Microfluidic device and flow velocity estimation method

Abstract

To provide a microfluidic device capable of easily measuring a flow velocity of a culture solution in an extremely narrow channel.SOLUTION: In a microfluidic device, a channel is connected to a first container, a culture solution flows through the channel, and a biological sample is disposed in the culture solution. A controller moves the culture solution in the channel to change a liquid amount of the culture solution in the first container. A first light source irradiates the culture solution with a first light passing through a liquid surface of the culture solution. A first light detector detects a first light intensity that is an intensity of the first light that has passed through the culture solution in the first container at a first timing, and a second light intensity that is an intensity of the first light that has passed through the culture solution in the first container at a second timing. An arithmetic unit estimates a temporal change amount between a first optical path length of the first light in the culture solution at the first timing and a second optical path length of the first light in the culture solution at the second timing, based on an optical density of the culture solution obtained from the first light intensity and an optical density of the culture solution obtained from the second light intensity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a microfluidic device and a method for estimating flow velocity. [Background technology]

[0002] Non-Patent Documents 1 to 4 disclose a microphysiological system (MPS). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Van Duinen, V. et al., "Perfused 3D angiogenic sprouting in ahigh -throughput in vitro platform", Angiogenesis, Vol.22, pp.157-165 (2019) [Non-patent document 2] Shinohara Marie et al., "Coculture with hiPS-derived intestinalcells enhanced human hepatocyte functions in a pneumatic-pressure-driventwo-organ microphysiological system", Scientific reports, 11.1, 5437(2021) Summary of the Invention [Problem to be solved by the invention]

[0004] In recent years, MPS has come to be used in cell culture or drug testing. In a typical configuration of an MPS, two containers containing culture medium are connected by a microchannel. Cells to be cultured or tested are placed in the microchannel. By moving the culture medium from one container to the other through the microchannel at a constant speed, the cells can be placed in a constant flow of culture medium.

[0005] Methods for moving the culture medium include using a pump or tilting the container and microchannel. In either method, it is necessary to continuously flow the culture medium at the desired flow rate. Because there is a risk that the microchannel may become clogged due to some factor, preventing the culture medium from flowing at the desired rate, it is desirable to continuously monitor the culture medium flow rate. However, the microchannels of MPSs are extremely narrow, with widths of, for example, 1 mm or less, making it difficult to measure the culture medium flow rate using conventional flow rate measurement methods.

[0006] An object of the present disclosure is to provide a microfluidic device and a flow rate estimation method that can easily measure the flow rate of a culture medium in an extremely narrow channel. [Means for solving the problem]

[0007] [1] A microfluidic device according to one embodiment of the present disclosure includes a first container, a flow channel, a control unit, a first light source, a first light detection unit, and a calculation unit. The first container contains a culture medium. One end of the flow channel is connected to the first container, and the culture medium flows through the flow channel, disposing a biological sample in the culture medium. The control unit moves the culture medium in the flow channel to change the volume of the culture medium in the first container. The first light source irradiates the culture medium in the first container with first light that passes through the surface of the culture medium. The first light detection unit detects a first light intensity, which is the intensity of the first light that has passed through the culture medium in the first container at a first timing, and a second light intensity, which is the intensity of the first light that has passed through the culture medium in the first container at a second timing different from the first timing. The calculation unit estimates a change over time between a first optical path length, which is the optical path length of the first light in the culture solution in the first container at a first timing, and a second optical path length, which is the optical path length of the first light in the culture solution in the first container at a second timing, based on the optical density of the culture solution in the first container obtained based on the first light intensity and the optical density of the culture solution in the first container obtained based on the second light intensity. The calculation unit estimates a flow velocity of the culture solution in the flow path based on the change over time.

[0008] In the microfluidic device described in [1] above, the calculation unit estimates the flow velocity of the culture medium in the flow channel based on the time change in the optical path length of the first light passing through the culture medium surface, i.e., the time change in the liquid surface position. The optical path length in the culture medium can be estimated based on the optical density of the culture medium, which is obtained based on the light intensity of the first light incident on the first light detection unit. This microfluidic device does not require a device for measuring flow velocity in an extremely narrow flow channel, making it easy to measure the flow velocity of the culture medium in an extremely narrow flow channel. Note that if the volume of the culture medium is excessive, the distance the first light travels in the culture medium becomes long, resulting in excessive attenuation of the first light and making it difficult to accurately estimate the flow velocity of the culture medium. This microfluidic device is suitable for measuring the flow velocity in an extremely narrow flow channel, such as a microchannel, where the volume of the culture medium is relatively small. [2] The microfluidic device of [1] above may further include a second container that contains a culture medium and is connected to the other end of the flow path. The control unit may move the culture medium between the first container and the second container through the flow path. This allows the culture medium to circulate between the first container and the second container, thereby reducing the amount of culture medium consumed.

[0009] [3] The microfluidic device of [2] above may further include a second light source and a second light detector. The second light source irradiates the culture solution in the second container with second light, the second light passing through the surface of the culture solution. The second light detector detects a third light intensity, which is the intensity of the second light passing through the culture solution in the second container at a first timing, and a fourth light intensity, which is the intensity of the second light passing through the culture solution in the second container at a second timing. The calculation unit may further estimate the time change based on the optical density of the culture solution in the second container obtained based on the third light intensity and the optical density of the culture solution in the second container obtained based on the fourth light intensity. In this case, even if the extinction coefficient of the culture solution is unknown, the time change in the optical path length can be estimated as long as the total length of the optical path length of the first light in the culture solution in the first container and the optical path length of the second light in the culture solution in the second container is known.

[0010] [4] In the microfluidic device of [3] above, the calculation unit may further estimate the amount of change over time based on the total length of the optical path length of the first light in the culture medium of the first container and the optical path length of the second light in the culture medium of the second container.

[0011] [5] In the microfluidic device of [4] above, the calculation unit may calculate the total length based on the total volume of the culture solution contained in the first container, the second container, and the flow path. For example, with this configuration, the total length of the optical path length of the first light in the culture solution in the first container and the optical path length of the second light in the culture solution in the second container can be easily determined.

[0012] [6] In the microfluidic device according to any one of [1] to [5] above, the optical axis of the first light source may be tilted with respect to the liquid surface of the culture medium in the first container. This allows the flow rate of the culture medium to be suitably estimated even when the first container is tilted to move the culture medium.

[0013] [7] The microfluidic device according to any one of [1] to [6] above may further include a mounting section for mounting the first container and a lid section for closing the opening at the top of the first container. The first light source may be disposed on the mounting section, and the first light detector may be disposed on the lid section. In this case, the reproducibility of the position of the optical axis within the first container is improved, and a decrease in the accuracy of estimating the first and second optical path lengths can be prevented. In addition, since the liquid surface of the culture medium has a curvature due to a meniscus generated by surface tension, the diameter of the first light changes as it passes through the liquid surface. By disposing the first light detector on the lid section, the distance between the first light detector and the liquid surface is reduced, allowing the first light to be incident on the first light detector with only a small change in the diameter of the first light.

[0014] [8] In the microfluidic devices of [1] to [7] above, the wavelength of the first light may be in the near-infrared range. The culture medium is mostly water, and water absorbs light in the near-infrared range. Therefore, by having the wavelength of the first light in the near-infrared range, the estimation accuracy of the first optical path length and the second optical path length can be improved.

[0015] [9] In the microfluidic devices of [1] to [8] above, the first light detection unit may include an organic photodiode. Organic photodiodes are inexpensive compared to other types of photodiodes, and can be easily disposed of along with the first container and the flow path. This prevents unwanted substances from being mixed into the culture solution (contamination). In addition, organic photodiodes can be designed to receive light of a desired wavelength. Therefore, a wavelength with a high absorption rate in the culture solution can be selected as the wavelength of the first light, thereby improving the accuracy of estimating the flow rate of the culture solution.

[0016]

[10] In the microfluidic devices of [2] to [9] above, the control unit may have an actuator that tilts the first container, the second container, and the flow path.

[11] Alternatively, the control unit may have a pressure pump that is provided in one or both of the first container and the second container and that changes the air pressure at the liquid surface of the culture solution.

[12] Alternatively, the control unit may have a flow pump that is provided in the flow path and moves the culture solution. For example, any of the configurations

[10] to

[12] above can move the culture solution in the flow path. Furthermore, with these configurations, if the flow path becomes clogged, the culture solution cannot flow at the desired flow rate, so the above microfluidic devices are particularly effective.

[0017]

[13] The microfluidic device according to any one of [1] to

[12] above may further include a third light source that irradiates the culture solution in the first container with a third light having a wavelength different from that of the first light. The first light detection unit may further detect a fifth light intensity, which is the intensity of the third light that has passed through the culture solution in the first container. The calculation unit may further estimate the hydrogen ion exponent (pH) of the culture solution based on the optical density of the culture solution in the first container obtained based on the fifth light intensity. In order to estimate changes in pH of the culture solution, a dye such as phenol red that changes color in response to changes in pH may be added to the culture solution. By measuring the optical density using third light having a wavelength that can measure the color change caused by this dye, the pH of the culture solution can be estimated in addition to the flow rate of the culture solution.

[0018]

[14] A flow rate estimation method according to an embodiment of the present disclosure is a flow rate estimation method for a microfluidic device. The microfluidic device includes a first container containing a culture medium and a channel connected to the first container, through which the culture medium flows and through which a biological sample is disposed in the culture medium. The flow rate estimation method includes a starting step, a detecting step of a first light intensity, a detecting step of a second light intensity, and a estimating step. The starting step involves moving the culture medium in the channel to start an operation of changing the volume of the culture medium in the first container. The detecting step involves irradiating the culture medium in the first container with a first light passing through the surface of the culture medium, and detecting the first light intensity, which is the intensity of the first light passing through the culture medium in the first container, at a first timing. The detecting step involves irradiating the culture medium in the first container with the first light, and detecting the second light intensity, which is the intensity of the first light passing through the culture medium in the first container, at a second timing different from the first timing. In the estimating step, a change over time between a first optical path length, which is the optical path length of the first light in the culture solution in the first container at a first timing, and a second optical path length, which is the optical path length of the first light in the culture solution in the first container at a second timing, is estimated based on the optical density of the culture solution in the first container obtained based on the first light intensity and the optical density of the culture solution in the first container obtained based on the second light intensity. In the estimating step, a flow velocity of the culture solution in the flow path is estimated based on the change over time.

[0019] In the flow velocity estimation method described in

[14] above, the flow velocity of the culture medium in a flow path is estimated based on the time change in the optical path length of the first light passing through the culture medium surface, i.e., the time change in the liquid surface position. The optical path length in the culture medium can be estimated based on the optical density of the culture medium, which is obtained based on the light intensity of the first light incident on the first light detector. This flow velocity estimation method does not require the installation of a device for measuring flow velocity in an extremely narrow flow path, making it easy to measure the flow velocity of the culture medium in an extremely narrow flow path. Note that if the volume of the culture medium is excessive, the distance the first light travels in the culture medium becomes long, resulting in excessive attenuation of the first light, making it difficult to accurately estimate the flow velocity of the culture medium. This flow velocity estimation method is suitable for measuring the flow velocity in an extremely narrow flow path, such as a microchannel, where the volume of the culture medium is relatively small.

[0020]

[15] In the flow rate estimation method according to

[14] above, the microfluidic device may further include a second container containing a culture solution and connected to the other end of the flow path. In the starting step, the culture solution may be moved between the first container and the second container through the flow path. In the detecting step, the culture solution in the second container may be irradiated with second light passing through the surface of the culture solution, and a third light intensity, which is the intensity of the second light having passed through the culture solution in the second container, may be further detected at a first timing. In the detecting step, the culture solution in the second container may be irradiated with second light, and a fourth light intensity, which is the intensity of the second light having passed through the culture solution in the second container, may be further detected at a second timing. In the estimating step, the time change may be further estimated based on an optical density of the culture solution in the second container obtained based on the third light intensity and an optical density of the culture solution in the second container obtained based on the fourth light intensity. In this case, even if the absorption coefficient of the culture medium is unknown, if the total length of the optical path length of the first light in the culture medium in the first container and the optical path length of the second light in the culture medium in the second container is known, the amount of change in the optical path length over time can be estimated.

[0021]

[16] In the flow velocity estimation method of

[15] above, in the estimating step, the time change may be further estimated based on the total length of the optical path length of the first light in the culture medium of the first container and the optical path length of the second light in the culture medium of the second container.

[0022]

[17] In the flow velocity estimation method according to

[16] above, in the estimating step, the total length may be calculated based on the total volume of the culture solution contained in the first container, the second container, and the flow path. For example, by using such a method, the total length of the optical path length of the first light in the culture solution in the first container and the optical path length of the second light in the culture solution in the second container can be easily determined.

[0023]

[18] In the flow velocity estimation methods of

[14] to

[17] above, the optical axis of the first light may be tilted with respect to the liquid surface of the culture medium in the first container. This makes it possible to suitably estimate the flow velocity of the culture medium even when the first container is tilted to move the culture medium.

[0024]

[19] In the flow velocity estimation methods of

[14] to

[18] above, the microfluidic device may further include a mounting section for mounting the first container and a lid section for closing the opening at the top of the first container. In the steps of detecting the first light intensity and detecting the second light intensity, the first light may be emitted from a light source disposed on the mounting section, and the intensity of the first light may be detected by a light detection section disposed on the lid section. In this case, as described above, the reproducibility of the position of the optical axis within the first container is improved, and a decrease in the estimation accuracy of the first optical path length and the second optical path length can be prevented. In addition, since the distance between the light detection section and the liquid surface is reduced, the first light can be incident on the light detection section with only a small change in the optical diameter of the first light.

[0025]

[20] In the flow velocity estimation methods

[14] to

[19] above, the wavelength of the first light may be included in the near-infrared range. In this case, as described above, it is possible to improve the estimation accuracy of the first optical path length and the second optical path length.

[0026]

[21] In the flow velocity estimation methods

[14] to

[20] above, an organic photodiode may be used to detect the first light intensity and the second light intensity. In this case, as described above, contamination of the culture solution can be prevented. In addition, the accuracy of estimating the flow velocity of the culture solution can be improved.

[0027]

[22] In the flow rate estimation methods of

[15] to

[21] above, in the starting step, the culture medium may be moved by tilting the first container, the second container, and the flow path.

[23] Alternatively, in the starting step, the culture medium may be moved by changing the air pressure at the surface of the culture medium in one or both of the first container and the second container.

[24] Alternatively, in the starting step, the culture medium may be moved using a flow pump provided in the flow path. For example, the culture medium in the flow path can be moved by any of the methods

[22] to

[24] . Furthermore, in these methods, if a blockage occurs in the flow path, the culture medium cannot flow at the desired flow rate, so the above flow rate estimation method is particularly effective.

[0028]

[25] The flow rate estimation method according to any one of

[14] to

[24] above may further include the steps of: irradiating the culture solution in the first container with a third light having a wavelength different from that of the first light; detecting a fifth light intensity, which is the intensity of the third light that has passed through the culture solution in the first container; and estimating the hydrogen ion exponent (pH) of the culture solution based on the optical density of the culture solution in the first container obtained based on the fifth light intensity. In this case, as described above, the pH of the culture solution can be estimated in addition to the flow rate of the culture solution. [Effects of the Invention]

[0029] According to the present disclosure, it is possible to provide a microfluidic device and a flow rate estimation method that can easily measure the flow rate of a culture medium in an extremely narrow channel. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is a perspective view schematically illustrating a microfluidic device according to an embodiment of the present disclosure. [Figure 2] FIG. 2 is a diagram for explaining a method for calculating the optical density of the culture medium in the first container. [Figure 3] FIG. 3 is a diagram illustrating an example of the hardware configuration of the calculation unit. [Figure 4] FIG. 4 is a diagram showing a state in which the optical axis of the first light source is inclined with respect to the liquid surface of the culture medium in the first container. [Figure 5] FIG. 5 is a graph showing the relationship between the reflectance and angle on the surface of the culture medium. [Figure 6] FIG. 6 is a flowchart illustrating a flow velocity estimation method according to one embodiment. [Figure 7] FIG. 7 is a diagram showing a specific example of the configuration of the peripheral structure of the first container and the second container. [Figure 8] FIG. 8 is a diagram showing a specific example of the configuration of the peripheral structure of the first container and the second container. [Figure 9] FIG. 9 is a diagram showing a specific example of the configuration of the peripheral structure of the first container and the second container. [Figure 10] FIG. 10 is a diagram showing a specific example of the configuration of the peripheral structure of the first container and the second container. DETAILED DESCRIPTION OF THE INVENTION

[0031] Specific examples of the present embodiment will be described with reference to the drawings as necessary. The present invention is not limited to these examples, but is defined by the claims, and is intended to include all modifications within the meaning and scope of the claims. In the following description, the same elements in the description of the drawings will be given the same reference numerals, and duplicate explanations will be omitted.

[0032] 1 is a perspective view schematically illustrating a microfluidic device 1 according to an embodiment of the present disclosure. The microfluidic device 1 includes a first container 3, a second container 4, a flow path 5, a control unit 6, a first light source 7, a second light source 8, a first light receiver 9 (first light detection unit), a second light receiver 10 (second light detection unit), and a calculation unit 22.

[0033] The first container 3 and the second container 4 contain a culture solution C. The first container 3 and the second container 4 are, for example, cylindrical containers having a bottom surface and an open top. The cross-sectional area of ​​each of the first container 3 and the second container 4 in a horizontal plane is constant in the depth direction. The first container 3 and the second container 4 are arranged next to each other in the horizontal direction. In one example, the capacity of the second container 4 is equal to the capacity of the first container 3. The capacity of the first container 3 and the second container 4 is, for example, within the range of 50 μl to 1000 μl.

[0034] The flow channel 5 connects the bottom of the first container 3 to the bottom of the second container 4. That is, one end of the flow channel 5 is connected to the bottom of the first container 3, and the other end of the flow channel 5 is connected to the bottom of the second container 4. The culture solution C flows from the first container 3 through the flow channel 5 to the second container 4. In the flow channel 5, a biological sample to be tested or cultured is placed in the culture solution C. The biological sample is, for example, a cell, tissue, cell aggregate, tissue, etc. The flow channel 5 is, for example, a microchannel. A microchannel is a flow channel having a cross-sectional dimension of less than 1 mm and greater than 1 μm. For example, when the cross section 51 of the flow channel 5, which is a cross section perpendicular to the direction in which the culture solution C flows, is square, the length of one side of the square is greater than 1 μm and less than 1 mm. In other words, the area of ​​the cross section 51 is 1 μm. 2 1mm larger 2 The shape of the cross section 51 is not limited to a square, but may be various other shapes.

[0035] The control unit 6 moves the culture solution C in the flow path 5 to change the amount of culture solution C in the first container 3 and the second container 4. The control unit 6 moves the culture solution C between the first container 3 and the second container 4 through the flow path 5. Therefore, the increase in the amount of culture solution C in the second container 4 is equal to the decrease in the amount of culture solution C in the first container 3. The control unit 6 has, for example, a pressure pump provided in one or both of the first container 3 and the second container 4 to change the air pressure at the liquid surface of the culture solution C. In this case, an air supply pipe 61 extends from the control unit 6 to the top of the first container 3, and an air intake pipe 62 extends from the control unit 6 to the top of the second container 4.

[0036] The control unit may have, instead of the pressure pump described above, an actuator that tilts the first container 3, the second container 4, and the flow path 5. In this case, the actuator tilts the first container 3, the second container 4, and the flow path 5, thereby moving the culture solution C from the first container 3 through the flow path 5 to the second container 4. Alternatively, the control unit may have, instead of the pressure pump and actuator described above, a flow pump that is provided in the flow path 5 and moves the culture solution C. In this case, the flow pump moves the culture solution C from the first container 3 through the flow path 5 to the second container 4.

[0037] The first light source 7 irradiates the culture solution C in the first container 3 with a first light L1 that passes through the surface of the culture solution C. The first light source 7 is, for example, a laser light source, and the first light L1 is, for example, laser light. The second light source 8 irradiates the culture solution C in the second container 4 with a second light L2 that passes through the surface of the culture solution C. The second light source 8 is, for example, a laser light source, and the second light L2 is, for example, laser light. The wavelengths of the first light L1 and the second light L2 are, for example, in the near-infrared range. The wavelength of the first light L1 may be equal to or different from the wavelength of the second light L2. The first container 3 and the second container 4 each have a light transmittance of, for example, 90% or more at the wavelengths of the first light L1 and the second light L2, respectively. In one example, the optical axes of the first light source 7 and the second light source 8 are perpendicular to the surface of the culture solution C. Alternatively, the optical axes of the first light source 7 and the second light source 8 may be inclined with respect to the surface of the culture solution C.

[0038] The first light receiver 9 detects a first light intensity and a second light intensity. The first light intensity is the intensity of the first light L1 passing through the culture solution C in the first container 3 at a first timing. The second light intensity is the intensity of the first light L1 passing through the culture solution C in the first container 3 at a second timing different from the first timing. The first timing and the second timing may be instantaneous or may include a certain period (e.g., required to detect light). The first light source 7 may irradiate the first light L1 only at the first timing and the second timing and not at other periods, or may irradiate the first light L1 for a continuous period including the first timing and the second timing. The first light receiver 9 includes a photodiode, and in one example, an organic photodiode. In the illustrated example, the first light source 7 is disposed on the bottom side of the first container 3 and the first light receiver 9 is disposed on the opening side of the top of the first container 3, but the arrangement of the first light source 7 and the first light receiver 9 may be reversed.

[0039] The second light receiver 10 detects a third light intensity and a fourth light intensity. The third light intensity is the intensity of the second light L2 that passed through the culture solution C in the second container 4 at the first timing. The fourth light intensity is the intensity of the second light L2 that passed through the culture solution C in the second container 4 at the second timing. The second light source 8 may irradiate the second light L2 only at the first timing and the second timing and not at other periods, or may irradiate the second light L2 for a continuous period including the first timing and the second timing. The second light receiver 10 includes a photodiode, and in one example, includes an organic photodiode. In the illustrated example, the second light source 8 is disposed on the bottom side of the second container 4 and the second light receiver 10 is disposed on the opening side of the top of the second container 4, but the arrangement of the second light source 8 and the second light receiver 10 may be reversed.

[0040] The calculation unit 22 estimates the first optical path length and the second optical path length. The first optical path length is the optical path length of the first light L1 in the culture solution C in the first container 3 at a first timing. The calculation unit 22 estimates the first optical path length based on the optical density of the culture solution C in the first container 3 obtained based on the first light intensity. The second optical path length is the optical path length of the first light L1 in the culture solution C in the first container 3 at a second timing. The calculation unit 22 estimates the second optical path length based on the optical density of the culture solution C in the first container 3 obtained based on the second light intensity.

[0041] FIG. 2 is a diagram illustrating a method for calculating the optical density of the culture solution C in the first container 3. As shown in FIG. 2, the absorption coefficient of the culture solution C is α (m), the optical path length in the culture solution C is L (m), the light intensity of the first light L1 before entering the culture solution C is l0, the light intensity of the first light L1 after emitting from the culture solution C is l1, the concentration of the culture solution C is c, and the molar absorption coefficient is ε. In this case, the optical density A is calculated by the following formula (1). Note that the light intensity l0 is known in advance, and the light intensity l1 can be determined from the first light intensity or the second light intensity.

number

[0042] The optical density of the culture solution C in the first container 3 at the first timing is A 1_ti The optical density of the culture solution C in the first container 3 at the second timing is A 1_t(i+1) The optical path length (first optical path length) in the culture solution C in the first container 3 at the first timing is L 1_ti (m), the optical path length (second optical path length) in the culture solution C in the first container 3 at the second timing is L 1_t(i+1) (m). At this time, the optical density A 1_ti and the first optical path length L 1_ti and optical density A 1_t(i+1) and the second optical path length L 1_t(i+1) The following relationship (2) holds between

number

number

[0043] The calculation unit 22 calculates the first optical path length L 1_ti and the second optical path length L 1_t(i+1) The flow velocity of the culture solution C in the flow channel 5 is estimated based on the amount of change over time (ΔL) between the first timing and the second timing. When the time difference between the first timing and the second timing is Δt=t(i+1)−ti (seconds), the flow velocity of the culture solution C in the flow channel 5 is m (m / s) is calculated using the following formula (4). m (m 3 / s) is the flow rate of flow channel 5 per unit time, and S m (m 2 ) is the area of ​​the cross section 51, and S is the area of ​​the liquid surface of the first container 3.

number

[0044] 3 is a diagram schematically illustrating an example of the hardware configuration of the calculation unit 22. As shown in FIG. 3, the calculation unit 22 may be physically configured as a typical computer including a processor (CPU) 23, main storage devices such as a nonvolatile memory (ROM) 24 and a volatile memory (RAM) 25, input devices 26 such as a keyboard, mouse, and touch screen, output devices 27 such as a display (including a touch screen), a communication module 28 such as a network card for transmitting and receiving data to and from other devices, and an auxiliary storage device 29 such as a hard disk. The input device 26 may receive input from a user regarding the extinction coefficient of the culture solution C or the total volume of the culture solution C.

[0045] The computer processor 23 can realize the functions of the calculation unit 22 by means of a flow velocity estimation program. In other words, the flow velocity estimation program causes the computer processor 23 to operate as the calculation unit 22. The flow velocity estimation program is stored in a storage device (storage medium) inside or outside the computer, such as the auxiliary storage device 29. The storage device may be a non-transitory recording medium. Examples of the recording medium include a flexible disk, a CD, a DVD, a recording medium such as a ROM, a semiconductor memory, a cloud server, etc.

[0046] The above description illustrates a case where the optical axes of the first light source 7 and the second light source 8 are perpendicular to the liquid surface of the culture solution C. However, the present invention is not limited to this. The optical axis of the first light source 7 may be inclined with respect to the liquid surface of the culture solution C in the first container 3. Similarly, the optical axis of the second light source 8 may be inclined with respect to the liquid surface of the culture solution C in the second container 4. FIG. 4 illustrates a state where the optical axis 3a of the first light source 7 is inclined with respect to the liquid surface of the culture solution C in the first container 3. This can occur when the bottom surface of the first container 3, which is normally horizontal, is inclined with respect to the horizontal. For example, this can occur when the first container 3, the second container 4, and the flow path 5 are inclined to move the culture solution C from the first container 3 through the flow path 5 to the second container 4. The angle θ formed between the normal vector of the liquid surface of the culture solution C and the optical axis 3a is shown in the figure.

[0047] FIG. 5 is a graph showing the relationship between the reflectance at the liquid surface of the culture solution C and the angle θ. In FIG. 5, the horizontal axis represents the angle θ (unit: degrees), and the vertical axis represents the reflectance (unit: %). As shown in FIG. 5, when the angle θ is greater than 25 degrees, the reflectance increases rapidly as the angle θ increases. As the reflectance increases, the light intensity detected by the first light receiver 9 decreases, so calculations must be performed taking the reflectance into account. In contrast, when the angle θ is less than 25 degrees, the reflectance remains almost unchanged compared to when the optical axis is perpendicular to the liquid surface of the culture solution C (i.e., angle θ = 0°). When the first container 3 is tilted to move the culture solution C, the resulting angle θ is at most 25 degrees. This makes it possible to avoid a decrease in the accuracy of flow velocity estimation.

[0048] Referring again to FIG. 1 , the microfluidic device 1 further includes a third light source 18. The third light source 18 irradiates the culture solution C in the first container 3 with third light L3, which has a wavelength different from that of the first light L1. The first light receiver 9 detects fifth and sixth light intensities. The fifth light intensity is the intensity of the third light L3 that has passed through the culture solution C in the first container 3 at a third timing. The sixth light intensity is the intensity of the third light L3 that has passed through the culture solution C in the first container 3 at a fourth timing that is different from the third timing. The first light receiver 9 may detect both the first light L1 and the third light L3 using a single light detection element, or may have a light detection element for detecting the third light L3 separate from the light detection element for detecting the first light L1. The third and fourth timings may be instantaneous or may include a certain period (e.g., a period required to detect the light). The third and fourth timings may be the same as or different from the first and second timings, respectively. Furthermore, the third light source 18 may irradiate the third light L3 only at the third and fourth timings and not at other times, or may irradiate the third light L3 for a continuous period including the third and fourth timings. In the illustrated example, the third light source 18 is disposed on the bottom side of the first container 3, and the first light receiver 9 is disposed on the top opening side of the first container 3. However, the third light source 18 and the first light receiver 9 may be disposed in the opposite order. The calculation unit 22 further estimates the hydrogen ion exponent (pH) of the culture solution C in the first container 3 based on the optical density of the culture solution C in the first container 3 obtained based on the fifth light intensity. Furthermore, the calculation unit 22 may further estimate a change in the hydrogen ion exponent (pH) of the culture solution C in the first container 3 based on the optical density of the culture solution C in the first container 3 obtained based on the fifth light intensity and the optical density of the culture solution C in the first container 3 obtained based on the sixth light intensity. In order to estimate the change in pH of the culture solution C, a dye such as phenol red, which changes color depending on the change in pH, may be added to the culture solution C. By measuring the optical density using a third light L3 having a wavelength that can measure the color change caused by this dye, it is possible to estimate the pH of the culture solution C and the change in pH, as well as the flow rate of the culture solution C.The method of calculating the optical density can be achieved by replacing the first light L1 with the third light L3 in the above-described FIG. 2 and its description.

[0049] Next, a flow velocity estimation method according to this embodiment will be described. The flow velocity estimation method according to this embodiment is a flow velocity estimation method for the microfluidic device 1 described above, and can be implemented using, for example, the microfluidic device 1. Fig. 6 is a flowchart showing the flow velocity estimation method according to this embodiment. As shown in Fig. 6, the flow velocity estimation method includes steps ST1 to ST7.

[0050] In step ST1, the control unit 6 starts operating to move the culture solution C in the flow path 5 and change the amount of culture solution C in the first container 3. At this time, the culture solution C is moved between the first container 3 and the second container 4 through the flow path 5. In this step ST1, the culture solution C may be moved by tilting the first container 3, the second container 4, and the flow path 5, or by changing the air pressure on the liquid surface of the culture solution C in one or both of the first container 3 and the second container 4, or the culture solution C may be moved using a flow pump provided in the flow path 5.

[0051] In step ST2, at a first timing, the culture solution C in the first container 3 is irradiated with first light L1 that passes through the liquid surface of the culture solution C, and a first light intensity that is the intensity of the first light L1 that has passed through the culture solution C in the first container 3 is detected. In step ST3, at a second timing, the culture solution C in the first container 3 is irradiated with first light L1, and a second light intensity that is the intensity of the first light L1 that has passed through the culture solution C in the first container 3 is detected. Note that in steps ST2 and ST3, the optical axis of the first light source 7 may be perpendicular to or inclined with respect to the liquid surface of the culture solution C in the first container 3.

[0052] Step ST4 includes steps ST41 and ST42. In step ST41, the first optical path length L1, which is the optical path length of the first light L1 in the culture solution C in the first container 3 at the first timing, is calculated. 1_tiand a second optical path length L, which is the optical path length of the first light L1 in the culture solution C of the first container 3 at the second timing. 1_t(i+1) The time change ΔL between 1_ti , the optical density A of the culture solution C in the first container 3 obtained based on the second light intensity 1_t(i+1) , and the known absorption coefficient α of the culture solution C, the first optical path length L is estimated by the above-mentioned formula (3). 1_ti and the second optical path length L 1_t(i+1) Based on the time change ΔL between m is estimated using the above-mentioned formula (4).

[0053] Next, the hydrogen ion exponent (pH) of the culture solution C and the amount of change in pH are estimated. First, in step ST5, the culture solution C in the first container 3 is irradiated with third light L3, and a fifth light intensity, which is the intensity of the third light L3 that has passed through the culture solution C in the first container 3, is detected at a third timing. Next, in step ST6, the culture solution C in the first container 3 is irradiated with third light L3, and a sixth light intensity, which is the intensity of the third light L3 that has passed through the culture solution C in the first container 3, is detected at a fourth timing different from the third timing. Next, in step ST7, the hydrogen ion exponent (pH) of the culture solution C is estimated based on the optical density of the culture solution C in the first container 3 obtained based on the fifth light intensity. Also, in step ST7, the amount of change in the hydrogen ion exponent (pH) of the culture solution C is estimated based on the optical density of the culture solution C in the first container 3 obtained based on the fifth light intensity and the optical density of the culture solution C in the first container 3 obtained based on the sixth light intensity. In step ST7, only the pH of the culture solution C may be estimated. In this case, step ST6 is not necessary.

[0054] The effects obtained by the microfluidic device 1 and flow rate estimation method according to the present embodiment described above will be described. In the microfluidic device 1 of the present embodiment, the calculation unit 22 estimates the flow rate of the culture solution C in the flow path 5 based on the time change ΔL of the optical path length of the first light L1 passing through the liquid surface of the culture solution C in the culture solution C, i.e., the time change in the liquid surface position. The optical path length in the culture solution C can be estimated based on the optical density of the culture solution C, which is obtained based on the light intensity of the first light L1 incident on the first light receiver 9. This microfluidic device 1 does not require a device for measuring the flow rate in the extremely narrow flow path 5, so the flow rate of the culture solution C in the extremely narrow flow path 5 can be easily measured. Note that if the volume of the culture solution C is excessive, the distance that the first light L1 travels in the culture solution C becomes long, resulting in excessive attenuation of the first light L1, making it difficult to accurately estimate the flow rate of the culture solution C. This microfluidic device 1 is an apparatus suitable for measuring the flow rate in an extremely narrow channel 5, such as a microchannel, in which the amount of culture solution C is relatively small.

[0055] Furthermore, in the flow rate estimation method of this embodiment, the flow rate of the culture solution C in the flow path 5 is estimated based on the amount of change over time in the optical path length of the first light L1 passing through the liquid surface of the culture solution C, i.e., the amount of change over time in the liquid surface position. The optical path length in the culture solution C can be estimated based on the optical density of the culture solution C, which is obtained based on the light intensity of the first light L1 incident on the first light receiver 9. According to this flow rate estimation method, there is no need to install a device for measuring the flow rate in the extremely narrow flow path 5, so the flow rate of the culture solution C in the extremely narrow flow path 5 can be easily measured. Note that this flow rate estimation method is also suitable for measuring the flow rate in an extremely narrow flow path 5, such as a microchannel, where the amount of culture solution C is relatively small.

[0056] As in this embodiment, the microfluidic device 1 may include a second container 4 that contains a culture solution C and is connected to the other end of the flow path 5. The control unit 6 may move the culture solution C between the first container 3 and the second container 4 through the flow path 5. This allows the culture solution C to circulate between the first container 3 and the second container 4, thereby reducing the amount of culture solution C consumed.

[0057] As in this embodiment, the wavelength of the first light L1 may be included in the near-infrared region. The majority of the culture solution C is water, and water absorbs light in the near-infrared region. Therefore, when the wavelength of the first light L1 is included in the near-infrared region, the first optical path length L 1_ti and the second optical path length L 1_t(i+1) In addition, in the microfluidic device 1, the volumes of the first container 3 and the second container 4 are extremely small, for example, 1 ml or less. In such small containers, the light in the near-infrared region is not completely absorbed by the culture solution C. Therefore, the first light L1 in the near-infrared region is used to measure the first optical path length L 1_ti and the second optical path length L 1_t(i+1) It is possible to estimate the following.

[0058] As in this embodiment, the first light receiver 9 may include an organic photodiode. Similarly, in the flow rate estimation method of this embodiment, an organic photodiode may be used to detect the first light intensity and the second light intensity. Because organic photodiodes are less expensive than other types of photodiodes, they can be easily disposed of along with the first container 3 and the flow path 5. This prevents unwanted substances from being mixed into the culture solution C (contamination). In addition, the organic photodiode can be designed to receive light of a desired wavelength. Therefore, a wavelength with a high absorption rate in the culture solution C can be selected as the wavelength of the first light L1, thereby improving the accuracy of estimating the flow rate of the culture solution C.

[0059] As in this embodiment, the control unit 6 may have an actuator that tilts the first container 3, the second container 4, and the flow path 5. Furthermore, in step ST1, the culture solution C may be moved by tilting the first container 3, the second container 4, and the flow path 5. Alternatively, the control unit 6 may have a pressure pump provided in one or both of the first container 3 and the second container 4 that changes the air pressure at the liquid surface of the culture solution C. Furthermore, in step ST1, the culture solution C may be moved by changing the air pressure at the liquid surface of the culture solution C in one or both of the first container 3 and the second container 4. Alternatively, the control unit 6 may have a flow pump provided in the flow path 5 that moves the culture solution C. Furthermore, in step ST1, the culture solution C may be moved using a flow pump provided in the flow path 5. For example, the culture solution C in the flow path 5 can be moved by any of these configurations. Furthermore, in an MPS, it is necessary to continuously flow the culture solution C at a desired flow rate. However, with these configurations, if the flow path 5 becomes clogged for some reason, the culture solution C cannot be flowed at the desired flow rate. The microfluidic device 1 and flow rate estimation method of this embodiment make it easy to continuously monitor the flow rate of the culture solution C, thereby enabling early detection of clogging in the flow channel 5. Therefore, the microfluidic device 1 and flow rate estimation method of this embodiment are particularly effective for these configurations.

[0060] As in the present embodiment, the calculation unit 22 may further estimate the pH of the culture solution C in the first container 3 based on the optical density of the culture solution C in the first container 3 obtained based on the fifth light intensity. Similarly, the flow rate estimation method may include step ST7 of estimating the pH of the culture solution C based on the optical density of the culture solution C in the first container 3 obtained based on the fifth light intensity. This makes it possible to estimate the pH of the culture solution C in addition to the flow rate of the culture solution C.

[0061] As described above, the optical axis 3a of the first light source 7 may be inclined with respect to the liquid surface of the culture solution C in the first container 3. In this case, even if the first container 3 is inclined to move the culture solution C, the flow velocity of the culture solution C can be suitably estimated. [First Modification]

[0062] The calculation unit 22 calculates the time change ΔL of the optical path length between the first timing and the second timing as a function of the optical density A of the culture solution C in the first container 3 obtained based on the first light intensity. 1_ti , and the optical density A of the culture solution C in the first container 3 obtained based on the second light intensity. 1_t(i+1) In addition, the estimation may be further based on the optical density of the culture solution C in the second container 4 obtained based on the third light intensity and the optical density of the culture solution C in the second container 4 obtained based on the fourth light intensity.

[0063] Specifically, the optical density of the culture solution C in the second container 4 at the first timing is A 2_ti The optical density of the culture solution C in the second container 4 at the second timing is A 2_t(i+1) The optical path length (third optical path length) in the culture solution C in the second container 4 at the first timing is L 2_ti (m), the optical path length (fourth optical path length) in the culture solution C in the second container 4 at the second timing is L 2_t(i+1) (m) Optical density A 2_ti is calculated by the above formula (1) based on the third light intensity. 2_t(i+1) is calculated by the above formula (1) based on the fourth light intensity. 2_ti and the third optical path length L 2_ti and optical density A 2_t(i+1) and the fourth optical path length L 2_t(i+1) The following relationship (5) holds between

number

[0064] In addition, the first optical path length L 1_ti , second optical path length L 1_t(i+1) , third optical path length L 2_ti , and the fourth optical path length L 2_t(i+1) The following relationship (6) holds between L total is the total optical path length within the culture medium C.

number

number

[0065] The calculation unit 22 calculates the flow velocity v of the culture solution C in the flow channel 5 based on the time change ΔL. m is estimated by the above-mentioned formula (4). In this case, the absorption coefficient α of the culture solution C is not required. total may be calculated based on the total volume of the culture solution C contained in the first container 3, the second container 4, and the flow path 5.

[0066] Furthermore, in the above-mentioned step ST2, simultaneously with detecting the first light intensity, the culture solution C in the second container 4 may be irradiated with second light L2 passing through the surface of the culture solution C, and a third light intensity, which is the intensity of the second light L2 that has passed through the culture solution C in the second container 4, may be further detected. Furthermore, in the above-mentioned step ST3, simultaneously with detecting the second light intensity, the culture solution C in the second container 4 may be irradiated with second light L2 passing through the surface of the culture solution C, and a fourth light intensity, which is the intensity of the second light L2 that has passed through the culture solution C in the second container 4, may be further detected. In that case, in step ST41, the time change ΔL is calculated by multiplying the optical density A 1_ti , optical density A 1_t(i+1) , optical density A 2_ti , optical density A 2_t(i+1) , and the total length L total In this case, the absorption coefficient α of the culture solution C is not required. In this step ST41, the total length L total may be calculated based on the total volume of the culture solution C contained in the first container 3, the second container 4, and the flow path 5.

[0067] According to this modification, even if the absorption coefficient of the culture solution C is unknown, the total length L of the optical path length of the first light L1 in the culture solution C in the first container 3 and the optical path length of the second light L2 in the culture solution C in the second container 4 can be calculated. total If is known, the time change ΔL can be estimated.

[0068] As described above, the calculation unit 22 and step ST4 calculate the total length L total may be calculated based on the total volume of the culture solution C contained in the first container 3, the second container 4, and the flow path 5. For example, with such a configuration, the total length L total can be easily known. [Specific configuration example]

[0069] 7 to 10 are diagrams showing specific examples of the configuration of the peripheral structure of the first container 3 and the second container 4. As shown in FIGS. 7 to 10, the microfluidic device 1 may include a base 11 or jig 15, a plate 12, and a lid 13. The base 11 is an example of a mounting portion in the present disclosure, and the plate 12 is mounted on the base 11. The jig 15 is a member that fixes the lid 13 to the plate 12 by sandwiching the plate 12 and the lid 13 from above and below. The portion located below the plate 12 is an example of a mounting portion in the present disclosure, and the plate 12 is mounted on the jig 15. The plate 12 is made of, for example, resin. The first container 3, the second container 4, and a flow path 5 are formed on the plate 12. The lid 13 is disposed on the plate 12 and closes the upper openings of the first container 3 and the second container 4.

[0070] In the configurations shown in FIGS. 7(a) and 7(b), the first light source 7 and the second light source 8 are disposed on a base 11. The first light receiver 9 and the second light receiver 10 are mounted on a substrate 17. The substrate 17 is, for example, a glass substrate or a plastic substrate. In the configuration shown in FIG. 7(a), the substrate 17 is disposed on the upper surface of the lid portion 13 (i.e., the surface opposite to the surface facing the first container 3 and the second container 4) and is adhered to the lid portion 13. The first light receiver 9 and the second light receiver 10 are mounted on the upper surface of the substrate 17. In this case, the lid portion 13 and the substrate 17 have a light transmittance of, for example, 90% or more at the wavelengths of the first light L1 and the second light L2. In the configuration shown in FIG. 7(b), the substrate 17 is disposed on the lower surface of the lid portion 13 (i.e., the surface facing the first container 3 and the second container 4) and is adhered to the lid portion 13. The first light receiver 9 and the second light receiver 10 are mounted on the lower surface of the substrate 17. In this case, the cover 13 and the substrate 17 do not need to be optically transparent and may block external light.

[0071] 7(c), the first light source 7 and the second light source 8 are disposed in a portion of the jig 15 that is located below the plate 12. The first light receiver 9 and the second light receiver 10 are disposed on the lid portion 13 and fabricated in a portion of the jig 15 that is located above the lid portion 13. In this case, the lid portion 13 has a light transmittance of, for example, 90% or more at the wavelengths of the first light L1 and the second light L2.

[0072] 7, in steps ST2 and ST3 of the flow velocity estimation method of the present disclosure, a first light source 7 and a second light source 8, respectively, disposed on the base 11 (or a portion of the jig 15 located below the plate 12), irradiate the first light L1 and the second light L2, respectively. Also, a first light receiver 9 and a second light receiver 10, respectively, disposed on the lid 13, detect the intensities of the first light L1 and the second light L2, respectively.

[0073] 7 simplifies the operation of the microfluidic device 1, reducing the user's workload. In addition, the reproducibility of the positions of the optical axes in the first container 3 and the second container 4 is improved. Therefore, the first optical path length L 1_ti and the second optical path length L 1_t(i+1) This prevents a decrease in the estimation accuracy of the first light L1. In addition, because the liquid surface of the culture solution C has a curvature due to a meniscus generated by surface tension, the light diameter of the first light L1 changes as it passes through the liquid surface. By disposing the first light receiver 9 and the second light receiver 10 on the lid portion 13, the distance between the first light receiver 9 and the second light receiver 10 and the liquid surface is reduced. Therefore, the first light L1 can be made incident on the first light receiver 9 with only a small change in the light diameter of the first light L1, and the second light L2 can be made incident on the second light receiver 10 with only a small change in the light diameter of the second light L2.

[0074] The present invention is not limited to the above example, and the first and second optical receivers 9 and 10 may be fabricated directly on the lid portion 13. In this case, the substrate 17 is not necessary. Instead of disposing the substrate 17, the first and second optical receivers 9 and 10 may be directly bonded to the lid portion 13 by a covalent bond, an ionic bond, a metallic bond, lamination, or an adhesive. The adhesive may be applied to the entire surfaces of the first and second optical receivers 9 and 10, or may be applied only to the ends of the first and second optical receivers 9 and 10. The first and second optical receivers 9 and 10 may be formed by directly depositing a film on the lid portion 13 by sputtering, vapor deposition, or coating.

[0075] 8, the first light source 7 and the first light receiver 9 are arranged in reverse, and the second light source 8 and the second light receiver 10 are arranged in reverse. That is, the first light source 7 and the second light source 8 are arranged on the lid portion 13 side, and the first light receiver 9 and the second light receiver 10 are formed on or attached to the base 11.

[0076] Specifically, in the configuration of FIG. 8(a), the first light source 7 and the second light source 8 are disposed above the lid portion 13 and spaced apart from the lid portion 13. The first light L1 and the second light L2 pass through the lid portion 13 and are irradiated onto the culture solution C. In the configuration of FIG. 8(b), the first light source 7 and the second light source 8 are fixed to the upper surface of the lid portion 13 by a jig 16 provided on the lid portion 13. In the configuration of FIG. 8(c), the first light source 7 and the second light source 8 are disposed above the lid portion 13, and are disposed in a portion of the jig 15 that is located above the lid portion 13.

[0077] 8, in steps ST2 and ST3 of the flow velocity estimation method of the present disclosure, a first light source 7 and a second light source 8, respectively, disposed on or above the lid 13, irradiate the lid 13 with a first light L1 and a second light L2. A first light receiver 9 and a second light receiver 10, respectively, disposed below the plate 12, detect the intensities of the first light L1 and the second light L2.

[0078] 9(a) to 9(c) show configurations obtained by omitting the second light source 8 and the second light receiver 10 from the configurations shown in FIGS. 7(a) to 7(c), respectively. FIG. 10(a) to 9(c) show configurations obtained by omitting the second light source 8 and the second light receiver 10 from the configurations shown in FIGS. 8(a) to 8(c), respectively. When estimating the flow velocity of the culture solution C using only the first light L1 without using the second light L2, as in the above embodiment, the configurations shown in FIGS. 9 and 10 may be used. Even in this case, the flow velocity of the culture solution C can be suitably estimated by the above formulas (3) and (4).

[0079] The microfluidic device and flow rate estimation method according to the present disclosure are not limited to the above-described embodiment, and various other modifications are possible. For example, in the above-described embodiment, the first container and the second container are connected to each other by a flow path. However, the second container may be omitted, and one end of the flow path may be connected to the first container, and the other end of the flow path may be open. Even in this case, the flow rate of the culture solution C can be suitably estimated using only the first light L1 according to the above formulas (3) and (4).

[0080] In the above embodiment, when the absorption coefficient α of the culture solution C is known, the first optical path length L obtained at the first timing is 1_ti and the first optical path length L obtained at the second timing. 1_t(i+1) Based on this (in other words, using only one of the first light L1 and the second light L2), the flow velocity of the culture solution C is estimated (see formulas (3) and (4)). This is not limited to this form, and if the total volume of the culture solution C is known, the first optical path length L obtained at the first timing can be used. 1_ti and the fourth optical path length L obtained at the second timing. 2_t(i+1) The flow velocity of the culture solution C may be estimated based on the second light path length L included in the formula (2) (in other words, using one of the first light L1 and the second light L2 at the first timing, and using the other of the first light L1 and the second light L2 at the second timing). 1_t(i+1) is the known total volume of the culture solution C (in other words, the known total length of the optical path length in the culture solution C in the first container 3 and the optical path length in the culture solution C in the second container 4) and the fourth optical path length L 2_t(i+1) It can be calculated based on the following.

[0081] In the above case, the microfluidic device has the following configuration: a first container and a second container for containing a culture medium; a flow channel having one end connected to the first container and the other end connected to the second container, through which the culture medium flows and through which a biological sample is placed in the culture medium; a control unit that moves the culture solution in the flow path to change the amount of the culture solution in the first container and the second container; a first light source that irradiates the culture solution in the first container with first light that passes through the liquid surface of the culture solution; a second light source that irradiates the culture solution in the second container with second light that passes through the liquid surface of the culture solution; a first light detection unit that detects a first light intensity that is the intensity of the first light that has passed through the culture solution in the first container at a first timing; a second light detection unit that detects a fourth light intensity, which is the intensity of the second light that has passed through the culture solution in the second container, at a second timing different from the first timing; a calculation unit that estimates a time change amount between an optical path length of the first light in the culture solution in the first container at the first timing and an optical path length of the first light in the culture solution in the first container at the second timing based on an optical density of the culture solution in the first container obtained based on the first light intensity, an optical density of the culture solution in the second container obtained based on the fourth light intensity, and a total volume of the culture solution, and that estimates a flow velocity of the culture solution in the flow path based on the time change amount; Equipped with.

[0082] In the above case, the flow velocity estimation method includes the following steps: The microfluidic device comprises: a first container and a second container for containing a culture medium; a flow channel having one end connected to the first container and the other end connected to the second container, through which the culture medium flows and through which a biological sample is placed in the culture medium; Equipped with The flow velocity estimation method includes: a step of starting an operation of moving the culture medium in the flow path to change the amount of the culture medium in the first container and the second container; irradiating the culture solution in the first container with first light that passes through a liquid surface of the culture solution, and detecting a first light intensity that is the intensity of the first light that has passed through the culture solution in the first container at a first timing; irradiating the culture solution in the second container with second light, and detecting a fourth light intensity that is the intensity of the second light that has passed through the culture solution in the second container at a second timing different from the first timing; estimating a time change amount between a first optical path length, which is an optical path length of the first light in the culture solution in the first container at the first timing, and a second optical path length, which is an optical path length of the first light in the culture solution in the first container at the second timing, based on an optical density of the culture solution in the first container obtained based on the first light intensity and an optical density of the culture solution in the second container obtained based on the fourth light intensity, and estimating a flow velocity of the culture solution in the flow path based on the time change amount; Includes: [Explanation of symbols]

[0083] 1...microfluidic device, 3...first container, 3a...optical axis, 4...second container, 5...flow path, 6...control unit, 7...first light source, 8...second light source, 9...first light receiver, 10...second light receiver, 11...base, 12...plate, 13...lid, 15, 16...jig, 17...substrate, 22...computing unit, 23...processor, 24...non-volatile memory, 26...input device, 27...output device, 28...communication module, 29...auxiliary storage device, 51...cross section, 61...air supply tube, 62...air intake tube, A...optical density, C...culture medium, L1...first light, L2...second light, θ...angle.

Claims

1. a first container containing a culture medium; a flow channel having one end connected to the first container, through which the culture medium flows and through which a biological sample is placed in the culture medium; a control unit that moves the culture solution in the flow path to change the amount of the culture solution in the first container; a first light source that irradiates the culture solution in the first container with first light that passes through a liquid surface of the culture solution; a first light detection unit that detects a first light intensity that is the intensity of the first light that has passed through the culture solution in the first container at a first timing, and a second light intensity that is the intensity of the first light that has passed through the culture solution in the first container at a second timing that is different from the first timing; a calculation unit that estimates a time change amount between a first optical path length, which is an optical path length of the first light in the culture solution in the first container at the first timing, and a second optical path length, which is an optical path length of the first light in the culture solution in the first container at the second timing, based on an optical density of the culture solution in the first container obtained based on the first light intensity and an optical density of the culture solution in the first container obtained based on the second light intensity, and that estimates a flow velocity of the culture solution in the flow path based on the time change amount; A microfluidic device comprising:

2. a second container that contains the culture medium and is connected to the other end of the channel; The microfluidic device according to claim 1 , wherein the control unit moves the culture medium between the first container and the second container through the flow channel.

3. a second light source that irradiates the culture solution in the second container with second light that passes through the liquid surface of the culture solution; a second light detection unit that detects a third light intensity that is the intensity of the second light that has passed through the culture solution in the second container at the first timing, and a fourth light intensity that is the intensity of the second light that has passed through the culture solution in the second container at the second timing; Further provided with 3. The microfluidic device according to claim 2, wherein the calculation unit estimates the amount of change over time further based on an optical density of the culture solution in the second container obtained based on the third light intensity and an optical density of the culture solution in the second container obtained based on the fourth light intensity.

4. 4. The microfluidic device according to claim 3, wherein the calculation unit estimates the amount of change over time based on a total length of an optical path length of the first light in the culture solution of the first container and an optical path length of the second light in the culture solution of the second container.

5. The microfluidic device according to claim 4 , wherein the calculation unit calculates the total length based on a total volume of the culture solution contained in the first container, the second container, and the channel.

6. 6. The microfluidic device according to claim 1, wherein the optical axis of the first light source is inclined with respect to the liquid surface of the culture medium in the first container.

7. The apparatus further includes a mounting portion for mounting the first container and a lid portion for closing an opening at the top of the first container, the first light source is disposed on the mounting portion; The microfluidic device according to any one of claims 1 to 5, wherein the first optical detection unit is disposed in the lid unit.

8. The microfluidic device according to any one of claims 1 to 5, wherein the wavelength of the first light is in the near-infrared range.

9. The microfluidic device according to any one of claims 1 to 5, wherein the first light detection unit includes an organic photodiode.

10. 6. The microfluidic device according to claim 2, wherein the control unit has an actuator that tilts the first container, the second container, and the channel.

11. The microfluidic device according to any one of claims 2 to 5, wherein the control unit has a pressure pump provided in one or both of the first container and the second container to change the air pressure at the liquid surface of the culture solution.

12. 6. The microfluidic device according to claim 2, wherein the control unit has a flow pump provided in the flow channel for moving the culture solution.

13. a third light source that irradiates the culture solution in the first container with third light having a wavelength different from that of the first light; the first light detection unit further detects a fifth light intensity that is the intensity of the third light that has passed through the culture solution in the first container; The microfluidic device according to any one of claims 1 to 5, wherein the calculation unit further estimates a hydrogen ion exponent (pH) of the culture solution based on an optical density of the culture solution in the first container obtained based on the fifth light intensity.

14. 1. A flow rate estimation method for a microfluidic device, comprising: The microfluidic device comprises: a first container containing a culture medium; a flow channel having one end connected to the first container, through which the culture medium flows and through which a biological sample is placed in the culture medium; Equipped with The flow velocity estimation method includes: a step of starting an operation of moving the culture medium in the flow path to change the amount of the culture medium in the first container; irradiating the culture solution in the first container with first light that passes through a liquid surface of the culture solution, and detecting a first light intensity that is the intensity of the first light that has passed through the culture solution in the first container at a first timing; irradiating the culture solution in the first container with the first light, and detecting a second light intensity, which is the intensity of the first light that has passed through the culture solution in the first container, at a second timing different from the first timing; estimating a time change amount between a first optical path length, which is an optical path length of the first light in the culture solution in the first container at the first timing, and a second optical path length, which is an optical path length of the first light in the culture solution in the first container at the second timing, based on an optical density of the culture solution in the first container obtained based on the first light intensity and an optical density of the culture solution in the first container obtained based on the second light intensity, and estimating a flow velocity of the culture solution in the flow path based on the time change amount; A flow velocity estimation method, comprising:

15. the microfluidic device further includes a second container that contains the culture medium and is connected to the other end of the channel; In the starting step, the culture medium is moved between the first container and the second container through the flow path; In the step of detecting the first light intensity, the culture solution in the second container is irradiated with second light that passes through a liquid surface of the culture solution, and a third light intensity that is the intensity of the second light that has passed through the culture solution in the second container is further detected at the first timing; In the step of detecting the second light intensity, the second light is irradiated onto the culture solution in the second container, and a fourth light intensity, which is the intensity of the second light that has passed through the culture solution in the second container, is further detected at the second timing; 15. The flow velocity estimation method according to claim 14, wherein in the estimating step, the amount of change over time is further estimated based on the optical density of the culture solution in the second container obtained based on the third light intensity and the optical density of the culture solution in the second container obtained based on the fourth light intensity.

16. 16. The flow velocity estimation method according to claim 15, wherein in the estimating step, the amount of change over time is further estimated based on a total length of an optical path length of the first light in the culture solution of the first container and an optical path length of the second light in the culture solution of the second container.

17. The flow velocity estimation method according to claim 16 , wherein in the estimating step, the total length is calculated based on a total volume of the culture solution contained in the first container, the second container, and the flow path.

18. The flow velocity estimation method according to any one of claims 14 to 17, wherein the optical axis of the first light is inclined with respect to the liquid surface of the culture medium in the first container.

19. the microfluidic device further includes a mounting portion for mounting the first container and a lid portion for closing an opening at an upper portion of the first container; 18. The flow velocity estimation method according to claim 14, wherein in the step of detecting the first light intensity and the step of detecting the second light intensity, a first light is irradiated from a light source arranged on the mounting portion, and the intensity of the first light is detected by a light detection unit arranged on the lid portion.

20. The flow velocity estimation method according to any one of claims 14 to 17, wherein the wavelength of the first light is included in the near-infrared region.

21. The flow velocity estimation method according to any one of claims 14 to 17, wherein an organic photodiode is used to detect the first light intensity and the second light intensity.

22. The flow velocity estimation method according to any one of claims 15 to 17, wherein in the starting step, the culture solution is moved by tilting the first container, the second container, and the flow path.

23. The flow velocity estimation method according to any one of claims 15 to 17, wherein in the starting step, the culture solution is moved by changing the air pressure at the liquid surface of the culture solution in one or both of the first container and the second container.

24. The flow velocity estimation method according to any one of claims 15 to 17, wherein in the starting step, the culture solution is moved using a flow pump provided in the flow path.

25. irradiating the culture solution in the first container with third light having a wavelength different from that of the first light, and detecting a fifth light intensity that is the intensity of the third light that has passed through the culture solution in the first container; estimating a hydrogen ion exponent (pH) of the culture solution based on an optical density of the culture solution in the first container obtained based on the fifth light intensity; The flow velocity estimation method according to any one of claims 14 to 17, further comprising:

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