JP2019178927A - Lipid bilayer membrane forming instrument - Google Patents

Abstract

To provide a highly portable lipid bilayer membrane forming instrument which is capable of holding a formed lipid bilayer membrane steady against vibrations and the like imparted thereto while being carried around or exchanging samples, and allows for exchanging samples without destroying the lipid bilayer membrane through simple manipulation.SOLUTION: A lipid bilayer membrane forming instrument is provided, comprising a well having a bottom and a sidewall, a through-hole formed through the bottom and designed to have a lipid bilayer membrane formed thereon, and a chamber facing a lower surface of the bottom and configured to be in communication with the chamber. The sidewall is provided with one or more vertical slits.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a device for forming a lipid bilayer membrane excellent in portability and sample exchangeability.

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

  It is also known that a protein is held in a lipid bilayer membrane and used as a sensor. For example, a receptor protein is held in a lipid bilayer membrane to form a sensor, or a specific binding substance that specifically binds to a test substance is added to the droplet when forming a lipid bilayer membrane by the droplet contact method. On the other hand, there is also a sensor that holds an ion channel protein in a lipid bilayer membrane, and in the presence of a test substance, the test substance binds to a specific binding substance to block the ion channel. (Patent Document 1). Patent Document 1 also describes that one of the droplets to be contacted in the droplet contact method is a hydrogel in order to facilitate detection of a test substance existing in the air.

JP-A-2017-83210

  The sensor having a lipid bilayer membrane described in Patent Document 1 and the like is also used for detection of chemical substances contained in the atmosphere or river water, for example. For this reason, there are not a few cases where the sensor is used outdoors, and in order to perform measurement at a site such as outdoors, it is required that the sensor be portable and that the sample can be easily exchanged at the measurement site. For the portability of the sensor, it is required that the formed lipid bilayer membrane is stably held against the movement accompanying the carrying and the vibration during the sample exchange.

  As a result of intensive studies, the inventors of the present invention have placed a through-hole in which a lipid bilayer membrane is formed at the bottom of the well and provided a slit in the side wall of the well, thereby stabilizing the formed lipid bilayer membrane. The present invention was completed by finding that the sample can be exchanged by simple operation without destroying the lipid bilayer membrane.

  That is, the present invention provides the following.

(1) a well having a bottom surface and a side wall; a through-hole provided on the bottom surface in which a lipid bilayer membrane is formed; and a chamber provided on a lower surface of the bottom surface and communicating with the through-hole. Is a lipid bilayer membrane forming device provided with one or more slits in the vertical direction.
(2) The device for forming a lipid bilayer membrane according to (1), further comprising a first electrode disposed in the well and a second electrode disposed in the chamber.
(3) The lipid bilayer membrane forming device according to (1) or (2), wherein the width of each of the one or more slits is 0.05 to 0.25 times the inner circumference of the well.
(4) The upper end portion of each of the one or more slits is the upper end portion of the well, and the lower end portion thereof is located above the bottom surface, according to any one of (1) to (3) A device for forming a lipid bilayer membrane.
(5) The device for forming a lipid bilayer membrane according to any one of (1) to (4), wherein the number of the slits is 1 to 4.
(6) The lipid bilayer membrane formation instrument according to any one of claims 1 to 5, wherein the chamber is in the form of a capillary.
(7) The device for forming a lipid bilayer according to any one of (1) to (6), wherein the inner surface of the well is coated with water / oil repellent coating.
(8) The lipid bilayer according to any one of (1) to (7), wherein the well has a dimension that allows water to be retained in the well by surface tension even if water is placed in the well and inverted. A device for film formation.
(9) The device for forming a lipid bilayer membrane according to (8), wherein the well has a substantially circular cross section, an inner diameter of 1 mm to 5 mm, and a depth of 1 mm to 5 mm.

  According to the device for forming a lipid bilayer membrane of the present invention, the formed lipid bilayer membrane is stably held against movement, etc. that accompanies carrying, sample vibration, and the like. It can be performed simply by immersing in a sample for a period of time. Therefore, the lipid bilayer membrane forming instrument of the present invention in which the lipid bilayer membrane of the present invention is formed can be applied to a sensor suitable for on-site measurement.

The typical disassembled perspective view of one preferable specific example of the lipid bilayer membrane formation instrument of this invention is shown. It is a typical perspective view which shows the state after assembling the lipid bilayer membrane formation instrument shown in FIG. It is a figure which shows typically the lipid bilayer membrane formation instrument produced in the following Example with the partial enlarged view. It is a figure which shows typically the state which formed the lipid bilayer membrane with the lipid bilayer membrane formation apparatus produced in the following Example, and (alpha) -hemolysin is not yet reconfigure | reconstructed in a membrane with the measurement result of an electric current value. . It is a figure which shows typically the state by which the lipid bilayer membrane was formed with the lipid bilayer membrane formation apparatus produced in the following Example, and (alpha) -hemolysin was reconfigure | reconstructed in a membrane with the measurement result of an electric current value. A lipid bilayer membrane was formed with the lipid bilayer membrane-forming device prepared in the following example, α-hemolysin was reconstituted in the membrane, and the state after addition of single-stranded DNA in the well was determined as the current value. It is a figure shown typically with the measurement result of. It is a figure which shows the measurement result of the electric current value with time measured by the sample exchange test performed in the following example. It is a figure which compares the frequency of the sharp electric current fall resulting from the single strand DNA observed in the sample exchange test performed in the following Example before and after sample exchange.

  A preferred embodiment of the present invention will be described below with reference to the drawings.

  FIG. 1 shows a schematic exploded perspective view of a preferred specific example of the device for forming a lipid bilayer membrane of the present invention, and FIG. 2 shows a perspective view after assembly.

A lipid bilayer membrane forming device 10 according to a preferred embodiment of the present invention includes a cylindrical well 12. The material of the well 12 is not particularly limited, but a plastic such as an acrylic resin is preferable because it can be easily manufactured. The well 12 does not necessarily have a cylindrical shape (that is, the cross-sectional shape is circular), and the cross-sectional shape may be another shape such as an ellipse or a polygon. In view of easiness, a cylindrical shape is preferable as in this specific example. The cylindrical well 12 includes a bottom surface 12a and a side wall 12b. A through-hole 14 in which a lipid bilayer is formed is provided at the center of the bottom surface 12a. The position where the through hole 14 is provided is not necessarily limited to the center of the bottom surface 12a. The diameter of the through hole 14 is not particularly limited, but is preferably about 50 μm to 200 μm from the viewpoint of the stability of the formed lipid bilayer membrane. The bottom surface 12a may be made of only plastic integrally formed with the side wall 12b of the well 12, but it is usually not easy to form a minute through hole having a diameter of 50 μm to 200 μm on a plastic plate having a thickness of about 1 mm. Therefore, the bottom surface 12a is preferably composed of two members. That is, a through hole 16 (see FIG. 1) having a diameter of about 0.5 mm to 1.5 mm, which communicates with a later-described capillary, is provided on a plastic bottom formed integrally with the side wall 12b, and the through hole 16 is formed on the upper surface thereof. It is preferable to dispose a film 18 to be coated and to form the through hole 14 in the film 18. In this case, the through hole 14 is disposed so as to communicate with the through hole 16, and the through hole 14 penetrates the bottom surface 12 a through the through hole 16. Here, as the film 18, a parylene (polyparaxylylene) resin film or the like can be preferably used. The parylene resin film normally used has a thickness of about 5 μm to 20 μm, and a fine through hole can be easily formed by photolithography. In addition, the lipid bilayer membrane formation instrument which formed the through-hole in the parylene resin film is already well-known (for example, patent 5345078 publication). The film 18 is preferably bonded with an adhesive in order to prevent water leakage from the surroundings.

The side wall 12b of the well 12 is provided with three slits 20 extending in the vertical direction. In the present specification and claims, “upper” and “lower” mean the direction in which the well 12 is placed with the bottom surface 12a of the well 12 in the vertical direction. Therefore, the bottom surface 12a is the lower side, and the opening side of the well 12 is the upper side. When the instrument 10 is used, the instrument 10 is often used upside down or horizontally, so that “upper” and “lower” when used are “upper” and “lower” when describing the configuration of the instrument 10. Does not necessarily match. The number of slits is not limited to three, and other numbers may be used as long as sample exchange described later can be performed without any problem, but one to four is preferable for smooth sample exchange. The width of each slit (the width in the horizontal direction (the direction perpendicular to the vertical direction)) is not particularly limited as long as the solution in the well 12 does not leak due to surface tension and the sample can be replaced without any problem. Is about 0.05 to 0.25 times the inner circumference, and preferably about 0.1 to 0.2 times. The preferred range of the width and number of each slit is as described above, but the combination of the slit width and number is a combination in which water does not leak out of the well due to surface tension when water is put into the well. Select. The upper end of each slit 20 is preferably the upper end of the well from the viewpoint of smooth sample exchange. Moreover, it is preferable that the lower end part of each slit 20 is above the said bottom face 12a from a viewpoint of preventing that a lipid bilayer is destroyed at the time of sample exchange. The vertical length of each slit 20 is preferably about 0.7 to 0.9 times the inner depth of the well 12. If the dimensions of the well 12 are set as described later, even if a solution is put into the well 12, the solution does not leak from the slit 20 due to the surface tension of the solution.

  A chamber is connected to the lower surface of the bottom surface 12 a of the well 12. In the embodiment shown, the chamber is in the form of a capillary 22. The chamber is not necessarily in the form of a capillary, but may be in a form that can accommodate a solution and an electrode 26 described later. When the chamber is the capillary 22, there is an advantage that the production is easy. The capillary 22 communicates with the through hole 14. The capillary 22 is preferably simply inserted and connected to the through-hole 16, so that the capillary 22 necessarily communicates with the through-hole 14.

  In the illustrated example, a first electrode 24 is disposed in the well 12 to measure the current passing through the lipid bilayer after the formation of the lipid bilayer, while the capillary 22 has an interior. A second electrode 26 is disposed. In the illustrated example, the first electrode 24 extends to the outside of the well 12 and can be grounded. On the other hand, the second electrode 26 is connected to an amplification circuit type measuring instrument (small amplifier excellent in portability, hereinafter referred to as “pocket amplifier”) for measuring a weak current, not shown.

  The inner surface of the well 12 is preferably coated with water / oil repellency (that is, both lyophobic) from the viewpoint of suppressing the sloshing of the solution in the well 12 and retaining the solution. The water / oil repellent coating can be easily performed by applying a commercially available fluorine-based coating agent or the like.

  The well 12 preferably has a dimension that allows water to be retained in the well due to surface tension even when water is put in the well and turned upside down. If this can be achieved, it is advantageous because the sample can be exchanged by inverting the well and immersing it in the sample solution, as specifically described in the examples below. The dimensions of the well 12 that can achieve this are as follows. When the cross section of the well 12 is substantially circular, the inner diameter is about 1 mm to 5 mm, preferably about 2 mm to 4 mm, and the depth is about 1 mm to 5 mm, preferably 1 mm to 3 mm. Degree.

  When forming a lipid bilayer membrane using the above-described lipid bilayer membrane forming device of one embodiment of the present invention, the lipid bilayer membrane is formed in the through-hole 14. The method of forming a lipid bilayer membrane in the through-hole is known per se, and can be performed by a known painting method or droplet contact method. For example, the operation of forming a lipid bilayer membrane by a painting method can be performed as follows. The operation procedure is not limited to the procedure described below.

  First, the capillary 22 is filled with an aqueous solution such as a potassium chloride-containing phosphate buffer. The second electrode 26 in the capillary 22 is connected to a pocket amplifier (not shown). On the other hand, the first electrode 24 is grounded outside the instrument. Then, a potassium chloride-containing phosphate buffer containing a target transmembrane protein that penetrates (ie, reconstitutes) the lipid bilayer membrane is filled into the well 12 from the upper opening of the well 12. Here, the transmembrane protein is appropriately selected according to the purpose of measurement. In the following embodiment, α-hemolysin having nanopores (ion channels) through which ions flow is used, but the present invention is not limited to this. For example, when an instrument is used as a sensor, it binds to a target chemical substance. The desired protein can be used. By the above operation, the first electrode and the second electrode are electrically connected. When a voltage is applied between the two electrodes, an ionic current flows and is measured by the pocket amplifier. Next, the lipid solution is applied to the vicinity of the through-hole 14 with a micropipette. Here, as the lipid solution, those conventionally used for forming a lipid bilayer membrane can be used, for example, an n-decane solution of diphytanoylphosphatidylcholine (DPhPC) or the like can be used. it can. By the above operation, a lipid bilayer membrane is spontaneously formed in the through hole 14. If left as it is for a while, the transmembrane protein spontaneously penetrates and reconstitutes the formed lipid bilayer, and an ionic current begins to flow through the ion channel of the protein. Since the ionic current at this time is proportional to the number of proteins reconstituted in the lipid bilayer membrane, the ionic current increases stepwise each time one molecule of protein is reconstituted.

  The device for forming a lipid bilayer membrane of the present invention has an excellent feature that the sample in the well 12 can be exchanged by a simple operation while maintaining the once formed lipid bilayer membrane. This feature is advantageous when it is desired to continue the measurement by quickly exchanging the sample without using a pipette or a pump at a measurement site such as outdoors.

When exchanging the sample, prepare a container with a new sample solution, and turn the instrument 10 upside down (as described above, if the dimensions of the well 12 are appropriately set, the container 10 is turned upside down. (Solution does not spill from well 12) Dip well 12 into the sample solution. At this time, the capillary 22 and the pocket amplifier 28, or the capillary 22 and the well 12 may remain connected. That is, the well 12 can be immersed in the sample solution with the whole being turned upside down while the instrument 10 is connected to the pocket amplifier 28. Alternatively, the sample liquid is placed in a container having an opening on the side surface (the sample liquid does not flow out of the opening due to surface tension), and the well 12 is placed horizontally from the upper opening of the well 12 to the sample liquid. The sample can also be exchanged by inserting it sideways. Then, the solution in the well 12 is exchanged in about 10 seconds to 30 seconds. In the example (comparative example) in which each of the slits 20 described above is useful for the smooth sample exchange and the slit 20 is not provided, the sample is hardly exchanged even if the above operation is performed.

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

1. Production of Lipid Bilayer Formation Device A lipid bilayer membrane formation device 10 shown in FIGS. 1 and 2 was produced as follows. The well 12 was produced by cutting an acrylic plate having a thickness of 4 mm. The cross section of the well 12 was circular, and the outer diameter was 4 mm, the inner diameter was 3 mm, and the depth was 2.2 mm. Three slits 20 were provided at equal intervals. The width of each slit was 1/9 times the inner circumference of the well 12 (that is, the central angle connecting each end of the cross section of each slit from the center of the cross section of the well 12 was 40 degrees). Each slit 20 extended in the vertical direction from the upper end portion of the well 12 and had a depth of 2 mm. A through-hole 16 having a diameter of 1.05 mm for fixing the glass capillary 22 was provided at the center of the bottom surface 12a of the well 12. Further, a hole having a diameter of 0.6 mm and a depth of 1.0 mm for fixing the first electrode 24 was provided in the vicinity of one slit 20. On the other hand, a parylene resin film 18 having a diameter of 2 mm and a thickness of 5 μm was prepared, and a through hole 14 having a diameter of 150 μm was formed at the center of the circle by photolithography. This parylene resin film 18 was affixed to the bottom surface 12a of the well 12 using an ultraviolet curable adhesive. The glass capillary 22 was inserted into the through-hole 16 from the lower side of the well 12 until the tip contacted the parylene resin film 18 and fixed with an instantaneous adhesive. The outer diameter of the glass capillary was 1 mm and the inner diameter was 0.6 mm. A silver wire having a diameter of 0.2 mm was inserted into the glass capillary. One end of a 1 mm diameter first electrode (Ag / AgCl electrode) was inserted into the hole, and the other end was grounded. Both the first and second electrodes were subjected to Ag / AgCl conversion by immersing a silver wire in sodium hypochlorite for the electrode part in contact with the aqueous solution.

2. Formation of lipid bilayer A glass capillary 22 was filled with a potassium chloride-containing phosphate buffer (1 M KCl, 10 mM phosphate buffer, pH 7). The solution was filled up to the lower surface of the parylene resin film 18. Next, the glass capillary 22 was connected to the pocket amplifier, and the second electrode was connected to the pocket amplifier terminal. To well 12, 13 μL of potassium chloride-containing phosphate buffer (1M KCl, 10 mM phosphate buffer, pH 7) containing α-hemolysin having a final concentration of 1 nM was added. As a result, the solution was filled up to the through-hole 14 of the parylene resin film 18, and the conductive state was established from the first electrode (ground electrode) to the pocket amplifier terminal. Next, 0.5 μL of a lipid solution (10 mg / mL DPhPC in n-decane) was applied to the vicinity of the through-hole 14 in the parylene resin film 18 using a micropipette. As a result, a lipid bilayer was spontaneously formed, and α-hemolysin was reconstituted into a lipid bilayer (data described later).

The instrument produced by the above operation is schematically shown in FIG. In FIG. 3, 28 is a pocket amplifier. The lipid bilayer membrane forming device 10 is connected to the top of the pocket amplifier 28, and an enlarged schematic cross-sectional view thereof is a diagram in the center of FIG. The right side of FIG. 3 is a schematic cross-sectional view of the lipid bilayer membrane 30 formed in the through hole 14. 32 is α-hemolysin.

3. Evaluation of Lipid Bilayer A 50 mV DC voltage was applied between the first electrode and the second electrode, and the current value between the two electrodes was measured. FIG. 4 and FIG. 5 are schematic diagrams for explaining the measurement results and what state they are in at that time. 4 and 5 are diagrams schematically showing the state of the instrument 10 , and the central diagram schematically shows the state of the lipid bilayer (both electrodes and a circuit are also shown together). The right figure shows the measurement result of the current value over time.

  FIG. 4 shows a state before α-hemolysin is reconstituted after the formation of the lipid bilayer membrane, and the current value is almost constant. FIG. 5 shows a state in which α-hemolysin is reconstituted into a lipid bilayer membrane, and the measured current value increases stepwise over time. Each time one molecule of α-hemolysin was reconstituted, the current value increased by about 50 pA, confirming the reconstitution of α-hemolysin.

  Next, single-stranded DNA (50-mer polythymine) having a final concentration of 10 μM was added to the solution in the well 12 and the applied DC voltage was set to 100 mV. Polythymine is linear in solution because it does not structurally cause intramolecular hybridization. The thickness of the single-stranded DNA is slightly smaller than the inner diameter of the nanopore of α-hemolysin and can pass through the nanopore, but while the single-stranded DNA is passing through the nanopore, the nanopore is single-stranded DNA. During that time, ions no longer flow through the nanopore, and the current drops. The state at this time is shown in FIG. The measurement result of the current value over time is shown in the right side of FIG. 6, where downward sharp sharp valleys are observed, indicating that single-stranded DNA sometimes passed through the nanopore. .

  As described above, a lipid bilayer membrane can be formed using the device prepared in this example, and a membrane protein is reconstituted in the formed lipid bilayer membrane, and the nanopore of the membrane protein is converted into a lipid bilayer. It was also confirmed that it opened to the double membrane. Therefore, it was confirmed that the lipid bilayer membrane forming instrument of the present invention can correctly form a lipid bilayer membrane used for measurement.

4). Evaluation of sample exchangeability (part 1)
Sample exchangeability was examined for the device prepared as described above (before formation of the lipid bilayer membrane). For comparison, a comparative device was produced in the same manner as described above except that no slit was formed. Water was added to each well. On the other hand, water colored with blue ink was put in a container. The apparatus was turned upside down, the opening at the top of the well was turned down, the whole well was immersed in colored water, maintained in that state for 30 seconds, pulled up and visually observed. As a result, in the comparative example in which no slit was formed, only the portion raised below the opening of the well was colored, but in the above embodiment of the present invention in which the slit was formed, the water inside the well was also colored. It was confirmed that the water inside the well was exchanged.

5. Evaluation of sample exchangeability (part 2)
As described above, a lipid bilayer membrane was formed, single-stranded DNA was added, and current measurement was continued. The sample solution was replaced with a potassium chloride-containing phosphate buffer at a frequency of once every several to 10 minutes. For sample exchange, a potassium chloride-containing phosphate buffer solution is placed in a container having an opening on the side surface (the sample solution does not flow out of the opening due to surface tension), and the sample is taken from the upper opening of the well 12 with the well 12 level. This was done by inserting the well 12 sideways into the solution. The holding time in the new sample solution was 30 seconds. The results are shown in FIG.

  The upper diagram in FIG. 7 shows measurement results of current values over time. The arrow in the figure indicates the time when the sample was exchanged. The lipid bilayer was destroyed after the sixth sample exchange. From this, the lipid bilayer membrane formed by the lipid bilayer membrane-forming instrument of the present invention was highly stable, withstood the sample exchange as many as 5 times, and maintained for 40 minutes. The lower part of FIG. 7 shows measurement results of current values over time before and after the first sample exchange. The left side shows the result before the sample exchange, and the right side shows the result after the sample exchange. Since the first sample contains single-stranded DNA, many sharp drops in current value due to the passage of the single-stranded DNA through the nanopore have been observed. On the other hand, after exchanging the sample with a potassium chloride-containing phosphate buffer, the frequency of sharp drop in the current value clearly decreases as a result of the significant decrease in the concentration of the single-stranded DNA. This frequency is shown in FIG.

  FIG. 8 is a diagram showing a comparison of the frequency of sharp current drop that occurred in each 1 second before and after sample replacement. After the sample exchange, the frequency of sharp drop in current value decreased drastically, indicating that the sample exchange was performed smoothly.

DESCRIPTION OF SYMBOLS 10 Lipid film forming instrument 12 Well 12a Well bottom surface 12b Well side surface 14 Through hole in parylene resin film 16 Through hole at bottom of well 18 Parylene resin film 20 Slit 22 Capillary 24 First electrode 26 Second electrode 28 Pocket amplifier 30 Lipid bilayer membrane 32 Transmembrane protein

Claims (9)

  A well having a bottom surface and a side wall; a through hole provided in the bottom surface; wherein a double membrane is formed; and a chamber provided in a lower surface of the bottom surface and communicating with the through hole. A device for forming a lipid bilayer membrane, wherein one or more slits are provided in the direction.   The device for forming a lipid bilayer membrane according to claim 1, further comprising a first electrode disposed in the well and a second electrode disposed in the chamber.   The lipid bilayer membrane forming device according to claim 1 or 2, wherein the width of each of the one or more slits is 0.05 to 0.25 times the inner circumference of the well.   The lipid bilayer membrane according to any one of claims 1 to 3, wherein an upper end portion of each of the one or more slits is an upper end portion of the well, and a lower end portion thereof is located above the bottom surface. Forming tool.   The lipid bilayer membrane formation instrument according to any one of claims 1 to 4, wherein the number of slits is 1 to 4. The device for forming a lipid bilayer according to any one of claims 1 to 5, wherein the chamber is in the form of a capillary.   The device for forming a lipid bilayer according to any one of claims 1 to 6, wherein an inner surface of the well is coated with water / oil repellent coating.   The device for forming a lipid bilayer according to any one of claims 1 to 7, wherein the well has a size that allows water to be retained in the well by surface tension even when water is put into the well and turned upside down.   The lipid bilayer membrane forming device according to claim 8, wherein the well has a substantially circular cross section, an inner diameter of 1 mm to 5 mm, and a depth of 1 mm to 5 mm.