JP2012233748A - Manufacturing method of biosensor device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of a biosensor device capable of stably forming an artificial lipid membrane.SOLUTION: A manufacturing method of a biosensor device prepares a body member (30) comprising: a first chamber (5); a second chamber (7) with an opening section (7a); and a communication section (6) positioned between the first chamber (5) and the second chamber (7). Then, the manufacturing method sequentially supplies the body member (30) with first electrolytic solution (2), lipid solution (1) to form an artificial lipid membrane and second electrolytic solution (3) through the opening section (7a) of the second chamber (7) so as to fill: the first chamber (5) with the first electrolytic solution (2); at least a part of communication section (6) with the lipid solution (1); and the second chamber (7) with the second electrolytic solution (3).

Description

  The present invention relates to a method for manufacturing a biosensor device, and more particularly to a method for manufacturing a biosensor device having an artificial lipid film.

  Biosensors in which a receptor is incorporated into an artificial lipid membrane using the excellent molecular recognition function of the receptor are known (see, for example, Patent Documents 1 and 2).

  The conventional biosensor is configured as a biosensor system 350 that measures the membrane potential of the artificial lipid membrane 301 formed on the sensor chip 300, as schematically shown in FIG. The artificial lipid membrane 301 of the sensor chip 300 has a structure shown in more detail in FIG. 10B, and this is manufactured by the method described in Patent Document 1 as follows. First, a sensor chip substrate 280 shown in FIG. 11A is prepared. In the sensor chip substrate 280, a sheet 310 having a plurality of wells 308 is disposed on the base material 311, an electrode 314 is formed on the bottom of the well 308, and a lead wire 306 is connected to the electrode 314 from the back side of the base material 311. Has been. Next, as shown in FIG. 11B, each of these wells 308 is filled with the reference solution 313, and the reference solution 313 is filled with the lipid film forming component 281. Thereafter, the entire substrate is cooled, A sensor chip precursor 290 is obtained. Then, as shown in FIG. 10 (a), the sample liquid 302 is filled in the test tank 303 as the liquid to be detected, and the sensor chip precursor 290 obtained above is immersed in the sample liquid 302, whereby a lipid membrane is obtained. The forming component 281 naturally changes to the artificial lipid film 301, thereby completing the sensor chip 300 having the artificial lipid film 301. At this time, for example, a membrane protein 312 (which was included in the lipid membrane forming component 281) is incorporated into the artificial lipid membrane 301 as a receptor. Similarly, the reference electrode 304 is immersed in the sample solution 302, and the sample solution 302 and the reference solution 313 are used by using a lead wire 306 (coated with an insulator) of the sensor chip 300 and a potential difference measuring device 307 connected to the reference electrode 304. As the membrane potential of the artificial lipid membrane 301 between the electrode 314 and the reference electrode 304 is measured. By using such a biosensor system 350, it is possible to detect a substance (molecule) contained in the sample liquid 302 and measure the amount by examining a change in the membrane potential of the artificial lipid membrane 301.

JP 2005-37207 A Japanese Patent Laid-Open No. 4-215052

  In the conventional manufacturing method, as described above with reference to FIGS. 10 to 11, the sensor chip precursor 290 is immersed in the sample solution 302 filled in the larger test tank 303, so that the lipid film forming component 281 is removed. An artificial lipid membrane 301 is formed. Since vibration is excited in the sample liquid 302 during the immersion, the liquid pressure applied to the lipid film-forming component 281 varies depending on the location and time, so that the lipid film-forming component 281 moves along the surface of the sheet 310, Even if the amount of components necessary to form the artificial lipid membrane 301 is insufficient and the artificial lipid membrane 301 is not formed or formed, the electrical characteristics (membranes) between the artificial lipid membranes 301 There has been a problem that variations in potential detection sensitivity occur.

  The present invention has been made to solve the problems of conventional manufacturing methods, and an object thereof is to provide a method of manufacturing a biosensor device capable of stably forming an artificial lipid film.

According to the present invention, a method for producing a biosensor device having an artificial lipid membrane,
Providing a body member having a first chamber, a second chamber having an opening, and a communication portion located between the first chamber and the second chamber;
The body member is supplied with the first electrolyte solution, the lipid solution for forming the artificial lipid membrane, and the second electrolyte solution sequentially from the opening of the second chamber, and the first chamber is filled with the first electrolyte solution, A method of manufacturing a biosensor device is provided that includes at least partially filling a communication portion with a lipid solution and filling a second chamber with a second electrolyte solution.

  The manufacturing method of the biosensor device of the present invention is fundamentally different from the conventional manufacturing method in which a test solution is filled with a sample solution and a sensor chip precursor is immersed in this to form an artificial lipid film. Is different. In the method for producing a biosensor device of the present invention, a body member having a predetermined structure is prepared, and a first electrolyte solution, a lipid solution for forming an artificial lipid film, and a second electrolyte solution are sequentially formed on the body member. Since the liquid is supplied, fluctuations in the fluid pressure applied to the lipid solution can be effectively reduced, whereby an artificial lipid membrane can be stably formed from the lipid solution at the communicating portion in the body member. .

  In the present invention, “filling” the first chamber with the first electrolyte means that the first electrolyte fills substantially the entire volume of the first chamber. Further, “at least partially filling” the communicating part with the lipid liquid means that the lipid liquid fills part or all of the volume of the communicating part, and can spread at least over the entire opening area of the communicating part. As long as it exists in, it will not specifically limit. Also, “filling” the second chamber with the second electrolyte means that the second electrolyte fills substantially the entire volume of the second chamber. Schematically, in the present invention, the first electrolyte solution, the second electrolyte solution, and the lipid solution are present corresponding to the first chamber, the second chamber, and the communicating portion, respectively, and the lipid solution is the first electrolyte solution. And the second electrolyte solution may be interposed. Therefore, the interface between the first electrolyte solution and the lipid solution does not need to be exactly the same as the boundary between the first chamber and the communication portion, and between the second electrolyte solution and the lipid solution. The interface does not need to be exactly coincident with the boundary between the second chamber and the communication portion.

  In such a method for producing a biosensor device of the present invention, the body member is arranged so that the second chamber is positioned above the first chamber, and the first electrolytic solution, from the opening of the second chamber, It can be carried out by quantitatively supplying the lipid solution and the second electrolyte solution sequentially.

  In one aspect of the present invention, the inner wall surface of the communicating portion in the body member has water repellency. According to this aspect, the artificial lipid membrane can be formed and held more stably at the communicating portion in the body member. In the present invention, water repellency means that the contact angle with water is, for example, 60 ° or more (less than 180 °), and preferably 70 ° to 120 °.

  In one aspect of the present invention, a notch is formed in the inner wall surface of the communicating portion in the body member. According to this aspect, since the lipid liquid supplied to the body member is likely to stay in the communicating portion due to this notch, the artificial lipid membrane can be more stably formed and held in the communicating portion in the body member. .

  In one aspect of the present invention, the second chamber has an inner overhang portion between the communication portion and the opening. According to this aspect, even if the lipid solution moves from the communicating portion to the second chamber along the inner wall surface of the second chamber, it can be prevented from further moving by the inner overhang portion, The artificial lipid membrane can be formed more stably at the communicating portion.

  In one aspect of the present invention, in the liquid supply step of the biosensor device manufacturing method, at least one liquid selected from the group consisting of a first electrolyte solution, a lipid solution, and a second electrolyte solution is used as a droplet. Supply in state. Such an embodiment is suitable for quantitatively supplying the liquid, and can suppress the mixing of air.

  According to the present invention, a body member having a predetermined structure is prepared, and a first electrolyte solution, a first electrolyte solution, a lipid solution for forming an artificial lipid film, and a second electrolyte solution are sequentially formed on the body member. By supplying, a method for producing a biosensor device capable of stably forming an artificial lipid film from a lipid solution at a communicating portion in the body member is provided.

It is a schematic sectional process drawing which shows the manufacturing method of the biosensor device in Embodiment 1 of this invention, Comprising: (a) is a preparation process of a body member, (b) is a 1st electrolyte solution supply process, (c). FIG. 4 is a diagram showing a lipid solution supply step, and FIG. 4D is a diagram showing a second electrolyte solution supply step. (A) is a schematic sectional drawing of the biosensor device manufactured in Embodiment 1 of this invention, (b) is a schematic sectional drawing explaining the use condition. (A) is a schematic sectional drawing which shows the body member used for the manufacturing method of the biosensor device in Embodiment 2 of this invention, (b) is a notch and the partially expanded schematic sectional drawing of the vicinity. It is a schematic sectional drawing which shows the biosensor device in Embodiment 2 of this invention. (A) is a schematic sectional drawing which shows the body member used for the manufacturing method of the biosensor device in the modification of Embodiment 2 of this invention, (b) is a notch and the partially expanded schematic sectional drawing of the vicinity. It is. It is a schematic sectional drawing which shows the body member used for the manufacturing method of the biosensor device in Embodiment 3 of this invention. It is a schematic sectional drawing which shows the biosensor device in Embodiment 3 of this invention. (A) shows the current response waveform of the biosensor device of Example 1 of the present invention, and (b) shows the current response waveform of the device of the comparative example. (A) shows the current response waveform of the biosensor device of Example 2 of the present invention, and (b) shows the current response waveform of the biosensor device of the reference example. It is a figure which shows the conventional biosensor system, Comprising: (a) shows the whole schematic perspective view of a biosensor system, (b) shows the expanded schematic sectional drawing of a sensor chip. It is a schematic sectional process drawing which shows the manufacturing method of the sensor chip precursor used for the conventional biosensor system.

  Various embodiments of the present invention are described in detail below with reference to the drawings.

(Embodiment 1)
The manufacturing method of the biosensor device in one embodiment of this invention is implemented as follows.

  First, a body member for a biosensor device is prepared. As shown in FIG. 1A, the body member 30 includes a first chamber 5, a second chamber 7 having an opening 7 a, and a communication portion 6 positioned between the first chamber 5 and the second chamber 7. (In FIG. 1A, the open areas of the first chamber 5, the second chamber 7, and the communication portion 6 are schematically shown by dotted lines).

  The 1st chamber 5, the communication part 6, and the 2nd chamber 7 prescribe | regulate the space which should be filled with the 1st electrolyte solution 2, the lipid solution 1, and the 2nd electrolyte solution 3 which are mentioned later, respectively. The first chamber 5 is sealed in a liquid-tight manner except for an opening region communicating with the communication portion 6. The second chamber 7 has an opening 7 a in addition to the opening region communicating with the communication part 6, and is sealed liquid-tight except for these. The first chamber 5 and the second chamber 7 communicate with each other through the communication part 6, but are spatially separated from each other by the communication part 6. The communication portion 6 has a smaller outer dimension than the first chamber 5 and the second chamber 7, and is formed in the body member 30 in the form of a narrowed portion (or neck portion) between the first chamber 5 and the second chamber 7. It is arranged.

  In the illustrated embodiment, the body member 30 is configured by laminating the base materials 10, 11, and 12 each having holes (or wells) formed on the base material 9. In such an embodiment, the first chamber 5 is defined (or surrounded) by the upper surface of the substrate 9, the inner wall surface of the substrate 10, and the lower surface of the substrate 11, and the communication part 6 is defined by the inner wall surface of the substrate 11. The second chamber 7 is defined by the upper surface of the substrate 11 and the inner wall surface of the substrate 12. The opening 7 a of the second chamber 7 coincides with the upper surface opening of the hole of the base material 12. And in the direction parallel to the main surface of the base material 9 (refer to FIG. 2, the direction perpendicular to the film thickness direction of the artificial lipid membrane 4 finally formed) The hole area of the substrate 11 is smaller than the outer dimension of the first chamber 5 (hole area of the substrate 10) and smaller than the outer dimension of the second chamber 7 (hole area of the substrate 12). Moreover, although the external dimension (hole area of the base material 10) of the 1st chamber 5 is the same as the external dimension (hole area of the base material 7) of the 2nd chamber 12, it is not limited to this, 1st The outer dimension of the chamber 5 may be smaller or larger than the outer dimension of the second chamber 7. In addition, the heights of the base materials 10, 11, and 12, and the shapes and sizes (as viewed from the film thickness direction) of the holes formed in the base materials 10, 11, and 12 are as desired. It may be determined as appropriate according to the dimensions and arrangement of the. The shape of the hole is not particularly limited, and may be, for example, a circle, an ellipse, a rectangle, or a polygon, and may be the same (similar) between the substrates or may be different.

  The substrates 9, 10, 12 can be composed of any suitable material having liquid tightness. For example, the base materials 9, 10 and 12 include polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyphenylene sulfide (PPS), acrylonitrile-butadiene-styrene copolymer resin (ABS resin), acrylic resin (for example, polymethyl). Methacrylate (PMMA)), cyclic polyolefin, polycarbonate (commonly called polycarbonate), and other organic materials. However, the present invention is not limited thereto, and the base materials 9, 10, and 12 may be made of an inorganic material such as glass, silicon, aluminum oxide, silicon oxide, or silicon nitride.

  Although the base material 11 may be comprised with the material similar to these base materials 9, 10, and 12, since it comprises the inner wall face of the communication part 6, it is preferable that at least an inner wall face has water repellency. For example, at least the inner wall surface (and possibly other surfaces, for example, the entire exposed surface of the base material 11) of the base material 11 may be subjected to a water repellent treatment with a fluorine resin or the like. Or the base material 11 may be comprised from the material which has water repellency, for example, a fluorocarbon resin, polytetrafluoroethylene (PTFE), and Teflon (trademark).

  Between these base materials 9, 10, 11, and 12, an adhesive layer (for example, a thermosetting film or the like, not shown) may be adhered. For example, when these base materials are composed of an organic material (organic film), the base materials are laminated, and the obtained laminated body is subjected to heat treatment to thermally bond the base materials to each other. May be bonded together.

  In addition, the body member 30 may have an electrode for measuring the membrane potential of the artificial lipid membrane 4 (see FIG. 2) to be finally formed. More specifically, electrodes 8 a and 8 a ′ are formed in the first chamber 5. In the illustrated embodiment, the electrodes 8a and 8a 'are formed by being routed to the back surface side of the substrate 8, but the present invention is not limited to this.

  Next, as shown in FIG. 1B, the first electrolytic solution 2 is supplied to the body member 30 prepared as described above from the opening 7 a of the second chamber 7 using the liquid supply device 51.

The 1st electrolyte solution 2 should just be what melt | dissolved the ionic substance in the polar solvent, and may be suitably selected according to the composition of the lipid solution 1, etc.
Examples of the ionic substance include sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride (MgCl ₂ ), sodium dihydrogen phosphate (NaH ₂ PO ₄ ), sodium monohydrogen phosphate (Na ₂ HPO ₄ ), and the like. Can be used alone or in combination, and potassium chloride is preferably used.
Examples of polar solvents include water, glycerin, sugar, sugar alcohol, ethanol, isopropyl alcohol, ethylene glycol, sorbitol, xylitol, dipropylene glycol, butylene glycol, polyethylene glycol, polyoxyethylene methyl glucoside, maltitol, mannitol, glucose, etc. Can be used alone or in combination.
The first electrolyte 2 preferably has an osmotic pressure within a range of 280 to 330 mOsm / kg-H ₂ O, but is not limited thereto. Alternatively, it is preferable to use a general solution used in electrophysiological experiments as the first electrolytic solution 2. Moreover, it is also preferable to adjust the viscosity of the 1st electrolyte solution 2 by adding an organic compound and a polymer | macromolecule.

  The supply of the first electrolytic solution 2 is performed so that the first chamber 5 is filled with the first electrolytic solution 2. Such supply can be performed by quantitatively supplying the first electrolytic solution 2 according to the volume of the first chamber 5. It is preferable to supply the first electrolyte solution 2 intermittently by dividing a predetermined amount of the first electrolyte solution 2 into a plurality of times. For example, it is preferable to supply the first electrolytic solution 2 in a droplet state by an ink jet method using an ink jet device as the liquid supply device 51. Thereby, it can suppress effectively that air mixes in the 1st chamber 5. FIG. When supplying the 1st electrolyte solution 2 in a droplet state, it can be set as the liquid volume of 10 pL or more and 300 pL or less per drop, for example. The ink jet method has a smaller amount of liquid per droplet than the other methods, and the supply amount can be adjusted in units of such a small amount of liquid, so the first electrolytic solution 2 is supplied quantitatively. It is suitable for. However, the present invention is not limited to this, and the first electrolytic solution 2 may be supplied by a dispensing method, a transfer method, or the like.

  Next, as shown in FIG. 1C, the lipid solution 1 is supplied from the opening 7 a of the second chamber 7 to the body member 30 to which the first electrolyte solution 2 has been supplied, as shown in FIG. To do. The liquid supply device 52 may be the same device as the liquid supply device 51 or a different device.

The lipid liquid 1 is preferably one in which lipid is dispersed or dissolved in a solvent.
Any appropriate lipid may be used as the lipid, and in particular, complex lipids containing phosphoric acid and sugar in the molecule, specifically, phospholipids, glycolipids, lipoproteins, sulfolipids, etc. are preferred, and phospholipids are preferred. Most preferred. Examples of phospholipids include glycerophospholipids (for example, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, diphytanoylphosphatidylcholine, dipalmitoylphosphatidylcholine, etc.) and sphingophospholipids (for example, sphingomyelin). The lipid may also be a simple lipid (eg glycerol monooleate) or a derived lipid. It may be a naturally occurring lipid such as azolectin (soybean phospholipid) or a synthetic lipid. Synthetic lipids are more preferred because they are easy to obtain highly pure and chemically stable. The fatty acid portion of the lipid is preferably a saturated or unsaturated fatty acid having 10 to 20 carbon atoms. Such lipid may be used individually by 1 type, or may be used in combination of 2 or more type.
The solvent can be appropriately selected depending on the lipid, and generally an organic solvent, preferably a saturated hydrocarbon is used. Examples include decane, hexadecane, hexane, chloroform and the like. Such a solvent may be used individually by 1 type, or may be used in combination of 2 or more type.
The lipid concentration with respect to the solvent is preferably 3 to 50 mg of lipid, more preferably 4 to 40 mg of lipid with respect to 1 mL of the solvent.

  The lipid liquid 1 is supplied so that the communication part 6 is at least partially filled with the lipid liquid 1. Such supply can be performed by quantitatively supplying the lipid solution 1 so that the lipid solution 1 spreads over the entire opening area of the communication portion 6 and covers the liquid surface of the first electrolyte solution 2. The lipid liquid 1 is preferably supplied intermittently by dividing a predetermined amount of the lipid liquid 1 into a plurality of times. For example, it is preferable to supply the lipid liquid 1 in a droplet state by an ink jet method using an ink jet device as the liquid supply device 52. Thereby, it can suppress effectively that air mixes in the communicating part 6. When the lipid liquid 1 is supplied in the form of droplets, it is preferable to form fine droplets, for example, a liquid volume of 10 pL or more and 200 pL or less per droplet. The ink jet method has a smaller amount of liquid per droplet than the other methods, and the supply amount can be adjusted in units of such a small amount of liquid, so that the lipid solution 1 can be quantitatively supplied. Is preferred. Since the supply amount of the lipid liquid 1 is small, an ink jet method is particularly preferably used. However, the present invention is not limited to this, and the lipid solution 1 may be supplied by a dispensing method, a transfer method, or the like.

  Next, as shown in FIG. 1 (d), the second electrolyte solution 3 is supplied to the body member 30 supplied with the lipid solution 1 from the opening 7 a of the second chamber 7 using the liquid supply device 53. To do. The liquid supply device 53 may use the same device as any of the liquid supply devices 51 and 52, or may use another device.

  As described above with respect to the first electrolytic solution 2, the second electrolytic solution 3 only needs to be obtained by dissolving an ionic substance in a polar solvent, and can be appropriately selected according to the composition of the lipid solution 1. The first electrolyte solution 2 and the second electrolyte solution 3 may be the same electrolyte solution or different electrolyte solutions. More specifically, the first electrolytic solution 2 and the second electrolytic solution 3 may be different or the same in any one or more of concentration, viscosity, and osmotic pressure.

  The supply of the second electrolytic solution 3 is performed so that the second chamber 7 is filled with the second electrolytic solution 3. Such supply can be performed by quantitatively supplying the second electrolytic solution 3 according to the volume of the second chamber 7. As for the supply of the second electrolytic solution 3, it is preferable that a predetermined amount of the second electrolytic solution 3 is intermittently supplied in a plurality of times. For example, it is preferable to use an ink jet device as the liquid supply device 53 and supply the second electrolytic solution 3 in a droplet state by an ink jet method. Thereby, it can suppress effectively that air mixes in the 2nd chamber 7. FIG. When supplying the 2nd electrolyte solution 3 in a droplet state, it can be set as the liquid volume of 10 pL or more and 300 pL or less per drop, for example. The ink jet method has a smaller amount of liquid per drop than the other methods, and the supply amount can be adjusted in units of such a small amount of liquid, so the second electrolytic solution 3 is supplied quantitatively. It is suitable for. However, the present invention is not limited to this, and the second electrolytic solution 3 may be supplied by a dispensing method, a transfer method, or the like.

  The lipid solution 1 supplied as described above becomes an artificial lipid membrane 4 existing between the first electrolyte solution 2 and the second electrolyte solution 3 as shown in FIG. Complete. The change from the lipid solution 1 to the artificial lipid membrane 4 is that a part of the lipid solution 1 spreads along the wall surface of the base material 11 after the second electrolyte solution 3 is supplied, and the lipid in the penetrating part 6 The liquid 1 becomes a thin film, which naturally occurs. It is necessary to supply the second electrolytic solution 3 after the lipid solution 1 is supplied and before the lipid solution 1 is completely evaporated. By supplying the second electrolytic solution 3 in a droplet state, vibration excited by the second electrolytic solution 3 can be reduced, and the artificial lipid membrane 4 can be formed more stably. The artificial lipid membrane 4 formed in this way comes into contact with the inner wall surface of the communicating portion 6 and can be held on this inner wall surface by the surface tension of the artificial lipid membrane 4 or the like.

  The artificial lipid membrane 4 is preferably a lipid bilayer membrane. The film thickness of the artificial lipid film 4 may be about 2 to 5 nm, for example. In the artificial lipid membrane 4, receptors (not shown) such as membrane proteins and ion channels can be incorporated depending on the substance to be detected.

  As described above, the biosensor device 40 shown in FIG. 2A is manufactured. In the manufacturing method of this embodiment, it is not necessary to immerse the sensor chip precursor in the sample solution in order to form the artificial lipid film as in the conventional manufacturing method. In the manufacturing method of the present embodiment, a body member 30 having a predetermined structure is prepared, and a lipid solution 1 and a second electrolyte solution 3 for forming the first electrolyte solution 2 and the artificial lipid membrane 4 on the body member 30. Can be effectively reduced, so that the artificial lipid membrane 4 can be stably stabilized from the lipid solution 1 at the communicating portion 6 in the body member 30. Can be formed.

  The biosensor device 40 obtained as described above can be used by immersing the electrodes 8b and 8b 'in the second electrolytic solution 3, for example, as shown in FIG. The electrodes 8a ′ and 8b ′ are connected to a power source (schematically indicated by the symbol “V” in the figure), and the electrodes 8a and 8b are connected to an ammeter (schematically indicated by the symbol “A” in the figure). Connected. More specifically, it is used as follows.

  First, in the absence of a substance to be detected, a voltage is applied to both surfaces of the artificial lipid membrane 4 by applying a voltage to the electrodes 8a ′ and 8b ′ connected to the power source. In this state, the artificial lipid membrane 4 The current flowing in the film thickness direction is measured with an ammeter as the current flowing between the electrodes 8a and 8b. If the artificial lipid membrane 4 is appropriately formed so that the first electrolytic solution 2 and the second electrolytic solution 3 are separated from each other, the current generally is insulative in the absence of the substance to be detected. It does not flow substantially.

  Next, a biosensor device is manufactured using the second electrolytic solution 3 prepared by dissolving or dispersing a predetermined amount of the substance to be detected, and the current flowing in the thickness direction of the artificial lipid membrane 4 is measured in the same manner as described above. To do. In the presence of the substance to be detected, the substance to be detected acts on the receptor incorporated in the artificial lipid membrane 4, whereby a current flows according to the amount of the substance to be detected. Therefore, calibration data can be obtained by varying the amount of the substance to be detected dissolved or dispersed in the second electrolytic solution 3 and collecting a plurality of measured current values.

  After obtaining calibration data in advance as described above, a biosensor device 40 is newly produced using the second electrolytic solution 3 prepared by mixing the sample solution, and the artificial lipid membrane 4 is produced in the same manner as described above. The current flowing in the film thickness direction is measured. From the measured current value, the presence / absence of the substance to be detected in the sample and the amount thereof can be examined.

  In the above method of use, a voltage is applied to both surfaces of the artificial lipid membrane 4 using the electrodes 8a ′ and 8b ′, and a current flowing in the film thickness direction of the artificial lipid membrane 4 is measured using the electrodes 8a and 8b. It was supposed to be. Such a configuration is particularly preferable for measuring a minute ion current with high accuracy. However, the present invention is not limited to this, and the number of electrodes may be one for each of the first electrolyte solution and the second electrolyte solution as long as the membrane potential of the artificial lipid membrane 4 can be measured. Any of the voltages may be measured.

(Embodiment 2)
The present embodiment is a modification of the biosensor device manufacturing method described in the first embodiment. Hereinafter, the modification point will be mainly described, and the same description as in the first embodiment applies unless otherwise specified.

  In the present embodiment, a body member 31 shown in FIG. In the body member 31, a notch 16 is formed on the inner wall surface of the communication portion 6. More specifically, in the body member 31, another base material 17 is used instead of the base material 11 in the body member 30 shown in FIG. 1A, and the inner wall surface that forms the communication portion 6 of the base material 17. A notch 16 is formed in. The notches 16 may be formed by grooving the inner wall surface of the base material 17.

  With reference to FIG.3 (b), the depth L of the notch 16 shall be 2-30% with respect to the external dimension of the communication part 6 (a diameter when the circular hole is formed in the base material 17). Good. More specifically, it is preferable to have a shape that is recessed at a depth L = 5 μm to 50 μm from the other inner wall surface portion (the inner end surface of the hole formed in the base material 17) that defines the communication portion 6. The width of the notch 16 is preferably 5 to 30 μm.

  The base material 17 is the same material as the base materials 9, 10, 12, for example, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyphenylene sulfide (PPS), acrylonitrile-butadiene-styrene copolymer resin (ABS resin). , And an organic material such as acrylic resin (for example, polymethyl methacrylate (PMMA)), cyclic polyolefin, and polycarbonate. However, it is not limited to these, The base material 17 may be comprised with inorganic type materials, such as glass, a silicon | silicone, aluminum oxide, a silicon oxide, silicon nitride, for example.

  The surface of the notch 16 preferably has water repellency. For example, as shown in FIG. 3B, the exposed surface 22 of the substrate 17 may be subjected to a water repellent treatment with a fluorine resin or the like. Or the base material 17 may be comprised from the material which has water repellency, for example, a fluorocarbon resin, polytetrafluoroethylene (PTFE), and Teflon (trademark).

  The biosensor device 41 shown in FIG. 4 is manufactured using the body member 31 as described above in the same manner as in the first embodiment. In the manufacturing method of this embodiment, since the lipid liquid 1 supplied to the communication part 6 enters the notch 16 and easily stays in the communication part 6 by the notch 16, the artificial lipid membrane 4 is formed. It can be formed stably.

  The present embodiment is not limited to the above aspect as long as a cutout is formed in the inner wall surface of the communication portion 6. For example, instead of the base material 17 in the body member 31, the base material 19, the intermediate material 20, and the base material 21 may be stacked and used as a body member 32 shown in FIGS. 5 (a) and 5 (b). . The intermediate material 20 positioned between the base material 19 and the base material 21 in a direction parallel to the main surface of the base material 9 (direction perpendicular to the film thickness direction of the artificial lipid membrane 4 finally formed) The hole area is larger than the hole area of the base material 19 and larger than the hole area of the base material 21. Thereby, the notch 18 is formed in the inner wall surface which comprises the communication part 6 of the base materials 19-21.

  With reference to FIG.5 (b), the depth L of the notch 18 shall be 2-30% with respect to the external dimension of the communication part 6 (diameter, when the circular hole is formed in the base material 17). Good. More specifically, it is a shape that is recessed at a depth L = 5 μm to 50 μm from the other inner wall surface part (the inner end surface of the hole formed in the base material 19 and the base material 21) that defines the communication part 6. Is preferred. The width of the notch 18 is equal to the thickness of the intermediate member 20 and is preferably 5 to 30 μm.

  The base materials 19 and 21 are made of the same material as the base materials 9, 10 and 12, for example, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyphenylene sulfide (PPS), acrylonitrile-butadiene-styrene copolymer resin (ABS). Resin), acrylic resin (for example, polymethyl methacrylate (PMMA)), cyclic polyolefin, polycarbonate, and other organic materials. Although the intermediate material 20 located between the base material 19 and the base material 21 may be comprised with such an organic type material, it is more preferable that it is a thermosetting adhesive sheet. However, it is not limited to these, The base materials 19 and 21 and the intermediate material 20 may be comprised with inorganic type materials, such as glass, a silicon | silicone, aluminum oxide, a silicon oxide, silicon nitride, for example.

  It is preferable that the surface of the notch 18 also has water repellency. For example, as shown in FIG. 5B, all exposed surfaces 23 of the base material 19, the intermediate material 20, and the base material 21 may be subjected to water repellent treatment with a fluorine-based resin or the like. Alternatively, the base materials 19 and 21 may be made of a material having water repellency, for example, a fluorocarbon resin, polytetrafluoroethylene (PTFE), or Teflon (registered trademark).

  Even when the body member 32 as described above is used, the same effects as those of the above-described embodiment can be obtained.

(Embodiment 3)
This embodiment is also a modification of the manufacturing method of the biosensor device described in the first embodiment. Hereinafter, the modification point will be mainly described, and the same description as in the first embodiment applies unless otherwise specified.

  In the present embodiment, a body member 33 shown in FIG. 6A is prepared. In the body member 33, the second chamber 7 includes the inner overhanging portion 24 between the communication portion 6 and the opening 7 a. In more detail, in the body member 33, the further base material 14 is laminated | stacked and used on the base material 12 in the body member 30 shown to Fig.1 (a), and the 2nd chamber 7 is the base material 11 used. , The inner wall surface of the substrate 12, the lower surface and the inner wall surface of the substrate 14, and the opening 7 a of the second chamber 7 coincides with the upper surface opening of the hole of the substrate 14. In the direction parallel to the main surface of the base material 9 (direction perpendicular to the film thickness direction of the artificial lipid membrane 4 finally formed), the hole area of the base material 14 is the hole area of the base material 12. It is smaller than (the maximum outer dimension of the second chamber 7). The height of the base materials 12 and 14 and the shape and size of the holes formed in each of these (as viewed from the film thickness direction) may be appropriately determined within a range in which the effect of the present embodiment can be obtained. The shape is preferably circular (in the case of a circular hole, the area of the hole is represented by the diameter of the hole). Specifically, the hole diameter of the base material 12 is larger than the hole diameter of the base material 14 and larger than the hole diameter of the base material 11. Preferably, the hole diameter of the base material 14 is the hole diameter of the base material 11. Same as or larger than the diameter. By making the hole diameter of the base material 14 equal to or larger than the hole diameter of the base material 11, the liquid supply becomes easy.

  The base material 14 is made of the same material as the base materials 9, 10, 11, 12 such as polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polyphenylene sulfide (PPS), acrylonitrile-butadiene-styrene copolymer resin (ABS). Resin), acrylic resin (for example, polymethylmethacrylate (PMMA)), cyclic polyolefin, and polycarbonate. However, it is not limited to these, The base material 14 may be comprised with inorganic type materials, such as glass, a silicon | silicone, aluminum oxide, a silicon oxide, silicon nitride, for example.

  The biosensor device 43 shown in FIG. 7 is manufactured using the body member 33 as described above in the same manner as in the first embodiment. For example, when the affinity between the lipid liquid 1 and the material forming the surface of the base material 11 is high, a part of the lipid liquid 1 supplied to the communicating portion 6 is along the upper surface of the base material 11. In the case of remarkable movement, there may be a concern that it may further move along the inner wall surface of the base material 12 like the lipid solution 15 outside the communicating portion illustrated in FIG. However, in the manufacturing method of this embodiment, since the inner overhanging portion 24 is provided on the inner wall surface of the second chamber 7 between the communication portion 6 and the opening 7a, the lipid solution 15 moves excessively. For this reason, since the lipid liquid 1 tends to stay in the communication part 6, the artificial lipid membrane 4 can be formed stably.

Example 1
In this example, the biosensor device 40 according to the first embodiment described above with reference to FIGS.

<1> Body member preparation process First, base materials 9, 10, 11, and 12 were prepared. For each substrate, a sheet made of polyethylene terephthalate (PET) was used. The thickness of the substrate 9 was 100 μm, the thickness of the substrate 10 was 100 μm, the thickness of the substrate 11 was 50 μm, and the thickness of the substrate 12 was 100 μm. A circular hole was formed in each of the base materials 10, 11, and 12, and the diameter of the hole in the base material 10 was 500 μm, the diameter of the hole in the base material 11 was 180 μm, and the diameter of the hole in the base material 12 was 500 μm. These holes were formed using a punching machine.

  Electrodes 8a and 8a 'were formed on the substrate 9. The electrodes 8a and 8a 'were formed by the following procedure. First, holes for electrode routing were formed in the base material 9 using a punching machine, and a conductive paste containing a Cu filler was filled in the holes. Next, a pattern made of Cu having a thickness of 12 μm was formed on these holes as an electrode base. Next, the outer peripheral surface of this pattern was plated with silver (Ag). Finally, the outer peripheral surface plated with silver was further coated with silver chloride (AgCl) using an aqueous sodium hypochlorite solution (5% by weight).

  The base materials 9, 10, 11, and 12 thus obtained are adhered to each other by aligning and laminating and thermocompression bonding with a thermosetting adhesive sheet having a thickness of 20 μm interposed therebetween. did.

  Thereby, the body member 30 shown to Fig.1 (a) was produced.

<2> Supply of First Electrolytic Solution Next, as shown in FIG. 1B, an ink jet device is used as the liquid supply device 51 for the body member 30 obtained as described above, and the opening portion of the second chamber 7. From 7a, 25.5 nL of the first electrolyte solution 2 was supplied in total. The total supply amount of the first electrolytic solution 2 was set to an amount corresponding to the volume of the first chamber 5. At this time, the amount of liquid discharged from the head part of the ink jet apparatus per one time was 170 pL, and the number of discharges was 150 times. As the first electrolytic solution 2, a 100 mmol / L potassium chloride (KCl) aqueous solution and glycerin mixed at a volume ratio of 50%: 50% were used.

<3> Supply of Lipid Solution Then, as shown in FIG. 1C, the first electrolyte solution supplied as described above from the opening 7 a of the second chamber 7 using another ink jet device as the liquid supply device 52. A total of 400 pL of lipid solution 1 was supplied onto 2. At this time, the amount of liquid discharged from the head part of the ink jet apparatus per one time was 20 pL, and the number of discharges was 20 times. As the lipid solution 1, a solution in which 10 mg of lecithin was dissolved in 1 mL of decane was used.

<4> Supply of Second Electrolytic Solution Next, as shown in FIG. 1 (d), as the liquid supply device 53, the same inkjet device as that used as the liquid supply device 51 is used to open the second chamber 7. From the part 7a, the second electrolyte solution 3 was supplied in a total amount of 30 nL on the lipid solution 1 supplied above. The total supply amount of the second electrolytic solution 3 is larger than the volume of the second chamber 7, but is maintained by the surface tension. At this time, the amount of liquid discharged from the head part of the ink jet apparatus per one time was 170 pL, and the number of discharges was 180 times. The same thing as said 1st electrolyte solution 2 was used for the 2nd electrolyte solution 3. FIG.

  As described above, the biosensor device 40 of this example was completed as shown in FIG. The artificial lipid membrane 4 can be naturally formed between the lipid solution 1 and the first electrolyte solution 2 and the second electrolyte solution 3 without requiring an additional step.

<5> Measurement of electrical characteristics of artificial lipid membrane As shown in FIG. 2 (b), the electrodes 8b and 8b 'are immersed in the second electrolyte solution 3, and the power source (symbol "V" in the figure schematically). A pulse voltage of 20 mV (pulse width 10 ms) is applied to the artificial lipid membrane 4 from the electrodes 8a ′ and 8b ′ connected to the electrode 8a ′, and the current response flowing in the thickness direction of the artificial lipid membrane 4 in this state is Measured with an ammeter connected to 8a and 8b (schematically indicated by symbol “A” in the figure). A patch clamp amplifier (manufactured by HEKA, model number EPC-10) was used as the power source and ammeter, and the measurement current was recorded over time. Even when the electrodes 8b and 8b ′ were immersed in the second electrolytic solution 3, the electrolytic solution was held by surface tension.

  On the other hand, as a comparative example, comparison was made in the same manner as in Example 1 except that the lipid solution 1 was not supplied (thus, the entire space in the device was filled with the first electrolyte solution = the second electrolyte solution). An example device was fabricated and the current response was measured for this device as described above.

FIG. 8A is a current response waveform of the biosensor device of Example 1, and FIG. 8B is a current response waveform of the device of the comparative example. In the biosensor device of this example, a current response waveform indicating insulation was observed with reference to FIG. On the other hand, in the device of the comparative example, with reference to FIG. 8B, a waveform in which a current always flowed during the application of the pulse voltage was observed. The variation (standard deviation) in the current peak value of the biosensor device of this example was about 10% when the number of samples n = 30.
From the above results, in the biosensor device of this example, the insulating artificial lipid membrane 4 can be stably formed with good reproducibility by separating the first electrolytic solution 2 and the second electrolytic solution 3 from each other. confirmed.

(Example 2)
In this example, the biosensor device according to the modified example of Embodiment 2 described above with reference to FIG. 5 was manufactured.

<1> Body Member Preparation Step First, a base material 19, an intermediate material 20, and a base material 21 were prepared. As the base materials 19 and 21, a sheet made of polyethylene terephthalate (PET) was used, and the thickness was set to 50 μm. Each of the base materials 19 and 21 was formed with a circular hole, and the diameter of each hole was 180 μm. These holes were formed using a punching machine. As the intermediate material 20, a thermosetting adhesive sheet having a thickness of 25 μm was used, a circular hole was formed, and the diameter of the hole was set to 200 μm.

  The base material 19, the intermediate material 20, and the base material 21 were aligned and laminated, and thermocompression bonded to produce a laminate having the notch 18. The depth L of the notch 18 was 10 μm.

  The other conditions were the same as in Example 1, and the laminate of the base material 19, the intermediate material 20 and the base material 21 prepared above was thermoset with a thickness of 20 μm together with the base materials 9, 10 and 12. The adhesive sheets were bonded to each other by being aligned and laminated and thermocompression bonded while being interposed between them.

  And the fluorine-type coating agent was apply | coated to the exposed surface 23 (including the surface of the notch 18) of the base material 19, the intermediate material 20, and the base material 21, and the exposed surface 23 was water-repellent-treated.

  As a result, the body member 32 shown in FIG.

<2> Supply of 1st electrolyte solution Next, 28 nL of 1st electrolyte solution was supplied to the body member 32 obtained by the above from the opening part 7a of the 2nd chamber 7 using the micropipette. The total supply amount of the first electrolyte solution was such that the volume of the first chamber 5 was satisfied and the liquid level of the first electrolyte solution coincided with the upper surface of the substrate 19. As the first electrolytic solution, a 100 mmol / L potassium chloride (KCl) aqueous solution and glycerin mixed at a volume ratio of 50%: 50% were used.

<3> Supply of Lipid Solution Then, using an ink jet device as a liquid supply device, a total amount of 400 pL of the lipid solution was supplied from the opening 7a of the second chamber 7 onto the first electrolyte solution supplied above. At this time, the amount of liquid discharged from the head part of the ink jet apparatus per one time was 20 pL, and the number of discharges was 20 times. As the lipid solution, a solution in which 10 mg of lecithin was dissolved in 1 mL of decane was used.

<4> Supply of 2nd electrolyte solution Next, 30 nL of 2nd electrolyte solution was supplied in total on the lipid solution supplied above from the opening part 7a of the 2nd chamber 7 using the micropipette. The total supply amount of the second electrolytic solution is larger than the volume of the second chamber 7, but is maintained by the surface tension. The same thing as said 1st electrolyte solution was used for the 2nd electrolyte solution 3. FIG.

  As described above, the biosensor device of this example similar to the biosensor device 41 shown in FIG. 4 was completed. The artificial lipid membrane 4 can be naturally formed between the lipid solution 1 and the first electrolyte solution 2 and the second electrolyte solution 3 without requiring an additional step.

<5> Measurement of electrical characteristics of artificial lipid membrane In the same manner as the biosensor device of Example 1, the biosensor device manufactured in this example was subjected to a pulse voltage of 20 mV (pulse width 10 ms) on the artificial lipid membrane 4. In this state, the current response flowing in the thickness direction of the artificial lipid membrane 4 was measured.

  On the other hand, as a reference example, the diameter of the hole of the intermediate member 20 was set to 180 μm similarly to the base materials 19 and 21 (therefore, the notch 18 was not formed). A device of a reference example was produced (therefore, the exposed surface without a notch was subjected to water repellent treatment, and 400 pL of lipid solution was supplied in total), and the current response of this device was measured in the same manner as described above. In addition, the biosensor device of this reference example also corresponds to what was manufactured according to the manufacturing method of the biosensor device of this invention.

FIG. 9A is a current response waveform of the biosensor device of Example 2, and FIG. 9B is a current response waveform of the biosensor device of the reference example. 9A and 9B, the current peak value of the biosensor device of this example was about 70% of the current peak value of the biosensor device of the reference example.
This is because the artificial lipid membrane formed by the biosensor device of this example having a notch on the inner wall surface of the communication part 6 is formed by the biosensor device of the reference example having no notch. It is thicker than the lipid membrane.
From the above results, in the biosensor device of this example, the lipid solution tends to stay in the communicating portion by the notch, and a thicker artificial lipid membrane is formed with the same amount of the lipid solution as in the case without the notch. It was confirmed that it can be formed. Therefore, it is understood that by providing a notch, an artificial lipid film having the same film thickness can be stably formed with less lipid solution.

  The biosensor device of the present invention can be widely used in the environment, food, housing, automobile, security field, etc., such as a biomolecule analyzer and an air pollutant analyzer. In addition, the biosensor device of the present invention can be widely used in the medical field such as lifestyle-related disease diagnosis apparatus, urine diagnosis apparatus, breath diagnosis apparatus, and stress measuring instrument, and in the healthcare field.

DESCRIPTION OF SYMBOLS 1 Lipid liquid 2 1st electrolyte solution 3 2nd electrolyte solution 4 Artificial lipid membrane 5 1st chamber 6 Communication part 7 2nd chamber 8a, 8a ', 8b, 8b' Electrode 9, 10, 11, 12, 13, 14, 17, 19, 21 Base material 15 Lipid liquid outside communication portion 16, 18 Notch 20 Thermosetting adhesive sheet 22, 23 Exposed surface 24 Inner overhang portion 30, 31, 32, 33 Body member 40, 41, 43 Biosensor Device 51, 52, 53 Liquid supply device 280 Sensor chip substrate 281 Lipid liquid 290 Sensor chip precursor 300 Sensor chip 301 Artificial lipid membrane 302 Sample liquid 303 Test tank 304 Reference electrode 306 Lead wire 307 Potentiometer 308 Well 310 Sheet 311 Base Material 312 Membrane protein 313 Reference solution 314 Electrode 350 Biosensor system

Claims (5)

A method for producing a biosensor device having an artificial lipid membrane,
Providing a body member having a first chamber, a second chamber having an opening, and a communication portion located between the first chamber and the second chamber;
The body member is supplied with the first electrolyte solution, the lipid solution for forming the artificial lipid membrane, and the second electrolyte solution sequentially from the opening of the second chamber, and the first chamber is filled with the first electrolyte solution, A method for manufacturing a biosensor device, comprising filling at least a portion of the communication portion with a lipid solution and filling the second chamber with a second electrolyte solution.   The manufacturing method of Claim 1 with which the inner wall surface of the communication part in a body member has water repellency.   The manufacturing method of Claim 1 or 2 with which the notch is formed in the inner wall face of the communication part in a body member.   The manufacturing method in any one of Claims 1-3 in which a 2nd chamber has an inner side overhang | projection part between a communication part and an opening part.   The manufacturing method in any one of Claims 1-4 which supplies at least any one liquid selected from the group which consists of a 1st electrolyte solution, a lipid solution, and a 2nd electrolyte solution in a droplet state.

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