperature using the measured light intensity and the temperature reference.
(Step 1)
The spectroscope 15 receives light transmitted through the microchannel 12 through which the liquid flows (S01).
(Step 2)
The detector 16 measures the intensity of the received light at at least one wavelength excluding the 470 nm wavelength in the wavelength range from 340 nm to 640 nm (S02).
(Step 3)
The liquid temperature calculator 18 outputs the temperature of the liquid flowing through the micro flow channel 12 using the temperature reference held in advance and the measured light intensity (S03).

Next, FIG. 12 shows another example of the temperature measurement method of the temperature measurement system 100 that outputs the temperature using the calculated absorbance and the temperature reference.
(Step 11)
Cresol red as a dye material is added to the liquid flowing through the micro flow channel 12 of the micro flow channel chip 11 (S11). The concentration of cresol red to be added to the fluid can be set to any concentration according to the sensitivity of the detector 16. Considering that the microchannel is used with a thickness of about 200 μm or less, the final concentration of cresol red to be added is preferably 200 μM or more. If added to a sample for PCR, about 200 μM is particularly preferable from the viewpoint of PCR yield. Since the absorbance changes depending on the concentration of cresol red (Lambert-Beer's law), the concentration is set to a predetermined concentration on a temperature basis.
(Step 12)
The light source 13 outputs light toward the microchannel 12 (S12). More specifically, the light source 13 outputs light toward cresol red in the fluid flowing through the microchannel 12. The light output from the light source 13 has a wavelength range of about 200 nm to 800 nm. The light output from the desirable light source 13 has a wavelength range excluding the wavelength of 470 nm out of the wavelengths of at least 340 nm to 640 nm.
(Step 13)
The spectroscope 15 receives the light from the micro flow channel 12 (S13).
(Step 14)
The detector 16 measures the intensity of the received light at at least one wavelength excluding the 470 nm wavelength in the wavelength range from 340 nm to 640 nm (S14).
(Step 15)
The liquid temperature calculator 18 calculates the absorbance from the measured light intensity, and calculates the temperature of the liquid in the micro flow channel 12 based on the calculated absorbance (S15).

In order to obtain high measurement accuracy, a light intensity I _{base serving} as a background is detected in advance in a system in which a fluid to which cresol red is not added is introduced, and a difference (I-I _{base) from} the light intensity I of the actual measurement is detected. ) Is desirably the effective light intensity. In addition, it is desirable to use a wavelength range in which the change with respect to the temperature is large among the absorption spectra obtained while changing the temperature using the temperature adjusting device 17. When calculating the temperature from the spectrum derived from cresol red as in the present embodiment, the wavelength used for temperature measurement is at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm to 640 nm. Good. Further, it is preferable that the temperature change of the absorptance is set to around 430 nm or 573 nm, which is relatively large.

(Modification 1)
In the method for measuring the temperature of the liquid in the microchannel, the temperature at a certain point is measured. On the other hand, in the method of detecting the temperature change of the liquid in the micro flow channel according to the modification, the temperatures at two different time points are measured.
Specifically, the method for detecting a change in the temperature of the liquid in the micro flow channel 12 is the same as the method for measuring the temperature of the liquid in the micro flow channel 12 twice. The second time, the temperature of the liquid in the micro flow channel 12 is calculated, and the temperature change of the liquid in the micro flow channel 12 is detected.

(Modification 2)
The method for detecting the temperature change of the liquid in the micro flow channel according to the second variation is specifically, the method for detecting the temperature change of the liquid in the micro flow channel, which detects light from the liquid containing cresol red in the micro flow channel. Receiving light;
Obtaining a first light intensity by measuring the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less for the received light;
Receiving the light and repeating the step of measuring the intensity of the light of at least one wavelength to obtain a second light intensity;
Detecting the temperature change of the liquid in the microchannel from the change in the light intensity by comparing the first light intensity with the second light intensity;
including.
In the method for detecting the temperature change of the liquid in the micro flow channel according to the second variation, in comparison with the method for detecting the temperature change of the liquid in the micro flow channel according to the first variation, the first light intensity is calculated without calculating the absorbance. The point is that the presence or absence of a temperature change is detected by directly comparing the second light intensity with the second light intensity. As described above, since the light intensity is compared without determining the absorbance, the presence or absence of a temperature change can be easily determined. This makes it possible to easily determine an error such as poor contact of the heater in the heating step or the cooling step.

(Embodiment 2)
<Temperature measurement system>
FIG. 13 is a schematic diagram showing a configuration of a temperature measuring system 100a including the temperature measuring device 20 for liquid in a microchannel according to the second embodiment. The temperature measurement system 100a is different from the temperature measurement system according to the first embodiment in that the temperature measurement system 100a receives reflected light instead of transmitted light from the microchannel. For this reason, the microchannel chip 21 also uses the upper surface of the microchannel 22 as a reflection surface. The liquid temperature measuring device 20 also differs from the liquid temperature measuring device according to the first embodiment in that a solid-state image sensor 26 is used as a light intensity measuring unit that measures light intensity.
The description of the same items as in the first embodiment is omitted.

<Micro channel chip>
In the second embodiment, light absorption occurring in the liquid introduced into the cover plate 211 of the microchannel chip 21 and the microchannel 22 is detected via the reflected light from the microchannel chip 21. It is preferable that the fluid introduced into the cover plate 211 of the microchannel chip and the inside 22 of the microchannel used in the measurement be made of a material through which light emitted from the light source 23 can be sufficiently transmitted. The upper surface of the microchannel chip 21 is preferably made of a material that reflects visible light, such as silicon or metal. In this case, the material of the cover plate 211 of the microchannel chip 21 is preferably a material having high transmittance to visible light, such as glass or plastic. In addition, cresol red is added to the liquid introduced into the micro flow channel 22 in advance. The arrangement and concentration of cresol red to be added are almost the same as in the first embodiment.
Further, the microchannel chip 21 has an advantage that the temperature controller 27 can be arranged on the back of the microchannel 22 to be heated, as shown in FIG.

<Light source>
The light source 23 is preferably a light source that covers visible light, and may be of any type such as a xenon lamp, a halogen lamp, natural light, and a white LED.

<Liquid temperature measurement device>
The liquid temperature measuring device 24 includes a lens 25, a solid-state image sensor 26, and a liquid temperature calculator 28.
The lens 25 may be of any form as long as it has a magnification sufficient to capture an image of the region of the microchannel 22 to be measured into the solid-state imaging device 26, and the type of the material is not particularly limited.

<Solid-state imaging device>
The solid-state imaging device 26 may be of any type, such as a CCD or a CMOS. In addition, as long as an arbitrary wavelength can be detected, an imaging function in a two-dimensional plane may not be provided, and a photomultiplier tube may be used. When calculating the temperature from the light intensity of the spectrum derived from cresol red, for example, the data acquired by the solid-state imaging device as in the second embodiment should be analyzed so that the detection rate near the wavelength of 573 nm is increased. Just fine.

  In particular, in the step of measuring the light intensity of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less, at least 470 nm or less of the RGB information acquired by the solid-state imaging device. Using a red filter that does not transmit light in the wavelength range, the signal intensity can be R (red wavelength range). Thereafter, the temperature is calculated from the signal intensity of R (red wavelength region). This makes it possible to eliminate the influence of the background signal (around 430 nm) generated from G (green wavelength region). Therefore, it is preferable from the viewpoint that a high temperature measurement range and measurement accuracy can be obtained.

In the temperature measurement, the step of heating or cooling a desired portion and the step of adjusting the heating or cooling to an arbitrary temperature can be performed by controlling the temperature of the microchannel chip by using this temperature measurement method. It is preferable from the viewpoint that can be used.
In the temperature measurement, the object of the temperature measurement is the polymerase chain reaction (PCR), which means that the control of the PCR is accurate, and an expensive optical device is introduced without going through a complicated manufacturing process. It is preferable from the viewpoint that it can be carried out without using.
The temperature measurement system 100a measures the temperature of the liquid by the same temperature measurement method as that of the temperature measurement system 100 of the first embodiment except that the light reflected from the microchannel 12 is used.

  Hereinafter, the present disclosure will be described more specifically. The following embodiments are examples for describing the present disclosure in more detail, and the present disclosure and embodiments are not limited to only the following examples.

(Example 1)
Example 1 will be described with reference to FIGS. 1 and 6 to 10.
In Example 1, as shown in FIG. 1, the detection of transmitted light by the fluid introduced into the microchannel chip 11 and the microchannel 12 was performed.
The microchannel chip 11 in FIG. 1 was manufactured by processing a glass substrate to form a microchannel 12.
The fluid flowing through the microchannel 12 was Tris-HCl (pH 8.0) having a concentration of 50 mM. The volume of the fluid was 300 μL.
60 μL of cresol red was added to the fluid flowing through the microchannel 12. Further, 840 μL of ultrapure water (840 μL) was added to the fluid, diluted and sealed. Further, in consideration of the sensitivity of the detector 16, the concentration of cresol red was adjusted to an appropriate concentration with respect to the thickness of the flow channel to be used. When the flow path thickness was 10 mm, the final concentration of cresol red was 50 μM.
As a comparative example, an experiment using three types of dye materials generally used as markers in gel electrophoresis in the same manner as cresol red in addition to cresol red was performed at the same time. For comparison, the final concentration was adjusted to be equal to that of this example (Table 1).

As the light source 13, a tungsten lamp was used.
As the spectroscopic detector 14, a diffraction grating type spectrometer 15 capable of separating light in a wavelength range of 200 nm or more and 800 nm or less, and a solid state detection element 16 composed of a photomultiplier tube were used.
The portion other than the measurement site of the microchannel chip 11 is heated by a hot-water bath type temperature controller 17, waits for 3 minutes until the temperature of the chip is uniformly and uniformly stabilized, and then measures the absorption spectrum with a spectroscopic detector. Was. The background signal was removed by measuring the absorption spectrum without adding each dye, taking the data value as _Ibase , and taking the difference (I- _base ) from the actual measurement value I.

The temperature of the liquid (25 ° C., 55 ° C., 70 ° C., and 95 ° C.) whose temperature was measured in advance was measured using a temperature measuring method.
FIG. 6 shows the results of measuring the relationship between the wavelength of light transmitted through a liquid to which cresol red as a coloring material is added and the absorptance. FIG. 7 shows the results of measuring the relationship between the wavelength of light transmitted through a liquid to which xenon cyanol, which is a coloring material, is added and the absorptance. FIG. 8 shows the result of measuring the relationship between the wavelength of light transmitted through a liquid to which bromophenol blue as a dye material is added and the absorptance. FIG. 9 shows the result of measuring the relationship between the wavelength and the absorptance of light transmitted through a liquid to which orange G as a coloring material is added.
As shown in FIGS. 7 to 9, in Comparative Examples 1 to 3, almost no change in absorbance was observed in the temperature range of 25 ° C. to 95 ° C.

From the measurement results of this example and Comparative Examples 1 to 3, the following was found for the absorption spectrum at 25 ° C. In this example using cresol red as the dye material, the strongest absorption derived from the dye was observed at a wavelength around 573 nm. In Comparative Example 1 using xenon cyanol as the dye material, the strongest absorption derived from the dye was observed at around 620 nm. In Comparative Example 2 using bromophenol blue as the dye material, the strongest absorption derived from the dye was observed at around 590 nm. In Comparative Example 3 using orange G as the dye material, the strongest absorption derived from the dye was observed at around 481 nm.
The absorption at each of the above wavelengths at 25 ° C. was set to 1, and the absorption was measured while changing the temperature. FIG. 10 shows the results of plotting the absorption rate (absorbance) at each temperature. More specifically, FIG. 10 shows the relationship between the absorptance of light of a predetermined wavelength and the temperature for each liquid to which four types of dye materials are added in Example 1.
From these results, the present inventor has found that when cresol red is added to a liquid as a coloring material, a predetermined wavelength is obtained in a temperature range from room temperature to about 100 ° C. as compared with Comparative Examples 1 to 3 using other coloring materials. It was confirmed that the light absorptance of the sample showed a very large temperature response. In other words, by measuring the absorptivity of light at least at one point in a wide wavelength range excluding 470 nm in the wavelength range of 340 nm to 640 nm including the wavelength of 573 nm, the temperature of the fluid in the microchannel 12 can be accurately determined. Can be calculated.
By observing the time change of the absorptance at two different points in time, the temperature change of the liquid can be detected.

(Example 2)
Second Embodiment A second embodiment will be described with reference to FIGS. 13, 14A to 14D and FIG.
In the second embodiment, as shown in FIG. 13, light absorption by a fluid introduced into the cover plate 211 of the microchannel chip 21 and the microchannel 22 is detected through reflected light from the microchannel chip 21. did. First, a 500 nm-thick silicon substrate is processed by reactive ion etching (RIE) to form a meandering microchannel 22 having a depth of about 250 μm and a width of about 500 μm on the front side, and a through hole on the back side. The microchannel chip 21 was formed. The surface of the microchannel 22 was sealed by bonding a 500 nm thick Pyrex glass cover plate 221 by anodization. After the fluid was introduced, the through hole on the back surface was sealed by pressing a silicone rubber sealing valve 212 with a force of 1N. A liquid for PCR prepared so that the final concentration of cresol red was 200 μM (see Table 2 for the composition) was sealed in the microchannel 22.

The sequence of primer 1 was (5'-ACGGGCTGCAGGCATACACT-3 '), and the sequence of primer 2 was (5'-GGC AGG TCC TGA ACC TC-3').
As the light source 23, a mixed light of a white LED and natural light was used. As the liquid temperature measuring device 24, a lens 25 having a magnification sufficient to capture an area (approximately 6 mm square) to be measured in the microchannel chip 11 and a solid-state image sensor 26 composed of a CCD for imaging the lens 25 are used. Was. In order to apply an arbitrary temperature to the microchannel 22, a temperature controller 27 made of a Peltier element was brought into contact with the back surface of the measurement area.

  14A, FIG. 14B, FIG. 14C, and FIG. 14D show the case where the temperature is changed to 25 ° C. (FIG. 14A), 60 ° C. (FIG. 14B), 68 ° C. (FIG. 14C), and 98 ° C. (FIG. 14D), respectively. 3 shows an image of the micro flow channel 22 captured by a solid-state imaging device 26 composed of a CCD. 14A to 14D are grayscale drawings of an image through a red filter. A change in color with increasing temperature was also observed visually. Further, of the RGB information acquired by the solid-state imaging device 25, the color distribution of 255 gradations and the signal intensity distribution of 255 gradations were mapped for the signal obtained in R (red wavelength region). Focusing on one arbitrary gradation (one gradation corresponding to around 573 nm in the present embodiment) in the color distribution, the difference between the signal intensity and the average signal intensity was calculated. The difference value was used as the detection rate. FIG. 15 shows the relationship between the detection rate and the temperature when the detection rate measured at 25 ° C. is set to 1. From these results, it was confirmed that even in such a simple measurement system, a response almost equivalent to that of Example 1 was obtained in a temperature range from room temperature to about 100 ° C. That is, as in Example 1, it was shown that the temperature of the fluid in the microchannel 12 can be calculated by measuring the change in the detection rate.

(Example 3)
Example 3 will be described with reference to FIG. Here, amplification of a desired DNA fragment (fragment length: 143 bp) containing the 114th base of exon 12 of the acetaldehyde dehydrogenase 2 (ALDH2) gene from a human genome sample was performed by PCR. The experiment was performed with the same configuration as in Example 2.

After holding at 98 ° C. for 2 minutes in the temperature controller 27, PCR was performed for 30 cycles at 98 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 30 seconds. Thereafter, the presence or absence of DNA amplification was confirmed by electrophoresis on acrylamide gel from the microchannel 22. FIG. 16 shows the results.
FIG. 16 is a gray scale drawing of an electrophoresis photograph of an amplification product subjected to PCR using the temperature measurement method of Example 3 and an amplification product of Comparative Example. The first lane is a ladder for reference, and the pitch between the ladders is 20 bp. In the second lane, PCR was performed without adding cresol red as a comparative example. The third lane shows the results of electrophoresis obtained from a product obtained by performing PCR with cresol red added in this example. Also in this example, it was confirmed that a sufficient amplification product was obtained.

  According to the method for measuring the temperature of a liquid in a microchannel according to the present disclosure, the temperature of the liquid in the microchannel can be measured easily and accurately, so that chemical synthesis, environmental analysis, and clinical sample analysis are performed. For temperature measurement and temperature calibration in a microchannel chip and a capillary for this purpose.

10, 20 Temperature measuring device 11, 21 Micro flow channel chip 12, 22 Micro flow channel 13, 23 Light source 15, 25 Spectroscope, lens (light receiving unit)
16, 26 Solid-state detection device, solid-state imaging device (light intensity measurement unit)
17, 27 Temperature controller 18, 28 Liquid temperature calculator 19a Inlet 19b Outlet 30 Cresol red carrier 100, 100a Temperature measuring system 211 Cover plate 212 Sealing valve

Claims (15)

Receiving light from a liquid containing cresol red in the microchannel,
Measuring the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less for the received light;
Calculating the temperature of the liquid in the microchannel based on the measured light intensity;
Including
The microchannel includes a cresol red carrying unit,
Further, before the step of receiving light, the cresol red carrying unit, by the step of supplying the cresol red to the liquid in the micro flow path,
A method for measuring the temperature of a liquid in a microchannel.   The micro flow according to claim 1, wherein in the step of calculating the temperature of the liquid, the absorbance is calculated from the measured light intensity, and the temperature of the liquid in the micro flow path is calculated based on the calculated absorbance. A method for measuring the temperature of a liquid in a road. In the step of measuring the intensity of light of at least one wavelength, the light intensity of two or more wavelengths in a wavelength range of 340 nm or more and less than 470 nm is measured,
In the step of calculating the temperature of the liquid in the microchannel, the temperature of the liquid in the microchannel is determined based on the sum or average of the light intensities of the two or more wavelengths in the wavelength range of 340 nm or more and less than 470 nm. The method for measuring the temperature of a liquid in a microchannel according to claim 1, wherein the temperature is calculated. In the step of measuring the intensity of light of at least one wavelength, the light intensity of two or more wavelengths in a wavelength range exceeding 470 nm and 640 nm or less is measured,
In the step of calculating the temperature of the liquid in the microchannel, the temperature of the liquid in the microchannel is based on the sum or average of the light intensities of the two or more wavelengths in a wavelength range exceeding 470 nm and not more than 640 nm. The method for measuring the temperature of a liquid in a microchannel according to claim 1, wherein the temperature is calculated. In the step of measuring the intensity of light of at least one wavelength, the light intensity of one wavelength in a wavelength range of 340 nm or more and less than 470 nm and the light intensity of one wavelength in a wavelength range of more than 470 nm and 640 nm or less. Measure the intensity and
In the step of calculating the temperature of the liquid in the micro flow channel, the light intensity of the one point wavelength in the wavelength range of 340 nm or more and less than 470 nm and the one point wavelength in the wavelength range of 470 nm or more and 640 nm or less. The method for measuring the temperature of a liquid in a microchannel according to claim 1, wherein the temperature of the liquid in the microchannel is calculated based on a difference between the light intensity of the microchannel and the light intensity of the liquid. In the step of calculating the temperature of the liquid in the micro flow channel, for a numerical value based on the calculated absorbance, based on a temperature reference indicating the relationship between absorbance and temperature stored in advance,
The method for measuring the temperature of a liquid in a microchannel according to claim 2, wherein the temperature of the liquid in the microchannel is calculated.   Further, with respect to a temperature reference indicating the relationship between the absorbance and the temperature, a temperature indicating the relationship between the absorbance and the temperature based on a numerical value based on the calculated absorbance and a separately measured temperature of the liquid in the microchannel. The method for measuring the temperature of a liquid in a microchannel according to claim 6, comprising calibrating a reference.   The step of measuring the intensity of light of at least one wavelength, wherein the received light is measured for the intensity of light transmitted through a red filter that does not transmit light in a wavelength range of at least 470 nm or less. The method for measuring the temperature of a liquid in a microchannel according to any one of 4, 6, and 7. The method for measuring the temperature of the liquid in the microchannel according to claim 1 twice, wherein the temperature of the liquid in the microchannel for the first time, the temperature of the liquid in the microchannel for the second time, Is calculated,
A method for detecting a temperature change of a liquid in a microchannel, wherein the method detects a temperature change of a liquid in the microchannel. The cresol red carrying section, in the supplying step, supplies a constant amount of the cresol red per unit time,
The method for measuring the temperature of a liquid in a microchannel according to claim 1. In the step of calculating the temperature of the liquid, an absorbance is calculated from the measured light intensity, and a numerical value based on the calculated absorbance is calculated based on a temperature reference indicating a relationship between the absorbance and the temperature stored in advance. Calculate the temperature of the liquid in the flow path,
The temperature reference corresponds to a certain amount per unit time supplied by the cresol red carrying unit,
The method for measuring the temperature of a liquid in a microchannel according to claim 10. The cresol red carrying section has cresol red surrounded by a film having solubility in the liquid,
In the supplying step, the liquid is supplied into the microchannel, and the film is dissolved, whereby the cresol red is supplied to the liquid in the microchannel.
The method for measuring the temperature of a liquid in a microchannel according to claim 1. A microchannel chip having a microchannel for holding a liquid,
At least one surface is made of a material that transmits light of at least one wavelength except for a wavelength of 470 nm in a wavelength range of 340 nm or more and 640 nm or less, and includes a microchannel including a channel that can be heated or cooled;
A cresol red carrying unit carrying cresol red to be supplied to the liquid in the microchannel,
A microchannel chip comprising:   14. The microchannel chip according to claim 13, wherein the cresol red carrying portion is provided with cresol red so as to supply cresol red to the liquid flowing in the microchannel in a constant amount per unit time. A light receiving unit that receives light from a liquid containing cresol red in the microchannel,
A light intensity measuring unit that measures the intensity of light of at least one wavelength excluding the wavelength of 470 nm in the wavelength range of 340 nm or more and 640 nm or less for the received light;
A liquid temperature calculation unit that calculates the temperature of the liquid in the microchannel based on the measured light intensity,
Including
The liquid temperature calculation unit calculates the absorbance from the measured light intensity, calculates the temperature of the liquid in the microchannel based on the calculated absorbance,
The liquid temperature calculator, for the calculated absorbance, based on a temperature reference indicating the relationship between the absorbance and the temperature stored in advance, calculates the temperature of the liquid in the micro flow channel,
The temperature reference corresponds to a certain amount of cresol red supplied per unit time to the micro flow channel by the cresol red carrying portion located in the micro flow channel,
A device for measuring the temperature of a liquid in a microchannel.