JPWO2018003856A1 - Micro-well plate for forming droplet array and method of manufacturing droplet array - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microwell plate and droplet array for droplet array formation capable of producing droplets at a simple and low cost and producing a two-dimensional droplet array having concentration gradient without performing precise pump and pipetting operations. Provide a manufacturing method of SOLUTION: A sample is added in the presence of a water component using a predetermined micro well plate in which micro wells are connected using a flow path, and the oil component is added before the concentration of the sample becomes constant. The addition of the oil makes it possible to produce a two-dimensional droplet array having a concentration gradient by closing the flow path with the sample or the sample having the concentration gradient. [Selected figure] Figure 1

Description

  The present invention relates to a microwell plate for droplet array formation and a method of manufacturing a droplet array.

  Microdroplets arrayed in two dimensions are expected to contribute to high throughput and cost reduction of biochemical assays and medical diagnostics. For example, microdroplets such as crystallization of protein in the field of drug discovery, evaluation of cell sensitivity to new drugs, digital PCR used for diagnosis of cancer and infection, and culture of microorganisms and cells are applied to various applications. There is a possibility that it will be (non-patent documents 1 to 3 below).

  In order to further develop two-dimensional droplet array technology, it is desirable to generate a droplet array having concentration gradients of reagents and samples without relying on pump and pipetting operations that require precise operations.

  Non-Patent Document 1 describes a technique for generating and arraying droplets in a microfluidic device.

  Non-Patent Document 2 describes a technique for generating a droplet array using a substrate on which a hydrophilic surface is patterned.

  Non-Patent Document 3 describes a slip tip. The slip chip is a technology of sliding a groove provided on two facing substrates to generate a droplet.

  In general, pipetting is required to generate a two-dimensional droplet array (see, for example, Non-Patent Document 3). In particular, to obtain a droplet array having a concentration gradient can not be achieved by any of the above methods, and it is necessary to use a human pipetting device and a device using a pump.

R. R. Pompano, et. al. , Annu. Rev. Anal. Chem. , 4, 59-81, 2011. E. Ueda et. Al. , Adv. Mater. , 25, 1234-1247, 2013. H. Yan, et. Al. , Chinese J. Anal. Chem. , 43, 10, 1520-1525, 2015

  An object of the present invention is to provide a microwell plate for forming a droplet array and a method for manufacturing a droplet array which can manufacture droplets simply and at low cost.

  An object of the present invention is to provide a microwell plate for droplet array formation and a method for producing a droplet array, which can produce a two-dimensional droplet array having a concentration gradient without performing precise pump and pipetting operations. I assume.

  The present invention is basically based on the use of a predetermined microwell plate in which microwells (wells) are connected using a flow channel, and if oil is added in the presence of a water component, the oil will be well. Based on the finding that it is easy to generate droplets by propagating the channel and blocking the channel.

  The present invention also adds the sample in the presence of the water component, and adds the oil component before the concentration of the sample becomes constant, thereby causing the oil to flow in a state where the concentration gradient of the sample is generated. Based on the finding that a two-dimensional droplet array with concentration gradient can be produced.

The present invention firstly provides a microwell plate for droplet array formation. The plate includes a substrate 11, a plurality of wells 13a, 13b, 13c, 13d, and a plurality of flow channels 17a, 17b, 17c.
The plurality of wells 13a, 13b, 13c and 13d are in the form of wells formed in a two-dimensional array on the substrate.
The microwell plate 15 for droplet array formation is a plate having a plurality of wells and for forming droplets in each of the plurality of wells.
The plurality of flow channels 17a, 17b, and 17c connect adjacent ones of the plurality of wells 13a, 13b, 13c, and 13d. This flow path is such that when there is no obstacle in the flow path, the liquid present in one well can move to the adjacent well.

  The microwell plate for forming a droplet array of the present invention has oil passages 19a, 19b, 19c, 19d and 19e. The oil passage is a portion to which oil components present in the plurality of wells and the plurality of channels move. The oil component is added to the plate (well, channel or oil channel) with the water component present in the plurality of wells and channels, whereby the oil component passes through the oil channel, the plurality of wells and Propagating multiple flow paths. On the other hand, in the plurality of channels 17a, 17b, and 17c, the water components are adjacent to each other by accommodating the oil components 23a and 23b in the plurality of channels 17a, 17b, and 17c, with the water component existing in the plurality of wells. To move to the well. Thus, the droplets 21a, 21b, 21c, 21d and the droplet array are formed.

  The plate may have grooves 23a, 23b, 23c, 23d. The groove is a groove existing through the plurality of wells and the plurality of channels. This groove functions as an oil passage or part of an oil passage.

  The oil passage of this plate may be a lipophilic portion provided on the lower wall surface or the bottom surface of the plurality of wells and the plurality of flow channels.

Next, the method of manufacturing the droplet array of the present invention will be described. This method uses the plate described above.
Water components are added to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
After the water component is added, the oil component is added to any of the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
The oil component propagates through the continuous oil passages 19a, 19b, 19c, 19d, 19e present in the plurality of wells 13a, 13b, 13c, 13d and the plurality of flow channels 17a, 17b, 17c.
Then, the oil components 23a and 23b are accommodated in the plurality of flow channels 17a, 17b and 17c in a state where the water component is present in the plurality of wells 13a, 13b, 13c and 13d. This prevents the water component from moving to the adjacent wells, and the droplets 21a, 21b, 21c, 21d containing the water component are formed.

  The method for producing a droplet array according to the present invention different from the above is after the step of adding the water component and before the step of adding the oil component, the target liquid is added to the plurality of wells 13a, 13b, 13c, It further includes the step of adding it to any of 13d and the plurality of flow paths 17a, 17b, 17c.

  In this case, the step of adding the oil component is preferably performed as follows. That is, the oil component is added before the concentration of the target solution becomes uniform in the plurality of wells 13a, 13b, 13c, 13d. Then, in a state where the target liquid has a concentration gradient in the plurality of wells 13a, 13b, 13c, 13d, the movement to the adjacent wells is hindered. In this way, a two-dimensional droplet array having a concentration gradient can be easily obtained.

  The present invention can provide a microwell plate for droplet array formation and a droplet array manufacturing method which can produce droplets simply and at low cost simply by adding an oil component while containing water components.

  The present invention adds a target solution in a state of containing a water component, and adds an oil component only before the concentration of the target solution becomes uniform, without performing a precise pump and pipetting operation. It is possible to provide a microwell plate for forming a droplet array and a method of manufacturing a droplet array, which can manufacture a two-dimensional droplet array having the

FIG. 1 is a conceptual view of a microwell plate for forming a droplet array. FIG. 2 is a diagram showing an example of the peripheral portion of one well. FIG. 3 is an example of a top view of the microwell plate. FIG. 3 (A) shows the microwell plate before droplets are formed, and FIG. 3 (B) shows the microwell plate with droplets formed. FIG. 4 is a diagram for explaining an oil passage. FIG. 5 is a top view and a cross-sectional view of a well portion having a groove. FIG. 5A shows a top view, and FIG. 5B shows a cross-sectional view. FIG. 6 is a conceptual diagram for explaining a method of manufacturing a droplet array. FIG. 7 is a conceptual view showing how wells, channels and droplets are formed after oil components are added. FIG. 8 is a conceptual view showing a manufacturing process of a two-dimensional droplet array having a concentration gradient. FIG. 9 is a conceptual view showing an example of a method of manufacturing a microwell plate. FIG. 10 is a photograph replacing the drawing of the manufactured micro well plate. FIG. 10 (A) shows an overall view of the manufactured micro well plate. FIG. 10 (B) shows a partially enlarged view of the manufactured micro-well plate. FIG. 11 is a photograph in place of a drawing showing the appearance of the microwell plate after addition of nomardecane. FIG. 11 shows the appearance of two adjacent wells when nomardecane is added. FIG. 11 (B) shows the appearance of two adjacent wells 15 seconds after adding nomardecane. FIG. 11C shows the appearance of two adjacent wells 30 seconds after adding nomardecane. FIG. 12 is a photograph in place of a drawing showing a micro-well plate in which droplets are formed. FIG. 13 is a photograph in place of a drawing showing that a droplet having a concentration gradient is formed. FIG. 13 (A) shows the appearance when nomardecane is added, and FIG. 13 (B) shows the appearance when droplets are formed after a predetermined time has elapsed. FIG. 14 is a photograph replacing the drawing showing the obtained droplets. FIG. 15 is a diagram showing the evaluation results of the obtained droplets. FIG. 16 is a graph in place of a drawing showing the rate at which droplets are formed. FIG. 17 is a graph in place of a drawing showing the width and height of the groove of the first device in the example and the ratio of droplet formation. FIG. 18 is a graph in place of a drawing showing the width and height of the groove of the second device in the example and the ratio of droplets formed. FIG. 19 is a drawing relating to the ninth embodiment. FIG. 19A is a conceptual view showing an array in the embodiment. FIG. 19B is a view for explaining the size of the well. FIG. 19C is a conceptual diagram for explaining the state of the well. FIG. 19D is a table in place of a drawing showing the results of droplet generation by n-decane based on Ag and Ap. FIG. 19E is a table in place of a drawing showing the results of droplet formation by n-hexane based on Ag and Ap. FIG. 19F is a table in place of a drawing showing the results of droplet formation by n-hexadecane based on Ag and Ap. FIG. 19G is a photograph in place of a drawing showing the appearance of generated droplets. FIG. 19H is a photograph replacing the drawing showing connected droplets. FIG. 19I is a photograph in place of a drawing showing that n-hexane is connected in a short period of time. FIG. 19 (J) is a photograph in place of a drawing showing that n-hexadecane has a lower droplet formation rate than other solvents. FIG. 20 is a drawing relating to the tenth embodiment. FIGS. 20A and 20B are photographs in place of drawings showing the state before and after droplet generation by n-decane for the case of (Ag = 10, Ap = 0). FIGS. 20C and 20D show the volumes of droplets generated in the microwell plate in the cases of (Ag = 10, Ap = 0) and (Ag = 15, Ap = 4), respectively. It is a graph replaced with the drawing shown. FIG. 21 is a drawing relating to an eleventh embodiment. FIG. 21 (A) is a schematic view showing how fluorescent molecule Rhodamine B (Rhodamine B), fluorescein (Fluorescein), and n-decane are dropped to a microwell plate having three reservoirs. FIG. 21 (B) is a photograph in place of a drawing showing a micro-well plate having three reservoirs. FIG. 21 (C) is a photograph in place of a drawing showing a situation in which a concentration gradient is generated by leaving for 2 hours after rhodamine B and fluorescein are dropped. FIG. 21 (D) is a photograph in place of a drawing showing a state in which n-decane is dropped to generate droplets after the state of FIG. 21 (C). FIG. 21E is a photograph replacing the drawing showing the concentration gradient of rhodamine B. FIG. 21F is a graph replaced with a drawing showing a concentration gradient of rhodamine B. FIG. 21 (G) is a photograph in place of a drawing showing the concentration gradient of fluorescein. FIG. 21H is a graph replaced with a drawing showing a concentration gradient of fluorescein. FIG. 22 is a fluorescence microscope picture replaced with a drawing showing that sf9 cells were encapsulated in the droplet.

  Hereinafter, an embodiment of the present invention will be described using the drawings. The present invention is not limited to the embodiments described below, and includes those appropriately modified by the person skilled in the art from the following embodiments within the obvious scope.

  FIG. 1 is a conceptual view of a microwell plate for forming a droplet array. FIG. 2 is a diagram showing an example of the peripheral portion of one well. FIG. 3 is an example of a top view of the microwell plate. FIG. 3 (A) shows the microwell plate before droplets are formed, and FIG. 3 (B) shows the microwell plate with droplets formed. As shown in FIG. 1, the microwell plate has a plurality of wells on a substrate. The microwell plate is a kind of detection instrument that can accommodate various reagents and samples in wells and is used, for example, for reagent evaluation, biological evaluation, and pathological evaluation. The microwell plate is already known as described in, for example, Patent Nos. 5848233, 5642551, 5592606, 4965095, and 4496962.

  In the example of the size of the part where the well exists in the microwell plate, one side is 3 mm or more and 50 cm or less, 3 mm or more and 20 cm or less, or 5 mm or more and 10 cm or less. The size of this portion may be square, rectangular, polygonal, circular or elliptical.

  The plate includes a substrate 11, a plurality of wells 13a, 13b, 13c, 13d, and a plurality of flow channels 17a, 17b, 17c. The material of the substrate is not particularly limited as long as it can form a well. A well-known thing may be suitably adopted as the material of the plate. Examples of plate materials are metals, resins and ceramics. Examples of metals are copper and aluminum. Examples of the resin are methacrylic, acrylic, polyethylene, polypropylene, polystyrene, polyethylene tephthalate, vinyl chloride, polyamide, polycarbonate, hot melt, phenol, bakelite, melamine, polytetrafluoroethylene, epoxy and mixed resins thereof. Among these, polypropylene, polystyrene, polycarbonate, polytetrafluoroethylene or polyethylene is preferred. An example of a specific substrate material is OSTEMER® crystal clear. The material of the substrate may be adjusted in consideration of the shapes of wells and flow channels, and lipophilicity and hydrophilicity.

  The substrate may be formed integrally with the well or the channel, or the substrate may be formed separately from the well or the channel. A portion of the surface of the substrate may be the bottom of the well, channel or groove. In this case, the surface of the substrate preferably has water repellency. If the surface of the substrate has water repellency, an oil passage can be formed effectively and droplets can be formed efficiently. An example of such a substrate using, for example, a fluorine-based resin is a polytetrafluoroethylene substrate. Alternatively, a water repellent layer may be provided on the surface of the substrate, and a well or a channel may be formed on the substrate.

  The plurality of wells 13a, 13b, 13c and 13d are in the form of wells formed in a two-dimensional array on the substrate. The number of wells may be appropriate according to the measurement object. Specifically, the number of wells is preferably 4 or more (2 × 2 or more) and 10000 or less, may be 25 or more and 1000 or less, 36 or more and 1000 or less, and 100 or more and 400 or less. It may be less than or equal to 225, more than 225 and less than 400, or more than 400 and less than 900.

  Each well may have the same shape or a different shape. If the shapes of the wells are different, for example, the size of the wells may be different (for example, smaller) for each row, the concentration gradient of the sample can be easily provided. Also, the size of the wells may be such that the size of the wells changes (for example, decreases) as it goes away from a certain point.

The shape of each well may be selected appropriately according to the physical properties of the sample to be treated and the water component. The shape seen from the top of the well is usually circular. On the other hand, it may be elliptical or polygonal. The area viewed from the top of the well is 0.01 mm ² or more and 10 mm ² or less, 0.01 mm ² or more and 4 mm ² or less, 0.05 mm ² or more and 2 mm ² or less, or 0.1 mm ² or more and 1.5 mm ² or less Good. An example of the depth of the well (the height from the top surface to the bottom surface of the well) is 0.1 mm or more and 1 cm or less, and may be 0.1 mm or more and 5 mm or less, or 0.3 mm or more and 2 mm or less. 5 mm or more and 1 mm or less may be sufficient. The cross-sectional shape of the well may be rectangular (or rectangular having a groove portion), or may be narrower (or wider) as it becomes deeper.

  The plurality of flow channels 17a, 17b, and 17c connect adjacent ones of the plurality of wells 13a, 13b, 13c, and 13d. Since there is no adjacent well at the end portion of the substrate 11, there may be no flow path, and a flow path is provided assuming that the arrayed wells exist at the end portion or end portion of the substrate. It may be done. In that case, the number of channels present at the end of the substrate may be one or more.

  The flow path is to connect adjacent wells. This flow path is such that when there is no obstacle in the flow path, the liquid present in one well can move to the adjacent well. In the example of the width of the flow path, the flow path is such that the oil component passes while the oil component passes. Therefore, the shape of the flow channel may be selected in consideration of physical force such as surface tension in the flow channel.

  The shape of the flow channel is usually rectangular. However, the top shape of the flow path may be smaller as it moves away from a certain point. Further, even if the shape of the flow channel is rectangular, the width of the flow channel may be designed to be smaller (or larger) as it goes away from a certain well. The cross-sectional shape of the flow channel is also generally rectangular (or a shape having a groove). On the other hand, the shape of the flow path may be such that the width becomes narrower (or wider) as it gets deeper.

  The length of the channel is at least one thousandth and one half of the width of the well (the maximum length of the well assuming that there is no channel) and at least one-half to three hundredths It may be 1 or less, 1/200 or more and 1/5 or less, 1/100 or more and 1/8 or less, or 1/50 or more and 1/10 or less. Specifically, the length of the flow path is 0.01 mm or more and 5 mm or less, and may be 0.01 mm or more and 2 mm or less, 0.01 mm or more and 0.5 mm or less, or 0. 05 mm or more and 0.5 mm or less may be sufficient, and 0.05 mm or more may be 0.3 mm or less.

  The width of the channel is not less than one hundredth and not more than one half of the width of the well (the maximum length of the well assuming that there is no channel), and at least one-fifty and a half It may be 1 or less, 1/20 or more and 1/5 or less, 1/15 or more and 1/8 or less, or 1/11 or more and 1/9 or less. An example of the width of the channel is one tenth of the width of the well. A specific example of the width of the flow path is 0.01 mm or more and 10 mm or less, may be 0.01 mm or more and 0.5 mm or less, or may be 0.03 mm or more and 0.2 mm or less, and 0.05 mm or more. It may be 1 mm or less.

  The depth of the channel may be the same as or different from the depth of the well. If the depth of the channel is the same as the depth of the well, the top surface of the channel may be at the same height as the top surface of the well. On the other hand, if the channel depth is different from the well depth, the channel depth may be shallower (even if the height is lower) or deeper (even if the height is higher) than the well depth. ) Good. In any case, the flow path preferably includes an oil path. The bottom of the channel and the bottom of the well may be at the same position, or the bottom of the channel may be lower than the bottom of the well. When the bottom of the flow channel and the bottom of the well are at the same position, the oil component passes through the flow channel smoothly. On the other hand, when the bottom of the channel is at a lower position than the bottom of the well, the oil component tends to stay in the channel.

  The plate may have grooves 23a, 23b, 23c, 23d. The groove is a groove existing through the plurality of wells and the plurality of channels. This groove functions as an oil passage or part of an oil passage. The cross-sectional shape of the groove may be rectangular, semicircular, or inverted triangular.

  The oil passages 19a, 19b, 19c, 19d and 19e are portions through which oil components present in the plurality of wells and the plurality of flow passages move. The oil passage may exist in the well and the flow passage, and may be a portion which does not change in physical property from the portion other than the oil passage, or may be a portion which differs in some physical property. The oil passage is preferably located at the bottom of the well and the flow passage, and the oil component moves through the oil passage. The microwell plate of the present invention has the above-described wells and channels, and when the oil components are added in the presence of water components in the wells and channels, the oil components move through the wells and channels Shape and physical properties that can be propagated. The part to which the oil component at this time moves is an oil passage. That is, the micro-well plate of the present invention is a micro-well plate for new use, and when oil components are added in the presence of water components in wells and channels, oil components move in the wells and channels And propagate, and this is a microwell plate for applications where droplets are formed.

  FIG. 4 is a diagram for explaining an oil passage. As shown in FIG. 4A, the oil passage 19a may be a well or a lower portion of the flow passage. The oil passage 19a shown in FIG. 4 (A) has the same physical properties as the portion other than the oil passage.

  As shown in FIG. 4 (B), the oil passage may be a portion of the well or flow path having physical properties different from those of other portions. In the example of the oil passage in this case, different surface processing is applied to the oil passage and portions other than the oil passage. For example, the portion other than the oil passage may be etched to make the surface roughness rough. Also, only the oil passage portion may be mirror-polished. The oil passage may be formed by applying a coating to the oil passage portion. The coating may be changed in physical properties from the parts other than the oil passage part, and any coating may be used as long as it functions as an oil passage. An example of the coating is a water repellent (hydrophobic agent). Examples of water repellents include waxes (eg paraffin wax and natural wax), resins (eg polypropylene resins, polyethylene resins, polyamide resins, and polyamine resins), fluorine resins (eg resins containing fluorine, poly Tetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyhexafluoropropylene, tetrafluoroethylene-hexafluoropropylene copolymer (FEP), polytetrafluoroethylene, and polychlorotrifluoroethylene, and silicone resin (polymethyl) Hydrogen siloxane and polydimethylsiloxane). These may be used alone or a plurality of them may be mixed and used. An example of a specific coating is CYTOP (types A to C) manufactured by Asahi Glass Co., Ltd. The oil passage may be formed by vapor deposition of metal on the oil passage portion. The oil passage thus formed may form a layer also referred to as a water repellent layer or a hydrophobic layer. Then, when the oil component is added, since the affinity with the oil component is higher than the water component, the oil component tends to be retained in the flow path, and the oil component is present on the bottom of the well. Droplets are likely to be formed.

  As shown in FIG. 4C, the oil passage 19c may be a groove present in the bottom of the well (and the bottom of the channel). Since this groove functions as an oil passage, it is preferable that the groove be provided through the bottom of the well and the bottom of the channel. The groove is a portion provided below the bottom of the well and having a shape recessed from the bottom. In the example shown in FIG. 4C, only the groove portion may function as an oil passage, or the groove portion and the well bottom surface (and the bottom surface of the flow passage) may function as an oil passage. Further, the entire groove portion may not function as an oil passage. In this case, the substrate surface may be the bottom of the groove or the substrate surface may be exposed through the groove. When the substrate surface has water repellency, good oil paths can be formed without coating the wells, and droplets can be preferably produced. Then, since it is not necessary to perform coating etc., productivity is enhanced.

  As shown in FIG. 4D, the oil passage 19d may be a groove present in the bottom of the well (and the flow passage), and may have a surface having physical properties different from those of the well and the flow passage surface. In this case, the surfaces having different physical properties are as described in the example of FIG. 4 (C). Even in this case, the oil does not have to move only through the groove portion, and the oil may move in contact with at least a part of the groove portion.

  As shown in FIG. 4E, the oil passage 19d is provided not only to the groove present on the bottom of the well (and the flow passage) but also to the well (and the flow passage) and has a physical property different from other portions. It may be. Also in this case, the oil does not have to move while adhering to the entire oil passage, as long as the oil contacts at least a part of the oil passage and moves.

  FIG. 5 is a top view and a cross-sectional view of a well portion having a groove. FIG. 5A shows a top view, and FIG. 5B shows a cross-sectional view.

When there is a groove, an example of the groove depth D _D is 1/25 or more of the well depth (height) D _W , and may be 1/20 or more and 1⁄2 or less. It may be one-half or more and one-third or less, one-sixth or more and one-quarter or less, or one-fifth. The width W _D of the groove may be the same as the width W _W of the flow path as shown in FIG. 5A, or may be smaller than the width of the flow path. When the width of the groove is smaller than the width of the flow channel, the width of the groove may be 1/3 or more and 2/3 or less of the width of the flow channel, or may be 1/2.

  In the microwell plate, the oil component is added to the plate (well, channel or oil channel) in a state where the water component is present in the plurality of wells and the plurality of channels, the oil component passes through the oil channel And propagate a plurality of wells and a plurality of channels. On the other hand, in the plurality of channels 17a, 17b, and 17c, the water components are adjacent to each other by accommodating the oil components 23a and 23b in the plurality of channels 17a, 17b, and 17c, with the water component existing in the plurality of wells. To move to the well. Thus, the droplets 21a, 21b, 21c, 21d and the droplet array are formed.

  Next, the method of manufacturing the droplet array of the present invention will be described. This method is a method of obtaining a droplet array using the plate described above. The method is briefly described as follows. Add the oil component while the water component is contained in the wells and channels. Then, while the oil component propagates through the oil passage of the well and the passage, it stays in the passage. The oil component thus staying in the flow path isolates the water component in the well and forms droplets. In the following description, S means step.

  FIG. 6 is a conceptual diagram for explaining a method of manufacturing a droplet array. FIG. 6A is a conceptual view showing the appearance of the plate before the addition of the water component. This example uses a plate having a groove at the bottom of the well. The lower part of FIG. 6 (A) is a schematic drawing of a top view and a sectional view of a certain plate.

  Water components are added to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c. The water component may be an aqueous solution or alcohol solution containing the target sample, or may be one in which the target sample is dispersed or one in which the target sample is dissolved. The plate is adapted to allow the wells to flow liquid through the channels. Therefore, for example, if a water component is added to a certain well, the water component is transmitted to the plurality of wells through the flow path. FIG. 6 (B) is a conceptual view showing the water component in the well and the channel. The water component may be added in an amount contained in the whole well, or an amount obtained by dividing the amount of components (for example, oil component) to be added later from the amount which can be contained in the wells and flow channels. You may add. When the water component is added in an amount to be contained in the whole well, the water component in the well swells and forms droplets after the oil component is added. For this reason, the water component may be added in an amount to be contained in the entire well.

  After the water component is added, the oil component is added to any of the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c. The oil component may be added from any one well. Also, the oil component may be added from any one flow path. Furthermore, the plate may be provided with an oil component addition unit for adding an oil component, and the oil component may be added from the oil component addition unit.

  A well-known oil component and an organic solvent can be used suitably if an oil component is a thing which does not mix completely with a water component. It is preferable that the oil component has higher affinity to the well and the channel than the water component. When the water component contains water as a main component, examples of the oil component are liquid alkanes having 4 to 20 carbon atoms, silicone oils, vegetable oils, essential oils, and esters (liquid). An example of the liquid alkane is a liquid alkane having 5 to 16 carbon atoms. Examples of vegetable oils are corn oil, palm oil and palm shell oil. Examples of esters are myristyl myristate, myristyl palmitate, myristyl stearate, myristyl isostearate, myristyl oleate, myristyl behenate, myristyl erucate, cetyl myristate, cetyl palmitate, cetyl stearate, cetyl isostearate, olein Acid cetyl, cetyl behenate, cetyl erucate, stearyl myristate, stearyl palmitate, stearyl stearate, stearyl isostearate, stearyl oleate, stearyl oleate, stearyl behenate, stearyl ercate, isostearyl myristate, isostearyl palmitate, stearin Isostearyl acid, isostearyl isostearate, isostearyl oleate, isostearyl behenate, isostearyl oleate, Oleyl lysylate, oleyl palmitate, oleyl stearate, oleyl isostearate, oleyl oleate, oleyl behenate, oleyl erbate, behenyl myristate, behenyl palmitate, behenyl stearate, behenyl isostearate, behenyl oleate, behenic acid Behenyl, behenyl erucate, erucyl myristate, erucyl palmitate, erucyl stearate, erucyl isostearate, erucyl oleate, erucyl behenate, and eruclic erucile. These may be used alone or in combination of two or more.

  The oil component propagates through the continuous oil passages 19a, 19b, 19c, 19d, 19e present in the plurality of wells 13a, 13b, 13c, 13d and the plurality of flow channels 17a, 17b, 17c. FIG. 6C is a schematic drawing of how the oil component propagates. The oil component exists in the lower portion of the well and propagates to the adjacent well through the groove connected to the flow path. Note that part of the oil component may propagate through the upper surface of the well. That is, since the affinity (permeability) with the surface of the substrate is higher than that of the water component, the oil component propagates through the substrate surface (including the well surface and the channel surface). Then, in a state in which the water component is present in the plurality of wells 13a, 13b, 13c, 13d, the oil component is accommodated in the plurality of flow paths 17a, 17b, 17c. This prevents the water component from moving to the adjacent wells, and the droplets 21a, 21b, 21c, 21d containing the water component are formed.

  FIG. 7 is a conceptual view showing how wells, channels and droplets are formed after oil components are added. As the oil component passes through the flow path, part of the oil component remains in the flow path. If the amount of oil component remains in the flow path is small, as shown in FIG. 7, the water component can flow in the adjacent wells. On the other hand, when a sufficient amount of oil component remains in the flow path, as shown in FIG. 7B, the water component is divided in the adjacent wells, and droplets are formed in each well. FIG. 7C is a conceptual view showing a state in which a droplet is formed.

The method of manufacturing the above-mentioned droplet array is
Adding a water component to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c;
Adding an oil component to any of the plurality of wells 13a, 13b, 13c, 13d and the channels 17a, 17b, 17c after the water component is added;
Including
The oil component propagates through the continuous oil passages 19a, 19b, 19c, 19d, 19e present in the plurality of wells 13a, 13b, 13c, 13d and the plurality of flow channels 17a, 17b, 17c,
In the state where the water component A exists in the plurality of wells 13a, 13b, 13c, 13d, the oil components 23a, 23b are accommodated in the plurality of flow paths 17a, 17b, 17c,
Water components are prevented from moving to the adjacent wells,
Form droplets 21a, 21b, 21c, 21d containing water components,
It is a method of manufacturing a droplet array.

  The method for producing a droplet array according to the present invention different from the above is after the step of adding the water component and before the step of adding the oil component, the target liquid is added to the plurality of wells 13a, 13b, 13c, It further includes the step of adding it to any of 13d and the plurality of flow paths 17a, 17b, 17c.

  In this case, the step of adding the oil component is preferably performed as follows. That is, the oil component is added before the concentration of the target solution becomes uniform in the plurality of wells 13a, 13b, 13c, 13d. Then, in a state where the target liquid has a concentration gradient in the plurality of wells 13a, 13b, 13c, 13d, the movement to the adjacent wells is hindered. In this way, a two-dimensional droplet array having a concentration gradient can be easily obtained.

  FIG. 8 is a conceptual view showing a manufacturing process of a two-dimensional droplet array having a concentration gradient. In this example, the first target solution and the second target solution are separately added to the wells present at the opposite corners. As described above, the target liquid may not be of one type but may be of plural types. In this case, each well has various concentrations and concentration ratios for a plurality of types of target solutions. The target liquid is a liquid containing a target sample. Examples of target samples include, but are not limited to, various reagents, markers, cells, microorganisms, genes, genetic material, peptides, proteins, and dyes.

Water components are added to the plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, 17c.
Next, after the water component is added, the target solution is added to any of the plurality of wells 13a, 13b, 13c, 13d and the plurality of flow channels 17a, 17b, 17c. In the example shown in FIG. 8A, the first target solution and the second target solution are separately added to the wells present at the opposite corners. Then, the first target liquid and the second target liquid propagate along the flow path to the adjacent wells.
The plurality of wells 13a, 13b, 13c, 13d and the flow paths 17a, 17b, and 17d are added after the target solution is added and before the concentration of the target solution becomes uniform in the plurality of wells 13a, 13b, 13c, 13d. Add the oil component to any of 17c.
The oil component propagates through the continuous oil passages 19a, 19b, 19c, 19d, 19e present in the plurality of wells 13a, 13b, 13c, 13d and the plurality of flow channels 17a, 17b, 17c.
Then, the oil components 23a and 23b are accommodated in the plurality of flow channels 17a, 17b and 17c in a state where the water component is present in the plurality of wells 13a, 13b, 13c and 13d. This prevents the water component from moving to the adjacent wells, and the droplets 21a, 21b, 21c, 21d containing the water component are formed.

  FIG. 8 (B) is a conceptual view showing that a plurality of droplets having concentration gradients are formed for two types of target liquids. Thus, in the state where the target liquid has a concentration gradient in the plurality of wells 13a, 13b, 13c, 13d, the target liquid can not move to the adjacent wells. In this way, it is possible to easily obtain a two-dimensional droplet array having a concentration gradient for the target liquid.

  Next, a method of manufacturing the microwell plate will be described. As described above, since the microwell plate itself is already known, the microwell plate may be manufactured using the already known method. In order to form a water repellent layer (hydrophobic layer), for example, a water repellent agent or a hydrophobic agent may be added to the well and the solvent may be evaporated. An example of a method of changing the surface roughness of the substrate, the well and a part of the flow path is to perform sandblasting with the part being masked. Thus, methods for producing physically or scientifically different sites of different physical properties are already known.

Production of Micro-Well Plate FIG. 9 is a conceptual view showing an example of a method for producing a micro-well plate.
In this example, first, a template to be a base of the microwell plate was prepared (FIG. 9 (A)). The mold was made of acrylic resin. The mold may be metal. Also, in this example, a groove was formed at the bottom of the well.
The resin used as the raw material of the substrate was injected into the mold (FIG. 9 (B)). In the example of FIG. 9 (B), OSTECC was used.
In this example, after the side glass serving as the substrate is mounted on the resin, ultraviolet light is irradiated to cure the OSTECC resin (FIG. 9C). Then, the resin on the mold was cured.
After that, the substrate was peeled off from the mold (FIG. 9 (D)). The substrate was made of OSTECC resin, so it was flexible (flexible) and could be peeled off from the mold.
Next, in order to form portions with different physical properties, CYTOP solution was added so as to be about 4 minutes to 6 minutes of the well (FIG. 9 (E)).
While the CYTOP solution was added, the substrate was heated to 100 ° C. to solidify the CYTOP solution (FIG. 9 (F)). Thus, the water repellent layer (hydrophobic layer) was formed.

The microwell plate produced in Example 1 was as follows.

  FIG. 10 is a photograph replacing the drawing of the manufactured micro well plate. FIG. 10 (A) shows an overall view of the manufactured micro well plate. FIG. 10 (B) shows a partially enlarged view of the manufactured micro-well plate. As shown in FIG. 10A, a microwell plate was manufactured.

  A microwell plate was manufactured in the same manner as in Example 1 except that there was no groove.

  A microwell plate was manufactured in the same manner as in Example 1 except that the coat layer by CYTOP was not provided.

  A microwell plate was manufactured in the same manner as in Example 1 except that a coating layer by CYTOP was provided up to the upper surface of the well.

Preparation of Droplet Array Ethanol was added as a water component to the microwell plate prepared in Example 1 to fill the wells and channels. After that, nomardecane was added as an oil component. FIG. 11 is a photograph in place of a drawing showing the appearance of the microwell plate after addition of nomardecane. FIG. 11A shows the appearance of two adjacent wells when nomardecane is added. FIG. 11 (B) shows the appearance of two adjacent wells 15 seconds after adding nomardecane. FIG. 11C shows the appearance of two adjacent wells 30 seconds after adding nomardecane. As shown in FIG. 11C, it can be seen that droplets were formed in the well. FIG. 12 is a photograph in place of a drawing showing a micro-well plate in which droplets are formed.

  It was examined whether droplets were also produced for the microwell plates produced in Examples 2 to 4. As a result, it had the groove, and the one in which the water repellent layer was partially formed formed the most excellent droplet. On the other hand, in the case without the groove and without the water repellent layer, the number of droplets formed was the smallest.

Preparation of Two-Dimensional Droplet Array Having a Concentration Gradient From the diagonal of the microwell plate prepared in Example 1, a solution containing red food pigment and blue food pigment is added as a target liquid, and the concentrations of the two target liquids are Before becoming uniform, nomardecane was added as an oil component. The situation is shown in FIG.
FIG. 13 is a photograph in place of a drawing showing that a droplet having a concentration gradient is formed. FIG. 13 (A) shows the appearance when nomardecane is added, and FIG. 13 (B) shows the appearance when droplets are formed after a predetermined time has elapsed. As shown in FIG. 13 (B), it can be seen that the droplets are formed in a two-dimensional array with a concentration gradient by using this method.

The microwell plate having 9 × 9 wells (81 in total) prepared in Example 1 was prepared. The microwell plate was impregnated with ethanol and then replaced with a 0.5% aqueous indigo carmine solution (blue No. 2). Subsequently, about 10 μl of any of alkanes of normal hexane (Hexane: C ₆ H ₁₄ ), nomar decane (Decane: C ₁₀ H ₂₂ ), and normal decadecane (Hexadecane: C ₁₆ H ₃₄ ) was dropped. The same type of plate was used for each alkane, and the formation of droplets was investigated three times each. Evaluation was as follows.
Droplet: The well is filled with an independent aqueous solution (droplet).
Connected droplet: An aqueous solution (droplet) is connected to the adjacent well or the outside.
Empty: the well is filled with alkane.
Broken well: There is damage to the well.

  FIG. 14 is a photograph replacing the drawing showing the obtained droplets. FIG. 15 is a diagram showing the evaluation results of the obtained droplets. As shown in FIG. 15, the number of droplets was counted and the ratio to the number of effective wells was taken. The results are shown in FIG. FIG. 16 is a graph in place of a drawing showing the rate at which droplets are formed. As shown in FIG. 16, the rate at which droplets are formed is 0.60 ± 0.05 (Hexane), 0.76 ± 0.13 (Decane), and 0.77 ± 0.03 ( Hexadecane).

  Two types of 9 × 9 micro well plates were prepared in the same manner as in Example 1 except that the width and height (step) of the grooves (lower flow channels) were changed. The widths and heights of the grooves of the prepared micro-well plate are represented as parameter values (mm) in the following table. The rate at which droplets are formed (number of droplets / number of wells) is shown in the following table. Also, the width and height of the groove of the first device, and the rate at which droplets are formed are shown in FIG. The groove width and height of the second device and the rate at which droplets are formed are shown in FIG. When the height (step) of the groove is 0, no groove is formed, and there is an example in which a channel having a width indicated by the parameter value exists. 17 and 18 are graphs of the contents of Table 2. The x-axis represents the width of the groove (channel), the y-axis represents the height of the groove (step), and the z-axis represents the droplet Indicates the rate of formation.

  From the above results, it can be seen that the width of the groove is 0.02 mm or more and 0.08 mm or less, and the rate of formation of droplets is high. Also, since droplets are sufficiently formed even when the groove width is 0.02 mm, droplets may be effectively formed even when the groove width is 0.02 or less. It can be said. Also, although no significant difference was found in droplet formation between the one without the groove and the one with the groove, the groove height (step) was 0.2 mm and 0.4 mm, When the width of is less than 0.08 mm, droplets were stably formed. Therefore, it can be seen that droplets are formed at a high rate when the groove height is 0.2 mm or more and 0.4 mm or less and the groove width is 0.08 mm or less. In the above example, the groove height was 0.2 mm and 0.4 mm, and both showed a good droplet formation rate. Therefore, the height of the groove is not limited to 0.2 mm or 0.4 mm, and it is considered that droplets can be formed even if the height of the groove is 0.2 mm or less or 0.4 mm or more. Be

Investigation of Droplet Generation Rate on Well Array Size and Type of Organic Solvent As shown in FIG. 19 (A), an array having 4 × 19 wells was prepared. At one end of the array, a reservoir for solvent introduction was fabricated. As shown in FIG. 19 (B), Ag = D / W and Ap = S / W are defined from the well diameter (D), width (W), height (H) and step height (S). The drop generation rate was investigated based on the value of. The D and H were fixed at 500 μm, the values of W and S were changed, and the values of Ag and Ap were changed. The residual aqueous solution in the wells after solvent introduction can be classified into three states: formed droplets, connected droplets, and no droplets, and the droplet generation rate is defined as the number of generated droplets divided by the number of effective wells. A blue 0.5% indigo carmine aqueous solution was used as the aqueous solution for observation. As solvents, n-decane, n-hexane and n-hexadecane immiscible with water were used.

  FIG. 19 (D) shows the result of droplet formation by n-decane based on Ag and Ap. When Ag = 15, droplet generation could be performed at a rate of 90% or more. FIG. 19 (G) and FIG. 19 (H) show the generated droplet and the connected droplet, respectively. As shown in FIG. 19 (E) and FIG. 19 (F), droplet formation could be performed using n-hexane and n-hexadecane. It was found that when N-hexane was used, the droplet formation rate was higher than when other solvents were used. It is considered that this is because n-hexane has low viscosity and can permeate widely, as compared with other solvents. However, since n-hexane is a volatile solvent, it was found that it would be connected in a few seconds as shown in FIG. 19 (I). When n-hexadecane was used, as shown in FIG. 19 (F), a lower droplet formation rate was observed compared to other solvents.

Volume evaluation of generated droplets in microwell plate Using microwell plate of two kinds of parameters (Ag = 10, Ap = 0, no lower channel) and (Ag = 15, Ap = 4, lower channel) As shown in FIG. 20 (A) and FIG. 20 (B), droplets were generated by n-decane. Rhodamine B (Rhodamine B), which exhibits red fluorescence, was included in the aqueous solution to enable fluorescence observation. The resulting droplets were immersed in n-decane, three-dimensional images were obtained with a confocal microscope, and the volume of the droplets was estimated every other column.

  FIGS. 20 (C) and 20 (D) are the cases of (Ag = 10, Ap = 0) and (Ag = 15, Ap = 4), and the droplets generated in the respective microwell plates Volume is shown. In (Ag = 10, Ap = 0), the volume of the droplet increases slightly as the number of columns increases, while in (Ag = 15, Ap = 4) such a tendency is not confirmed. From this result, it was found that the volume change of each row can be suppressed by providing the lower channel.

Demonstration of Array Having Concentration Gradient by Fluorescence Intensity Measurement As shown in FIGS. 21 (A) and 21 (B), a microwell plate having three reservoirs was fabricated. The fluorescent molecule Rhodamine B (Rhodamine B), which emits red fluorescence, and fluorescein (Fluorescein), which emits green fluorescence, are dropped to these reservoirs, respectively. After the rhodamine B and fluorescein were dropped, it was allowed to stand for 2 hours to generate a concentration gradient (FIG. 21 (C)), and then n-decane was dropped to generate droplets (FIG. 21 (D)).

  As shown in FIG. 21 (E) to FIG. 21 (H), the concentration gradient of rhodamine B and fluorescein was successfully contained in the droplet.

Cell entrapment experiment We performed entrapment experiment of cell called Sf9. First, sf9 cells stained with a substance that emits green fluorescence due to an intracellular esterase reaction, such as Calcein (AM), were prepared. Subsequently, the microwell plate was filled with the medium containing sf9 cells, followed by droplet generation by dropping n-decane. As a result of observation under a fluorescence microscope, it was confirmed that sf9 cells were encapsulated in the droplet as shown in FIG.

  The present invention can be used in the field of scientific and chemical equipment industry. The present invention can be used in new drug development, biological experiments, and chemical experiments.

11 substrates; 13a, 13b, 13c, 13d wells;
15 micro well plates; 17a, 17b, 17c channels;
19a, 19b, 19c, 19d, 19e oil passages;
21a, 21b, 21c, 21d droplets;
23a, 23b, 23c, 23d grooves

Claims (5)

Substrate (11),
A plurality of wells (13a, 13b, 13c, 13d) provided on the substrate and formed in a two-dimensional array;
A microwell plate (15) for forming a droplet array,
It further has a plurality of flow paths (17a, 17b, 17c) connecting adjacent ones of the plurality of wells (13a, 13b, 13c, 13d),
In the plurality of wells (13a, 13b, 13c, 13d) and the plurality of channels (17a, 17b, 17c), an oil component is present in a state in which water components exist in the plurality of wells and the plurality of channels. It has a continuous oil passage (19a, 19b, 19c, 19d, 19e) through which the oil component propagates through the plurality of wells and the plurality of channels by being added;
The plurality of flow paths (17a, 17b, 17c) accommodate oil components (23a, 23b) in the plurality of flow paths (17a, 17b, 17c) in a state where the water component is present in the plurality of wells By doing so, the water component is prevented from moving to the adjacent wells, and droplets (21a, 21b, 21c, 21d) are formed,
Microwell plate for droplet array formation (15). A micro-well plate (15) for droplet array formation according to claim 1, comprising
It further has grooves (23a, 23b, 23c, 23d) present through the plurality of wells (13a, 13b, 13c, 13d) and the plurality of flow channels (17a, 17b, 17c),
Microwell plate for droplet array formation (15). A micro-well plate (15) for droplet array formation according to claim 1, comprising
The oil passages (19a, 19b, 19c, 19d) are provided on lower wall surfaces or bottom surfaces of the plurality of wells (13a, 13b, 13c, 13d) and the plurality of flow passages (17a, 17b, 17c). Oily part,
Microwell plate for droplet array formation (15). Substrate (11),
A plurality of wells (13a, 13b, 13c, 13d) provided on the substrate and formed in a two-dimensional array;
A plurality of flow paths (17a, 17b, 17c) connecting adjacent ones of the plurality of wells;
Using the microwell plate (15),
Adding a water component to the plurality of wells (13a, 13b, 13c, 13d) and the flow path (17a, 17b, 17c);
Adding an oil component to any of the plurality of wells (13a, 13b, 13c, 13d) and the flow path (17a, 17b, 17c) after the water component is added;
Including
The oil component propagates through continuous oil passages (19a, 19b, 19c, 19d, 19e) present in the plurality of wells (13a, 13b, 13c, 13d) and the plurality of flow passages (17a, 17b, 17c) The
The oil components (23a, 23b) are accommodated in the plurality of flow channels (17a, 17b, 17c) in a state where the water components are present in the plurality of wells (13a, 13b, 13c, 13d),
The water component is prevented from moving to the adjacent well,
Forming droplets (21a, 21b, 21c, 21d) containing the water component,
Method of manufacturing a droplet array. 5. A method of manufacturing a droplet array according to claim 4,
After the addition of the water component and before the addition of the oil component, the target solution is formed of the plurality of wells (13a, 13b, 13c, 13d) and the plurality of flow channels (17a, 17b, 17c). Further including the step of adding to either
The oil component is added before the concentration of the target fluid becomes uniform in the plurality of wells (13a, 13b, 13c, 13d), whereby the target fluid is added to the plurality of wells (13a, 13b, 13c). , 13d) with a concentration gradient, which prevents movement to adjacent wells,
Method of manufacturing a droplet array.

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