JP7464285B2 - Microfluidic Devices - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Research on the microfluidic chip invented by Toshio Fukuda and Taiga Shiga was published in the proceedings of the Robotics and Mechatronics Symposium 2021 in Osaka (https://robomech2021.org) on June 6, 2021, and at the Robotics and Mechatronics Symposium 2021 in Osaka (https://robomech2021.org) on June 7, 2021.

The present invention relates to a microfluidic device.

Patent Document 1 describes a cancer detection method that utilizes the chemotaxis of nematodes, or nematodes. Nematodes have a chemotaxis that shows a repulsive behavior toward urine from healthy people and an attractive behavior toward urine from cancer patients. The chemotaxis detection method of nematodes used in this cancer detection method is as follows. First, a sample of urine is placed at a predetermined location near the periphery of a petri dish. Next, 100 nematodes are placed in the center of the petri dish and allowed to move on their own for about one hour. After that, the number of nematodes is counted on the side where the sample urine was placed and on the opposite side, with the center of the petri dish as the boundary. The number of nematodes on the side where the sample urine was placed is N(+), and the number of nematodes on the opposite side is N(-), and these are applied to the following calculation formula.
Calculation formula: N(+) - N(-) / total number of individuals The value obtained by the calculation formula ranges from +1 to -1. If the value of the calculation formula is positive, it is evaluated that nematodes have been attracted to the urine sample, and the subject can be determined to have the possibility of cancer.

JP 2020-31649 A

However, the nematode chemotaxis detection method shown in Patent Document 1 takes a long time to detect chemotaxis. In addition, this chemotaxis detection method may be less accurate and precise because the nematode's attraction behavior may weaken over time due to the diffusion of the urine sample.

The present invention has been made in consideration of the above-mentioned conventional situation, and aims to provide a microfluidic device that can detect an attractant substance early by utilizing the chemotaxis of nematodes and obtain detection results with high accuracy and precision.

The microfluidic device of the present invention comprises:
a first port for disposing the nematode;
a second port for disposing a substance to be tested;
a channel communicating between the first port and the second port;
Equipped with
The channel has a reverse motion suppression region that allows the nematode to move freely from the first port toward the second port and suppresses the nematode's movement freely from the second port toward the first port.

In this microfluidic device, nematodes that are attracted to the substance being tested and move from the first port through the channel to the second port are prevented from moving back from the second port to the first port by the reverse migration inhibition region of the channel, so the number of nematodes that are attracted to the substance being tested and move into the second port can be accurately determined.

Therefore, the microfluidic device of the present invention can utilize the chemotaxis of nematodes to detect attractants early and obtain detection results with high accuracy and precision.

FIG. 1 is a plan view showing a microfluidic device according to a first embodiment. FIG. 2 is a plan view showing a nematode self-propelled in a channel of the microfluidic device of Example 1. FIG. 2 is a plan view showing an example of a trajectory of a nematode self-propelled in a channel of the microfluidic device of Example 1. FIG. 13 is a plan view showing an example of a trajectory of a nematode self-propelled in a channel of a microfluidic device of a comparative example. FIG. 4 is a plan view showing a first line and a second line used in the evaluation method of Example 1. (A) is a graph showing the time progression of the nematode population in the second port of a channel having a first connection, and (B) is a graph showing the time progression of the nematode population in the second port of a channel not having a first connection. 13 is a graph showing the time course of the number of nematodes in the second port in an experiment to confirm the chemotaxis of nematodes to test substances including the urine of cancer patients and the urine of healthy individuals, where (A) shows the results of the second experiment and (B) shows the results of the first experiment. 13 is a graph showing the maximum number of nematodes in the second port in an experiment to confirm the chemotaxis of nematodes to test substances including urine of cancer patients and urine of healthy individuals. This is a graph showing the ratio of the maximum number of nematodes in the second port to the number of nematodes in the second port at each time in an experiment to confirm the chemotaxis of nematodes to test substances including urine of cancer patients and urine of healthy individuals. FIG. 1 shows a plan view of a three-port microfluidic device. 1A to 1C are plan views showing microfluidic devices having first connecting parts with different shapes.

A preferred embodiment of the present invention will be described.

The channel of the microfluidic device of the present invention may have a self-propelling assistance region that assists the nematode in self-propelling from the first port to the second port. In this case, the microfluidic device allows the nematode attracted to the test substance to quickly self-propel from the first port to the second port within the channel. Therefore, the microfluidic device can detect the attractant substance early by utilizing the chemotaxis of the nematode.

In the microfluidic device of the present invention, the reverse migration suppression region may be disposed closer to the second port than the self-propelled assistance region. In this case, the microfluidic device allows the nematodes attracted to the substance to be tested within the channel to rapidly self-propel from the first port toward the second port, and can accurately grasp the number of nematodes in the second port by suppressing the reverse migration of the nematodes in the second port attracted to the substance to be tested toward the first port. Therefore, the microfluidic device can detect the attractant substance early by utilizing the chemotaxis of the nematodes, and can obtain detection results with good accuracy and precision.

The channel of the microfluidic device of the present invention has a pair of side walls with flat side surfaces that extend in opposing directions, and the reverse migration suppression region may include a plurality of first connecting parts that are connected at both ends to the respective side surfaces, have a central axis perpendicular to the respective side surfaces, and have a substantially circular shape with a concave side surface on the second port side in a cross section cut by a virtual plane perpendicular to the central axis. In this case, when the head of the nematode enters the concave part of the side surface of the first connecting part, the head of the nematode is unlikely to escape from this concave part, and reverse migration of the nematode toward the first port can be suppressed. Therefore, this microfluidic device can obtain accurate and precise detection results in detecting an attractant that utilizes the chemotaxis of nematodes.

The multiple first connectors of the microfluidic device of the present invention are arranged at equal intervals on multiple second virtual lines perpendicular to a first virtual line extending straight from the first port to the second port, and the first connectors arranged on adjacent second virtual lines may overlap with their central axes shifted when viewed along the first virtual line. In this case, the arrangement of the multiple first connectors can assist the nematode attracted to the substance to be tested in moving on its own from the first port to the second port. This microfluidic device can therefore utilize the chemotaxis of the nematode to detect an attractant substance at an early stage.

The channel of the microfluidic device of the present invention has a pair of side walls with flat side surfaces that extend in an opposing manner, and the self-propelled assistance region is provided with a plurality of second connecting parts, both ends of which are connected to the respective side surfaces, with a central axis perpendicular to the side surfaces, and a cross section cut by a virtual plane perpendicular to the central axis being circular, and the plurality of second connecting parts are arranged at equal intervals on a plurality of second virtual lines perpendicular to a first virtual line extending straight from the first port to the second port, and the second connecting parts arranged on adjacent second virtual lines may overlap with the central axis shifted when viewed along the first virtual line. In this case, the arrangement of the plurality of second connecting parts can assist the self-propelling of the nematode attracted to the test substance when the nematode self-propels from the first port to the second port. For this reason, this microfluidic device can detect the attractant substance early by utilizing the chemotaxis of the nematode. The circular shape referred to here does not have to be a perfect circle, but may be any round shape, including an elliptical shape.

Next, we will explain Example 1, which embodies the microfluidic device of the present invention, with reference to the drawings.

Example 1
As shown in Fig. 1, the microfluidic device 1 of the first embodiment includes a first port 10, a second port 20, and a channel 30. The microfluidic device 1 is formed by bonding PDMS (dimethylpolysiloxane) and a glass plate, and is transparent. The first port 10, the second port 20, and the channel 30 are formed between a pair of sidewall portions having planar side surfaces that extend in an opposing relationship. The opposing side surfaces respectively constitute the upper and lower surfaces of the first port 10, the second port 20, and the channel 30, and the distance between them is 70 µm.

The first port 10 is used to place a nematode X (see FIG. 2) when conducting a test utilizing the chemotaxis of a nematode X. The second port 20 is used to place a test substance when conducting a test utilizing the chemotaxis of the nematode X. The channel 30 communicates between the first port 10 and the second port 20. The channel 30 has a length _L1 of 3200 μm and a width _L2 of 1880 μm.

The channel 30 has a plurality of first connecting parts 310 and a plurality of second connecting parts 320. Each of the first connecting parts 310 and each of the second connecting parts 320 connects both upper and lower ends to the upper and lower surfaces of the channel 30. The central axis of each of the first connecting parts 310 and each of the second connecting parts 320 is perpendicular to the upper and lower surfaces of the channel 30.

1 and 5, the first connecting parts 310 are arranged at equal intervals on three second imaginary straight lines S1 to S3 that are arranged perpendicular to a first imaginary straight line FL that extends straight from the first port 10 toward the second port 20. The interval _W2 between the central axes of the first connecting parts 310 arranged on the same second imaginary straight lines S1 to S3 is 260 μm. The multiple first connecting parts 310 arranged on each of the second imaginary straight lines S1 to S3 are disposed across the entire width of the channel 30.

The second connecting parts 320 are arranged at equal intervals on ten second virtual straight lines S4 to S13 that are arranged perpendicular to the first virtual straight line FL. The interval _W2 between the central axes of the second connecting parts 320 arranged on the same second virtual straight line S4 to S13 is 260 μm. The multiple second connecting parts 320 arranged on each of the second virtual straight lines S4 to S13 are disposed across the entire width of the channel 30.

The three second virtual straight lines S1 to S3 along which the multiple first connecting parts 310 are aligned are disposed closer to the second port 20 than the ten second virtual straight lines S4 to S13 along which the multiple second connecting parts 320 are aligned. The three second virtual straight lines S1 to S3 along which the multiple first connecting parts 310 are aligned and the ten second virtual straight lines S4 to S13 along which the multiple second connecting parts 320 are aligned, totaling thirteen second virtual straight lines S1 to S13, are aligned at equal intervals over the entire length direction of the channel 30. The interval _W1 between every other second virtual straight line S1 to S13 is 500 μm.

The cross-sectional shape of the first connecting part 310 cut by a virtual plane perpendicular to the central axis of the first connecting part 310 (hereinafter referred to as the "cross-sectional shape of the first connecting part 310") is a substantially circular shape with a concave side surface on the second port 20 side. The cross-sectional shape of the first connecting part 310 is a circle with a diameter of 200 μm, with the side surface on the second port 20 side cut out in a rectangular shape. In other words, each first connecting part 310 has both ends connected to the respective side surfaces of the pair of side wall parts, is a substantially cylindrical shape with a central axis perpendicular to each side surface, and has a groove 311 extending along the central axis formed on the side surface on the second port 20 side. In the cross-sectional shape of first connecting portion 310, bottom surface portion 311A extending linearly parallel to second virtual straight lines S1-S3 of the notched portion has length _L3 of 120 μm, and side surface portions 311B extending linearly from both ends of bottom surface portion 311A toward the circumferential surface of first connecting portion 310 has length _L4 of 50 μm. In the cross-sectional shape of first connecting portion 310, corner portions 311C formed by bottom surface portion 311A and side surface portions 311B extending from both ends of bottom surface portion 311A are right angles.

Six first connecting parts 310 are lined up on the first second virtual straight line S1 and the third second virtual straight line S3 from the second port 20 side. Seven first connecting parts 310 are lined up on the second second virtual straight line S2 from the second port 20 side. When viewed along the first virtual straight line FL, the central axes of the first connecting parts 310 lined up on the first second virtual straight line S1 and the third second virtual straight line S3 from the second port 20 side coincide and completely overlap.

When viewed along the first virtual line FL, the first connecting parts 310 arranged on adjacent second virtual lines S1 to S3 overlap with the central axis of one first connecting part 310 arranged on the other second virtual line S1 to S3 being located in the center between the central axes of the two first connecting parts 310 arranged on one second virtual line S1 to S3. In the channel 30, the area where the multiple first connecting parts 310 arranged on the first second virtual line S1 to the third second virtual line S3 from the second port 20 side are arranged is the reverse running suppression area R1. The reverse running suppression area R1 is formed at one end of the channel 30 on the second port 20 side.

The cross-sectional shape of the second connecting portion 320 cut by a virtual plane perpendicular to the central axis of the second connecting portion 320 is a circle with a diameter of 200 μm. In other words, each second connecting portion 320 is connected at both ends to each side surface of a pair of side wall portions, and has a cylindrical shape with a central axis perpendicular to each side surface.

On the 4th to 13th second imaginary straight lines S4 to S13 from the second port 20 side, seven second coupling parts 320 are lined up on the even-numbered second imaginary straight lines S4, S6, S8, S10, S12. On the 4th to 13th second imaginary straight lines S4 to S13 from the second port 20 side, six second coupling parts 320 are lined up on the odd-numbered second imaginary straight lines S5, S7, S9, S11, S13. When viewed along the first imaginary line, the central axes of the second coupling parts 320 lined up on the even-numbered second imaginary straight lines S4, S6, S8, S10, S12 from the second port 20 side coincide with each other and completely overlap. The second connecting parts 320 arranged on the odd-numbered second virtual straight lines S5, S7, S9, S11, and S13 from the second port 20 side, which are the fourth to thirteenth lines, have the same central axis and completely overlap when viewed along the first virtual line.

When viewed along the first virtual line FL, the second connecting parts 320 arranged on adjacent second virtual lines S4 to S13 overlap with the central axis of one second connecting part 320 arranged on one second virtual line S4 to S13 being located in the center between the central axes of the two second connecting parts 320 arranged on the other second virtual line S4 to S13. In the channel 30, the region where the multiple second connecting parts 320 arranged on the fourth second virtual line S4 to the thirteenth second virtual line S13 from the second port 20 side are arranged is the self-propelled assistance region R2.

The arrangement of each first connecting portion 310 and each second connecting portion 320 was determined based on the wavy trajectory of the nematode X, which has a total length of 500 μm to 600 μm, moving on a plane. The average wavelength of this trajectory is 462±132 μm, and the average amplitude is 158±54 μm. As shown in FIG. 2, each first connecting portion 310 and each second connecting portion 320 serve as a catch for the nematode X when the nematode X, attracted by the substance to be tested, moves on its own from the first port 10 to the second port 20, allowing the nematode X to move on its own smoothly.

As shown in FIG. 5, both widthwise edges of the channel 30 are curved outward and protrude along the outer periphery of the first connecting portion 310 and the second connecting portion 320 at the portions where the even-numbered second virtual straight lines S2, S4, S6, S8, S10, and S12 cross from the second port 20 side. Both widthwise edges of the channel 30 are curved inward and recessed at the portions where the odd-numbered second virtual straight lines S1, S3, S5, S7, S9, S11, and S13 cross from the second port 20 side. In other words, both widthwise edges of the channel 30 have curved projections and recesses that extend alternately and continuously.

<Chemotaxis test>
Using the microfluidic device 1 of Example 1 described above, a chemotaxis test of the nematode X was carried out as follows.
First, the first port 10, the second port 20, and the channel 30 of the microfluidic device 1 are filled with pure water. Next, 2 μl of M9 buffer containing nematode X is dropped into the first port 10, and 2 μl of a test solution of a test substance is dropped into the second port 20 at approximately the same time. The nematode X is C. elegans (Caenorhabditis elegans). The test solution is butanone that attracts nematode X. A test image was recorded, and measurements were made based on the image.

<Example of nematode X trajectory>
An example of the trajectory of the nematode X when the microfluidic device 1 of Example 1 is used is shown in Fig. 3. In this case, the nematode X smoothly self-propels from the initial position P1 toward the second port 20 through the self-propelled assistance region R2 and reaches the second port 20. After that, when the nematode X enters the groove 311 on the side surface of the first connecting portion 310, the head of the nematode X is difficult to escape from this groove 311, and the nematode X is prevented from moving backward toward the first port 10, and remains in the second port 20 as shown at position P2 after about 15 seconds.

As a comparative example, a microfluidic device 2 was prepared in which the first connecting portion 310 was not provided and the second connecting portion 320 was arranged over the entire channel 30, and an example of the trajectory of the nematode X when this device was used is shown in FIG. 4. In the microfluidic device 2, the second connecting portion 320 is arranged over the entire channel 30, and the entire channel 30 is the self-propelled assistance region R2. In this case, the nematode X smoothly self-propels from the initial position P1 toward the second port 20 through the self-propelled assistance region R2 and reaches the second port 20. The nematode X then smoothly reverses through the self-propelled assistance region R2 toward the first port 10, and travels back and forth between the first port 10 and the second port 20 in a short time, as shown at position P2 after about 15 seconds.

<Evaluation method>
As shown in Figure 5, the number of nematodes X in the second port 20 to the right of the first line L1 is Nr, the number of nematodes X moving back along the first line L1 from the second port 20 toward the first port 10 is Ng, and the number of nematodes X moving back along the second line L2 from the second port 20 toward the first port 10 is Np.

The first evaluation index represents the number E of nematodes X in the second port 20 as shown in Equation 1, and chemotaxis evaluation is performed based on the number E of nematodes X in the second port 20 every 10 seconds.
E = Nr ... Formula 1

The second evaluation index is calculated as a reverse-running prevention rate G from the number of individuals E who passed the first line L1 and the second line L2 in the wrong direction, as shown in Equation 2. The reverse-running prevention rate G represents the ratio of the number of individuals who did not pass the second line L2 to the number of individuals who passed the first line L1. It can be said that the larger the value of the reverse-running prevention rate G, the stronger the reverse-running prevention effect.
G = (Ng - Np) / Ng ... formula 2
A similar evaluation was also performed on a microfluidic device 2 as a comparative example in which the first connecting portion 310 was not present and the second connecting portion 320 was disposed over the entire channel 30 .

As shown in FIG. 6(A), the microfluidic device 1 of Example 1 shows a strong attraction reaction of the nematode X within 20 to 30 seconds after 2 μl of M9 buffer containing the nematode X is dropped into the first port 10 and 2 μl of the test substance, butanone, is dropped into the second port 20 at approximately the same time. After about 50 seconds after the dropping, the population E of the nematode X in the second port 20 is maintained to a certain extent, and even 180 seconds after the dropping, the population E of the nematode X in the second port 20 is maintained.

As shown in FIG. 6(B), the microfluidic device 2 of the comparative example shows a strong attraction reaction of the nematode X within 20 to 30 seconds after 2 μl of M9 buffer containing the nematode X is dropped into the first port 10 and 2 μl of the test substance butanone is dropped into the second port 20 at approximately the same time. 180 seconds after the dropping, the concentration of the dropped butanone becomes uniform within the microfluidic device 2, and the nematode X starts to move backwards violently, resulting in a decrease in the number E of nematode X in the second port 20.

The reverse flow blocking rate G of the microfluidic device 1 having the first connecting portion 310 of Example 1 and the microfluidic device 2 having no first connecting portion 310 of the comparative example is shown in Table 1. While the reverse flow blocking rate of the microfluidic device 2 of the comparative example was about 19%, the microfluidic device 1 of Example 1 was able to increase the reverse flow blocking rate to about 58%.

The first and second evaluation indices revealed that the shape of the first connecting portion 310 and the reverse migration suppression region R1 in which multiple first connecting portions 310 are arranged are effective in suppressing reverse migration of the nematode X.

<Chemotaxis to urine>
Next, in a cancer testing experiment, the chemotaxis of nematode X was confirmed for test substances including urine of cancer patients and urine of healthy individuals. A mutant (odr-3) of nematode X with only olfactory organs was used. 36 μl of pure water, 4 μl each of urine of cancer patients and healthy individuals were dropped onto a petri dish, and the pure water and each urine were thoroughly mixed to prepare test substances, and 2 μl of the test substance was dropped into the first port 10. For each test substance including urine, the population E of nematode X in the second port 20 was counted twice every 10 seconds.

As shown in Figures 7 (A) and (B), the results of two experiments showed that the time to reach a steady state differed for the test substance containing the urine of healthy subjects. After 2 μl of M9 buffer containing nematode X was dropped into the first port 10 and the test substance was dropped into the second port 20 at roughly the same time, a significant difference between the test substance containing the urine of cancer patients and the test substance containing the urine of healthy subjects was confirmed within 30 seconds. A strong attraction response was observed for the test substance containing the urine of cancer patients within 50 seconds of dropping.

In the first experiment, as shown in FIG. 8, for the test substance containing the urine of a cancer patient, a maximum of 51 nematodes X migrated to the second port 20 within 180 seconds after the substance was dropped. In the first experiment, for the test substance containing the urine of a healthy subject, a maximum of 35 nematodes X migrated to the second port 20 within 180 seconds after the substance was dropped.

In the first experiment, the maximum number of nematodes X in the second port 20 and the ratio of the number E of nematodes X in the second port 20 at each time are shown in Figure 9 and Table 2. There is a clear difference in the number of nematodes X moving between 30 and 40 seconds after dropping. The attraction behavior toward the test substance, which contains the urine of cancer patients, is almost over 60 seconds after dropping.

As described above, the microfluidic device 1 of Example 1 includes a first port 10, a second port 20, and a channel 30. The first port 10 drips M9 buffer containing nematode X. The second port 20 drips a test substance. The channel 30 connects the first port 10 to the second port 20. The channel 30 has a reverse migration inhibition region R1 that allows the nematode X to move independently from the first port 10 toward the second port 20 and inhibits the nematode X from moving in the reverse direction from the second port 20 toward the first port 10.

In this microfluidic device 1, nematodes X are attracted to the substance to be tested and move from the first port 10 through the channel 30 to the second port 20 on their own. The reverse migration inhibition region R1 of the channel 30 inhibits reverse migration from the second port 20 to the first port 10, so that the number of nematodes X attracted to the substance to be tested and moved into the second port 20 can be accurately determined.

Therefore, the microfluidic device 1 of Example 1 can utilize the chemotaxis of the nematode X to detect an attractant substance early and obtain detection results with high accuracy and precision.

The channel 30 of Example 1 has a self-propelling assistance region R2 that assists the self-propelling of the nematode X from the first port 10 toward the second port 20. Therefore, in this microfluidic device 1, the nematode X attracted to the test substance within the channel 30 can quickly self-propel from the first port 10 toward the second port 20. Therefore, this microfluidic device 1 can detect the attractant substance early by utilizing the chemotaxis of the nematode X.

In the microfluidic device 1 of Example 1, the reverse migration suppression region R1 is disposed closer to the second port 20 than the self-propelled assistance region R2. Therefore, in this microfluidic device 1, the nematode X attracted to the substance to be tested in the channel 30 can quickly move on its own from the first port 10 toward the second port 20, and the number of nematodes X in the second port 20 attracted to the substance to be tested can be accurately grasped by suppressing reverse migration toward the first port 10 of the nematode X in the second port 20. Therefore, this microfluidic device 1 can detect the attractant substance early by utilizing the chemotaxis of the nematode X, and can obtain detection results with good accuracy and precision.

The channel 30 of the first embodiment has a pair of side walls whose flat side faces extend in an opposing manner. The reverse migration suppression region R1 has a plurality of first connecting parts 310. Each first connecting part 310 has both ends connected to each side and a central axis perpendicular to each side. Each first connecting part 310 has a substantially circular shape with a concave side on the second port 20 side in a cross section cut by a virtual plane perpendicular to the central axis. Since the reverse migration suppression region R1 has a plurality of first connecting parts 310, when the head of the nematode X enters the concave part of the side of the first connecting part 310, the head of the nematode X is unlikely to escape from the concave part, and reverse migration of the nematode X toward the first port 10 can be suppressed. Therefore, this microfluidic device 1 can obtain accurate and precise detection results in detecting an attractant using the chemotaxis of the nematode X.

The first connecting parts 310 of the first embodiment are arranged at equal intervals on a plurality of second virtual straight lines S1 to S3 that are perpendicular to a first virtual straight line FL that extends straight from the first port 10 to the second port 20. The first connecting parts 310 arranged on adjacent second virtual straight lines S1 to S3 overlap with their central axes shifted when viewed along the first virtual straight line FL. This arrangement of the first connecting parts 310 can assist the self-propulsion of the nematode X attracted to the test substance when it moves from the first port 10 to the second port 20. Therefore, this microfluidic device 1 can detect the attractant substance at an early stage by utilizing the chemotaxis of the nematode X.

The channel 30 of the first embodiment has a pair of side walls with flat side surfaces that extend in an opposing manner. The self-propelled assistance region R2 is provided with a plurality of second connecting parts 320, each of which has both ends connected to each side surface, a central axis perpendicular to the side surface, and a cross section cut by a virtual plane perpendicular to the central axis that is circular. The plurality of second connecting parts 320 are arranged at equal intervals on a plurality of second virtual straight lines S4 to S13 perpendicular to the first virtual straight line FL that extends straight from the first port 10 to the second port 20. The second connecting parts 320 arranged on adjacent second virtual straight lines S4 to S13 overlap with their central axes shifted when viewed along the first virtual straight line FL. Such an arrangement of the plurality of second connecting parts 320 can assist the self-propelled nematode X when the nematode X attracted to the test substance self-propels from the first port 10 to the second port 20. Therefore, this microfluidic device 1 can detect attractants at an early stage by utilizing the chemotaxis of nematode X.

The present invention is not limited to the first embodiment described above with reference to the drawings, and the following embodiments are also included within the technical scope of the present invention.
(1) The microfluidic device of Example 1 has two ports, but may have three or more ports. When the microfluidic device has three ports, as shown in FIG. 10, the microfluidic device 3 may have a first port 40 in which a nematode is placed, and a second port 50 and a third port 60 in which a test substance is placed. In this case, the second channel 70 extending from the first port 40 is connected to the center of the first channel 80 that connects the second port 50 and the third port 60. A plurality of first connecting parts 310 are arranged at both ends of the first channel 80 that are continuous with the second port 50 and the third port 60, and a plurality of second connecting parts 320 are arranged in the other regions of the first channel 80 and the second channel 70. A reverse migration suppression region R1 is formed at both ends of the first channel 80 by multiple first connecting portions 310, so that when the nematodes dropped into the first port 40 reach the second port 50 and the third port 60, the nematodes can be prevented from migrating reversely from the second port 50 and the third port 60.

(2) The first connecting portion of the microfluidic device of Example 1 was cut out in a rectangular shape in cross section, but the cutout shape of the first connecting portion may be other shapes, such as an arc shape, as shown in FIG. 11 .
(3) As shown in FIG. 11, the microfluidic device 4 may have a reverse flow suppression region R1 in which a first connecting portion 350 having a different cross-sectional shape is arranged.
(4) In Example 1, the reverse flow suppression area is formed at one end of the channel on the second port side. However, it may be formed not only at one end of the channel, but also in the middle part or the other end on the first port side.
(5) Although the channel in the first embodiment has a self-propelled assistance region, the channel may not have a self-propelled assistance region if it does not have a second connecting portion.
(6) The channel in the first embodiment has a reverse running suppression region and a free-running assistance region, but the entire channel may be the reverse running suppression region.
(7) In Example 1, the microfluidic device is used for cancer testing. However, this microfluidic device can be used for testing that utilizes the chemotaxis of nematodes, and is not limited to cancer testing.

1...Microfluidic device 10...First port 20...Second port 30...Channel 310...First connection part 320...Second connection part FL...First virtual straight line R1...Reverse motion suppression region R2...Self-propelled assistance region S1 to S13...Second virtual straight line X...Nematode (nematode)

Claims (5)

a first port for disposing the nematode;
a second port for disposing a substance to be tested;
a channel communicating between the first port and the second port;
Equipped with
The channel has a reverse motion suppression region that allows the nematode to move freely from the first port to the second port and suppresses the nematode from moving freely from the second port to the first port ,
The channel has a pair of side walls with planar sides extending in opposite directions,
The reverse flow suppression region has both ends connected to each of the side surfaces, a central axis perpendicular to each of the side surfaces, and a cross-sectional shape cut by a virtual plane perpendicular to the central axis is approximately circular with a concave side surface on the second port side . The microfluidic device according to claim 1, wherein the channel has a self-propelling assistance region that assists the nematode in self-propelling from the first port to the second port. The microfluidic device according to claim 2, wherein the reverse motion suppression region is disposed closer to the second port than the self-propelled assistance region. 4. The microfluidic device according to claim 1, wherein the first connecting portions are arranged at equal intervals on a plurality of second imaginary lines perpendicular to a first imaginary line extending straight from the first port toward the second port, and the first connecting portions arranged on adjacent second imaginary lines overlap with their central axes shifted when viewed along the first imaginary line. The channel has a pair of side walls having planar sides extending in opposite directions,
The self-propelled assistance region includes a plurality of second connecting parts, each of which is connected to each of the side surfaces at both ends, has a central axis perpendicular to the side surfaces, and has a cross-sectional shape cut by a virtual plane perpendicular to the central axis that is circular,
4. The microfluidic device according to claim 2, wherein the second connecting portions are arranged at equal intervals on a plurality of second imaginary lines perpendicular to a first imaginary line extending straight from the first port toward the second port, and the second connecting portions arranged on adjacent second imaginary lines overlap with the central axes shifted when viewed along the first imaginary line.

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