JP7635799B2 - Microwell arrays and microfluidic devices - Google Patents

Description

The present invention relates to microwell arrays and microfluidic devices .

In recent years, microwell arrays having various types of fine flow channel structures formed using techniques such as etching and photolithography used in the manufacture of semiconductor circuits have been investigated.
The wells of these microwell arrays are used as chemical reaction vessels for carrying out various biochemical or chemical reactions in tiny volumes of fluid.

Materials used for manufacturing microfluidic systems include hard substances such as silicon and glass, and soft substances such as various polymeric resins such as PDMS (polydimethylsiloxane) and silicone rubber.
For example, Patent Documents 1 to 3 describe the use of such microfluidic systems as various microchips and biochips.

In recent years, attention has been focused on a technique for testing biological materials by carrying out an enzyme reaction in a very small space having a very small volume.
For example, one of the new approaches in the detection and quantification of nucleic acids is the digital PCR technique.
Digital PCR technology is a technique in which a mixture of reagents and nucleic acid is divided into countless tiny droplets for PCR amplification, and signals such as fluorescence are detected from the droplets containing the nucleic acid, and quantification is performed by counting the droplets in which a signal is detected.

Methods under consideration for producing microdroplets include a method in which microdroplets are produced by fragmenting them with a sealing liquid, and a method in which microdroplets are produced by pouring a reagent into a hole formed on a substrate and then pouring a sealing liquid into the hole (see, for example, Patent Document 3).

Patent No. 4911592 Patent No. 5337324 International Publication No. 2014/034781

However, with conventional microwell arrays, it can be difficult to encapsulate samples, such as aqueous liquid PCR reaction solutions, within the wells.

Therefore, an object of the present invention is to provide a microwell array and a microfluidic device that make it easy to seal an aqueous liquid in the wells.

The present invention is a microwell array comprising a plurality of wells having hydrophobic portions on the well surface.
The plane area S2 (10 ⁻¹² m ² ) of the well opening is 1.1 to 1.8 times the well bottom area S1 (10 ⁻¹² m ² ), and the angle θ formed by the side of the well with respect to a plane including the well opening is 77° to 90°.

The present invention allows for easy analysis of biomolecules.

FIG. 1A is a perspective view showing a first embodiment of a microfluidic device according to the present invention, and FIG. 1B is a cross-sectional view taken along line bb in FIG. 1A and 1B are a cross-sectional view and a perspective view showing a first embodiment of a microwell array according to the present invention. 1A to 1C are cross-sectional views showing a first embodiment of a method for producing a microwell array according to the present invention. FIG. 1 is a cross-sectional view showing an experimental example of a microwell array according to the present invention. FIG. 1 is a cross-sectional view showing a state in which an aqueous liquid is sealed in the microwell arrays of the microfluidic devices of Experimental Examples 1 to 3. 1 is a photograph showing the result of observing the microwell array of the microfluidic device of Experimental Example 1 with a fluorescent microscope from the substrate side. 1 is a photograph showing the result of observing the microwell array of the microfluidic device of Experimental Example 2 with a fluorescent microscope from the substrate side. 13 is a photograph showing the result of observing the microwell array of the microfluidic device of Experimental Example 3 with a fluorescent microscope from the substrate side. FIG. 11 is a cross-sectional view showing the state after an aqueous liquid and an oil are delivered to the microwell arrays of the microfluidic devices of Experimental Examples 4 and 5. 13 is a photograph showing the result of observing the microwell array of the microfluidic device of Experimental Example 4 with a fluorescent microscope from the substrate side. 13 is a photograph showing the result of observing the microwell array of the microfluidic device of Experimental Example 5 with a fluorescent microscope from the substrate side.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment of a microwell array, a microfluidic device, a method of sealing an aqueous liquid in wells of a microwell array, and a method of manufacturing a microwell array according to the present invention will now be described with reference to the drawings.
Fig. 1 is a perspective view (a) showing a microfluidic device in this embodiment, and a cross-sectional view (b) taken along line b-b in (a), Fig. 2 is a cross-sectional view (a) and a perspective view (b) showing a microwell array in this embodiment, and Fig. 3 is a cross-sectional view showing a method for producing a microwell array in this embodiment. Note that the dimensional ratios in each figure are exaggerated in some places for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[Microwell array]
In this embodiment, the present invention provides a microwell array comprising a plurality of wells having hydrophilic and hydrophobic portions on the well surface.
As will be described later, the microwell array of this embodiment makes it extremely easy to encapsulate an aqueous liquid in the wells.

Here, encapsulation means that an aqueous liquid is introduced into the wells that constitute the microwell array, and further, the liquids introduced into each well are isolated so that they do not mix with each other.
An example of a method for isolating the wells is to introduce an aqueous liquid into the wells and then form an oil layer on the top of the wells.
According to the microwell array of this embodiment, aqueous liquid can be easily filled in 90% or more, for example 95% or more, for example 99% or more, for example 100% of the wells constituting the microwell array.

The microwell array of this embodiment has a volume per well of, for example, 1 femtoliter (fL) to 6 nanoliters (nL), preferably 1 fL to 5 picoliters (pL), and more preferably 1 fL to 300 fL.
When the capacity per well is within this range, enzymatic reactions carried out in a small space, such as digital PCR or invader reactions, can be carried out suitably.
Digital PCR can be used to detect, for example, gene mutations.

The microwell array of this embodiment is capable of retaining the aqueous liquid enclosed within the wells even when the temperature of the liquid is changed, for example, for detecting gene mutations.
The range of the temperature to be changed is, for example, 0°C to 100°C, preferably 0°C to 80°C, and more preferably 20°C to 70°C.
When the aqueous solution enclosed in the well is in this range, enzymatic reactions carried out in a microspace, such as digital PCR or invader reaction, can be carried out suitably.

When an enzyme reaction is utilized in an analysis using a microwell array, it is preferable that the material of the microwell array does not inhibit the enzyme reaction.
Alternatively, the surface of the material of the microwell array can be coated with a substance that does not inhibit the enzyme reaction.

There are no particular limitations on the shape or size of the wells of the microwell array, but for example, when various biochemical reactions are carried out in microdroplets, it is preferable that the wells have a shape and size capable of accommodating one to several biomolecules or carriers.
The carrier may be, for example, a bead, and a biomolecule serving as a sample may be bound to the carrier.

The shape of the well may be, for example, a cylinder, a polyhedron composed of multiple faces (e.g., a rectangular parallelepiped, a hexagonal prism, an octagonal prism, etc.), an inverted cone, an inverted pyramid (an inverted triangular pyramid, an inverted square pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, an inverted polygonal pyramid with seven or more sides), etc., or a combination of two or more of these shapes.

For example, one portion may be cylindrical and the other portion may be inverted cone shaped.
In the case of an inverted cone or pyramid, the bottom of the cone or pyramid serves as the entrance (opening) of the well, but the well may have a shape in which a portion is cut off from the top of the inverted cone or pyramid.
In this case, the bottom of the microwell is flat.

When the well shape is cylindrical or rectangular, the bottom of the microwell is usually flat, but may be curved (convex or concave).
The bottom of the microwell can be curved, even when the shape is an inverted cone or pyramid with a portion cut off from the apex.

When the shape of the well is, for example, a truncated cone, as shown in FIG. 2, if its volume is V (10 ⁻¹⁸ m ³ ), then the bottom area S1 (10 ⁻¹² m ² ) of the well is, for example, 1 to 4.5 times V, preferably 2 to 4.3 times, and more preferably 3 to 4.2 times.
When the bottom area of the wells is within this range relative to the volume per well, the microwell array of this embodiment makes it possible to easily enclose aqueous liquid in 90% or more, for example 95% or more, for example 99% or more, for example 100% of the wells constituting the microwell array.
As described below, the inventors have discovered that such a configuration makes it extremely easy to encapsulate an aqueous liquid in the wells of a microwell array.

The shape of the well can also be controlled by, for example, controlling the planar area of the well.
For example, as shown in FIG. 2, the plane area S2 (10 ⁻¹² m ² ) of the opening of the well is 0.5 to 2.2 times, preferably 1 to 2 times, and more preferably 1.1 to 1.8 times, the bottom area S1 of the well.
When the bottom area of the wells is within this range relative to the volume of each well, the microwell array of this embodiment makes it very easy to enclose an aqueous liquid.

For example, as shown in FIG. 2, the maximum diameter D2 (μm) of the opening of the well is 0.5 to 1.5 times, preferably 0.7 to 1.4 times, and more preferably 1 to 1.3 times, the maximum diameter D1 (μm) of the bottom of the well.
When the maximum diameter of the bottom surface of the well is within this range relative to the maximum diameter of the opening of the well, the microwell array of this embodiment makes it very easy to enclose an aqueous liquid.

For example, as shown in FIG. 2, the angle θ of the side surface formed from the bottom surface of the well is, for example, 30° or more and 103° or less, preferably 60° or more and 103° or less, and more preferably 90° or more and 103° or less.
In addition, the side angle θ may be 77° or more, or the cube root of the volume V (10 ⁻¹⁸ m ³ ) of the well may be 3.5 or more.
When the angle of the side surface formed from the bottom surface of the well is within this range, the microwell array of this embodiment makes it very easy to enclose an aqueous liquid.

For example, as shown in FIG. 2, the height H (μm) from the bottom of the well is 0.2 to 10 times the maximum diameter D1, preferably 0.4 to 5 times, and more preferably 0.6 to 2 times.
When the angle of the side surface formed from the bottom surface of the well is within this range, the microwell array of this embodiment makes it very easy to enclose an aqueous liquid.

When the well is cylindrical, for the purpose of enclosing an aqueous liquid containing biomolecules, the maximum diameter and height H of the well may be, for example, 10 nm to 100 μm, preferably 100 nm to 10 μm, and more preferably 1 μm to 10 μm.
The dimensions of the wells are appropriately determined, taking into consideration the amount of aqueous liquid to be contained in the wells and the suitable ratio between the size of the carriers, such as beads to which biomolecules are attached, and the dimensions of the wells, so that one or several biomolecules can be contained in one microwell.

The detection target to be detected using the microwell array of this embodiment may be, for example, a sample collected from a living body such as blood, a PCR product, or an artificially synthesized compound.
For example, when DNA, which is a biomolecule, is to be detected, the well may have a shape and size that allows one DNA molecule to be placed therein.

The microwell array of this embodiment has a well density of, for example, 100,000 to 10 million/ ^cm2 , preferably 100,000 to 5 million/cm2, and more preferably 100,000 to 1 million/ ^cm2 .
When the well density is within this range, the operation of sealing the aqueous liquid sample in a predetermined number of wells can be easily performed.
Furthermore, the wells can be easily observed to analyze the experimental results.

For example, when measuring mutations in cell-free DNA, if the proportion of the mutation to be detected relative to the wild type is about 0.01%, it is preferable to use, for example, 1 to 2 million wells.

In the microwell array of this embodiment, each well can be produced by a process of depositing a hydrophilic or hydrophobic material on a substrate to form a hydrophilic or hydrophobic layer, and excavating the hydrophilic or hydrophobic layer to form a plurality of wells.
However, as long as the conditions are such that this embodiment can be realized, the microwells may be made of the same material as the substrate and formed by molding or cutting.

(substrate)
The microwell array of this embodiment may be formed on a substrate.
The substrate may or may not be electromagnetically transparent. Examples of electromagnetic waves include X-rays, ultraviolet rays, visible light, and infrared rays.
When a microwell array is formed on an electromagnetically transparent substrate, it becomes possible to use electromagnetic waves to analyze the results of experiments performed on the microwell array.
For example, fluorescence, phosphorescence, etc., which are generated as a result of irradiation with electromagnetic waves, can be measured from the substrate side. For detection of fluorescence, phosphorescence, etc., a fluorescence microscope, etc., can be used.
In this case, the electromagnetic waves may be irradiated, for example, from the substrate side, or from the inlet side of the well.

For example, when detecting fluorescence with a peak in the visible light range of 400 to 700 nm in a microwell array, a substrate that has good transparency to at least light in the visible light range can be used.

Examples of substrates having electromagnetic wave transparency include glass and resin.
Examples of resin substrates include ABS resin, polycarbonate resin, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene resin, polyethylene resin, polypropylene resin, polyvinyl acetate, PET (polyethylene terephthalate), PEN (polyethylene naphthalate), and the like.
These resins may contain various additives, and a plurality of resins may be mixed.

From the viewpoint of using fluorescence or phosphorescence to detect the experimental results, it is preferable that the above-mentioned substrate is substantially free of autofluorescence. Here, "substantially free of autofluorescence" means that the substrate does not have any autofluorescence at the wavelength used to detect the experimental results, or even if it does, the autofluorescence is so weak that it does not affect the detection of the experimental results.
For example, if the intensity of the autofluorescence is less than 1/2 or 1/10 of the fluorescence to be detected, it can be said to be weak enough not to affect the detection of the experimental results.

An example of a material that is electromagnetically transparent and does not emit any autofluorescence is quartz glass.
Materials that have weak autofluorescence and do not interfere with the detection of experimental results using electromagnetic waves include low-fluorescence glass, acrylic resin, COC (cycloolefin copolymer), COP (cycloolefin polymer), and the like.

Fluorescence used to detect biomolecules can be derived from, for example, fluorescent beads containing fluorescent molecules, or from intercalators such as SYBR Green that specifically enter the double helix of DNA and emit fluorescence.

Fluorescence emitted from the biomolecules contained in the wells can be observed from the substrate side of the microwell array, for example, using an inverted microscope.
By observing the fluorescence and counting the number of microwells that emit a specific fluorescence, the number of target molecules can be identified.

For example, before being placed in a well, the biomolecule may be treated with a fluorescent label that specifically labels the target molecule.
Alternatively, beads that specifically recognize the target molecule may be used to capture the target molecule, and the beads may then be placed in a well and contacted with a fluorescent label that can specifically label the target molecule, thereby fluorescently labeling the target molecule in the well.

The thickness of the substrate can be determined as appropriate, but when observing the fluorescence from the substrate side using a fluorescence microscope, it is preferable that the thickness is, for example, 5 mm or less, for example, 2 mm or less, or 1.6 mm or less.

In observing biomolecules using the microwell array of this embodiment, other than fluorescence, for example, turbidity can also be used.
The turbidity can be measured, for example, by the transmittance of light having a wavelength of about 400 to 1000 nm.

(hydrophilic resin)
The resin that forms the hydrophilic portion (hereinafter may be referred to as "hydrophilic resin") is not particularly limited as long as it exerts the effects of the present invention, but examples thereof include those in which the molecules of the constituent components of the resin have hydrophilic groups that exhibit hydrophilicity.
Examples of the hydrophilic group include a hydroxyl group, a carboxyl group, a sulfone group, a sulfonyl group, an amino group, an amide group, an ether group, and an ester group.

More specifically, the resin may be appropriately selected from siloxane polymers; epoxy resins; polyethylene resins; polyester resins; polyurethane resins; polyacrylamide resins; polyvinylpyrrolidone resins; acrylic resins such as polyacrylic acid copolymers; polyvinyl alcohol resins such as cationized polyvinyl alcohol, silanolated polyvinyl alcohol, and sulfonated polyvinyl alcohol; polyvinyl acetal resins; polyvinyl butyral resins; polyethylene polyamide resins; polyamide polyamine resins; cellulose derivatives such as hydroxymethyl cellulose and methyl cellulose; polyalkylene oxide derivatives such as polyethylene oxide and polyethylene oxide-polypropylene oxide copolymers; maleic anhydride copolymers; ethylene-vinyl acetate copolymers; styrene-butadiene copolymers; and combinations of the above resins.
The hydrophilic resin may be a thermoplastic resin, a thermosetting resin, a resin that is cured by active energy rays such as electron beams or UV light, or an elastomer.

The hydrophilic portion (the material of the hydrophilic portion) may have a contact angle of less than 70 degrees measured according to the sessile drop method specified in JIS R3257-1999.

Furthermore, in the case where the above-mentioned substrate is present, it may also have a function of bonding the substrate and the hydrophobic layer together.
For example, a hydrophilic layer may be formed by applying a thermosetting silane coupling agent or the like to the substrate and thermally curing the agent to form a siloxane polymer.

(Hydrophobic resin)
The resin forming the hydrophobic portion (hereinafter sometimes referred to as "hydrophobic resin") is not particularly limited as long as it exerts the effects of the present invention, but may be appropriately selected from, for example, novolak resins; acrylic resins; methacrylic resins; styrene resins; vinyl chloride resins; vinylidene chloride resins; polyolefin resins; polyamide resins; polyimide resins; polyacetal resins; polycarbonate resins; polyphenylene sulfide resins; polysulfone resins; fluororesins; silicone resins; urea resins; melamine resins; guanamine resins; phenolic resins; cellulose resins; and combinations of the above resins, and may be used that have a contact angle of 70 degrees or more measured in accordance with the sessile drop method specified in JIS R3257-1999.
The hydrophobic resin may be a thermoplastic resin, a thermosetting resin, a resin that is cured by active energy rays such as electron beams or UV light, or an elastomer.

The hydrophobic portion may be formed of, for example, a resist. The resist may be a photoresist, which is easy to form a fine structure. The photoresist may be, for example, a photosensitive novolac resin.

[Microfluidic Devices]
In this embodiment, the present invention provides a microfluidic device comprising a channel and the above-mentioned microwell array disposed within the channel.
The use of such microfluidic devices facilitates the containment of aqueous liquids within the wells of a microwell array.

Fig. 1(a) is a perspective view showing one embodiment of a microfluidic device, and Fig. 1(b) is a cross-sectional view taken along line bb in Fig. 1(a).
The microfluidic device 1000 of this embodiment comprises a bottom member 1100, a cover member 1200, a flow channel 1300, a flow channel inlet 1210 provided at one end of the flow channel 1300, a flow channel outlet 1220 provided at the other end of the flow channel 1300, a sealing member 1400 forming the side of the flow channel 1300, and a microwell array m arranged inside the flow channel 1300.
When sending a fluid to the flow channel 1300, for example, a syringe or the like may be used to introduce the fluid from the flow channel inlet 1210 and discharge the fluid from the flow channel outlet 1220.

The structure of the microwell array m will be described later.
The substrate 110 of the microwell array m and the bottom member 1100 of the microfluidic device 1000 may be integral.

In addition, the hydrophilic layer 1120 and the hydrophobic layer 1130 of the microwell array m may be integrally molded on the bottom member 1100 of the microfluidic device 1000.
That is, the microwell array m may be formed on the bottom member 1100 of the microfluidic device 1000.

The materials for bottom member 1100 and cover member 1200 can be the same as those for the substrate of the microwell array described above.
The material of the sealing member 1400 is not particularly limited, but examples thereof include a double-sided adhesive tape in which an acrylic adhesive is laminated on both sides of a core film made of silicone rubber or acrylic foam.

[Method of Encapsulating Aqueous Liquid in Wells of a Microwell Array]
In this embodiment, the present invention provides a method for sealing an aqueous liquid in a well of a microwell array, the method comprising the steps of: delivering an aqueous liquid to a flow path of a microfluidic device comprising a flow path and the above-mentioned microwell array disposed within the flow path, and introducing the aqueous liquid into a well of the microwell array; and delivering a sealing liquid to the flow path, forming a layer of sealing liquid on top of the aqueous liquid introduced into the well, thereby sealing the aqueous liquid in the well.

Here, the aqueous liquid refers to water, biological samples such as buffer solutions containing biomolecules to be detected, enzyme reaction solutions, and the like.
A biological sample is generally an aqueous liquid.
The aqueous liquid may be, for example, a PCR reaction solution using a biological sample as a template and containing SYBR Green as a detection reagent.
Furthermore, the aqueous liquid may contain a surfactant or the like to facilitate sealing the liquid within the wells.

Furthermore, the sealing liquid refers to a liquid used to isolate the liquids introduced into each well of the microwell array so that they do not mix with each other, and for example, oils can be used.
As the oil, for example, Sigma's product name "FC40", 3M's product name "HFE-7500", mineral oil used in PCR reactions, etc. can be used.
In conventional microwell arrays, it can be difficult to use mineral oil as a sealing liquid, but with the microwell array described above, it is possible to seal an aqueous liquid in the wells even when mineral oil is used as the sealing liquid.

The sealing liquid preferably has a contact angle of 5 to 80 degrees with respect to the material of the hydrophobic layer of the microwell array.
When the contact angle of the sealing liquid is in this range, an aqueous liquid can be sealed in the wells of the microwell array.
The contact angle of the sealing liquid may be measured, for example, according to the sessile drop method defined in JIS R3257-1999, using the sealing liquid instead of water.

By appropriately selecting the combination of the material forming the hydrophobic layer of the microwell array and the sealing liquid, liquids other than the specific oils mentioned above can also be used as the sealing liquid.

Next, a method for sealing an aqueous liquid in the wells of a microwell array will be described with reference to FIG.
First, an aqueous liquid such as a PCR reaction solution or an invader reaction solution is delivered from the flow channel inlet 1210 of the microfluidic device 1000 using a syringe or the like.
As a result, the aqueous liquid is introduced into each well of the microwell array m disposed inside the flow channel 1300 .

Next, a sealing liquid is delivered from the flow channel inlet 1210 using a syringe or the like.
As a result, a layer of sealing liquid is formed near the inlet of each well of the microwell array m disposed inside the channel 1300, and the aqueous liquid is sealed in the well.
Since the microwell array m has the above-mentioned configuration, the method of this embodiment makes it possible to easily seal an aqueous liquid in each well of the microwell array m.

After the aqueous liquid is sealed in the wells of the microwell array, an enzymatic reaction such as a PCR reaction or an invader reaction can be carried out using a thermal cycler or the like.

[Method of manufacturing microwell array]
First Embodiment
In this embodiment, a method for manufacturing a microwell array is provided, which includes a step of forming a hydrophobic layer by depositing a hydrophobic material on an electromagnetic wave transparent substrate, and a step of excavating the hydrophobic layer to form a plurality of wells.

A method for producing the microwell array 100 of this embodiment will now be described with reference to FIG.
3A to 3C are cross-sectional views illustrating a method for manufacturing the microwell array 100 of the first embodiment.

In the method of manufacturing microwell array 100 of this embodiment, first, hydrophobic layer 120 is formed on surface 115 of substrate 110, as shown in FIG.
The substrate 110 may be, for example, a glass substrate.
At this time, a solution containing HMDS (hexamethyldisilazane) may be applied to the surface of the glass substrate.
This improves adhesion of the hydrophobic layer 120 to the substrate 110 .

As the hydrophobic resin, a photoresist may be used.
In this case, the photoresist is applied onto the hydrophobic layer 120 by spin coating or the like. After spin coating, the photoresist is heated to perform pre-baking and solidified to form the hydrophobic layer 120.

The hydrophobic layer 120 is then excavated to form the wells, which can be done, for example, by developing a photoresist.
Specifically, a pattern mask is attached onto the hydrophobic layer 120, and ultraviolet light or the like is irradiated from above to expose the pattern mask.

For example, a positive photoresist is used. A positive photoresist is a type that decomposes when exposed to light and dissolves in a developer. Therefore, a pattern mask is used that has holes of the desired pattern in the areas where the microwells are to be formed.
The exposure time and conditions may be appropriately determined by referring to the data sheet of the photoresist to be used.

Subsequently, development is carried out using a developer suitable for the photoresist used to form the microwell array 100. The development time is appropriately determined by referring to the data sheet for the photoresist used.
After development, the development is stopped and the substrate is washed with a rinse solution, such as pure water.
Subsequently, post-baking is performed as necessary to stabilize the photoresist.
By the above operations, microwell array 100 of this embodiment as shown in FIG. 2 can be manufactured.

The above-mentioned well forming step may be a step of forming a plurality of wells by performing dry etching and excavating the hydrophobic layer 120. Dry etching is a method of etching an object by bombarding an object with ions at high speed and utilizing the kinetic energy of the ions.
By the above method, a microwell array having wells exposed on the surface of substrate 110 can be manufactured.

[Method of Analyzing Aqueous Liquid]
In this embodiment, a method for analyzing an aqueous liquid is provided, comprising the steps of irradiating electromagnetic waves to wells of the above-mentioned microwell array in which an aqueous liquid is sealed, and detecting fluorescence or phosphorescence emitted from the wells.

The analysis method of the present embodiment makes it possible to measure how many of the wells constituting a microwell array are emitting fluorescence or phosphorescence.
For example, a PCR reaction is carried out in a microwell array, and the percentage of wells in which amplification is observed can be calculated by detecting the fluorescence of SYBR Green in wells in which PCR amplification is observed.

The analysis method of this embodiment may be performed using, for example, a fluorescence microscope.
Furthermore, irradiation with electromagnetic waves may be performed from the substrate side of the microwell array, from the well side, or from any other arbitrary direction.
Furthermore, the fluorescence or phosphorescence generated as a result of irradiation with electromagnetic waves may be detected from the substrate side of the microwell array, from the well side, or from any other direction. However, when detecting fluorescence or phosphorescence using a fluorescence microscope, for example, it is convenient to detect it from the substrate side of the microwell array.

The present invention will now be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

<Experimental Example 1>
A microwell array of Experimental Example 1 was fabricated according to the manufacturing method of the first embodiment described above.
FIG. 4 is a cross-sectional view showing the structure of the microwell array of Experimental Example 1.

The microwell array of Experimental Example 1 had truncated conical wells with an opening diameter D2 of about 4.92 μm, a bottom diameter D1 of about 3.82 μm, and a height H of about 3 μm. A glass substrate was used as the substrate 110.
As the hydrophobic resin 120, a positive photoresist (model number "PFI89-B4") manufactured by Sumitomo Chemical Co., Ltd. was used.

<Experimental Example 2>
A microwell array of Experimental Example 2 was prepared in the same manner as in Experimental Example 1.
The microwell array of Experimental Example 2 had truncated conical wells with an opening diameter D2 of about 6.96 μm, a bottom diameter D1 of about 6.46 μm, and a height H of about 3 μm, and a glass substrate was used as substrate 110.
As the hydrophobic resin 120, a positive photoresist (model number "PMER P-HA 100PM", manufactured by Tokyo Ohka Kogyo Co., Ltd.) was used.
The adhesive used was HMDS.

<Experimental Example 3>
A microwell array of Experimental Example 3 was prepared in the same manner as Experimental Examples 1 and 2.
The microwell array of Experimental Example 3 had truncated conical wells with an opening diameter D2 of about 5.32 μm, a bottom diameter D1 of about 4.08 μm, and a height H of about 3 μm. A glass substrate was used as the substrate 110.
As the hydrophobic resin 120, a positive photoresist (model number "MICRO POSIT S1818" manufactured by Rohm and Haas Company) was used.

Microfluidic devices including the microwell arrays 100 of Experimental Examples 1, 2, and 3 were fabricated.
Next, an aqueous liquid containing an appropriate amount of FITC, a fluorescent substance, dissolved therein and an appropriate amount of a surfactant was delivered to the microfluidic device using a syringe.
Thereafter, oil (model number "FC40", manufactured by Sigma) was delivered to the microfluidic device using a syringe.

FIG. 5 is a cross-sectional view showing the state after oil has been delivered to the microfluidic device of this experimental example.
The microfluidic device 1000 included a bottom member 1110 that holds a microwell array, a cover member 1200, and a microchannel 1300.
In this example, the bottom member 1110 is integral with the substrate 110 of the microwell array 100 of Example 1.
That is, the microwell array 100 of Experimental Example 1 was formed using a bottom member 1110 as a substrate 110 .

When an FITC solution containing an appropriate amount of surfactant was delivered as the aqueous liquid 440 to the microfluidic device 1000 of Experimental Examples 1, 2, and 3, the aqueous liquid 440 was introduced into the wells of the microwell array 100.

Next, oil 450 was delivered to microfluidic device 1000, and as shown in FIG. 5, a layer of oil 450 was formed near the well entrance of the well while retaining aqueous liquid 440 within the well of microwell array 100.
That is, aqueous liquid 440 was sealed in the wells of microwell array 100 .
Each well functions as a chemical reaction vessel for carrying out various biochemical or chemical reactions in a minute volume of aqueous liquid 440 .

FIG. 6 is a photograph showing the result of observing the microwell array of Experimental Example 1 with a fluorescence microscope from the substrate side after oil was delivered.
As a result, as shown in FIG. 6, FITC fluorescence was observed in a regular pattern in the wells of the microwell array of Experimental Example 1.
In FIG. 6, the white parts are the wells emitting fluorescence, and the black parts are the substrate.
This result indicates that the FITC solution could be enclosed in each well of the microwell array of Experimental Example 1.

In addition, in the microwell array of Experimental Example 1, the novolac resin used as the hydrophobic resin has autofluorescence, but the fluorescence value is sufficiently low and there is a sufficient difference between the fluorescence value of the FITC to be detected, so there is no problem in detecting the fluorescence of FITC.

FIG. 7 is a photograph showing the result of observing the microwell array of Experimental Example 2 with a fluorescence microscope from the substrate side after oil was delivered.
As a result, as shown in FIG. 7, FITC fluorescence was observed in a regular pattern in the wells of the microwell array of Experimental Example 2.
In FIG. 7, the white parts are the wells emitting fluorescence, and the black parts are the substrate.
This result indicates that the FITC solution could be enclosed in each well of the microwell array of Experimental Example 2.

FIG. 8 is a photograph showing the results of observing the microwell array of Experimental Example 3 with a fluorescence microscope from the substrate side after oil was delivered.
As a result, as shown in FIG. 8, FITC fluorescence was observed in a regular pattern in the wells of the microwell array of Experimental Example 3.
In FIG. 8, the white parts are the wells emitting fluorescence, and the black parts are the substrate.
This result indicates that the FITC solution was successfully enclosed in each well of the microwell array of Experimental Example 3.

<Experimental Example 4>
A microwell array of Experimental Example 4 was prepared in the same manner as Experimental Examples 1 to 3.
The microwell array of Experimental Example 4 had truncated conical wells with an opening diameter D2 of about 4.10 μm, a bottom diameter D1 of about 2.32 μm, and a height H of about 3 μm, and a glass substrate was used as substrate 110.
As the hydrophobic resin 120, a negative photoresist (model number "ZPN1150-90" manufactured by Zeon Corporation) was used.

<Experimental Example 5>
The microwell array of Experimental Example 5 was prepared in the same manner as Experimental Examples 1 to 4.
The microwell array of Experimental Example 5 had truncated conical wells with an opening diameter D2 of about 4.34 μm, a bottom diameter D1 of about 2.88 μm, and a height H of about 3 μm. A glass substrate was used as the substrate 110.
As the hydrophobic resin 120, a negative photoresist (model number "ZPN1150-90") was used.

A microfluidic device equipped with the microwell array 100 of Experimental Examples 4 and 5 was assembled in the same manner as Experimental Examples 1 to 3.

Next, an aqueous liquid containing an appropriate amount of FITC, a fluorescent substance, dissolved therein and an appropriate amount of a surfactant was delivered to the microfluidic device using a syringe.
Thereafter, oil (model number "FC40", manufactured by Sigma) was delivered to the microfluidic device using a syringe.

FIG. 9 is a cross-sectional view showing the state after oil has been delivered to the microfluidic device of this experimental example.
FIG. 10 is a photograph showing the results of observing the microwell array of Experimental Example 4 with a fluorescence microscope from the substrate side after oil was delivered.
As a result, as shown in FIG. 10, in the microwell array of Experimental Example 4, fluorescence was observed across multiple wells, and regular FITC fluorescence along the well pattern was not observed.
This result indicates that the FITC solution could not be enclosed in the microwell array of Experimental Example 4.

FIG. 11 is a photograph showing the results of observing the microwell array of Experimental Example 5 with a fluorescence microscope from the substrate side after oil was delivered.
As a result, as shown in FIG. 11, in the microwell array of Experimental Example 5, fluorescence was observed across multiple wells, and regular FITC fluorescence along the well pattern was not observed.
This result indicates that the FITC solution could not be enclosed in the microwell array of Experimental Example 5.

This result indicates that the FITC solution could not be enclosed in the microwell arrays of Experimental Examples 4 and 5.
In other words, it was revealed that when microwells of a certain size or larger are formed whose bottoms are not adhered to the delivered liquid, it becomes difficult to retain aqueous solution, and when oil is delivered, all of the aqueous solution in the wells is replaced by oil.

We also show that a microwell array with wells in which a volume V of solution in the well is in contact with the bottom surface of the well at a certain size or greater is very useful for enclosing an aqueous solution in the well.

The dimensions for each experimental example are shown in Table 1.

As for the shape of the well, as shown in Table 1, the following was found for the volume V ( ^{10-18 m3} ), bottom area S1 ( ^{10-12 m2} ), opening area S2 ( ^{10-12 m2} ), maximum diameter D2 (μm) of the opening, maximum diameter D1 (μm) of the bottom, angle θ of the side formed from the bottom of the well, and height H (μm) shown in Figure 2.

It is preferable that the volume V is 1 fL to 6 nL/well, and the mantissa of the volume V is 1 to 4.5 times, 2 to 4.3 times, or 3 to 4.2 times the mantissa of the bottom area S1 (10 ⁻¹² m ² ).

The planar area S2 (10 ⁻¹² m ² ) is preferably 0.5 to 2.2 times, more preferably 1 to 2 times, even more preferably 1.1 to 1.8 times, the bottom area S1.

It is preferable that the maximum diameter D2 (μm) of the opening is 0.5 to 1.5 times, 0.7 to 1.4 times, or 1 to 1.3 times the maximum diameter D1 (μm) of the bottom surface.

It is preferable that the side angle θ is 30° or more and 103° or less, 60° or more and 103° or less, or 90° or more and 103° or less. Alternatively, the side angle θ may be 77° or more, and the cube root of the mantissa of the well volume V (10 ⁻¹⁸ m ³ ) may be 3.5 or more.

It is preferable that the height H (μm) is 0.2 to 10 times the maximum diameter D1, 0.4 to 5 times, or 0.6 to 2 times.

According to the present invention, it is possible to easily encapsulate an aqueous liquid in a well.
It is also possible to provide a method for producing the above-mentioned microwell array, a microfluidic device, a method for sealing an aqueous liquid in wells of a microwell array, and a method for analyzing an aqueous liquid.
For example, in cases where diagnosis is performed by detecting DNA, RNA, or the like derived from a living organism, it becomes possible to place nucleic acids together with a reagent in a minute space.

As an example of how the present invention can be used, by detecting nucleic acids in a very small space, genetic mutations can be detected with higher accuracy than ever before. This technology makes it possible to detect disease-causing genes early and perform more accurate diagnoses.

100, m... Microwell array 110, 1100... Substrate 115... Surface 1120... Hydrophilic layer 120, 1130... Hydrophobic layer 1000... Microfluidic device 1100... Bottom member 1200... Cover member 1300... Microchannel 1210... Channel inlet 1220 Channel outlet 1400... Sealing member m... Microwell array 440... Aqueous liquid 450... Oil

Claims (10)

A plurality of wells having hydrophobic portions on the well surface,
the plane area S2 (10 ⁻¹² m ² ) of the opening of the well is 1.1 to 1.8 times the bottom area S1 (10 ⁻¹² m ² );
The angle θ formed by the side surface of the well with respect to a plane including the opening of the well is 77° or more and 90° or less;
Microwell array. The maximum diameter D2 (μm) of the opening of the well is 1.5 times or less than the maximum diameter D1 (μm) of the bottom surface of the well;
The microwell array of claim 1 . The height H (μm) from the bottom surface of the well is 5 times or less than the maximum diameter D1 (μm) of the bottom surface of the well;
The microwell array of claim 1 . The mantissa of the well volume V (10 ⁻¹⁸ m ³ ) is 4.5 times or less the mantissa of the well bottom area S1 (10 ⁻¹² m ² );
The microwell array of claim 1 . The volume of the well is 1 fL to 6 nL/well;
The microwell array of claim 1 . The density of the wells is 100,000 to 10 million/ ^cm2 ;
The microwell array of claim 1 . The well is provided on an electromagnetically transparent substrate.
The microwell array of claim 1 . The electromagnetic wave is an X-ray, an ultraviolet ray, a visible light ray, or an infrared ray.
The microwell array of claim 7 . The substrate is substantially free of autofluorescence.
The microwell array of claim 7 or claim 8 . A flow path;
The microwell array of claim 1 disposed within the flow channel;
Equipped with
Microfluidic devices.

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