JPWO2020054704A1 - Analytical device - Google Patents

Abstract

Abstract An analytical device is disclosed that has a simple structure, is highly portable, and can change the liquid used for measurement without using a pump or external power. The analysis device is one or a plurality of communicating the substrate, the detection well provided on the substrate, the liquid storage well provided on the substrate, the liquid absorbing material arranged in the substrate, and the detection well and the liquid storage well. The first flow path of the above and one or a plurality of second flow paths communicating the detection well and the liquid absorbing material are provided. The liquid contained in the liquid storage well flows through the first flow path, the detection well, and the second flow path in this order to reach the liquid absorbing material, and the flow velocity of the liquid flowing into the detection well at this time and The flow rate of the liquid flowing out of the detection well is substantially equal.

Description

The present invention relates to an analytical device, and more particularly to an analytical device which is excellent in portability and can exchange a liquid to be used for measurement without using electric power.

The cells that make up an organism, various organelles such as mitochondria, Golgi apparatus, and endoplasmic reticulum, and cell nuclei that exist inside the cells are covered with a biological membrane on the outside, and this biological membrane is basically a lipid bilayer membrane. It is composed of. Various proteins having physiological activity, that is, receptors, enzymes, etc., are retained on the lipid bilayer membrane in a form that penetrates the lipid bilayer membrane. These transmembrane proteins play an important role in vivo. In particular, it is known that various receptors existing on the cell membrane trigger various physiological reactions by binding to ligands existing in the living body. For this reason, various ligands that enhance the function of the receptor, inhibitors that inhibit the function of the receptor, etc. are used as pharmaceuticals, and natural or artificial ligands and inhibitors that can be used as new pharmaceuticals are being studied. ing.

It is also known that a protein is retained in a lipid bilayer membrane and used as a sensor. For example, a specific binding substance that specifically binds to a test substance is contained in a droplet when a receptor protein is held in a lipid double membrane to serve as a sensor, or when a lipid double membrane is formed by a droplet contact method. On the other hand, we also know a sensor that retains the ion channel protein in the lipid double membrane so that the test substance binds to the specific binding substance and blocks the ion channel when the test substance is present. Has been done.

In such a lipid bilayer sensor, if (1) a mechanism that can automatically wash away the analyte after measurement and exchange the solution, and (2) a compact solution-driven system that operates without external power is constructed, the sensor will be used. It is thought that it will be possible to add the function of "on-site time-lapse measurement (time-lapse measurement)".

Regarding (1) above, as previous research, a case where a lipid double membrane was formed in the microchannel and the solution was exchanged using one syringe pump, and a detection unit with an artificial cell membrane using two pumps. There are cases where the inflow and outflow of a solution into (droplets) are matched and the solution is exchanged while keeping the amount of droplets constant (Non-Patent Document 1 and Non-Patent Document 2). Regarding (2) above, there are no reports of previous studies on lipid bilayer membranes, but there are cases where the suction power of water-absorbent polymers is used as a solution drive source and applied to immune chips and sweat analysis sensors ( Patent Document 1, Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). Regarding the immune chip, the solution moves on the fiber sheet due to the suction force of the water-absorbent polymer, and the flowing antigen binds to the antibody at the detection site to enable detection. In addition, the sweat analysis sensor samples and detects sweat, which is an analytical substance, with a water-absorbent polymer.

JP-A-2015-132473 Japanese Unexamined Patent Publication No. 2017-026621 Japanese Unexamined Patent Publication No. 2012-081405

Tsuji, et al., Lab Chip, 2013, 13, 1476 Kawano et al., Small, 2010, 6, 2100. Oyama et al., Sens. Actuators B Chem, 2017, 240, 881. Curto et al., Sens. Actuators B Chem. 2012, 175, 263.

When an analytical device provided with a lipid bilayer membrane is used as a sensor, it is mostly used in a place other than the laboratory, so that portability and a simple structure are required. Further, by making the liquid used for measurement exchangeable, it is advantageous because on-site time-lapse measurement becomes possible as described above. Further, it is advantageous that it can operate without using external power.

An object of the present invention is to provide an analytical device having a simple structure, excellent portability, and capable of exchanging a liquid to be used for measurement without using a pump or external power.

As a result of diligent research, the inventors of the present application can move an amount of liquid capable of exchanging liquid in the detection well by the liquid absorption phenomenon by the liquid absorbing material, and flow into the detection well. We found that it is possible to make the flow velocity of the liquid flowing out of the detection well equal to the flow velocity of the liquid flowing out of the detection well, and realized that the liquid in the detection well can be exchanged by utilizing this phenomenon, and completed the present invention. bottom.

That is, the present invention provides the following.
(1) The substrate, the detection well provided on the substrate, the liquid storage well provided on the substrate, the liquid absorbing material arranged in the substrate, and the detection well and the liquid storage well communicate with each other. The liquid contained in the liquid storage well is provided with one or more first flow paths and one or more second flow paths communicating the detection well and the liquid absorbing material. The flow velocity of the liquid that flows through the flow path 1, the detection well, and the second flow path in this order to reach the liquid absorbing material, and flows into the detection well at this time, and the liquid that flows out from the detection well. An analytical device whose flow velocity is substantially equal to that of.
(2) The analytical device according to (1), wherein the detection well is in the form of a double well chamber separated by a lipid bilayer membrane.
(3) The capacity of the detection well for liquid exchange is 10 μL to 500 μL, the length of the first flow path is 4 mm to 200 mm, the width of the first flow path is 0.2 mm to 2 mm, and the first flow. The depth of the road is 0.1 mm to 1 mm, the length of the second flow path is 4 mm to 200 mm, the width of the second flow path is 0.2 mm to 2 mm, and the depth of the second flow path is 0.1 mm. The analytical device according to (1) or (2), which is ~ 1 mm.

The analytical device of the present invention has a simple structure and is excellent in portability, and the liquid to be used for measurement can be exchanged without using a pump or external power. Further, as specifically shown in the following examples, the liquid volume and flow rate of the solution exchange can be set to desired values, and it takes a relatively long time so as not to destroy the lipid bilayer membrane. It is also possible to change the liquid slowly.

It is an exploded perspective view of one specific example of this invention. It is a schematic diagram which shows the double well chamber which can be adopted as a detection well in this invention. It is a figure which shows the relationship between the width and depth of the 1st flow path and the flow velocity measured in the following Example. It is a figure which shows the relationship between the water level of a detection well, and the flow velocity when the length of the 1st flow path is changed, which measured in the following Example. It is a figure which shows the relationship between the water supply amount and the flow velocity when the width, depth, and length of the 2nd flow path are changed, which were measured in the following Examples. It is a figure which shows the relationship between the elapsed time after the start of liquid exchange and the ink density | concentration in a detection well measured in the following Example. It is a figure which shows the relationship between the elapsed time after the start of liquid exchange and the fluorescent substance concentration in a detection well measured in the following Example. It is a figure which shows the lower substrate, the middle substrate and the upper substrate of the analysis device produced in the following Example. It is a figure which shows the relationship between the time and the current value measured in the following Example. It is a figure which shows the histogram of the current value before and after the addition of cyclodextrin measured in the following Example. It is a figure which shows the lower substrate, the middle substrate and the upper substrate of the analysis device produced in the following Example. It is a figure which shows the relationship between time and voltage when hypochlorous acid is often added, which was measured in the following Example. It is a figure which shows the relationship between the time and the current value at the time of performing continuous measurement for 3 hours in the following Example. In the following examples, it is a figure which shows the relationship between the block frequency (the number of current drops which occur per unit time with respect to one nanopore formed by hemoricin), and the concentration of cyclodextrin. It is a figure which shows the relationship between time and the concentration of cyclodextrin at the time of performing continuous measurement for 3 hours measured in the following Example. It is a figure which shows the relationship between the time and the current value measured in the following comparative example.

Hereinafter, a preferred specific example of the present invention will be described with reference to the drawings.

FIG. 1 is an exploded perspective view of an analytical device which is a preferable specific example of the present invention. The analytical device shown in FIG. 1 includes a lower substrate 10, a middle substrate 12, and an upper substrate 14. These three substrates are laminated and integrated to form a substrate for an analytical device as a whole. The reason why the substrate is formed from the above three substrates is to facilitate the production of the analysis device, and each component described later may be formed on one substrate from the beginning.

..
The lower substrate 10 is provided with an electrode 16 connected to a circuit for measurement or two through holes through which wiring connected to the electrode is passed. The central substrate 12 is formed with a detection well 18, a liquid storage well 20, and a liquid absorbing well 22 for accommodating a liquid absorbing material (not shown). Electrodes for measurement are usually arranged in the detection well 18. A first flow path 24 that communicates the detection well 18 and the liquid storage well 20 is provided, and a second flow path 26 that communicates the detection well 18 and the liquid absorption well 22 is provided. The first flow path 24 and the second flow path 26 may be one or a plurality of each. In the case of a plurality of pieces, it is meaningless if there are too many pieces, and the production becomes complicated, so 3 pieces or less is preferable. As specifically described in the following examples, the first flow path 24 and the second flow path 26 can form the analysis device of the present invention one by one without any problem. It is convenient and preferable to make a book. In the specific example shown in FIG. 1, there are three first flow paths 24 and one second flow path 26. Each flow path may be just a groove. Further, the flow path does not necessarily have to be a straight line, and a curved portion may be formed or a hairpin shape may be formed in order to appropriately set the flow path length (see the following examples). The detection well 18, the liquid storage well 20, the liquid absorption well 22, the first flow path 24, and the second flow path 26 may or may not penetrate independently of each other. When penetrating, the upper surface of the lower substrate 10 forms the well or the bottom surface of the flow path.

The upper substrate 14 is provided with through holes of the same shape and size at positions corresponding to the detection well 18, the liquid storage well 20, and the liquid absorption well 22, respectively, and when laminated with the middle substrate 12, each of them is provided. Form the top of the well. The upper substrate 14 may be omitted, but in the presence of the upper substrate 14, the upper substrate 14 forms the upper part of the side wall of each well, the depth of each well becomes deeper, and a large amount of liquid is accommodated accordingly. Further, when a predetermined amount of liquid is put into each well, the upper surface of the upper substrate 14 can be made higher than the height of the liquid level, which is advantageous because the liquid is less likely to spill from each well. The analytical device of the present invention is excellent in portability and suitable for carrying, but it is possible to prevent liquid from spilling from each well even when carrying. The upper part of the first and second flow paths may also be an opening (see Examples below). In this case, the liquid can be dropped directly into each flow path.

The lower substrate 10, the middle substrate 12, and the upper substrate 14 are laminated and integrated into one substrate. It is convenient to manufacture the apparatus that each substrate is made of a plastic plate such as an acrylic plate, but the material of the substrate is not limited as long as it does not react with the liquid to be subjected to the reaction. In addition, the substrates can be easily integrated by crimping, bonding with an adhesive, or the like.

In the specific example shown in FIG. 1, the first flow path 24 and the second flow path 26 are formed in the central substrate 12, but these flow paths are provided as specifically described in the following examples. It can also be formed on the lower substrate 10. In this case, the end of each flow path and each well are three-dimensionally connected, that is, the end of each flow path opens to the bottom surface of each well.

The liquid absorbing material (not shown) contained in the liquid absorbing well 22 is not particularly limited as long as it has a liquid absorbing property, and is formed of, for example, a cotton-like fiber formed from a polymer fiber, a non-woven fabric, or a polymer. A porous substance such as a sponge can be used. The polymer is not particularly limited, and for example, a super absorbent polymer such as a highly water-absorbent resin (Aqualic (trade name) CA, Nippon Shokubai Co., Ltd.) can be used.

The thickness of the substrate (in this specific example, the substrate in which the above three substrates are integrated) is not particularly limited as long as it can form each well and each flow path, but is usually 0.5 mm to 50 mm. The degree, preferably about 1 mm to 30 mm. The volume of the detection well 18 is not particularly limited and may be any volume suitable for carrying out the reaction, but is usually about 1 μL to 4000 μL, preferably about 3 μL to 1000 μL, and more preferably about 10 μL to 500 μL. .. The capacity of the liquid storage well 20 is not particularly limited as long as it can store the amount of liquid required for liquid exchange, but is usually about 0.1 mL to 36 mL, preferably about 1 mL to 5 mL. .. The capacity of the liquid absorbing well 22 is not particularly limited as long as it can accommodate a required amount of liquid absorbing material, but is usually about 0.2 mL to 37 mL, preferably about 2 mL to 6 mL. The amount of the liquid absorbing material to be stored may be more than the amount required for the liquid exchange, and is appropriately set according to the desired amount of the liquid to be subjected to the liquid exchange, the liquid absorbing property of the liquid absorbing material, and the like. Usually, it is about 1 mg to 1000 mg, preferably about 10 mg to 500 mg.

The cross-sectional shape of each flow path is not particularly limited, but a rectangle is convenient and preferable in terms of fabrication. The length, width and depth of each flow path affect the flow velocity of the liquid flowing through the flow path. The length of the first flow path 24 is appropriately set, but is usually about 2 mm to 300 mm, preferably about 4 mm to 200 mm. The width of the first flow path 24 is appropriately set, but is usually about 0.05 mm to 10 mm, preferably about 0.1 mm to 5 mm, and more preferably about 0.2 mm to 2 mm. The depth of the first flow path 24 is appropriately set, but is usually about 0.01 mm to 4 mm, preferably about 0.05 mm to 2 mm, and more preferably about 0.1 mm to 1 mm. The length of the second flow path 26 is appropriately set, but is usually about 2 mm to 300 mm, preferably about 4 mm to 200 mm. The width of the second flow path 26 is appropriately set, but is usually about 0.05 mm to 10 mm, preferably about 0.1 mm to 5 mm, and more preferably about 0.2 mm to 2 mm. The depth of the second flow path 26 is appropriately set, but is usually about 0.01 mm to 4 mm, preferably about 0.05 mm to 2 mm, and more preferably about 0.1 mm to 1 mm.

The liquid contained in the liquid storage well 20 flows through the first flow path 24, the detection well 18, and the second flow path 26 in this order, reaches the liquid absorbing material, and flows into the detection well 18 at this time. The length, width, and depth of each flow path and the amount of the liquid absorbing material are appropriately set so that the flow velocity of the liquid and the flow velocity of the liquid flowing out from the detection well 18 are substantially equal. Here, "substantially equal" means that the amount of the reaction solution in the detection well 18 does not change to such an extent that it adversely affects the reaction, and the flow velocity (μL) of the solution flowing into the detection well 18 is not changed. When (/ min) is 1, the flow velocity (μL / min) of the liquid flowing out from the detection well 18 is preferably in the range of 0.85. To 1.15. This range is even more preferably 0.9 to 1.1, even more preferably 0.95 to 1.05, even more preferably 0.99 to 1.01, and most preferably 1.00. As specifically described in the examples below, the length, width, and depth of each flow velocity affect these flow velocities. That is, the longer the length, the lower the flow velocity, and the larger the depth, the higher the flow velocity, and the width gives a peak of the flow velocity to some extent. Therefore, by adjusting the length, width, and depth of each flow path. , The flow velocity of the liquid flowing into the detection well 18 and the flow velocity of the liquid flowing out of the detection well 18 can be made substantially equal. The flow velocity can be measured by the method specifically described in the following examples, and the length, width, and depth of each flow path can be set by the design method specifically described in the following examples. It can be done easily. In addition, the liquid absorption property of the liquid absorbing material and the amount used also affect the flow velocity. It was found that the liquid absorbing material loses its liquid absorbing property when it absorbs the liquid, but the flow velocity does not fluctuate when the amount of the liquid absorbing is small. That is, as specifically described in the following Examples, when 100 mg of the water-absorbent polymer was used, the flow velocity hardly changed in the range of the water absorption amount of 600 μL or less. When limiting each flow velocity to a desired range, it is preferable to use an amount of liquid absorbing material in which the flow velocity hardly changes, because the flow velocity can be easily controlled. Further, each flow velocity (μL / sec) is preferably about 0.1% to 20%, more preferably about 0.2% to 10%, still more preferably about 0.4% to 8% of the volume of the detection well 18. It is preferable to enable liquid exchange within an appropriate time. Further, since the static pressure differs depending on the water level of the liquid stored in the liquid storage well and the detection well, the water level also affects the flow velocity. The water level can be appropriately selected according to the desired amount of liquid and the sizes of the liquid storage well and the detection well. The width, depth, etc. can be set.

The detection well 18 may be a simple well depending on the type of reaction, but the detection well 18 may be a double-well chamber separated by a lipid bilayer membrane. Since the lipid bilayer membrane is fragile and easily broken, it is considered that liquid exchange is not easy. However, in the analysis device of the present invention, the external noise is reduced and the liquid absorbing material is used without using a pump. Since the liquid is exchanged slowly, it is particularly effective when applied to an analytical device that uses a lipid bilayer membrane.

The double well chamber separated by the lipid bilayer membrane and the measurement method itself using the double well chamber are well known and are described in, for example, Patent Document 3. A schematic plan view and cross-sectional view of the double-well chamber are shown in FIG. 2 (similar to FIG. 1 of Patent Document 3). That is, in the double well chamber, two cylindrical wells are arranged adjacent to each other, and the boundary portion where the two wells are in contact with each other is a gap (for example, about several mm on each side). FIG. 2A is a plan view, and FIG. 2B is an end view of the b-b'line cut portion in FIG. 2A. Note that FIG. 2 schematically shows a double-well chamber for understanding the invention, and the dimensional ratio of each component is significantly different from the actual one.

In the specific example schematically shown in FIG. 2, two wells 32 and 34 are formed in the substrate 28, and their boundaries are separated by a partition wall 30. The partition wall 30 is provided with a through hole 36 (see FIG. 2B). In the example shown in FIG. 2, the planar shape of the well is basically circular, and only the boundary portion where the two circles meet is straight, but the shape of the well is not limited, and the through hole is formed. There is no problem with other shapes as long as they are separated by the partition wall. The size of the well is not particularly limited, but the pore size is more preferably about 2 mm to 8 mm so that the lipid solution is easily pressed by the droplet when the droplet of water or the aqueous solution is formed in the lipid solution as described later. Is about 3 mm to 5 mm, and the depth is preferably about 50% to 200% of the pore diameter, more preferably about 50% to 100%, but it can be used in the analytical device of the present invention regardless of whether it is larger or smaller than this range. It is possible. The number of through holes provided in the partition wall may be one or a plurality, and is usually 20 or less, preferably about 1 to 15. When there are a plurality of through holes, a lipid bilayer film is not always formed in all the through holes, and a lipid bilayer film is formed only in the through holes where the lipid solution is strongly pressed by the droplets. .. The size of the through hole may be any size as long as the lipid bilayer membrane can be formed, and the diameter is usually about 500 nm to 1000 μm. When the partition wall is formed of a parylene resin film, it is preferable that a small through hole is easily formed, but the partition wall is not necessarily limited to the parylene resin. Further, an electrode for measuring a current is arranged in each well.

In the analytical device of the present invention, when the above-mentioned double-well chamber is used as the detection well 18, the well for liquid exchange may be any one of the two wells of the double-well chamber. However, when it is desired to exchange liquids in both wells, each well can be used as a detection well, and a liquid storage well, a liquid absorbing well, and first and second flow paths can be provided for each well. Therefore, a plurality of liquid storage wells, liquid absorption wells, and first and second flow paths can be provided on one substrate.

When the analytical device of the present invention is used, first, the required reaction solution is put into the detection well as usual, and the reaction and measurement are carried out. When the reaction solution is desired to be exchanged, the exchange solution and the liquid absorbing material are put into the liquid absorbing well. Then, the liquid flows from the liquid storage well through the first flow path 24 into the detection well 18, and further, the liquid flows from the detection well 18 through the second flow path 26 into the liquid absorbing material in the liquid absorbing well 22. Is absorbed by. After the lapse of a predetermined time, the liquid in the detection well 18 is almost completely replaced. Alternatively, the replacement liquid may be put into the liquid storage well before the reaction and measurement are performed in advance, and the liquid absorbing material may be put into the liquid absorbing well when the reaction is desired. Further, the exchange liquid and the liquid absorbing material may be put into the liquid absorbing well in the liquid storage well before the reaction and the measurement, and the solution may be exchanged continuously. Further, if necessary, the first flow velocity and the second flow velocity may be staggered for use. In addition, in order to facilitate the flow of the liquid, the liquid may be dropped in the vicinity of the liquid absorbing material, or the liquid may be dropped directly into the first flow path and / or the second flow path (the following examples). reference). As the liquid in this case, a buffer solution or the like that does not affect the reaction is preferable. Further, the vicinity of the liquid absorbing material may be hydrophilized (see Examples below).

As described above, the analytical device of the present invention has a simple structure and is excellent in portability, and the liquid to be used for measurement can be exchanged without using a pump or external power. Further, as specifically shown in Examples and Comparative Examples below, the analytical device of the present invention capable of liquid exchange has a lipid bilayer membrane as compared with a conventional analytical device having no liquid exchange function. It is maintained unbroken for a long time. It is considered that the reason for this is that the solution is constantly supplied from the storage well to the detection well, and the amount of droplets in the detection well is always kept constant. When the solution is not supplied, it is difficult to keep the amount of droplets constant due to the influence of drying and the like, which causes instability of the lipid bilayer membrane.

Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of analytical device 1. Analytical device design
(1) Examination of control of inflow rate A square liquid storage well with a side of 20 mm and a cylindrical detection well with a diameter of 4 mm (both have a depth of 4 mm) were formed on the acrylic plate. A flow path was provided to communicate the liquid storage well and the detection well. Various simulated devices with various changes in the length, width, and depth of the flow path were prepared, and an experiment was conducted in which the replacement liquid was placed in the liquid storage well and the water level in the detection well was measured. Before pouring water into the reservoir well, from 1 second to 2000 seconds after pouring water, take a picture of the inside of the detection well from the side of the detection well every 0.1 to 20 seconds, and change the water level in the detection well. Was measured. The flow velocity flowing into the detection well was calculated from the change in water level and the capacity of the detection well. The results are shown in FIGS. 3 and 4.

FIG. 3 is a diagram showing the relationship between the flow path width, the flow path depth and the flow velocity when the flow path length is 5 mm, and FIG. 4 is a diagram when the flow path lengths are 5 mm, 20 mm, 40 mm, 60 mm and 80 mm. , It is a figure which shows the relationship between the water level of a detection well, and the flow velocity. The measurement results plotted in FIG. 4 are in ascending order of the flow path length from the top, and when the flow path length is 60 mm and 80 mm, the plots are substantially overlapped.

From the above results, it was clarified that the inflow velocity into the detection well can be controlled to 0.03 to 32 μL / sec by adjusting the length, width, depth, and water level of the detection well.

(2) Examination of control of outflow rate Various simulated devices similar to the above (1) were manufactured, and the liquid storage well in (1) was used as the liquid absorption well. In the liquid absorbing well, 100 mg of a water absorbing polymer (specifically, Aquaric (trade name) CA, Nippon Shokubai Co., Ltd.) was placed near the outlet of the flow path as a liquid absorbing material. The vicinity of the outlet of the flow path was hydrophilized with Cell Face Coat WG-R1 (Marusho Sangyo Co., Ltd.). Fill the detection well with 45 μL of water, and in the same manner as in (1) above, take photographs from the side of the detection well every 0.2 to 20 seconds over time to measure the change in water level, and measure the amount of water absorbed and outflow. The speed was calculated. The results are shown in FIG.

The indication of "wdl" in the upper right of FIG. 5 indicates the width (mm), depth (mm) and length (mm) of the flow path. Therefore, for example, the description that wdl is "0.2 0.1 5" indicates that the width of the flow path is 0.2 mm, the depth is 0.1 mm, and the length is 5 mm. The display of "wdl" may also be used in the following description. From the results shown in FIG. 5, the outflow rate can be controlled in the range of about 0.04 μL / sec to 8.8 μL / sec, and when the water absorption amount is 600 μL or less, the flow velocity hardly changes depending on the amount of liquid absorption. You can see that. Table 1 shows an example of unifying the first flow velocity and the second flow velocity, and the solution exchange duration and the expected exchange time.

As shown in Table 1, by selecting wdl, a wide range of sustainability (time (minutes) until the total flow rate reaches 600 μL) and a wide range of sustainability after equalizing the first flow rate and the second flow rate. It is considered that the time required for exchanging the detection well solution (time (minutes) for exchanging 20 μL of the solution to 1/10 concentration) can be achieved.

(3) Examination of liquid exchange Based on the results of the preliminary experiments in (1) and (2) above, an analytical device with inflow rate and outflow rate set to approximately 0.24 μL / sec was prepared, and liquid exchange in the detection well was examined. bottom. The diameter of the detection well was 4 mm and the depth was 4 mm. The wdl of the first flow path was set to 0.2 0.2 15 and the wdl of the second flow path was set to 0.2 0.1 20. The first flow path and the second flow path were set to one each.

2 μL of the blue ink solution was placed in the detection well. 3 mL of water was put into the liquid storage well, and 100 mg of a water-absorbing polymer (specifically, Aquaric (trade name) CA, Nippon Shokubai Co., Ltd.) as a liquid-absorbing material was put into the liquid-absorbing well. The change in ink density in the detection well was qualitatively measured every 2 seconds. The ink density was measured by taking a picture with a microscope. The results are shown in FIG.

FIG. 6 shows time-series photographs before and after ink addition. As shown in FIG. 6, it is confirmed that the ink is poured after the ink is dropped. After 180 seconds, it can be seen that the liquid in the detection well was almost completely replaced. As a result, it was confirmed that the analytical device of the present invention using the liquid absorbing material without using a pump can exchange the liquid.

Furthermore, an experiment using a fluorescent substance was conducted. That is, a calcein solution was placed in the detection well as a fluorescent substance. 3 mL of water was put into the liquid storage well, and 100 mg of a water-absorbing polymer (specifically, Aquaric (trade name) CA, Nippon Shokubai Co., Ltd.) as a liquid-absorbing material was put into the liquid-absorbing well. The calcein concentration in the detection well was fluorescently measured every 10 to 60 seconds. The calcein concentration was measured by measuring the fluorescence brightness. The results are shown in FIG.

The horizontal axis of FIG. 7 is time (minutes), and the vertical axis is the concentration of calcein. As shown in FIG. 7, after about 400 seconds, the calcein concentration became almost 0, and it can be seen that the liquid in the detection well was almost completely replaced. As a result, it was confirmed that the analytical device of the present invention using the liquid absorbing material without using a pump can exchange the liquid.

2. Preparation of Analytical Device Based on the results of the preliminary experiment described in 1 above, an analytical device equipped with a double-well chamber was prepared. That is, as shown in FIG. 8, an acrylic plate having a thickness of 1 mm was used as a lower substrate, an acrylic plate having a thickness of 3 mm was used as a middle substrate, and an acrylic plate having a thickness of 4 mm was used as an upper substrate. A first flow path and a second flow path were formed on the lower substrate. The first flow path has a width of 0.2 mm, a depth of 0.2 mm, and a length of 15 mm. The second flow path has a width of 0.2 mm, a depth of 0.1 mm, and a length of 20 mm. The diameter of each well in the double well chamber was 4 mm. The first flow path and the second flow path communicate with only one well of the double well chamber (the well on the side where the silver chloride electrode described below is used as the working electrode). Other sizes are as shown in FIG. 8 (unit: mm). The lower substrate and the middle substrate were laminated and crimped with a crimping machine, and then the upper substrate was laminated on the middle substrate and crimped with a crimping machine to prepare an analysis device. In this analytical device, each channel is formed on a lower substrate, so that each channel is connected to each well at the bottom of each well. Further, silver chloride paste was embedded in the through holes formed in the lower part of each well provided on the lower substrate to serve as an electrode for measurement. One of the electrodes was grounded and the other was connected to a current measuring circuit (Patent Document 3) as a working electrode. Further, a parylene resin film having a thickness of 5 μm having a plurality of through holes having a diameter of 100 μm was inserted between each well as a partition wall and adhered.

3. 3. 5 μl of a 20 mg / mL 1,2-difitanoyl-sn-glycero-3-phosphocholine (DphPC) n-decane solution was added dropwise to the well on the ground side where the measurement channel was not arranged. At this time, it was dropped so as to be applied to the vicinity of the partition wall portion. 10 μl of an electrolyte solution (1 M KCl, adjusted to potassium phosphate buffer solution pH 7.4) was added dropwise to the flow path near the site where the absorbent material was installed, and the second flow path was filled with the solution. Similarly, 10 μl of the electrolyte was added dropwise to the first flow path on the liquid storage well side, and the first flow path was filled with the solution. In the same manner as above, 20 mg / ml DphyPC was added dropwise to the well on the working electrode side. A solution prepared by dissolving hemolysin (10 nM) in an electrolyte solution was added dropwise to the ground side well in an amount of 21 μl. 21 μl of the electrolytic solution was added dropwise to the well on the working electrode side. 3 mL of electrolyte solution was placed in the reservoir well (liquid height 7.5 mm in the reservoir well). 100 mg of a water-absorbing polymer (specifically, Aquaric (trade name) CA) as a liquid-absorbing material was placed in the vicinity of the end of the second flow path of the liquid-absorbing well. Current measurement was started using an amplifier. The applied voltage was 60 mV. Membranes were formed until a nanopore-derived current due to hemoricin was detected. After the nanopore-derived current was detected, a model analyzer, cyclodextrin (methyl β-cyclodextrin 500 μM), was added dropwise to a well with a 5 μl working electrode. For data analysis, a histogram of the current value was created, Gaussian fitting was performed on the peak, and the area derived from nanopores by hemoricin and the area of the occlusion signal of cyclodextrin were quantified.

The measurement results are shown in FIGS. 9, 10 and 2. As shown in FIG. 9, before the addition of the cyclodextrin-containing solution, the current increased stepwise, and each time the hemolysin was reconstituted into the lipid bilayer, the current was transmitted through the hemolysin ion channel. It was confirmed that it was flowing. That is, it was confirmed that a lipid bilayer membrane was formed in the through hole, and hemolysin was reconstituted in this lipid bilayer membrane (hemolysin was retained through the lipid bilayer membrane). It was observed that the addition of cyclodextrin-containing solutions often reduced the current. This is a phenomenon that occurs because cyclodextrin sometimes blocks the ion channels of hemolysin. Furthermore, since the solution flows in and out of the detection well from the first flow path and the second flow path continuously, the frequency of the current drop decreases with the passage of time, and the liquid is exchanged. I understand. Further, as shown in FIG. 10, the current value decreased significantly 3 minutes after the addition of cyclodextrin, but the current recovered to the original value 18 minutes after the addition of cyclodextrin, and the liquid exchange was carried out. It was confirmed that it was done. Furthermore, as shown in Table 2, the area ratio was 0 before the addition of cyclodextrin, but the area of the occlusion signal of cyclodextrin increased significantly 3 minutes after the addition of cyclodextrin, while the area derived from hemolysin nanopores. The area ratio became 0.23 because of the decrease. Eighteen minutes after the addition of cyclodextrin, the area ratio decreased to 0.03, confirming that liquid exchange had taken place.

Example 2
1. 1. Fabrication of Analytical Device As shown in FIG. 11, an acrylic plate having a thickness of 1 mm was used as a lower substrate, an acrylic plate having a thickness of 4 mm was used as a middle substrate, and an acrylic plate having a thickness of 4 mm was used as an upper substrate. Only through holes for inserting electrodes were made in the lower substrate. A detection well with a diameter of 3 mm, a liquid storage well with a length and width of 12 mm x 10 mm, and a liquid absorption well with a diameter of 11 mm x 11 mm were formed as through holes in the central substrate. Further, one first flow path and one second flow path were formed. The first flow path has a width of 0.2 mm, a depth of 0.1 mm, and a length of 4 mm. The second flow path has a width of 0.2 mm, a depth of 0.1 mm, and a length of 10.5 mm. Other sizes are as shown in FIG. 11 (unit: mm). The lower substrate and the middle substrate were laminated and crimped with a crimping machine. At this time, the central substrate was arranged so that the flow path faces downward. Next, the upper substrate was laminated on the central substrate and crimped with a crimping machine to prepare an analysis device. Further, a platinum electrode having a diameter of 0.3 mm was arranged in the through hole provided in the lower substrate. The platinum electrodes were arranged so as to be exposed in the liquid storage well and the detection well, respectively. The platinum electrode was fixed with an adhesive.

2. Measurement The same electrolyte solution as in Example 1 was placed in an 800 μL reservoir well. 20 mg of the same water-absorbing polymer as in Example 1 was placed near the end of the second flow path in the liquid-absorbing well. 5 μL of the electrolyte solution was placed in the detection well. It was confirmed that the potential difference became 0 V in the closed circuit state. I switched to a voltmeter and started voltage measurement. An aqueous sodium hypochlorite solution having a concentration of 3.1 mM was added dropwise to a 5 μL detection well. When the voltage returned to 0 V, the sodium hypochlorite aqueous solution was dropped again in the same manner. The results are shown in FIG.

As shown in FIG. 12, immediately after the addition of the sodium hypochlorite aqueous solution, an electromotive force is generated due to the concentration gradient, but the electromotive force generated due to the flushing effect decreases and returns to the initial value of 0 V. Similar behavior is seen when sodium hypochlorite is added again. It can be seen that sodium hypochlorite can be detected and washed out continuously.

Example 3
The experiment was carried out as follows using the analytical device prepared in Example 1. A solution of DPhPC in decans (20 mg / ml) was placed in both wells in an amount of 3 μL each. Buffer (1 M KCl, 10 mM phosphate buffer pH, 7.6) was added to the detection wells in 20 μL. 1 nM of hemoricin was dissolved in the above buffer and 20 μL was added dropwise to the well on the ground side. Then, electrical measurement was started. The measurement conditions were applied voltage: 60 mV, sampling frequency: 5 KHz, and the measurement results were processed by a 1 kHz Bessel low pass filter. 3 mL of buffer was added dropwise to the liquid storage well and 100 mg of water absorbing polymer was added dropwise to the liquid absorbing well. After the hemolysin is reconstituted in the membrane to form nanopores, a 1 mM cyclodextrin compound (heptakis (6-O-sulfo) -β-cyclodextrin, hereinafter simply referred to as "cyclodextrin") is applied at time intervals. It was dropped into the detection well where the solution was exchanged multiple times after opening (see FIG. 13). The dropping amount was 4 μL only for the first time and 2 μL thereafter. The results are shown in FIG.

As shown in FIG. 13, each time cyclodextrin was added dropwise, the hemolysin channel was blocked and the current decreased, and the hemolysin-reconstituted lipid bilayer membrane was maintained for 3 hours. Was confirmed.

Furthermore, as follows, the relationship between the measurement time and the cyclodextrin concentration in the detection well in the above experiment was investigated. A calibration curve was prepared by acquiring a blocking signal under the condition that no solution exchange was performed. That is, the cyclodextrin signal was obtained without adding the solution of the water-absorbing polymer and the liquid storage well. The block frequency (the number of current drops that occur per unit time per nanopore) and the concentration of cyclodextrin were plotted to create a calibration curve (see FIG. 14). From the slope of the graph, 0.003 μM ^-1 s ^-1 was calculated. From the obtained calibration curve, the vertical axis of the graph shown in FIG. 13 was converted into a concentration. Specifically, the block frequency that occurs at a certain time was calculated, and the concentration was calculated by dividing ^{the acquisition blocking frequency by the slope (0.003 μM -1} s ^-1). In the above experiment, the final concentration of the dropped cyclodextrin was 200 μM for the first time (the leftmost arrow in FIG. 15) and 100 μM after that. The results are shown in FIG.

As shown in FIG. 15, it was shown that the cyclodextrin concentration increased with each dropping of cyclodextrin and decreased with time. That is, it was confirmed that the solution was exchanged.

Comparative Example 1
The same experiment as in Example 3 was performed except that no solution exchange was performed, that is, no buffer was placed in the liquid storage well and no water absorbing polymer was placed in the liquid absorbing well. In Comparative Example 1, the analytical device itself used was the one produced in Example 1, but since the buffer was not put in the liquid storage well and the water-absorbing polymer was not put in the liquid absorption well, the liquid storage well and the liquid absorption well were not put. This is similar to the experiment using an analytical device that does not have a liquid absorption well. The experiment was conducted as follows. A solution of DPhPC in decans (20 mg / ml) was placed in both wells in an amount of 3 μL each. The buffer (1M KCl, 10 mM phosphate buffer pH 7.6) was then placed in 20 μL in the detection wells. Hemoricin 1-10 nM was dissolved in buffer and 20 μL was added dropwise to the well on the gland side. Then, electrical measurement was started. The measurement conditions were applied voltage: 60 mV, sampling rate: 5 KHz, and the measurement results were processed by a 1 kHz Bessel low pass filter. Each time the membrane was destroyed, the lipid bilayer membrane was reformed and the durability time was determined. The results are shown in FIG.

As shown in FIG. 16, the lipid bilayer membrane was destroyed in 2 minutes at the earliest and 20 minutes at the longest without the solution exchange.

10 Lower substrate 12 Middle substrate 14 Upper substrate 16 Electrode 18 Detection well 20 Liquid storage well 22 Liquid absorption well 24 First flow path 26 Second flow path 28 Board 30 Partition wall 32 well 34 well 36 Through hole

Claims (3)

The substrate, the detection well provided on the substrate, the liquid storage well provided on the substrate, the liquid absorbing material arranged in the substrate, and the detection well and the liquid storage well communicating with each other 1 or The liquid contained in the liquid storage well is provided with a plurality of first flow paths and one or a plurality of second flow paths communicating the detection well and the liquid absorbing material, and the liquid contained in the liquid storage well is the first flow. The flow velocity of the liquid flowing through the path, the detection well, and the second flow path in this order to reach the liquid absorbing material and flowing into the detection well at this time, and the flow velocity of the liquid flowing out of the detection well. Analytical devices that are substantially equal. The analytical device according to claim 1, wherein the detection well is in the form of a double well chamber separated by a lipid bilayer membrane. The capacity of the detection well for liquid exchange is 10 μL to 500 μL, the length of the first flow path is 4 mm to 200 mm, the width of the first flow path is 0.2 mm to 2 mm, and the depth of the first flow path. The length is 0.1 mm to 1 mm, the length of the second flow path is 4 mm to 200 mm, the width of the second flow path is 0.2 mm to 2 mm, and the depth of the second flow path is 0.1 mm to 1 mm. The analytical device according to claim 1 or 2.

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