JP5744211B2 - Isoelectric focusing cell and isoelectric focusing device - Google Patents

Description

  The present invention relates to an electrophoresis cell and an electrophoresis apparatus.

  As a technique for analyzing proteins, DNA, and the like, an analytical technique using electrophoresis is widely used in fields such as biochemistry and molecular biology. As one of the methods of electrophoresis, isoelectric focusing is known as described in, for example, Patent Document 1 and Patent Document 2 below.

  Isoelectric focusing is a method of electrophoresis that uses the difference in isoelectric point depending on the type of protein to separate and analyze proteins. The charge at the amino acid side chain, amino terminal, and carboxyl terminal constituting the protein varies depending on the pH condition, and the pH value at which the total charge becomes zero is called the isoelectric point.

  In isoelectric focusing, a solution containing an electrolyte is placed in an electrophoresis cell of an electrophoresis apparatus, and a voltage is applied to the solution to form a pH gradient. When a sample containing multiple types of proteins is added to this solution and an electric field is applied, each protein moves through a solution with a pH gradient toward the same pH as the isoelectric point unique to each protein. However, it remains at the same pH position as the isoelectric point for each protein. Thus, proteins can be separated according to the isoelectric point of each protein using the pH gradient in the solution. For example, a sample dilution solution containing carrier ampholite or surfactant and a sample such as a protein mixture, cell extract, tissue extract, or serum are added to an electrophoresis gel having a pH gradient, and approximately several thousand. When a high voltage of volt is applied to create an electrical gradient, each sample moves to a pH position corresponding to its own isoelectric point.

JP 2000-55879 A JP 2011-22238 A

  When using an isoelectric focusing apparatus and separating each sample according to the difference in isoelectric point of the sample containing protein, the degree of electroosmotic flow greatly affects the separation accuracy. Electroosmotic flow refers to the flow of solution that occurs with the apparent charging of the solution. For example, when a conventional silica electrophoresis cell as described in Patent Document 1 is used, the inner wall of the pipeline is dissociated by the dissociation of silanol groups present on the inner wall of the pipeline in which the solution is sealed. Is negatively charged. Due to this charging, the solution in the electrophoresis cell is apparently positively charged, and when the electric field is applied, the entire solution flows to the negative electrode side. This electroosmotic flow is generated not only in silica and the like but also in various conventionally used materials such as quartz glass, and deteriorates the accuracy of sample separation in the solution. For example, in Patent Document 2, the silanol group is chemically modified by pre-treating the inner wall surface in the electrophoresis cell with a linear acrylamide polymer, polyvinyl alcohol, or the like so that an electroosmotic flow is not generated by isoelectric focusing. Although described, the linear acrylamide polymer, polyvinyl alcohol, and the like were also charged due to the influence of the hydroxyl group in the molecule, and the electroosmotic flow could not be sufficiently suppressed. In addition, the use of several tens of times deteriorates the polymer component deposited by the above treatment and causes peeling, so that the number of times it can be used repeatedly is reduced.

  An object of the present invention is to provide an isoelectric focusing cell and an electrophoresis apparatus that can suppress the generation of an electroosmotic flow and can separate a plurality of samples with relatively high accuracy.

The present invention is an isoelectric focusing cell used in an isoelectric focusing device, comprising a conduit filled with a solution that can be electrophoresed, and the inner peripheral surface of the conduit is An isoelectric focusing cell comprising a sapphire single crystal is provided.

Also provided is an isoelectric focusing apparatus comprising the electrophoresis cell and a voltage applying means for applying a voltage to the solution filled in the conduit.

  According to the present invention, the generation of electroosmotic flow can be satisfactorily suppressed and the separation accuracy of the sample can be improved.

It is a schematic perspective view explaining one Embodiment of the isoelectric focusing apparatus of this invention. It is a figure explaining the electrophoresis cell with which the isoelectric focusing apparatus shown in FIG. 1 is provided, (a) is a schematic perspective view of an electrophoresis cell, (b) and (c) each comprise an electrophoresis cell. It is a schematic perspective view of a member. It is sectional drawing which expands and shows a part of isoelectric focusing apparatus shown in FIG. It is a figure explaining other embodiment of an electrophoresis cell, (a) is a schematic perspective view of an electrophoresis cell, (b) and (c) are schematic perspective views of the member which comprises an electrophoresis cell, respectively. .

  Hereinafter, an embodiment of an electrophoresis cell of the present invention and an embodiment of an electrophoresis apparatus using the electrophoresis cell will be described in detail with reference to the drawings.

  The embodiment is not limited to the following examples, and can be appropriately changed in design without departing from the gist of the present invention. In particular, conventionally known techniques can be used as appropriate for general electrophoresis.

  FIG. 1 is a schematic configuration diagram of an isoelectric focusing apparatus 1 (hereinafter also simply referred to as an apparatus 1) which is an embodiment of the isoelectric focusing apparatus of the present invention. The apparatus 1 shown in FIG. 1 is an isoelectric focusing apparatus that migrates a measurement target sample (hereinafter also simply referred to as a sample) contained in a solution 11 (not shown in FIG. 1) according to a pH gradient. . As the solution 11 used in the present embodiment, a solution obtained by mixing a plurality of samples each containing a protein with a mixed solution containing an electrolyte is used. As the mixed solution, for example, a mixed solution of a gelling agent solution that gels by light and a carrier ampholite is used. As the mixed solution, for example, a mixed solution of a gelling agent solution that gels by heat and a carrier ampholite may be used. In the following embodiment, the case where the solution 11 having a relatively low viscosity before gelation is subjected to isoelectric focusing and then the solution 11 after isoelectric focusing is gelled by light or heat will be described. ing.

  The apparatus 1 applies a voltage to the electrophoresis cell 10 provided with a conduit 16 (not shown in FIG. 1) filled with a solution 11 to be electrophoresed, and the solution 11 filled in the conduit 16. Voltage application means 30, power supply 40 for electrophoresis, light irradiation means 50 for gelation, and analysis unit 60.

  2A and 2B are diagrams for explaining an electrophoresis cell provided in the isoelectric focusing device shown in FIG. 1, wherein FIG. 2A is a schematic perspective view of the electrophoresis cell 10, and FIGS. 2B and 2C are electrophoresis. It is a schematic perspective view of the member which comprises a cell. The electrophoresis cell 10 is an electrophoresis cell used in an electrophoresis apparatus, and includes a pipe line 16 filled with a solution 11 that can be electrophoresed. An inner peripheral surface 16a of the pipe line 16 has a sapphire unit. A region composed of crystals is provided. The electrophoresis cell 10 is formed by combining four sapphire substrates. In the present embodiment, a first substrate 21 made of sapphire having a planar one main surface 21a (hereinafter also referred to as a first one main surface 21a) and a planar one main surface 22a (hereinafter referred to as a second one). Two main substrates 22 made of sapphire, each having two step portions 23 on both sides of the second first main surface 22a. The two first substrates 21 are arranged so that the first main surfaces 21a face each other in parallel, and constitute a first substrate pair 20α that is a combination of the two first substrates 21. FIG. 2B shows the first substrate 21 arranged on the upper side in FIG. 2A among the two first substrates 21 shown in FIG. The two second substrates 22 are also arranged so that the second first main surfaces 22a face each other in parallel, and constitute a second substrate pair 20β that is a combination of the two second substrates 22. .

  FIG. 2C shows the second substrate 22 arranged on the left side in FIG. 2A among the two second substrates 22 shown in FIG. The inner peripheral surface 16a of the conduit 16 of the electrophoresis cell 10 is composed of two first first main surfaces 21a of the first substrate pair 20α and two second first main surfaces 22a of the second substrate pair 20β. ing. The step 23 of the second substrate 22 includes a side surface 23 a perpendicular to the second one main surface 22 a of the second substrate 22, and the side surface 23 a faces the first one main surface 21 a of the first substrate 21 in parallel. The first one main surface 21a and the side surface 23a are joined via a joining member (not shown) such as an adhesive.

  Thus, the electrophoresis cell 10 has two substrates (the first substrate 21 and the first substrate 21) made of a sapphire single crystal in which one main surface (the first one main surface 21a and the second one main surface 22a) is opposed to each other. 2 substrate 22), a pair of substrates (first substrate pair 20α and second substrate pair 20β), and the inner peripheral surface 16a of the duct 16 has two main surfaces (first The main surface 21a and the second first main surface 22a) are configured to include at least a part of each.

  In addition, one main surface of one of the two pairs of substrates (first substrate pair 20α and second substrate pair 20β) is perpendicular to one main surface of the other substrate pair.

Both the first substrate 21 and the second substrate 22 are sapphire single crystal substrates, and the content ratio of alumina (Al ₂ O ₃ ) is 95% by mass or more. The first first main surface 21a of the first substrate 21 and the second first main surface 22a of the second substrate 22 are substantially parallel to, for example, one of crystal planes (for example, A plane or C plane) in the sapphire single crystal. ing. That is, the crystal surface of the sapphire single crystal appears on the inner peripheral surface 16a of the pipe line 16, and the electrical characteristics of the inner peripheral surface 16a of the pipe line 16 are made uniform with high accuracy, such as surface roughness, There is little variation in mechanical properties. The expression of the crystal plane of the sapphire single crystal means that the angle between the principal plane and the crystal plane is 5 ° or less in absolute value.

  As the first substrate 21 and the second substrate 22 made of sapphire single crystal, for example, a relatively large single crystal wafer in which the C-plane or A-plane of the sapphire single crystal appears on the surface is predetermined. It can be produced by cutting into a size. The step portion 23 of the second substrate 22 can be formed by grinding the end portion after cutting. The first substrate 21 and the second substrate 22 have portions corresponding to the inner peripheral surface 16a of the pipe line 16, that is, the first first main surface 21a and the second first main surface 22a are mirror surfaces. Yes. The mirror surface means that the surface arithmetic average roughness Ra is 0.8 μm or less.

  In each of the first substrate 21 and the second substrate 22, the thickness in the direction orthogonal to the inner peripheral surface of the region corresponding to the inner peripheral surface 16a of the pipe line 16 is set to 0.1 mm or more and 10 mm or less. ing. A sapphire substrate (first substrate 21 and second substrate 22) made of sapphire single crystal has relatively high mechanical strength, and even if the thickness is 0.1 mm or more and 10 mm or less, Damage such as chipping is unlikely to occur.

  In the electrophoresis cell 10 of the present embodiment, the first substrate 21 and the second substrate 22 are 75 mm in the length direction and have thicknesses in the directions orthogonal to the first first main surface 21a and the second first main surface 22a, respectively. Are both 0.5 mm. Further, the length of the first substrate 21 in the horizontal direction in FIG. 2B is 3 mm. Further, the entire length of the second substrate 22 in the vertical direction in FIG. 2C is 4.5 mm. The inner peripheral surface 16a of the conduit 16 of the electrophoresis cell 10 has a longitudinal length of 1.5 mm in FIG. 2A and a lateral length of 2 mm in FIG. .

  As shown in FIG. 2, sealing members 18 for sealing the pipeline 16 are disposed at both ends of the electrophoresis cell 10. The sealing member 18 is made of, for example, conductive plastic. The sealing member 18 is bonded to the end surfaces of the first substrate 21 and the second substrate 22 via a conductive adhesive or the like. As the sealing member 18, for example, a conductive resin film or the like may be used. In this case, the conductive resin film may be fixed to the end of the sealing member 18 by being bound by an elastomer band or the like.

  FIG. 3 is an enlarged cross-sectional view showing a part of the electrophoresis apparatus 1 shown in FIG. In the apparatus 1, voltage applying means 30 for applying a voltage to the solution 11 filled in the pipe line 16 is provided at both ends of the electrophoresis cell 10 in the length direction. The voltage applying means 30 includes an electrode liquid tank 32 filled with an electrode liquid 35 containing an electrolyte, and an electrode 31 provided on the wall surface of the electrode liquid tank 32 and in electrical contact with the electrode liquid 35. The electrode liquid tank 32 is provided with an opening 33 on one of its wall surfaces. The end of the electrophoresis cell 10 is inserted into the opening 33, and the opening 33 is closed by the electrophoresis cell 10. A seal member 37 made of resin or the like is provided on the inner peripheral surface of the opening 33. The electrode liquid 35 stored in the electrode liquid tank 32 is in contact with the electrode 31 and the sealing member 18 in the electrode liquid tank 32. That is, the electrode 31 and the sealing member 18 are electrically connected via the electrode liquid 35 in the electrode liquid tank 32. The solution 11 filled in the pipe line 16 is electrically connected to the electrode 31 through a sealing member that seals the pipe line 16. In addition, it is good also as a structure which the electrode member 35 and the solution 11 with which the inside of the pipe line 16 contacted using the sealing member provided with several through-holes, such as a porous member, for example.

  Each electrode 31 disposed at both ends of the electrophoresis cell 10 is connected to a power supply 40 for electrophoresis. The electrophoresis power source 40 sets the two electrodes 31 to different potentials and forms an electric field between the two electrodes 31, thereby forming a pH gradient in the solution 11 filled in the pipe line 16.

  Each sample contained in the solution 11 in the pipeline 16 moves the solution 11 in which a pH gradient is formed toward the same pH as the isoelectric point specific to the protein contained in each sample. It stays at the same pH position as the electrical point.

In the electrophoresis cell 10, the inner peripheral surface 16 a of the pipe line 16 is made of sapphire single crystal, and electroosmotic flow is less generated when a voltage is applied by the power supply 40 for electrophoresis. This is because the bonding between the atoms of Al ₂ O ₃ constituting the sapphire single crystal is relatively strong, and charging due to adhesion of OH groups or the like is very little.

  Since the electrophoretic cell 10 using sapphire generates less electroosmotic flow and the sample is less moved and separated by the electroosmotic flow, the sample can be separated at a specific pH position with high accuracy. Further, since the electroosmotic flow is small, even when the viscosity of the solution 11 is relatively small, minute position fluctuations of the sample according to the electroosmotic flow are suppressed, and a relatively high separation accuracy can be secured. When the electrophoresis cell 10 of this embodiment is used, the viscosity of the solution 11 can be made relatively small, so that the moving speed of the sample in the solution 11 is high, and the separation time by electrophoresis can be shortened. it can. In addition, since the sample easily moves with a small resistance even at a small voltage, the voltage at the time of separation can be reduced. When the electrophoresis cell 10 of the present embodiment is used, the sample can be separated with high accuracy in a short time without gelling the solution 11.

  In addition, sapphire has high thermal conductivity and can efficiently release the heat of the solution 11. That is, the temperature rise of the solution 11 accompanying voltage application can be suppressed, and the occurrence of measurement error factors accompanying the temperature rise such as protein denaturation accompanying the temperature rise of the solution 11 can be suppressed. As described above, when the electrophoresis cell 10 is used, the thermal conductivity is high, so that the temperature of the solution 11 being measured can be stabilized and stable separation can be obtained.

  In addition, after separating the sample for a short time in a relatively low viscosity state without gelling the solution 11 as described above, the solution 11 is gelled, so that the separated state is maintained even after the voltage application is stopped. Can be maintained for a long time. The gelation light irradiation means 50 is a light irradiation device that irradiates the solution 11 in the conduit 16 with light for gelation via the sapphire substrate 20. As described above, the solution 11 used in the present embodiment is mixed with, for example, a gelling agent solution that gels by light. From the light irradiation means 50 for gelation, light having a wavelength and intensity suitable for gelation of the gelling agent (indicated by an arrow 51 in FIG. 1) is irradiated toward the electrophoresis cell 10. The gelation light irradiation means 50 may be a known light emitting device such as a light emitting device using an LED light emitting element or a light emitting device using a laser diode element. By gelling, it becomes easy to handle the sample after separation, such as partially cutting out the region corresponding to the desired pH, and the degree of freedom of measurement after separation can be improved. The solution after gelation can be phase-transferred again and returned to the liquid phase. After cutting out, the solution may be returned to the liquid phase and the desired component may be taken out from the liquid phase.

  The electrophoresis cell 10 is made of a sapphire single crystal and is excellent in light transmittance. Further, the sapphire single crystal has high mechanical strength. For example, the thickness in the direction orthogonal to the first one main surface 21a and the main surface 22a corresponding to the inner peripheral surface 16a of the duct 16 is relatively 0.1 to 10 mm. It has been made smaller. The sapphire substrate constituting the electrophoresis cell 10 has a high light transmittance, and the solution 11 can be gelled by light irradiation for a short time. When light for gelation is irradiated for a long time, the protein of the sample to be measured may be damaged. When the electrophoresis cell 10 made of a sapphire single crystal is used, irradiation with light for gelation can be made relatively short, so that damage to proteins contained in the solution 11 can be made relatively small.

  Note that the gelation method of the solution 11 is not limited to gelation by light irradiation, and gelation by heat may be used. In the case of gelation by heat, isoelectric focusing may be performed using a mixed solution of a gelling agent solution that gels by heat and a carrier ampholite. Instead of the gelation light irradiation means 50, the apparatus 10 may be provided with a heat application means (not shown) that heats the entire electrophoresis cell 10 from the outside to raise the temperature of the solution 11 in the conduit 16. . As the heat application means, for example, a known infrared irradiation device or a heater device provided with a heating resistor may be used. In this case, sapphire has high thermal conductivity, and the heat applied from the heat application means is quickly transferred to the solution 11 in the pipe line 16, so that the gelation speed is extremely fast and the components separated during the gelation. Is less disturbed.

  The apparatus 1 can measure the characteristics of each sample after the solution 11 is gelled. The apparatus 1 includes an analysis unit 60. The analysis unit 60 uses a light irradiation unit 62 for irradiating light to the solution 11 filled in the electrophoresis cell 10, and a solution according to the irradiation light 67 from the light irradiation unit 62. A light detection unit 64 that detects light emitted from the light source 11, and the light irradiation unit 62 transmits the irradiation light 67 to the solution 11 through the electrophoresis cell 11. The analysis unit 60 also includes a calculation unit 66 that receives the signal output from the light detection unit 64, performs signal processing, and calculates a desired physical quantity. In the apparatus 1 of the present embodiment, analysis using so-called Raman spectroscopy is performed.

  The light irradiation unit 62 includes a laser light source 52 that emits laser light of a predetermined wavelength, and a laser that can move the laser light source 52 along the length direction of the conduit 16 and includes a motor, a rail, and the like. Light source moving means 54. The light irradiation unit 62 moves the laser light source 52 by the laser light source moving means 54, and applies laser light (irradiation) to the desired pH position of the solution 11 in the conduit 16 of the electrophoresis cell 10 after electrophoresis. Light 67) can be irradiated through the sapphire substrate 20. When the irradiation light 67 is irradiated on the sample contained in the solution 11 at the desired pH position (that is, separated at this position), the irradiation light 67 is scattered by the sample at the desired pH position, and scattered. Light exits from the solution 11. The scattered light includes Rayleigh scattering (elastic scattering) in which light having the same wavelength as the incident light is scattered, and Raman scattering (inelastic scattering) in which light is scattered to a wavelength different from incident light by molecular vibration.

  The light detection unit 64 outputs a signal corresponding to the wavelength distribution of the intensity of the Raman scattered light generated when the irradiation light 67 is irradiated onto the sample contained in the solution 11. The light detection unit 64 includes a Rayleigh light removal filter 53 for cutting the Rayleigh scattered light, a diffraction grating 55 that separates the Raman scattered light, and a CCD [Charge Coupled Device] that detects and outputs the dispersed light for each wavelength. And an optical sensor 57 including elements and the like. The Rayleigh light removal filter 53, the diffraction grating 55, and the optical sensor 57 are placed on the stage 59. The light detection unit 64 includes stage driving means 68 that moves the stage 59 along the longitudinal direction of the pipe line 16, and the positions of the Rayleigh light removal filter 53, the diffraction grating 55, and the optical sensor 57 are adjusted together with the stage 59. Thus, the scattered light emitted from the sample at the desired pH position can be received efficiently. The optical sensor 57 outputs an electrical signal corresponding to the received light intensity for each wavelength, and sends this electrical signal to the computing unit 66.

  The calculation unit 66 is a computer having a known CPU, receives an electric signal from the optical sensor 57, and stores various information representing physical property values of the sample at the pH position irradiated with the irradiation light 67 such as a Raman shift value. The calculated information is output from output means such as a monitor or a printer (not shown).

  The electrophoresis cell 10 is made of a sapphire single crystal and is excellent in light transmission, and transmits the irradiation light 67 irradiated from the irradiation unit 62 satisfactorily. In addition, the scattered light 69 that has been irradiated with the irradiation light 67 and is scattered and emitted by the sample contained in the solution 11 also transmits well.

  In addition, as described above, there is little fluctuation in the position of the sample after separation due to electroosmotic flow, and there is little reduction in measurement accuracy due to movement of the sample position during analysis. The isoelectric focusing device of this embodiment has a relatively high measurement accuracy by light irradiation. For example, when quartz is used as the electrophoresis cell, the inner surface may be coated with an acrylamide polymer or the like to suppress electroosmotic flow. In this case, this coating layer absorbs light of a specific wavelength. The irradiation light 67 is reflected at the interface between the coating layer and quartz. Therefore, an extra loss is likely to occur in the irradiation light 67 and the scattered light 69, and the analysis accuracy is lowered due to this loss. When the electrophoretic cell 10 is made of a sapphire single crystal, it is possible to simultaneously suppress the generation of electroosmotic flow and reduce the loss of irradiation light and scattered light. The electrophoresis apparatus of the present invention can perform various types of analysis such as so-called infrared absorption analysis as well as the above-described Raman spectroscopic analysis.

  Moreover, the electrophoresis cell 10 of this embodiment is configured by combining four sapphire substrates. In the electrophoresis cell 10 of the present embodiment, these four sapphire substrates can be disassembled and individually washed after the sample separation operation. The electrophoresis cell 10 has a small amount of residual components after analysis in the entire inner periphery 16a of the pipe line 16 including, for example, a region corresponding to a corner portion of the inner peripheral surface 16a of the pipe line 16 where the residual components are relatively easily attached. can do. In addition, a sapphire substrate made of a sapphire single crystal is less likely to be scratched on the surface, and the substrate 20 is less damaged during cleaning. The electrophoresis cell 10 of the present embodiment can be easily and accurately regenerated and reused many times.

  As the above-mentioned gelling agent that gels by light irradiation, a photopolymerizable hydrogelling agent that is water-soluble and has no charge can be used.

  Such a photopolymerizable hydrogelator solution includes, for example, a solution containing at least a polymerizable monomer, a solution containing at least a polymerizable monomer and a photopolymerization initiator, a polymerizable monomer, a crosslinkable monomer, water , Polyhydric alcohol, photopolymerization initiator, aminocarboxylic acid derivative, electrolyte, acrylic acid / methacrylic acid copolymer, pH adjuster, antiseptic, bactericidal agent, antifungal agent, rust inhibitor, antioxidant, stabilizer , Fragrances, surfactants, colorants and the like. Examples of the polymerizable monomer include a non-electrolytic acrylamide derivative, an electrolytic acrylic derivative, a non-electrolytic acrylic derivative, and examples of the cross-linkable monomer include a polyfunctional acrylamide derivative, a polyfunctional acrylic ester, and a polyfunctional allyl derivative. Is available. Moreover, not only the said example but a conventionally well-known photopolymerizable hydrogelator can also be utilized suitably.

  In addition, as a gelling agent, you may use the gelling agent which gelatinizes with temperature. As a gelling agent that gels when the temperature changes, a thermoreversible hydrogelling agent that is water-soluble and has no charge can be used. In addition, a single type of hydrogelator may be used, or a plurality of types may be used in combination. Examples of such thermoreversible hydrogelators include hydroxypropyl methylcellulose, agarose, poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, copolymers thereof, polyvinyl methyl ether, and polyvinyl alcohol partially acetylated products. Can be mentioned. Examples of thermoreversible hydrogelators include water, polyhydric alcohols, photopolymerization initiators, aminocarboxylic acid derivatives, electrolytes, acrylic acid / methacrylic acid copolymers, pH adjusters, preservatives, bactericides, An antifungal agent, an antirust agent, an antioxidant, a stabilizer, a fragrance, a surfactant, a colorant, and the like may be appropriately mixed.

  4A to 4C are diagrams for explaining an electrophoresis cell 110, which is another embodiment (second embodiment) of the electrophoresis cell. The electrophoresis cell 110 is joined to the first substrate 121 made of a sapphire single crystal having the concave portion 123 provided on the one main surface 121a, and the one main surface 121a of the first substrate 121. A second substrate 122 made of a sapphire single crystal having one main surface 122a that closes the opening of the first substrate 121, and the inner peripheral surface of the duct 126 is the same as the inner peripheral surface of the concave portion 123 of the first substrate 121. It is formed by a part of one main surface 122a of the second substrate 122. The one main surface 121a of the first substrate 121 and the one main surface 122a of the second substrate 122 are joined by an adhesive (not shown).

  The pipe line 126 is closed by a conductive sealing member 128. Also in the electrophoresis cell 120, the thickness of the first substrate 121 and the second substrate 122 in the region corresponding to the inner peripheral surface of the pipe 126 is perpendicular to the inner peripheral surface is 0.1 mm or more and 10 mm. It is as follows. In the sapphire substrate 120, the part corresponding to the inner peripheral surface of the pipe 126 is mirror-polished.

  Also in the electrophoresis cell 60 of the second embodiment, the electroosmotic flow is hardly generated, and the sample can be separated with high accuracy by the electric field. Moreover, the electrophoresis cell 60 has high light permeability, and can perform desired analysis with high accuracy by light irradiation. The electrophoresis cell 60 of the second embodiment has a relatively high mechanical strength and a relatively high long-term reliability performance.

  As described above, isoelectric focusing can be performed with high accuracy and in a short time by using the electrophoresis cell of the present embodiment.

  The isoelectric focusing described above is used as the first-dimensional electrophoresis, and then, for example, the second-dimensional sample is separated according to the molecular weight by so-called SDS-PAGE [Sodium dodecyl sulfate-polyacrylamide gel electrophoresis]. Separation may be performed. The electrophoresis cell 10 of the present embodiment can also be suitably used for the first-dimensional separation when performing such second-dimensional separation.

  In the case of using the electrophoresis cell 10 in which the inner peripheral surface of the pipe line 16 is sapphire, in the first-dimensional isoelectric focusing, the sample is separated with high accuracy while maintaining a low viscosity solution without gelation. can do.

  For example, when the first-dimensional electrophoresis is performed using a conventional electrophoresis cell made of quartz or the like, the separation accuracy is suppressed by suppressing excessive migration due to electroosmotic flow during the first-dimensional isophoresis. In order to increase the viscosity, the solution was gelled so that the viscosity was relatively high. When performing second-dimensional electrophoresis on a solution that has been gelled to such a high viscosity, the viscosity of the solution is reduced by, for example, complicated treatment with various chemical solutions so that the solution has a certain low viscosity suitable for second-dimensional electrophoresis. It was necessary to make adjustments. In such adjustment of the viscosity, for example, it was necessary to remove the electrophoresis cell from the apparatus and handle it by human hands to sequentially perform various processes. In the electrophoresis apparatus of the present embodiment, the first-dimensional electrophoresis is performed in a short time using the solution 11 having a relatively low viscosity, and the second-dimensional electrophoresis is performed as it is using the solution 11 having a low viscosity. Can also be done. By using the electrophoresis apparatus of the present embodiment, the first-dimensional electrophoresis and the second-dimensional electrophoresis can be continuously performed without performing complicated chemical solution processing by hand.

  Electrophoresis was performed using the electrophoresis apparatus shown in FIG. A solution containing 5% agarose, 2% HPMC [hydroxypropyl methyl cellulose], and pI-Marker [Marker Protein for pH Gradient Determination of polyacrylamide Gel Isoelectric Separation] is filled in an electrophoresis cell made of a sapphire single crystal shown in FIG. Electrophoresis was performed. The applied voltage applied to the solution 11 in the electrophoresis cell 10 is 100 V applied at the start of electrophoresis, increased by 100 V per minute to 500 V after 5 minutes, and maintained at 500 V until 40 minutes after starting. did. When the change from 10 minutes to 40 minutes after the start of electrophoresis was observed for the central position of the sample assembly position confirmed by the color development of the marker, when the electrophoresis cell 10 using a sapphire single crystal was used, The variation of ± 1 mm or more in the assembly position could not be confirmed.

  On the other hand, when an electrophoresis apparatus having the same configuration as in FIG. 1 was used, only the electrophoresis cell was changed to an electrophoresis cell made of transparent plastic, and electrophoresis was performed under the same conditions as above, it was confirmed by the color of the marker. The central position of the sample collection position continued to fluctuate slowly without being fixed at a specific position over a range of ± 1 cm from 10 minutes to 40 minutes after the start of electrophoresis. That is, in the conventional electrophoresis cell, the sample position continued to fluctuate due to the influence of electroosmotic flow.

  As mentioned above, although one Embodiment of this invention was described in detail, this invention is not limited to the said embodiment. For example, in the above embodiment, the isoelectric focusing device has been described as an example. However, the present invention is not limited to isoelectric focusing, and various types of electrophoresis can be performed. Single crystal sapphire has little electroosmotic flow, and the influence of electroosmotic flow can be reduced in any electrophoresis. Of course, various improvements and modifications may be made without departing from the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 Apparatus 10,110 Electrophoresis cell 16,126 Pipe line 18 Sealing member 20 Sapphire substrate 21 1st board | substrate 21a One main surface (1st one main surface)
22a One main surface (second one main surface)
22 Second substrate 23 Stepped portion 23a Side surface 30 Voltage application means 31 Electrode 32 Electrode liquid tank 33 Aperture 40 Power supply device for electrophoresis 50 Light irradiation means for gelation 60 Analysis unit 62 Light irradiation unit 64 Light detection unit 66 Calculation unit

Claims (8)

An isoelectric focusing cell used in an isoelectric focusing apparatus,
A cell for isoelectric focusing, comprising a conduit filled with a solution that can be electrophoresed, wherein the inner peripheral surface of the conduit is made of a sapphire single crystal. A substrate pair consisting of a combination of two substrates made of sapphire single crystals with one main surface facing each other,
2. The isoelectric focusing cell according to claim 1, wherein an inner peripheral surface of the conduit includes at least a part of each of the two opposing main surfaces of the pair of substrates.   3. The isoelectric focusing cell according to claim 1, wherein the substrate has a thickness of 0.1 mm or more and 10 mm or less perpendicular to the main surface.   The pair of substrate pairs, wherein the main surface of one of the two substrate pairs is perpendicular to the main surface of the other substrate pair. 4. An isoelectric focusing cell according to 3. On the other hand, a first substrate made of a sapphire single crystal provided with a recess on the main surface;
A second substrate provided with one main surface bonded to the one main surface of the first substrate and closing the opening of the concave portion,
2. The inner peripheral surface of the conduit is formed by an inner peripheral surface of the concave portion of the first substrate and a part of the one main surface of the second substrate. The isoelectric focusing cell as described.   The thickness of the first substrate and the second substrate perpendicular to the inner peripheral surface in a portion corresponding to the pipe line is 0.1 mm or more and 10 mm or less. Cell for isoelectric focusing. The electrophoresis cell according to any one of claims 1 to 6,
An isoelectric focusing device comprising: a voltage applying means for applying a voltage to the solution filled in the conduit. A light irradiation unit for irradiating the solution filled in the electrophoresis cell with light through the electrophoresis cell;
The isoelectric focusing device according to claim 7, further comprising: a light detecting unit that detects light emitted from the solution through the electrophoretic cell in accordance with irradiation light from the light irradiation unit. apparatus.

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