Patent Application
20200038860
The present invention relates to a microreactor chip and a manufacturing method for the same.
JP 2015-040754 A (Patent Literature 1) discloses a high-density minute chamber array that includes a flat substrate, a plurality of minute chambers formed so as to be regularly arranged in a high density by a hydrophobic substance on a surface of the substrate and having a capacity of 4000×10−18 m3 or less, and a lipid bilayer membrane formed to seal a test aqueous solution in openings of the plurality of minute chambers filled with the test aqueous solution.
Development of applied technology based on the conventional high-density minute chamber array has been desired.
A microreactor chip according to an aspect of the present disclosure includes:
a substrate; and
a hydrophobic layer that is a layer provided on the substrate and made of a hydrophobic substance and is formed so that openings of a plurality of chambers are arranged regularly on a main surface of the layer, wherein
each chamber is provided with a first lipid bilayer membrane and a second lipid bilayer membrane that are disposed with a gap therebetween in a depth direction so as to fractionate the chamber in the depth direction.
In various biomolecular reactions occurring through a lipid bilayer membrane, for example, membrane transport processes, membrane permeation reactions, enzyme reactions on a membrane surface, and the like, since it takes a long time to diffuse a reaction product and a change in the substance concentration with the enzyme activity is very gradual, it is difficult to detect the various biomolecular reactions occurring through the lipid bilayer membrane with high sensitivity. When a capacity of a chamber is large, the concentration change in the chamber becomes small, and detection as the concentration change becomes difficult. When the number of chambers is small, the measurement throughput is lowered. Therefore, there is a need for a high-density minute chamber array in which a large number of minute chambers with the extremely small capacity sealed with the lipid bilayer membrane are formed in a high density. Patent Literature 1 described above discloses the high-density minute chamber array. However, there is an unexamined part about the applied technology.
The inventors have performed an intensive examination to find out the applied technology of the conventional high-density minute chamber array. As a result, the following knowledge is obtained. The following knowledge is only a trigger for the present invention, and does not limit the present invention.
That is, the high-density minute chamber array is developed, so that it is possible to efficiently perform measurement such as transmembrane-type substance transport using membrane proteins. Incidentally, if each chamber can be further segmented in the high-density minute chamber array, the detection sensitivity of the activity can be improved, and the properties of the membrane proteins may be clarified in more detail.
The inventors have established technology for forming two layers of lipid bilayer membranes in each chamber by developing a new protocol for forming the lipid bilayer membranes, in the conventional high-density minute chamber array, on the basis of the above insight. That is, the inventors have succeeded in segmenting each chamber by the lipid bilayer membranes. Further, in the above technology, it is possible to quantitatively control an interval between the two layers of lipid bilayer membranes to be formed, and a volume of each fraction that has been segmented can be controlled (greatly reduced).
Further, use of the above technology not only significantly improves the conventional membrane protein activity detection sensitivity with the reduction of the reactor capacity by fractionation, but also artificially constructs bilayer membrane organelles or bacterial cell membranes in vitro, and a path to function analysis of the membrane proteins present in the bilayer membrane organelles or the bacterial cell membranes in which measurement is difficult in the past is pioneered. That is, the development of the technology is an innovation in the function analysis of the membrane proteins.
Embodiments described below have been created on the basis of such knowledge.
A microreactor chip according to a first aspect of an embodiment includes:
a substrate; and
a hydrophobic layer that is a layer provided on the substrate and made of a hydrophobic substance and is formed so that openings of a plurality of chambers are arranged regularly on a main surface of the layer.
Each chamber is provided with a first lipid bilayer membrane and a second lipid bilayer membrane that are disposed with a gap therebetween in a depth direction so as to fractionate the chamber in the depth direction.
According to the above aspect, since each chamber is segmented by the two layers of lipid bilayer membranes, a volume of the reactor is greatly reduced. As a result, a concentration change of a reaction product or a reaction substrate in the reactor due to the reaction of one biomolecule can be increased, detection sensitivity at the time of detection as the concentration change can be increased, and even if the reaction of the biomolecule is extremely slow, the reaction of the biomolecule can be detected with high sensitivity. Further, the bilayer membrane organelles or the bacterial cell membranes can be artificially constructed in vitro, and function analysis of the membrane proteins present in the bilayer membrane organelles or the bacterial cell membranes in which measurement is difficult in the past can be performed
Further, according to the above aspect, each chamber is fractionated in the depth direction by the two layers of lipid bilayer membranes. For this reason, when light emitted from a fluorescent substance included in a liquid in the reactor is detected using a confocal laser microscope placed under the substrate, an fluorescent image is suppressed from being distorted by the lens action in the fractionated reactor, and quantitative observation can be performed.
A microreactor chip according to a second aspect of the embodiment is the microreactor chip according to the first aspect, wherein
a capacity of each chamber is 4000×10−18 m3 or less.
A microreactor chip according to a third aspect of the embodiment is the microreactor chip according to the first or second aspect, wherein
an interval between the first lipid bilayer membrane and the second lipid bilayer membrane is 10 μm or less.
According to the above aspect, it is possible to reproduce a membrane interval of the bilayer membrane organelles or the bacterial cell membranes in vitro.
A microreactor chip according to a fourth aspect of the embodiment is the microreactor chip according to any one of the first to third aspects, wherein
at least one of the first lipid bilayer membrane and the second lipid bilayer membrane holds a membrane protein.
A microreactor chip according to a fifth aspect of the embodiment is the microreactor chip according to any one of the first to fourth aspects, wherein
each chamber is provided with a third lipid bilayer membrane that is disposed with a gap in the depth direction with respect to the first lipid bilayer membrane and the second lipid bilayer membrane so as to further fractionate the chamber in the depth direction.
A method for manufacturing a microreactor chip according to a sixth aspect of the embodiment includes:
a step of preparing the microreactor chip before lipid bilayer membrane formation, the microreactor chip including a substrate and a hydrophobic layer that is a layer provided on the substrate and made of a hydrophobic substance and is formed so that openings of a plurality of chambers are arranged regularly on a main surface of the layer; a step of forming a first lipid bilayer membrane in the opening of the chamber;
a step of introducing a liquid having a higher concentration than a liquid filled into the chamber into a liquid flow passage with the main surface of the hydrophobic layer as a bottom surface and pushing down the first lipid bilayer membrane to the inner side of the chamber by an osmotic pressure; and
a step of forming a second lipid bilayer membrane in the opening of the chamber.
According to the above aspect, each chamber can be segmented by the two layers of lipid bilayer membranes. As a result, the volume of the reactor can be greatly reduced. As a result, a concentration change of a reaction product or a reaction substrate in the reactor due to the reaction of one biomolecule can be increased, detection sensitivity at the time of detection as the concentration change can be increased, and even if the reaction of the biomolecule is extremely slow, the reaction of the biomolecule can be detected with high sensitivity. Further, according to the above aspect, the bilayer membrane organelles or the bacterial cell membranes can be artificially constructed in vitro, and the function analysis of the membrane proteins present in the bilayer membrane organelles or the bacterial cell membranes in which measurement is difficult in the past can be performed.
A method for manufacturing a microreactor chip according to a seventh aspect of the embodiment is the method for manufacturing a microreactor chip according to the sixth aspect, wherein
in the step of forming the first lipid bilayer membrane, in a state where the chamber is filled with a first liquid, an organic solvent containing lipid is flown to the liquid flow passage to form an inner lipid monolayer membrane with a lipid hydrophilic group facing the first liquid side of the chamber in the opening of the chamber, and a membrane formation aqueous solution is flown to the liquid flow passage to form an outer lipid monolayer membrane with a lipid hydrophobic group facing the side of the inner lipid monolayer membrane so as to overlap the inner lipid monolayer membrane.
According to the above aspect, the first lipid bilayer membrane can be efficiently formed in the opening of the chamber.
A method for manufacturing a microreactor chip according to an eighth aspect of the embodiment is the method for manufacturing a microreactor chip according to the sixth or seventh aspect, wherein
in the step of forming the second lipid bilayer membrane, in a state where the opening side of the first lipid bilayer membrane of the chamber is filled with a second liquid, an organic solvent containing lipid is flown to the liquid flow passage to form an inner lipid monolayer membrane with a lipid hydrophilic group facing the second liquid side of the chamber in the opening of the chamber, and a membrane formation aqueous solution is flown to the liquid flow passage to form an outer lipid monolayer membrane with a lipid hydrophobic group facing the side of the inner lipid monolayer membrane so as to overlap the inner lipid monolayer membrane.
According to the above aspect, the second lipid bilayer membrane can be efficiently formed in the opening of the chamber.
A method for manufacturing a microreactor chip according to a ninth aspect of the embodiment is the method for manufacturing a microreactor chip according to any one of the sixth to eighth aspects, and further includes:
a step of introducing a liquid having a higher concentration than a liquid filled between the first lipid bilayer membrane and the second lipid bilayer membrane into the liquid flow passage and pushing down the second lipid bilayer membrane to the inner side of the chamber by an osmotic pressure; and
a step of forming a third lipid bilayer membrane in the opening of the chamber.
A method according to a tenth aspect of the embodiment is
a method for recovering a reaction product from a reactor defined between a first lipid bilayer membrane and a second lipid bilayer membrane of a microreactor chip, the microreactor chip including a substrate and a hydrophobic layer that is a layer provided on the substrate and made of a hydrophobic substance and is formed so that openings of a plurality of chambers are arranged regularly on a main surface of the layer, each chamber being provided with the first lipid bilayer membrane and the second lipid bilayer membrane that are disposed with a gap therebetween in a depth direction so as to fractionate the chamber in the depth direction, wherein
a recovery aqueous solution having a lower concentration than a test aqueous solution filled into the reactor is introduced into a liquid flow passage with the main surface of the hydrophobic layer as a bottom surface, the second lipid bilayer membrane is pushed up to the outer side of the chamber by an osmotic pressure and destroyed, the reaction product in the test aqueous solution is transferred to the recovery aqueous solution, and the reaction product is recovered from the liquid flow passage together with the recovery aqueous solution.
According to the above aspect, the reaction product in the reactor defined between the first lipid bilayer membrane and the second lipid bilayer membrane can be easily recovered in a batch.
A method according to an eleventh aspect of the embodiment is
a method for controlling a volume of a reactor defined between a first lipid bilayer membrane and a second lipid bilayer membrane of a microreactor chip, the microreactor chip including a substrate and a hydrophobic layer that is a layer provided on the substrate and made of a hydrophobic substance and is formed so that openings of a plurality of chambers are arranged regularly on a main surface of the layer, each chamber being provided with the first lipid bilayer membrane and the second lipid bilayer membrane that are disposed with a gap therebetween in a depth direction so as to fractionate the chamber in the depth direction, wherein
a volume control aqueous solution having a higher concentration than a test aqueous solution filled into the reactor is introduced into a liquid flow passage with the main surface of the hydrophobic layer as a bottom surface, and the second lipid bilayer membrane is pushed down to the inner side of the chamber by an osmotic pressure.
According to the above aspect, the osmotic pressure is controlled, so that it is possible to quantitatively control the interval between the two layers of lipid bilayer membranes, and the volume of each reactor that has been segmented can be controlled (greatly reduced).
Hereinafter, specific examples of embodiments will be described in detail with reference to the accompanying drawings. In the individual drawings, components having the same functions are denoted by the same reference numerals, and detailed description of the components having the same reference numerals is not repeated.
As illustrated in
The substrate 22 has a light transmitting property and is flat. The substrate 22 can be made of, for example, glass, acrylic resin, or the like. A material, a thickness, a shape, and the like of the substrate 22 are not particularly limited as long as light incident on the substrate 22 from below the substrate 22 can transmit the substrate 22 and enter a chamber 26, and light incident on the substrate 22 from the inside of the chamber 26 can transmit the substrate 22 and escape below the substrate 22. Specifically, for example, the thickness of the substrate 22 may be 0.1 mm to 5 mm, 0.3 mm to 3 mm, or 0.7 mm to 1.5 mm. A size of the substrate 22 in plan view is not particularly limited.
The hydrophobic layer 24 is a layer made of a hydrophobic substance. Examples of the hydrophobic substance include a hydrophobic resin such as a fluororesin and a substance other than a resin such as glass. A thickness of the hydrophobic layer 24 can be appropriately adjusted according to a capacity of the chamber 26 to be described later. Specifically, for example, the thickness may be 10 nm to 100 μm, 100 nm to 5 μm, or 250 nm to 1 μm.
In the hydrophobic layer 24, openings of a plurality of minute chambers 26 are provided on a main surface of the hydrophobic layer 24 so as to be regularly arranged in a high density. The capacity of the chamber 26 is 4000×10−18 m3 or less (4000 μm3 or less). The capacity of the chamber 26 may be, for example, 0.1×10−18 m3 to 4000×10−18 m3, 0.5×10−18 m3 to 400×10−18 m3, or 1×10−18 m3 to 40×10−18 m3.
The depth of the chamber 26 may be, for example, 10 nm to 100 μm, 100 nm to 5 μm, or 250 nm to 1 μm.
The opening of the chamber 26 can be circular, for example. A diameter of a circle in the case of the circle may be, for example, 0.1 μm to 100 μm, 0.5 μm to 5 μm, or 1 μm to 10 μm.
The “regular” means that the chambers are arranged on the substrate in a lattice shape, a matrix shape, a staggered shape, or the like as viewed from the thickness direction of the substrate, for example. The “regular” can mean that the chambers are arranged at a constant interval in a plurality of rows, for example.
The “high density” means that the number of chambers per square mm (1 mm2) may be 0.1×103 to 2000×103, 1×103 to 1000×103, or 5×103 to 100×103. When the number of chambers is converted into the number of chambers per 1 cm2 (1×104 m2) , the number of chambers may be 10×103 to 200×106, 100×103 to 100×106 or 0.5×106 to 10×106.
In the microreactor chip 20, the plurality of chambers 26 can be formed so that a depth is 100 μm or less and a diameter at the time of conversion into a circle is 100 μm or less, can be formed so that the depth is 2 μm or less and the diameter at the time of conversion into the circle is 10 μm or less, or can be formed so that the depth is 1 μm or less and the diameter at the time of conversion into the circle is 5 μm or less. In this way, it is possible to relatively easily manufacture the microreactor chip 20 before lipid bilayer membrane formation by using a method for forming a thin membrane made of the hydrophobic substance on the surface of the substrate 22 and forming the plurality of minute chambers 26 on the thin membrane. The “diameter” “at the time of conversion into the circle” means a diameter of a circle having the same area as a shape of a cross-section perpendicular to a depth direction. For example, when the cross-section is a square of 1 μm square, the diameter at the time of conversion into the circle is 2/√π≈1.1 μm.
The chamber 26 can be formed as a thin membrane made of a hydrophobic substance having a predetermined thickness range including a thickness of 500 nm so as to have a predetermined diameter range including a diameter of 1 μm at the time of conversion into the circle. If the magnitude of a reaction rate of a biomolecule to be tested or the content of the biomolecule is considered and ease of production is considered, it is considered that the depth or diameter of the chamber 26 is preferably several hundred nm to several μm. Here, the “predetermined thickness range” can be, for example, a range of 50 nm, that is 0.1 times 500 nm, to 5 μm, that is 10 times 500 nm, or a range of 250 nm, that is 0.5 times 500 nm, to 1 μm, that is 2 times 500 nm. The “predetermined diameter range” can be, for example, a range of 100 nm, that is 0.1 times 1 μm, to 10 μm, that is 10 times 1 μm, or a range of 500 nm, that is 0.5 times 1 μm, to 2 μm, that is twice 1 μm.
In one example, each chamber 26 is formed to have a diameter R of 5 μm in the hydrophobic layer 24 having a thickness D of 1 μm. Therefore, a capacity L of each chamber 26 is L=π(2.5×10−6)2×1×10−6 m3≈19.6×10−18 m3. If the chambers 26 are arranged at intervals of 2 μm vertically and horizontally in plan view, an area S required for one chamber 26 is a square having a side of 7 μm, and the area S is calculated as S=(7×10−6)2 m2=49×10−12 m2. Therefore, about 2×106 (20×103 per square mm) chambers 26 per 1 cm2 (1×10−4 m2) are formed on the glass substrate 22.
As shown in
An interval between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 is 10 μm or less. The interval between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 may be, for example, 0.1 nm to 10 μm, 0.5 nm to 5 μm, or 1 nm to 1 μm.
In the microreactor chip 20, since the interval between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 is 10 μm or less, it is possible to reproduce a membrane interval of bilayer membrane organelles or bacterial cell membranes in vitro.
An internal space of each chamber 26 fractionated by the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 is filled with a test aqueous solution. The test aqueous solution is not particularly limited as long as it is a liquid capable of forming the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32.
In the first lipid bilayer membrane 31, an inner lipid monolayer membrane 31a with a lipid hydrophilic group facing the inner side of the chamber 26 (lower side in
As the lipid configuring the inner lipid monolayer membrane 31a and 32a or the outer lipid monolayer membranes 31b and 32b, natural lipid such as being derived from soybeans and Escherichia coli and artificial lipid such as dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG) can be used.
One or both of the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 can hold a membrane protein. In this way, the microreactor chip 20 can be used for detection of biomolecular reactions or the like through various membrane proteins. A method for holding (reconfiguring) the membrane protein in the lipid bilayer membrane 30 will be described later.
Since the chamber 26 is fractionated in the depth direction by the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32, the microreactor chip 20 is used for detection of the biomolecular reaction, so that the volume of the fraction defined between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 can be reduced. As a result, a concentration change of a reaction product or a reaction substrate in the microreactor due to the reaction of one biomolecule can be increased, detection sensitivity at the time of detection as the concentration change can be increased, and even if the reaction of the biomolecule is extremely slow, the reaction of the biomolecule can be detected with high sensitivity. Further, according to the above aspect, the bilayer membrane organelles or the bacterial cell membranes can be artificially constructed in vitro, and the function analysis of the membrane proteins present in the bilayer membrane organelles or the bacterial cell membranes in which measurement is difficult in the past can be performed. In particular, if the bacterial cell membrane can be reproduced in vitro, it is expected that it is possible to perform function analysis of a drug efflux membrane protein derived from multi-drug resistant bacteria, which is difficult in the past. That is, the corresponding technology is a pharmacologically very important technology.
Although illustration is omitted, an electrode may be provided in each chamber 26 (for example, an inner surface or a bottom surface of the chamber 26). The electrodes may be electrically connected to each other. The electrode may be made of a metal, for example, copper, silver, gold, aluminum, chromium, or the like. The electrode may be made of a material other than the metal, for example, indium tin oxide (ITO), a material containing indium tin oxide and zinc oxide (IZO), ZnO, a material containing indium, gallium, zinc, and oxygen (IGZO), or the like.
The thickness of the electrode may be, for example, 10 nm to 100 μm, 100 nm to 5 μm, or 250 nm to 1 μm.
In such a configuration, light incident on the substrate 22 from below the substrate 22 transmits the substrate 22 and enters the chamber 26, and light incident on the substrate 22 from the inside of the chamber 26 transmits the substrate 22 and escapes below the substrate 22.
[Method for Manufacturing Microreactor Chip]
Hereinafter, a method for manufacturing the microreactor chip 20 according to the first embodiment will be described.
As shown in
1. Preparation of Microreactor Chip Before Lipid Bilayer Membrane Formation
First, as shown in
Next, as shown in
Next, as shown in
Next, as shown in
Next, as shown in
The plurality of chambers 26 may be formed in the thin membrane made of the hydrophobic substance using a method other than the dry etching, for example, a method such as nanoimprinting. In the case of the dry etching, the inner surface of the chamber 26 becomes hydrophilic due to the action of O2 plasma, and it becomes easier to fill the chamber 26 with the test aqueous solution at the time of forming the lipid bilayer membrane to be described later. Therefore, the dry etching is preferable.
2. Formation of First Lipid Bilayer Membrane
First, as shown in
Next, as shown in
If the organic solvent containing the lipid 35 is introduced from the liquid introduction hole 46 into the liquid flow passage 48, in a state where the chamber 26 is filled with the first test aqueous solution, the inner lipid monolayer membrane 31a with the hydrophilic group of the lipid 35 facing the side of the first test aqueous solution of the chamber 26 is formed so as to seal the opening of the chamber 26.
Next, the membrane formation aqueous solution to form the first lipid bilayer membrane 31 is introduced from the liquid introduction hole 46 into the liquid flow passage 48 (step S123). As the membrane formation aqueous solution, specifically, for example, the buffer solution A diluted to 60% can be used.
If the membrane formation aqueous solution is introduced from the liquid introduction hole 46 into the liquid flow passage 48, the outer lipid monolayer membrane 31b with the hydrophobic group of the lipid 35 facing the side of the inner lipid monolayer membrane 31a is formed so as to overlap the inner lipid monolayer membrane 31a. Thereby, the first lipid bilayer membrane 31 is formed in the opening of the chamber 26.
After the step of forming the first lipid bilayer membrane 31, a step of reconfiguring the membrane protein in the first lipid bilayer membrane 31 may be provided. The reconfiguration step may be a step of introducing any one of cell membrane fragments including the membrane protein, a lipid bilayer membrane with embedded protein, water-soluble protein, liposome incorporating protein, and protein solubilized with surfactants into the first lipid bilayer membrane 31 and incorporating protein into the first lipid bilayer membrane 31 to form a membrane protein. As a method for incorporating the protein into the lipid bilayer membrane, a membrane fusion or the like can be used in the case of the liposome, and a thermal fluctuation or the like can be used in the case of the protein solubilized with the surfactant.
3. Pushing Down of First Lipid Bilayer Membrane
First, as shown in
During the incubation, as illustrated in
An amount by which the first lipid bilayer membrane 31 is pushed down can be quantitatively controlled. Specifically, for example, in order to push down the first lipid bilayer membrane 31 to half the depth of the chamber 21 in a state where the chamber 26 is filled with a liquid including an electrolyte of 100 mM, a liquid including an electrolyte of 200 mM is introduced into the liquid flow passage 48. In this case, the first lipid bilayer membrane 31 is pushed down to half the depth of the chamber 21 by the osmotic pressure so that the volume of the space of the inner side of the first lipid bilayer membrane 31 of the chamber 26 is reduced to ½ and the concentration of the electrolyte in the liquid of the inner side of the first lipid bilayer membrane 31 becomes 200 mM.
4. Formation of Second Lipid Bilayer Membrane
First, as illustrated in
When the concentration of the second test aqueous solution is higher than the concentration of the liquid of the inner side of the first lipid bilayer membrane 31, the second test aqueous solution is introduced from the liquid introduction hole 46 into the liquid flow passage 48 and then incubated for 5 minutes, for example, so that the first lipid bilayer membrane 31 can be further pushed down to the inner side of the chamber 26 by the osmotic pressure.
Next, as shown in
If the organic solvent containing the lipid 35 is introduced from the liquid introduction hole 46 into the liquid flow passage 48, in a state where the opening side of the first lipid bilayer membrane 31 of the chamber 26 is filled with the second test aqueous solution, the inner lipid monolayer membrane 32a with the hydrophilic group of the lipid 35 facing the side of the second test aqueous solution of the chamber 26 is formed so as to seal the opening of the chamber 26.
Next, the membrane formation aqueous solution to form the second lipid bilayer membrane 32 is introduced from the liquid introduction hole 46 into the liquid flow passage 48 (step S143). As the membrane formation aqueous solution, specifically, for example, the buffer solution A diluted to 60% can be used.
If the membrane formation aqueous solution is introduced from the liquid introduction hole 46 into the liquid flow passage 48, the outer lipid monolayer membrane 32b with the hydrophobic group of the lipid 35 facing the side of the inner lipid monolayer membrane 32a is formed so as to overlap the inner lipid monolayer membrane 32a. Thereby, the second lipid bilayer membrane 32 is formed in the opening of the chamber 26.
After the step of forming the second lipid bilayer membrane 32, a step of reconfiguring the membrane protein in the second lipid bilayer membrane 32 may be provided. The reconfiguration step may be a step of introducing any one of cell membrane fragments including the membrane protein, a lipid bilayer membrane with embedded protein, water-soluble protein, liposome incorporating protein, and protein solubilized with surfactants into the second lipid bilayer membrane 32 and incorporating protein into the second lipid bilayer membrane 32 to form a membrane protein. As a method for incorporating the protein into the lipid bilayer membrane, a membrane fusion or the like can be used in the case of the liposome, and a thermal fluctuation or the like can be used in the case of the protein solubilized with the surfactant.
By the method described above, it is possible to manufacture the microreactor chip 20 in which each chamber 26 has been segmented by the two layers of lipid bilayer membranes 31 and 32, as illustrated in
Here, light incident on the substrate 22 from below the substrate 22 transmits the substrate 22 and enters the chamber 26, and light incident on the substrate 22 from the inside of the chamber 26 transmits the substrate 22 and escapes below the substrate 22. When the membrane protein is reconfigured in the first lipid bilayer membrane 31 or the second lipid bilayer membrane 32, a function of the membrane protein can be analyzed by detecting light emitted from a fluorescent substance included in the test liquid accommodated in the chamber 26 using a confocal laser microscope. A vertical illumination type confocal microscope may be used as the microscope.
In the present embodiment, each chamber 26 is fractionated in the depth direction by the two layers of lipid bilayer membranes 31 and 32. For this reason, when the light emitted from the fluorescent substance included in the test liquid in the chamber 26 is detected using the confocal laser microscope placed under the substrate 22, a fluorescent image is suppressed from being distorted by the lens action in the fractionated reactor, and quantitative observation can be performed.
[Method for Controlling Volume of Reactor Defined Between First Lipid Bilayer Membrane and Second Lipid Bilayer Membrane]
Next, a method for controlling the volume of the reactor defined between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 in the microreactor chip 20 according to the first embodiment will be described with reference to
First, as shown in
During the incubation, as illustrated in
An amount by which the second lipid bilayer membrane 32 is pushed down can be quantitatively controlled. Specifically, for example, in order to push down the second lipid bilayer membrane 32 until the volume of the reactor decreases to ½, in a state where the reactor between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 is filled with the liquid including the electrolyte of 100 mM, the liquid including the electrolyte of 200 mM is introduced into the liquid flow passage 48. In this case, the second lipid bilayer membrane 32 is pushed down by the osmotic pressure until the volume of the reactor decreases to ½ so that the concentration of the electrolyte of the liquid in the reactor becomes 200 mM.
According to the above method, the osmotic pressure is controlled, so that it is possible to quantitatively control the interval between the two layers of lipid bilayer membranes 31 and 32, and the volume of each reactor that has been segmented can be controlled (greatly reduced).
[Method for Recovering Reaction Product from Reactor Defined Between First Lipid Bilayer Membrane and Second Lipid Bilayer Membrane]
Next, a method for recovering a reaction product from the reactor defined between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 in the microreactor chip 20 according to the first embodiment will be described with reference to
First, as shown in
During the incubation, as illustrated in
According to the above method, the reaction product in the reactor can be easily recovered in a batch.
In the microreactor chip 20 according to the first embodiment, the method for recovering the reaction product from the reactor defined between the first lipid bilayer membrane 31 and the second lipid bilayer membrane 32 is not limited to the above method. For example, the second lipid bilayer membrane 32 may be pierced with a needle and the reaction product may be recovered from the reactor.
In the first embodiment described above, an example in which a chamber 26 is fractionated in a depth direction by two layers of lipid bilayer membranes 31 and 32 has been described. On the other hand, in the second embodiment, as illustrated in
An internal space of each chamber 26 fractionated by the three layers of lipid bilayer membranes 31 to 33 is filled with a test aqueous solution. The test aqueous solution is not particularly limited as long as it is a liquid capable of forming the lipid bilayer membranes 31 to 33. Since the chamber 26 is fractionated by the three layers of lipid bilayer membranes 31 to 33, a relation between three types of liquids can be observed.
[Method for Manufacturing Microreactor Chip]
Next, a method for manufacturing a microreactor chip 20 according to the second embodiment will be described. FIG. 14 is a flowchart illustrating an example of a method for manufacturing the microreactor chip 20 according to the second embodiment.
As shown in
5. Pushing Down of Second Lipid Bilayer Membrane
First, as shown in
During the incubation, as illustrated in
6. Formation of Third Lipid Bilayer Membrane
First, as illustrated in
When the concentration of the third test aqueous solution is higher than the concentration of the liquid of the inner side of the second lipid bilayer membrane 32, the third test aqueous solution is introduced from the liquid introduction hole 46 into the liquid flow passage 48 and then incubated for 5 minutes, for example, so that the second lipid bilayer membrane 32 can be further pushed down to the inner side of the chamber 26 by the osmotic pressure.
Next, as shown in
If the organic solvent containing the lipid 35 is introduced from the liquid introduction hole 46 into the liquid flow passage 48, in a state where the opening side of the second lipid bilayer membrane 32 of the chamber 26 is filled with the third test aqueous solution, an inner lipid monolayer membrane 33a with the hydrophilic group of the lipid 35 facing the side of the third test aqueous solution of the chamber 26 is formed so as to seal the opening of the chamber 26.
Next, a membrane formation aqueous solution to form the third lipid bilayer membrane 33 is introduced from the liquid introduction hole 46 into the liquid flow passage 48 (step S163).
If the membrane formation aqueous solution is introduced from the liquid introduction hole 46 into the liquid flow passage 48, an outer lipid monolayer membrane 33b with the hydrophobic group of the lipid 35 facing the side of the inner lipid monolayer membrane 33a is formed so as to overlap the inner lipid monolayer membrane 33a. Thereby, the third lipid bilayer membrane 33 is formed in the opening of the chamber 26.
After the step of forming the third lipid bilayer membrane 33, a step of reconfiguring the membrane protein in the third lipid bilayer membrane 33 may be provided. The reconfiguration step may be a step of introducing any one of cell membrane fragments including the membrane protein, a lipid bilayer membrane with embedded protein, water-soluble protein, liposome incorporating protein, and protein solubilized with surfactants into the third lipid bilayer membrane 33 and incorporating protein into the third lipid bilayer membrane 33 to form a membrane protein. As a method for incorporating the protein into the lipid bilayer membrane, a membrane fusion or the like can be used in the case of the liposome, and a thermal fluctuation or the like can be used in the case of the protein solubilized with the surfactant.
By the above method, it is possible to manufacture the microreactor chip 20 in which each chamber 26 has been segmented by the three layers of lipid bilayer membranes 31 to 33, as illustrated in
Similarly, a step of forming a new lipid bilayer membrane in the opening of the chamber 26 after pushing down a lipid bilayer membrane of an uppermost layer to the inner side of the chamber 26 by the osmotic pressure is repeated, so that four or more layers of lipid bilayer membranes can be provided in each chamber 26.
The description of the embodiments and the modifications described above and the disclosure of the drawings are merely an example for explaining the invention described in claims, and the invention described in the claims is not limited by the description of the embodiments and the modifications or the disclosure of the drawings. The components of the embodiments and the modifications described above can be arbitrarily combined without departing from the gist of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-040664 | Mar 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/007927 | 3/2/2018 | WO | 00 |
