JP2008232927A - Protein concentrating device, protein measuring device, and air conditioner using these - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for easily and rapidly concentrating the protein contained in liquid to be inspected. <P>SOLUTION: This device for concentrating the protein contained in the liquid to be inspected comprises a first space having a large volume, a second space having a volume smaller than that of the first space, a partition for partitioning the first space and the second space and passing the protein, an electric field generating means for generating a direct current electric field from the first space toward the second space, and a discharge port for discharging liquid contained in the second space. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein concentrator that concentrates a test solution containing a specific protein into a high-concentration protein solution.

  Immunoassay using antigen-antibody reaction is known as a useful analysis / measurement method in the medical field, biochemistry field, measurement field such as allergen, etc., but conventional immunoassay is complicated in operation. The problem is that analysis requires a long time.

  In order to solve this problem, Patent Document 1 proposes a technique for applying a micro-channel device having a micro-order channel formed on a substrate to immunoassay. However, since the technique of Patent Document 1 uses a thermal lens method as a detection method, there is a problem that the device becomes large.

  On the other hand, Patent Document 2 proposes a technique using an electrochemical detection method instead of the thermal lens method. The technique of Patent Document 2 has an advantage that the microchannel device can be easily downsized. An electrochemical detection type microchannel device according to this technique will be described with reference to the drawings.

  As shown in FIG. 13, a microchannel injection hole 504A, a discharge hole 505A, a microreaction vessel 503, and microchannels 504 and 505 are formed on the surface of the substrate 501, and their depth is about 100 μm, The width of the channel is about 200 μm. Between the microreaction vessel 503 and the flow path 505, a damming portion 508 having a flow path having a width smaller than the diameter of the fine particles 502 is formed so that the fine particles 502 immobilizing the antibody are dammed. is there. In addition, an electrode 506, a connection pad 507, and a wiring 509 that electrically connects them are formed in a desired pattern on the microchannel 505.

  A detection method using this device will be described. For example, a test solution containing an allergen is injected from an injection hole 504A using an external pump or the like, and an antigen-antibody reaction is performed with the microparticle 502 on which an antibody that specifically reacts with the allergen is immobilized, thereby capturing the allergen on the surface of the microparticle. .

  After washing with a buffer solution, a solution containing an antibody labeled with an enzyme is injected from the injection hole 504A to form a complex composed of an immobilized antibody-allergen-enzyme-attached antibody on the surface of the fine particles.

  After washing with a buffer solution, a substrate material that is converted into an electrochemically active substance by an enzyme is flowed, and converted into an electrochemically active substance by an enzyme of the complex. By detecting the electrochemically active substance in the region where the electrode 506 is formed from the connection pad 507 as a change in current or potential, the concentration of the substance to be detected in a small amount of sample liquid can be known.

  Such an antigen or antibody to be subjected to immunoassay is extracted from a specimen containing them. For example, in the case of pollen, Patent Document 3 proposes a technique related to an automatic pollen collecting device. Moreover, the technique which extracts allergen from the collected pollen is proposed by patent document 4 or 5. FIG.

JP 2001-4628 A JP 2003-285298 A JP-A-5-215653 JP-A-6-329553 Special table 2003-507432 gazette

  Next, pollen collection, protein extraction, and protein detection methods according to the prior art will be described. Collect pollen on a glass slide with petrolatum thinly and evenly on the surface or on an adhesive tape wrapped around a drum. The collected pollen is put into a buffer solution and the antigen is extracted from the pollen. Thereafter, pollen is separated by centrifugation, and a buffer solution (supernatant) containing the antigen is taken out and used as an analysis sample.

  Then, the analysis sample is injected into the electrochemical detection type microchannel device, and the amount of the electrochemically active substance corresponding to the amount of allergen is measured by the above method.

  In the measurement method according to the conventional technique described above, a large amount (about 10 to 1000 ml) of the extraction solution is used when extracting the protein from the specimen, so that the protein concentration of the extract is lowered. For this reason, when this extract is injected into a microchannel device as a test liquid (usually 1 to 500 μl) and the amount of protein in the test liquid is measured, the protein concentration is too low for accurate measurement. There is a case.

  In order to solve this problem, a method of increasing the absolute amount of protein by injecting a large amount of protein extract into the microchannel device may be considered, but the microchannel device is usually used at a flow rate of about 6 μl / min. However, if a protein extract of about 10 to 1000 ml is caused to flow through the microchannel, it will take about 30 to 2800 hours to finish the flow. Therefore, this method is not practical.

  Therefore, there is a need for a technique for simply and quickly concentrating a low concentration protein extract without heating or the like.

  In the prior art, a sample is collected by a method in which a substance containing allergen floating in the air (for example, pollen) is attached to the surface of a glass slide or an adhesive tape, and then put into an extraction solution to extract proteins. However, this method requires a long time to measure the protein. For this reason, in order to perform simple and quick measurement, it is necessary to automate a series of these steps, but no protein measuring apparatus that automates these steps has yet been provided.

  Therefore, it is desired to develop an apparatus capable of automatically performing from sample collection to antigen extraction, protein concentration, and measurement.

  A series of the present invention aims to solve the above-mentioned problems. 1st invention for solving the said subject is the apparatus which concentrates the protein in a test liquid, Comprising: The 1st space where volume is large, The 2nd space where volume is smaller than the said 1st space, A partition that separates the first space and the second space and allows protein to pass through, an electric field generating means that generates a direct current electric field from the first space toward the second space, and the second space And a discharge port for discharging the liquid contained in the protein concentration device.

  The partition in the above configuration allows the protein to pass through, but also allows the protein extraction solution to pass through along with the protein. This is because a protein extraction solution has an overwhelmingly smaller molecular weight than protein. The first space and the second space are partitioned through such a partition. In the state where no electric field is applied, both protein and solution can pass through the partition, so that the protein concentration in the first space and the second space gradually becomes uniform, but the electric field generating means in the above configuration is driven. In the state, the electric field acting from the first space toward the second space accelerates the movement of the protein to the second space. Therefore, protein concentration in the second space is increased.

  This will be described in more detail. Since a protein is charged in a solution having a pH other than its isoelectric point, when a direct current electric field is applied to the protein in this state, the protein moves in the direction of the opposite charge (protein electrophoresis). Since this movement is a movement in one direction controlled by an electric field, the protein concentration in one space (second space) can be increased with the partition as a boundary. That is, when a direct current electric field is applied from the first space to the second space so that the electric field generating means acts on the second space side, the charge opposite to the charge of the protein that is the target of concentration is applied to the first space. Since the protein in the protein extraction solution present in is moved to the second space, the protein concentration in the second space is higher (concentration) than in the first space. Moreover, in the above configuration, since the volume of the second space is smaller than the volume of the first space, the concentration effect by the application of the electric field is efficiently exhibited.

  From the above, according to the above configuration, the protein extract can be concentrated very efficiently without using means such as heating and decompression. By using this concentrated protein extract as a test solution for protein detection, accuracy can be improved. High protein content can be measured.

  In the first invention, the electric field generating means may include a pair of electrodes arranged in a state of facing the first space and the second space.

  When a pair of electrodes arranged in a state of facing the first space and the second space are used, the movement distance of protein molecules in the first space can be reduced. Therefore, protein molecules in the first space move efficiently to the second space side.

  In the first invention group, the volume ratio of the second space to the first space may be 1/20 to 1/1000000.

  With this configuration, since there is a sufficient difference in the space volume between the first space and the second space, an extremely high concentration effect can be obtained.

  In the first invention group, the volume of the first space may be 10 to 1000 ml, and the volume of the second space may be 2 to 60 μl.

  With this configuration, since both space volumes are sufficiently small, it is easy to exert an electric field effect, and the time required for concentration can be shortened. Here, in order to reliably extract protein from the protein-containing substance, it is preferable that the amount of the extraction solution is 2 ml or more. However, even if the amount of the extraction solution is larger than 600 ml, the extraction rate is almost improved. However, as the amount of the extracted solution increases, the cost increases and the handleability deteriorates. In order to increase the protein detection efficiency, it is preferable to fill the first space with the extraction solution. Therefore, the volume of the first space is preferably 10 to 1000 ml, more preferably 20 to 200 ml.

  In addition, in order to properly detect a protein using a microchannel device, it is appropriate that the volume of the test solution used for detection is 500 μl or less, and therefore the volume of the second space is preferably 500 μl or less. And On the other hand, if the volume of the second space is smaller than 1 μl, the volume of the second space is too small, so that the protein that can move to the second space is reduced and it is difficult to perform sufficient concentration. Furthermore, the volume of the second space is preferably 1 to 500 μl, more preferably 5 to 20 μl.

  In the first invention group, the partition may have a plurality of holes having a hole diameter of 0.001 to 100 μm.

  If the pore diameter is 0.001 μm, protein and extraction solution can pass through, and if the pore diameter is 100 μm or less, insoluble matter such as pollen shells can be sufficiently blocked. It is possible to concentrate the protein extraction solution while preventing contamination. The optimum hole diameter for preventing insoluble matter from mixing into the second space should be determined by the size of the target specimen. Usually, the hole diameter of the partition is preferably 1 to 100 μm, more preferably 0. .01 to 20 μm. Examples of the partition through which proteins and extraction solutions can pass and can block insoluble matters such as pollen shells include agarose gel and polyacrylamide gel, and membrane filters.

In the first invention group, the second space may be formed of a micro flow channel having an area of a cross section perpendicular to the flow direction of 400 to 250,000 μm ² .

When the second space is constituted by a micro flow channel having a cross-sectional area of 400 to 250,000 μm ² , a protein concentrating device that is excellent in concentration efficiency and easy to take out the concentrated solution is realized.

  In the first invention group, the first space may be configured to have a function of extracting a protein by immersing a specimen in an extraction solution.

  In the above configuration, the partition may have a filter function that prevents a specimen from passing therethrough.

  If the sample is mixed in the second space, the detection sensitivity may be lowered in the subsequent detection. Therefore, it is preferable that the partition has a function of preventing the specimen from moving to the second space.

  The second aspect of the present invention for solving the above-described problem is that a movable carrier that carries a specimen collected from an external space and a protein extracted from the specimen carried on the carrier into a solution. , A protein concentration apparatus comprising: an extraction concentration unit for concentrating the protein extraction solution; and a moving means for moving the carrier to the extraction concentration unit, wherein the extraction concentration unit has a first volume increase. A partition that divides the space, the second space having a volume smaller than that of the first space, the first space, and the second space and allows protein to pass from the first space to the second space. On the other hand, it is a protein concentrating device having an electric field generating means for generating a DC electric field and a discharge port for discharging the liquid contained in the second space.

  According to the above configuration, the sample collected from the external space is carried by the carrier, and this is carried to the first space by the moving means. Since the volume of the first space is sufficiently large, the protein can be extracted from the specimen by allowing the protein extraction solution to exist in the first space. The extracted protein is divided into the first space, the second space, and the like. And is concentrated on the second space side by the cooperative action of the partition for allowing the protein to pass through and the electric field generating means. Therefore, with this configuration, operations from collection of a sample from an external space to protein extraction and protein concentration can be performed with one apparatus.

  In the configuration of the second invention, the extraction and concentration unit includes an extraction unit that extracts the protein contained in the specimen into a solution, a concentration unit that concentrates the protein extraction solution extracted by the extraction unit, and the extraction unit. And a flow path connecting the concentrating part.

  In this configuration, the extraction unit and the concentration unit are provided separately, and both are connected by a flow path. However, with this structure, the extraction unit has a means for increasing the protein extraction efficiency and the protein concentration efficiency. There is an advantage that it becomes easy to incorporate additional means such as means. Additional embodiments will be described later.

  In the second aspect of the invention, the movably supported carrier can also serve as the partition.

  With this configuration, the device structure can be simplified.

  In the second invention group, the extraction and concentration unit includes a first container having an opening and a second container having an opening and an openable / closable discharge port, and at least one of the first container and the second container One is movable, and the openings of both containers are in contact with each other, or the openings of both containers are integrated with each other in contact with each other with the carrier interposed therebetween. The first space and the second space are formed, and the partition may be provided in the second container.

  In order to extract protein from a sample carried on a carrier, it is convenient to immerse the whole carrier in a protein extraction solution. However, if liquid leakage occurs, problems such as a decrease in extraction rate may occur. . On the other hand, if the carrier itself is hermetically fixed in the space, the carrier cannot be moved. Therefore, in the above-described configuration, the first space and the second space of the extraction and concentration unit can be formed by the first container and the second container, at least one of which is movable.

  In this configuration, the carrier part carrying the specimen is moved from time to time to be positioned at the opening of the second container, and the specimen is removed from the carrier into the second container for sample withdrawal. By moving it with a jig or the like, and then moving and contacting the first container, a sealed space can be formed in a state in which the specimen is put. In this configuration, the extraction solution may be placed in the second container in which the specimen is placed in advance.

  Further, in the case of another configuration, the carrier part carrying the specimen is moved as needed, and is positioned at the part where the openings of the first container and the second container face each other. In this state, the first container and the second container By moving either one or both of the containers in a direction in which the openings are in contact with each other, a sealed space can be formed in a state in which the carrier portion carrying the specimen is included.

  In any configuration, if the protein extraction solution is put into the first space formed as described above (or put in advance), the protein can be extracted without leakage. . The concentrated solution can be collected by opening the openable / closable outlet of the second container.

  In the configuration using the first container and the second container, the carrier interposed between the first container and the second container may also serve as the partition. With this configuration, the apparatus can be simplified and it is not necessary to provide a partition in the minute second container, so that the second container can be easily created.

  In the configuration in which the first container and the second container are provided, container driving means for moving the first container and / or the second container is provided in order to bring the openings into contact with each other or to separate the two from each other. be able to. Moreover, in order to improve the sealing degree of the internal space formed by contact | abutting, the member which improves adhesiveness, such as a rubber material, can be arrange | positioned in both openings.

  In the second invention group, the extraction unit includes a cutting unit that cuts the carrier, an adsorption plate that adsorbs the cut carrier, and a vibration unit that vibrates the adsorption plate. can do.

  According to this configuration, the extraction efficiency of the protein from the specimen can be increased.

  In the second invention group, the extraction unit may include an ultrasonic generator.

  According to this configuration, the protein extraction efficiency from the specimen is further increased.

  Moreover, the said 2nd invention group WHEREIN: The said extraction part shall be equipped with a micro heater.

  According to this configuration, the extraction efficiency of the protein from the specimen is increased.

  In the second invention group, the protein concentrating device further includes pH adjusting means for injecting a pH adjusting solution into a flow path connecting the extracting unit and the concentrating unit to adjust the solution pH in the concentrating unit. can do.

  As the extraction solution, it is preferable to use a molecule having a high extraction efficiency or a solution having a pH. However, the pH of the extraction solution having a high extraction efficiency may be close to the isoelectric point of the protein. Electrophoresis cannot be performed efficiently. With this configuration, the pH of the extraction solution can be adjusted to a pH different from the isoelectric point of the protein after extraction, so that the protein is surely charged in the concentration section, and the concentration efficiency can be increased. . Examples of the pH adjusting means include means for adding acid or alkali to the solution while monitoring the pH of the extraction solution.

  In the second invention group, the protein concentrating device further includes an electric field increasing substance adding means for adding a charge increasing substance that increases the charge of the protein to a flow path connecting the extraction section and the concentration section. be able to.

  Some proteins have little charge in solution, and such proteins are difficult to concentrate using electrophoresis. When a charge-enhancing substance is added to such a protein, the charge-enhancing substance is adsorbed on the protein and the charge is increased. Thereby, since electrophoresis can be caused, protein can be concentrated. Examples of the charge enhancing substance include an anionic surfactant.

  Moreover, in the said 2nd invention group, the said support body is a strip | belt shape, The said moving means shall be provided with the roller which advances the said strip | belt-shaped support body, and the motor which drives the said roller. Can do.

  When the shape of the carrier is a belt and the carrier is moved by a roller equipped with a motor, the efficiency from sample collection to extraction can be improved and automation is facilitated. However, the configuration is not limited to this, and for example, a system in which a carrier is placed on a movable belt may be used.

  In the second invention group, the protein concentrating device further comprises: marking means for marking the band-shaped carrier for specifying a specimen-carrying region; and mark detection means for detecting the marking. It can be set as the structure provided.

  In order to extract protein from the specimen carried on the carrier, it is necessary to reliably move the specimen carrying area of the carrier to the extraction concentration unit or the extraction unit. As a method for this purpose, there is a method of controlling the moving speed and time to regulate the moving distance, but it is more certain that the method is to mark the specimen carrying area and detect it. In the marking method, a plurality of specimens can be collected continuously by intermittently moving the belt-shaped carrier, and a plurality of specimen types (for example, specimen 1 and specimen 2) can be selected by the mark detection means. Identify and correlate protein extraction and measurement.

  In the second invention group, the protein concentrating device has a container driving means for driving the first container and / or the second container, and further controls operations of the container driving means and the moving means. An operation control unit, and when the marking is detected by the mark detection unit, the operation control unit stops the operation of the moving unit, and drives the first container and / or the second container to drive the first container. And the second container can be integrated.

  In this configuration, since the operation control unit controls the container driving unit and the moving unit in an integrated manner, the convenience of the apparatus is significantly improved. The above control can be performed by arranging a computer in the operation control unit and programming and storing the operation in this computer in advance.

  In the second invention group, the same configuration as in the first invention group can be added. That is, the electric field generating means may include a pair of electrodes disposed in a state of facing the first space and the second space.

  The volume ratio of the second space to the first space may be 1/20 to 1/1000000.

  The volume of the first space may be 10 to 1000 ml, and the volume of the second space may be 1 to 500 μl.

  Further, the partition may have a plurality of holes having a hole diameter of 0.001 to 100 μm.

In addition, the second space may have a microchannel structure in which an area of a cross section perpendicular to the flow direction is 400 to 250,000 μm ² .

The third aspect of the present invention for solving the above-described problems relates to a protein measuring apparatus incorporating the protein concentrating apparatus according to the above-described second invention group. The configuration is as follows.
A protein concentrator having any one of the configurations of the second invention group, and a microchannel device for measuring the type or amount of the protein concentrated by the protein concentrator, the microchannel device comprising: A protein measuring apparatus, comprising: a reaction channel; a reactant fixed in the reaction channel; and a detection unit that detects the amount of protein.

  According to this configuration, operations from sample collection from external space to protein extraction, protein concentration, and protein detection can be performed with a single device, so protein type identification and content measurement The work efficiency is greatly improved.

  As the microchannel device, for example, a device including a reaction channel, a reactant fixed in the reaction channel, and a detection unit (electrode) as shown in FIG. 13 can be used.

The 4th present invention for solving the above-mentioned subject relates to the air conditioner incorporating the protein measuring device concerning the 3rd above-mentioned invention. The configuration is as follows.
The protein measuring apparatus according to the third aspect of the present invention, a blowing means for sending outside air outside the apparatus to the inside of the apparatus, and the amount of blowing of the blowing means is increased or decreased according to the amount of protein contained in the outside air measured by the protein measuring apparatus. An air conditioner comprising: an air blowing control means.

  According to this configuration, the amount of blast can be changed according to the amount of detected protein. For example, when the amount of allergen protein is large, the amount of the sample in the outside air can be reduced by controlling the air flow rate so that the sample containing the allergen protein is adsorbed to the filter in the air conditioner. Moreover, when there is little allergen protein, the ventilation volume can be reduced and the running cost of an apparatus can be reduced.

The fifth present invention for solving the above-mentioned problems relates to a further aspect of an air conditioner incorporating the above-described protein measuring apparatus according to the third invention. The configuration is as follows.
The protein measuring device according to the third invention, the protein-containing material removing unit for removing the protein-containing material, and the removal for controlling the operation of the protein-containing material removing unit according to the protein amount measured by the protein measuring device An air conditioner comprising: an operation control unit.

  According to this configuration, the operation of the sample removing unit is changed according to the amount of the detected protein, so that the protein removing operation is made appropriate. The specimen removing unit includes, for example, a negative ion generation unit or plasma generation unit and an adsorption unit that adsorbs negative ion ions or plasma. When the protein measuring device detects the protein, the removal operation control means controls the negative ion generator to generate negative ions. As a result, the protein-containing substance is negatively charged and is adsorbed and removed by the negative ion adsorption portion. Further, the removal operation control means controls the negative ion generator so as not to generate negative ions, for example, when no allergen protein is detected, thereby reducing the running cost of the apparatus.

  As described above, according to the protein concentration apparatus of the present invention, a protein solution can be efficiently concentrated in a short time. Moreover, since the protein concentration apparatus of the present invention does not use the heat evaporation method or the reduced pressure evaporation method when concentrating the protein solution, protein denaturation does not occur. Such a protein concentration apparatus of the present invention is suitable as a pretreatment apparatus for identifying or quantifying proteins.

  Moreover, according to the protein measuring apparatus of the present invention, it is possible to continuously collect samples such as pollen and microorganisms, extract proteins, concentrate proteins, and identify and quantify proteins. According to such a protein measuring apparatus of the present invention, from sample collection to protein identification or quantification can be performed simply, rapidly and with high accuracy.

  Moreover, according to the air conditioner of the present invention, it is possible to adjust the air in the room or the like while monitoring the presence / absence and the abundance of protein, and thereby, for example, an effect that a comfortable living environment can be maintained. can get.

  The best mode for carrying out the present invention will be described below in detail with reference to the drawings.

[Embodiment 1]
A protein concentration apparatus according to this embodiment will be described with reference to FIG.

  As shown in FIG. 1 (a), the protein concentrator 100 includes a first space 1 having a volume of 10 to 1000 ml, a second space 2 having a volume of 1 to 500 μl, a first space 1 and a second space 2. And a partition 3 for partitioning. The first space 1 and the second space 2 are provided with electrodes 4 and 5, respectively, and the electrodes 4 and 5 are connected to a constant voltage generator 6 that applies a DC voltage by a wiring 7. These electrodes 4, 5, constant voltage generator 6 and wiring 7 constitute an electric field generating means.

  The protein concentrating device 100 includes a discharge channel (discharge port) 8 that discharges the concentrated protein solution from the second space, and an injection channel 9 that sends the liquid to the second space. The injection channel 9 is provided with valves 11 and 12, respectively. A liquid container 15 is provided upstream of the injection channel 9.

  The protein concentrator 100 also includes a protein solution injection channel (injection port) 13 for injecting a protein solution into the first container, and the protein solution injection channel 13 is also provided with a valve 14.

  Here, the relationship between the first space and the second space will be described. The first space is a space containing a protein extract before concentration, and the second space is a space containing a concentrated protein concentrate. In order to efficiently concentrate proteins, it is necessary to make the volume of the second space smaller than the volume of the first space. The volume ratio of the second space to the first space is preferably 1/20 to 1/1000000. If the volume ratio of the two is less than 1/20, sufficient concentration cannot be achieved. On the other hand, if the volume ratio exceeds 1/1000000, the second space is too small and it becomes difficult to form a device, and the volume difference is On the contrary, the concentration efficiency becomes worse because it is too large. For these reasons, the volume ratio of the two is more preferably 1/1000 to 1/100000, and even more preferably 1/2000 to 1/10000.

  Further, the specific contents of the apparatus will be described. FIG. 1B is a cross-sectional view taken along the line A-A ′ of FIG. As shown in this cross-sectional view, the protein concentrating device 100 can be configured by a device main body 101 and a lid 102. Resin, glass, or the like can be used as the material of the apparatus main body and the lid. As the constant voltage generator 6 and the wiring 7, known ones can be used. Moreover, as the partition 3, an agarose gel, polyacrylamide gel, a filter, etc. can be used. Here, since it is difficult to directly fix the agarose gel or the polyacrylamide gel to the apparatus main body 101 and the lid 102, a resin partition is fixed between the partition 3, the apparatus main body 101, and the lid 102. It is preferable to interpose the jig 16.

  Next, the usage method of this protein concentration apparatus is demonstrated. First, the valve 11 is closed, the valves 12 and 14 are opened, and the casein solution is injected into the protein concentrator 100 from the liquid container 15 through the injection channel 9. At this time, the casein solution passes through the partition 3 and is also injected into the first space 1. As a result, the entire interior of the protein concentrating device 100 is washed, and a nonspecific adsorption preventing film for preventing nonspecific adsorption of proteins is formed by casein. Thereafter, the valve 14 is closed, the valve 11 is opened, air is sent from the injection channel 9, and the casein solution in the protein concentrator 100 is discharged.

  The valve 12 is closed, the valves 11 and 14 are opened, and Tris (tris (hydroxymethyl) aminomethane) hydrochloric acid buffer (pH about 6) containing Cry-J1 (cedar pollen allergen protein) is supplied from the protein injection channel 13 to the first. Put in space 1. At this time, the buffer solution passes through the partition 3 and the second space 2 is also filled with the buffer solution.

  Here, the isoelectric point of Cry-J1 is about 9, and Cry-J1 is negatively charged in this buffer solution.

  Next, after closing the valves 11 and 14 and sealing the apparatus, a DC voltage of 0.1 to 10 kV is applied using the constant voltage generator 6 so that the electrode 5 becomes a positive electrode and the electrode 4 becomes a negative electrode. By this voltage application, Cry-J1 negatively charged in the Tris hydrochloric acid buffer moves toward the electrode having the opposite polarity, that is, toward the positive electrode 5 side (second space side). As a result, the amount of Cry-J1 included in the first space 1 decreases, and the amount of Cry-J1 included in the second space 2 increases. That is, Cry-J1 is concentrated in the second space 2 having a smaller volume than the first space.

  This voltage application may be continuous or intermittent, but is preferably intermittent from the viewpoint of the structural stability of the protein. The voltage application time may be about 5 to 60 minutes.

  Thereafter, the valve 11 is opened, and the Cry-J1 solution concentrated in the second space is taken out.

  As described above, according to the present embodiment, the protein solution can be concentrated by a simple method. When this concentrated solution is used for detection by a known method, the amount of protein can be detected reliably in a short time.

[Embodiment 2]
In Embodiment 2, an extraction solution containing a specimen is used instead of the protein solution, and a partition having a function of preventing the specimen from passing is used. Specifically, in this embodiment, cedar pollen was planned as a specimen (target for protein extraction). Therefore, a membrane filter having a mesh size of 24 μm through which cedar pollen having a general size does not pass was used as a partition. Since other than this is the same as in the first embodiment, the apparent structure of the protein concentrator is the same as in the first embodiment. Therefore, the present embodiment will be described below with reference to FIG.

  First, the valve 11 is closed, the valves 12 and 14 are opened, and the casein solution is injected into the protein concentrator 100 from the liquid container 15 through the injection channel 9. At this time, the casein solution passes through the partition 3 and is also injected into the first space 1. As a result, the entire interior of the protein concentrating device 100 is washed, and a nonspecific adsorption preventing film for preventing nonspecific adsorption of proteins is formed by casein. Thereafter, the valve 14 is closed, the valve 11 is opened, air is sent from the injection channel 9, and the casein solution in the protein concentrator 100 is discharged.

  Next, the valve 12 is closed, the valves 11 and 14 are opened, and a mixed solution of an ammonium hydrogen carbonate solution containing an allergen protein of cedar pollen and a Tris (tris (hydroxymethyl) aminomethane) hydrochloric acid buffer (pH about 6), The protein is introduced into the first space 1 from the protein injection channel 13. At this time, the buffer solution passes through the partition 3 and the second space 2 is also filled with the buffer solution.

  Allergen proteins contained in cedar pollen are particularly easily extracted into an ammonium bicarbonate solution. Therefore, allergen protein (for example, Cry-J1) is extracted into the extraction solution. Other proteins are extracted at the same time. The isoelectric point of Cry-J1 is about 9, and Cry-J1 is negatively charged in this buffer solution.

  Next, the valve 11 is closed, and a voltage of 1 to 20 kV is applied using the constant voltage generator 6 so that the electrode 5 becomes a positive electrode and the electrode 4 becomes a negative electrode. By this voltage application, the negatively charged Cry-J1 moves toward the opposite polarity electrode, that is, the positive electrode 5 side. As a result, the amount of Cry-J1 included in the first space 1 decreases, and the amount of Cry-J1 included in the second space 2 increases. That is, Cry-J1 is concentrated in the second space 2 having a smaller volume than the first space. As in the case of the first embodiment, this voltage application may be continuous or intermittent. The voltage application time may be about 5 to 60 minutes.

  Thereafter, the valve 11 is opened, and the solution containing Cry-J1 concentrated in the second space is taken out.

  As described above, according to the second embodiment, proteins can be extracted and concentrated from a specimen by a simple method. When this concentrated solution is used for detection by a known method, the amount of protein can be detected reliably in a short time.

[Embodiment 3]
A protein concentration apparatus according to the third embodiment will be described with reference to FIGS.

  In Embodiment 3, pollen will be described as an example of a sample, but it is needless to say that the present invention can be applied to samples other than pollen (for example, airborne substances such as mites).

  As shown in FIGS. 2 and 3, the protein extraction and concentration apparatus 300 includes a pollen collection unit 50, an extraction and concentration unit 60, a carrier 10 that carries pollen, and a moving unit 70 a to move the carrier. 70c. The shape of the carrier 10 is a belt shape (belt shape) and is wound around the roller 70a. And when this strip | belt-shaped carrier 10 is wound up by the roller 70c via the roller 70b, the carrier 10 moves.

  The pollen collecting unit 50 includes a carrier 10 that collects the pollen 20 and a roller 70a around which the carrier 10 is wound.

  As the carrier 10, filter paper, film, mesh (materials such as stainless steel, copper, nickel, nylon, polyester, and carbon can be used) can be used. An agent may be coated. A hole may be formed in the carrier 10, but a hole having a size through which pollen passes is not preferable. Since the particle diameter of cedar pollen is about 30 μm, the pore diameter is preferably less than 30 μm.

  In the present embodiment, the belt-like carrier 10 has a role of pollen adhesion and also serves as a carrier carrier that is wound around a roller. However, the carrier and a carrier belt that moves the carrier are used. , May be formed separately. In this case, for example, a section-shaped carrier having an appropriate area is attached to or placed on the belt surface.

  The conveying belt only needs to be able to move the carrier efficiently, and any shape may be used as long as this is the case. For example, the conveyor belt can be a mesh-like one.

  Also, for example, the conveyor belt is a rubber or resin belt wider than the container opening, and a section-like carrier smaller than the container opening area is placed on or attached to the belt in the container opening region. In this state, the container is overlapped so that the openings of both containers sandwich the belt. With this method, since a rubber band is interposed in the overlapping portion of both containers, a highly sealed space without liquid leakage can be easily formed.

  The extraction and concentration unit 60 includes a first container 31 and a second container 32, and the carrier 10 passes between the first container 31 and the second container 32. The carrier 10 is installed and conveyed along a guide groove 45 (see FIG. 3) provided in the first container 31. The conveying speed is 0 (stopped state) during pollen collection and protein extraction, and when it is conveyed from the pollen collection unit 50 to the concentration extraction unit 60, it may be conveyed at an arbitrary speed. However, it should be noted that the carrier 10 may be broken if the conveyance speed is increased too much. Preferably, it is set to about 1 to 1000 mm / second.

  Moreover, the 1st space 1 is formed when the 1st container 31 and the 2nd container 32 overlap. The second container 32 is provided with a second space 2 and a partition 3 that partitions the first space 1 and the second space 2. The first space 1 and the second space 2 are provided with electrodes 4 and 5 facing each other, and are wired to a constant voltage generator (not shown) that applies a DC voltage to the electrodes 4 and 5. (Not shown).

The extraction and concentration unit 60 includes a discharge flow path (discharge port) 8 that discharges the protein solution concentrated from the second space, and an injection flow path 9 that sends the liquid to the second space. The injection channel 9 is provided with valves 11 and 12, respectively.
The extraction concentration unit 60 includes an extraction liquid injection channel 18 that injects an extraction liquid into the first container, and the extraction liquid injection channel 18 is also provided with a valve 19.

  The partition 3 only needs to be able to pass at least allergen without passing through pollen, and the material can be polyacrylamide gel, agarose gel, filter, mesh, etc. (materials include stainless steel, copper, nickel, nylon, polyester, carbon, etc.) ) Can be used. Further, a fence-like filter having a width of 0.002 to 30 μm and a width of 20 μm to 1 mm may be used. In the case of a filter, the hole diameter of the hole formed in the filter may be 0.002 to 30 μm.

  As shown in FIGS. 2 and 3, in the present invention, the volume of the first space 1 is designed to have a capacity for containing an extraction solution necessary for allergen extraction from pollen, preferably 10 to 1000 ml. On the other hand, the volume of the second space 2 is preferably designed to be 1 to 500 μl.

  Here, since the volume of the 2nd space 2 is very small, there exists a possibility that the distance of the partition 3 and the electrode 5 may become very small. In order to ensure a sufficient distance between the partition 3 and the electrode 5, it is preferable to employ a structure in which the side surface of the second space 2 is inclined to reduce its internal volume (see FIG. 2).

Further, in order to reduce the volume of the second space 2 and reliably discharge the protein solution concentrated in the second space to the discharge channel 8, the second space has a size of 20 to 5000 μm as shown in FIG. A meandering microchannel 23 having a width of 20 to 50 μm (cross-sectional area of 400 to 250,000 μm ² ) may be used. In FIG. 4, the electrode 5 is formed in a portion other than the microchannel 23, but the electrode 5 may be formed only on the bottom surface of the microchannel 23. As a method for forming the channel 23 and the electrode 5, a conventional photolithography processing method, a hot embossing method, or the like can be used. When the second space 2 is formed with the configuration shown in FIGS. 2 and 3, it is necessary to increase the design values of the electrode 5 area and the upper area of the second container 32 to some extent. However, if the configuration in which the second space 2 is used as a microchannel as shown in FIG. 4 is adopted, the second space with a minute volume can be easily obtained. There is a merit that the design of the second container 32 can be afforded and the processing becomes easy.

  In the vicinity of the pollen collection unit 50, a pollen collection region marker forming unit 22a is provided, and in the vicinity of the extraction and concentration unit 60, a pollen collection region marker detector 22b is provided. The pollen collection region marker forming unit 22a performs marking for specifying the region (pollen adhering portion) 21 where the pollen is collected on the surface of the carrier 10 by a method such as making a hole in the carrier 10 or applying colored ink. . The marker thus formed is detected by the pollen collection region marker detector 22b provided in the vicinity of the extraction and concentration unit 60, and as will be described later, the operation control unit 35 and the carrier driving unit 42 as shown in FIG. Then, the motor 33 is stopped to ensure that the pollen adhering portion 21 is taken into the extraction and concentration unit.

  This marker can include information such as the date and time of pollen collection and the number of times. For example, information is recorded according to the number of holes to be formed, and the information is also read at the time of detection and recorded in the operation control unit, so that a correlation between the extracted and concentrated liquid and pollen collection date and time described later can be obtained. .

  Next, the steps of collecting pollen, extracting and condensing allergen using the protein extraction and concentration apparatus according to the present embodiment will be described with reference to FIGS. The following operations are programmed in advance in the operation control unit 35 in FIG.

(Collecting pollen)
By operation of a switch (not shown) or the like, a signal for driving the motor 33 is input from the input unit 34 to the operation control unit 35, and the motor 33 is driven from the operation control unit 35 via the carrier driving unit 42, and pollen The carrier 10 to which no pollen is attached is transported to a predetermined position where the pollen is taken in. After driving for a predetermined time, a motor stop signal from the operation control unit 35 enters the motor 33 via the carrier driving unit 42 and stops the motor 33. When the motor 33 is stopped, in order to identify the area where pollen adheres, the carrier 10 located in the vicinity of the pollen collection part 50 is marked using a separately provided pollen collection area marker (area detector) 22a. . As a method of attaching the mark, a conventional method such as coloring the carrier with ink or the like or making a hole in the carrier can be used. Thereafter, air is taken in for a predetermined time, and pollen in the air is adhered to the carrier 4 (FIG. 2).

(Conveyance to extraction and concentration unit)
Upon receiving a signal from the operation control unit, the motor 33 is driven through the carrier driving unit 42, the carrier 10 is wound around the roller 70c, and the pollen adhering portion 21 is sent to the extraction and concentration unit 60 through the roller 70b. It is done.

  When the pollen 20 attached to the carrier 10 enters the extraction / concentration unit 60, the pollen collection region marker detector (region detector) 22b detects the mark attached by the pollen collection region marker device (region detector) 22a. Then, the signal enters the operation control unit 35 through the marker detection unit 40, a stop signal is input from the operation control unit 35 to the motor 33 through the carrier driving unit 42, and the roller 70c stops (see FIG. 6A). ).

(Unit integration)
After the roller 70c is stopped, a signal is input from the operation control unit 35 to the first container driving unit 41, the driving motor 45 (not shown in FIG. 2) is operated, and the first container 31 of the extraction and concentration unit 60 is driven. Thus, the apparatus is in close contact with the second container 32 and the apparatus is closed. The timing at which the motor 33 is stopped and the first container 31 is closed may be the mark as described above, and is managed by the conveyance time calculated from the conveyance speed and the distance between the adhesion region 21 and the extraction concentration unit installation position. May be. The driving of the first container 31 can be controlled using a conventional sensor or the like, for example, by a method of detecting a change in driving force when the container is in a closed state.

(Injection of extraction solution)
A signal from the operation control unit 35 is sent to the valve drive unit 37, the valve 12 is opened, and the extraction solution is injected into the container from the injection flow path 9. At this time, the valve 19 is opened simultaneously, air is discharged from the first space 1 and the second space 2, and the container is filled with the extraction solution. When the container is filled with the extraction solution, a signal from the operation control unit 35 is sent to the valve drive unit 37, and the valves 12 and 19 are closed. By leaving in this state for 5 to 120 minutes, the allergen 24 is extracted from the pollen into the extraction solution (see FIG. 6B). The opening / closing timing of the valves 19 and 20 is controlled by time.

  It goes without saying that proteins other than allergen 24 are simultaneously dissolved in the extraction solution. In addition, the extraction solution is previously adjusted from the pollen and adjusted to a pH different from the isoelectric point of the allergen 24, or is different from the isoelectric point of the allergen 24 by adding acid or alkali after extraction. Adjust the pH. As a result, the allergen 24 is charged in the extraction solution.

(Protein concentration)
Next, a signal from the operation control unit 35 is sent to the voltage application unit 36, and a DC voltage is applied to the electrodes 4 and 5. At this time, a polarity opposite to the charge of the allergen 24 is applied to the electrode 5. Then, all the proteins having the same polarity as the allergen 24 and the allergen 24 in the extraction solution are moved from the first space 1 to the second space 2 through the partition 3 by the electrophoresis, and the second space 2 In this state, the allergen 24 is concentrated (see FIG. 4C). The voltage is preferably 0.1 to 10 kV, and the application time is 5 to 60 minutes. However, since these conditions depend on the protein to be concentrated, they should be set accordingly. In addition, when protein movement is slow, heat may be generated and side reactions may occur. In this case, it is preferable to apply intermittently.

  While applying the voltage or with the application stopped, a signal from the operation control unit 35 is sent to the valve driving unit 37, the valves 11 and 12 are opened, the buffer solution is injected from the injection channel 9, and the discharge flow A buffer containing a high concentration of allergen 24 is taken out from the path 8. The volume of the allergen high concentration buffer is substantially the same as the volume of the second space 2.

  That is, when the apparatus of the present invention is used, the volume of the allergen-containing buffer solution injected into the analysis microchannel device is about 1 to 500 μl, and when the flow rate is 6 μl / min, the volume is fixed to the analysis microchannel device. The time required to pass through the antibody region is about 0.2 to 90 minutes, which is advantageous in that it is significantly shortened compared to the conventional case.

(Washing the container)
A signal from the operation control unit 35 is sent to the valve drive unit 37, the valves 12 and 19 are opened, a cleaning buffer solution is injected from the injection flow path 9, and the first container 1 and the second container 2 are cleaned. To do. Thereafter, a signal from the operation control unit 35 is sent to the air introduction pump drive unit 43, and air is sent from the air introduction pump 44 into the container to dry the first container 1 and the second container 2. This completes a series of operations of pollen collection, protein extraction, and protein concentration.

  In the conventional method, pollen collection, protein extraction, and protein concentration need to be performed, respectively, and it is necessary to use a large apparatus such as a centrifuge, and there is a problem that automation is difficult. Then, by previously programming the device operation procedure in the operation control unit 35, it is possible to repeatedly perform pollen collection and concentration as described above. Further, by utilizing the pollen collection area marker information, it is possible to associate the collection time with the concentrate.

  Also, from the time when the roller stops, pollen can be collected next time and a sample can be obtained over time.

[Embodiment 4]
As shown in FIG. 7, the present embodiment has a configuration in which the carrier 10 is used as a partition, and the rest is the same as in the third embodiment.

  As shown in FIG. 7, the protein extraction and concentration apparatus 300 includes a pollen collection unit 50, an extraction and concentration unit 60, a carrier 10 that carries pollen, and moving means 70a to 70c that move the carrier, It has. The shape of the carrier 10 is a belt shape and is wound around the roller 70a. And when this strip | belt-shaped carrier 10 is wound up by the roller 70c via the roller 70b, the carrier 10 moves.

  The pollen collecting unit 50 includes a carrier 10 that collects the pollen 20 and a roller 70a around which the carrier 10 is wound.

  The carrier 10 can be made of filter paper, film, mesh (materials such as stainless steel, copper, nickel, nylon, polyester, carbon, etc.) can be used, and an adhesive is coated on the surface to facilitate the application of pollen. May be. However, since the carrier 10 has a configuration that also serves as a partition, it needs to have a property that pollen does not pass through and protein passes through. In the present embodiment, the carrier 10 serves as a conveyance belt and pollen adhesion, but the conveyance belt may be formed separately. For example, the transport belt may have a mesh shape.

  The extraction and concentration unit 60 includes a first container 31 and a second container 32, and the carrier 10 passes between the first container 31 and the second container 32. The carrier 10 is installed and conveyed along a guide groove 45 (not shown) provided in the first container 31. The conveying speed is 0 (stopped state) during pollen collection and protein extraction, and when it is conveyed from the pollen collection unit 50 to the concentration extraction unit 60, it may be conveyed at an arbitrary speed. However, it should be noted that the carrier 10 may be broken if the conveyance speed is increased too much. Preferably, it is set to about 1 to 1000 mm / second.

  In the extraction and concentration unit 60, when the first container 31 and the second container 32 are integrated, the carrier 10 serves as a partition to form the first space 1 and the second space 2. Electrodes 4 and 5 are provided in a state of being opposed to each other in the first space and the second space, and a constant voltage generator (not shown) for applying a DC voltage to the electrodes 4 and 5 is provided. They are connected by wiring (not shown).

  The extraction and concentration unit 60 includes a discharge channel 8 that discharges the concentrated protein solution from the second space, and an injection channel 9 that sends the liquid to the second space. Are provided with valves 11 and 12, respectively. The extraction concentration unit 60 includes an extraction liquid injection channel 18 that injects an extraction liquid into the first container, and the extraction liquid injection channel 18 is also provided with a valve 19.

  The method of using the apparatus according to the fourth embodiment may be the same as that of the third embodiment.

  In the configurations described in the third and fourth embodiments, the carrier 10 is taken into the space 1 and the allergen 24 is extracted from the pollen 20 into the extraction solution. However, depending on the material of the carrier 10, the allergen 24 is used. May cause non-specific adsorption to the carrier 10 and the amount of allergen extracted into the extraction solution may be reduced. In order to prevent non-specific adsorption, the pollen removal jig 49 provided in the first container 31 is vibrated as in the apparatus shown in FIG. 8A to remove the pollen 20 attached to the carrier 10. After dropping into the first space 1 of the second container 32, the upper part of the second container is then closed with a support 48 provided with the electrode 4 as shown in FIG. It can be made the structure which produces.

  In this apparatus, the extraction solution is the next pollen at the timing when the carrier 10 with pollen is carried to the extraction and concentration unit, for example, when the marker is detected by the pollen collection region marker detector 22b and the carrier 10 is stopped. May be injected into the second container 32 from the injection channel 18 before entering the operation of removing the pollen, or may be injected after the pollen 20 is dropped into the second container 32. In this apparatus structure, since the carrier 10 does not contact the extraction solution, the allergen 24 does not adsorb nonspecifically to the carrier 10.

  After the first space 1 is sealed, if the extract is not contained in the first container in advance, extraction and concentration are performed in the steps after (injection of extraction solution) described in the third embodiment. Further, even when the extract is preliminarily contained, after the extract is further injected and the first space and the second space are filled with the extract according to (Injection of extraction solution) of the third embodiment, According to the process, an extraction and concentration process is performed. The difference from the configuration of Embodiment 3 (injection of extraction solution) onward is only the absence of the carrier 10 in the second container and the position of the injection flow path 18.

[Embodiment 5]
As shown in FIG. 9, the present embodiment is substantially the same as the third embodiment except that the extraction unit and the concentration unit are separately formed.

  As shown in FIG. 10, the protein extraction and concentration apparatus 300 includes a pollen collection unit 50, an extraction unit 80, a concentration unit 90, a carrier 10 that carries pollen, and a moving means 70a that moves the carrier. ~ 70c. The shape of the carrier 10 is a belt shape and is wound around the roller 70a. And when this strip | belt-shaped carrier 10 is wound up by the roller 70c via the roller 70b, the carrier 10 moves.

  The pollen collecting unit 50 includes a carrier 10 that collects the pollen 20 and a roller 70a around which the carrier 10 is wound.

  The carrier 10 can be made of filter paper, film, mesh (materials such as stainless steel, copper, nickel, nylon, polyester, carbon, etc.) can be used, and an adhesive is coated on the surface to facilitate the application of pollen. May be. Although holes may be formed in the carrier 10, the hole diameter needs to satisfy the condition that pollen does not pass through.

  As shown in FIG. 9, the extraction unit 80 includes a first container 31 and a second container 32, and the carrier 10 passes between the first container 31 and the second container 32. The first container 31 is provided with a suction plate 25 that cuts the carrier 10 into a circular shape or a quadrilateral shape and sucks the cut portion, and the second container 32 is provided with a base 28. However, these are provided for the purpose of increasing extraction efficiency, and are not necessary for extraction solutions, specimens, and allergens that can be extracted quickly.

  The concentrating unit 90 has the same configuration as that of the third embodiment except that the concentrating container 26 is integrally formed, and the first space 1 and the second space 2 are provided across the partition 3. ing.

  The extraction unit 80 and the concentration unit 90 are connected by a flow path 27 including a valve 29.

FIG. 10 shows a process of performing extraction and concentration using the apparatus according to this embodiment. The following operations are controlled by the operation control unit (see FIG. 5).
First, the region where the pollen of the carrier 10 is attached is carried to the extraction unit 70 (see FIG. 10A).

  The 1st container 31 and the 2nd container 32 are match | combined (refer FIG.10 (b)).

  The extraction solution is sent to the extraction unit. At the same time as this work or after this work, the area where the pollen is attached is cut out by the suction plate 25 (see 9 (c)).

  The cut portion 10a of the carrier 10 is pulled to the suction plate 25 (see FIG. 10D).

  At this time, for example, by causing the adsorption plate to vibrate up and down, pollen adhering to the carrier 10a is dispersed in the buffer solution, and the buffer solution is also stirred, so that the allergen extraction efficiency is improved. is there. In this case, the suction effect may be enhanced by driving the suction plate such as vibration.

  The allergen 24 thus extracted is injected together with the pollen 3 into the concentrating unit 90 through the injection flow path 27 (see FIG. 10 (e)).

  A DC voltage is applied to the electrode, and the protein is concentrated by electrophoresis (see FIG. 10F). In this case, the detection time shortening effect due to the concentration is a value close to the volume ratio of the first space 1 and the second space 2 of the concentration unit.

  In the configuration in which the extraction unit 80 and the concentration unit 90 are separated as described above, the extraction unit 80 can be provided with means for increasing protein extraction efficiency, such as an ultrasonic generator or a micro heater.

  Further, since another means can be incorporated between the extraction unit and the concentration unit, for example, when the pH of the buffer solution with high extraction efficiency and the isoelectric point of the allergen are close, the extraction unit and the concentration unit PH adjusting means for adjusting the pH of the solution can be connected to the flow path connecting the two.

  In addition, in normal electrophoresis, in order to enable movement of a protein with a small amount of charge or no charge, a charge-enhancing substance adding means for adding a protein and an anionic surfactant such as sodium dodecyl sulfate (SDS) Can also be incorporated.

[Embodiment 6]
In the present embodiment, a protein measuring apparatus in which the protein concentrating device according to the third embodiment and a protein detection microchannel device are integrated will be described.

  FIG. 11 is a schematic diagram of the protein measuring apparatus according to the present embodiment. The protein measuring apparatus has a configuration in which a protein extraction and concentration apparatus 300 and a protein detection microchannel device 500 are connected by a pipe.

  This protein concentrator 300 is the same as that in the third embodiment. Therefore, the description of the structure and usage is omitted.

  This protein detection microchannel device 500 is formed by superposing a substrate 501 on which a channel 504 is formed, an injection hole 504A, a discharge hole 505A, and a substrate 510 on which an electrochemical detection electrode 506 is formed. .

  The flow path 504 is filled with beads 502 to which an antibody is immobilized, and a damming portion 508 for blocking the beads 502 is formed. In addition, a pipe connected to the sample collection and protein extraction and concentration apparatus 300 is fixed to the injection hole 504A by a joint 511.

  In addition, a pipe 302 connected to the cleaning liquid, the labeled antibody solution, and the substrate solution storage unit 301 is connected to the pipe.

  A method of using this apparatus will be described. First, in the same manner as in the third embodiment, sample collection, allergen extraction, and allergen concentration are performed.

  Thereafter, the allergen concentrate is injected from the injection hole 504A of the microchannel device 500. Thereby, the allergen contained in the allergen concentrate and the antibody fixed to the beads 502 react to capture the allergen.

  Thereafter, the cleaning liquid is flowed to clean the flow path 504.

  Thereafter, the enzyme-labeled antibody solution is injected from the injection hole 504A of the microchannel device 500. As a result, the enzyme-labeled antibody reacts with the antibody-antigen complex immobilized on the beads 502, and the enzyme-labeled antibody is captured.

  Thereafter, the cleaning liquid is flowed to clean the flow path 504.

  Thereafter, the substrate solution is injected from the injection hole 504A of the microchannel device 500. Thereby, a substrate and an enzyme react and an electrochemically active substance is produced.

The amount of the electrochemically active substance is detected by the electrode 506.
In addition, in the sample collection and protein extraction and concentration apparatus 300, the microchannel device 500, and the channel that connects them, the surface of the wall in contact with the protein-containing solution is subjected to a film or treatment for preventing nonspecific adsorption of the protein. Needless to say.

  According to the present embodiment, the operation from the collection of the specimen to the detection of the allergen amount can be performed with one apparatus, and the working efficiency is dramatically improved.

[Embodiment 7]
In the present embodiment, the protein measuring apparatus according to the sixth embodiment described above is incorporated into an air cleaner.

  FIG. 12 is a schematic diagram of the air cleaner according to the present embodiment. The air cleaner in FIG. 12 has a main body 201.

  As shown in FIG. 12, the interior of the main body 201 is partitioned forward and backward by a partition plate 202. As a result, a first compartment 203 is formed in front of the main body 201 (upward from the partition plate 202), and a second compartment 204 is formed in the rear (downward from the partition plate 202). ing. The first compartment 203 and the second compartment 204 communicate with each other via a single or a plurality of openings provided in the partition plate 202. In addition, an air inlet 205 for air from the outside is provided in front of the main body 201, and an air outlet 206 for discharging the sucked air to the outside is provided on the rear upper surface of the main body 201.

  The suction port 205 includes a plurality of suction ports. In the present embodiment, the suction port 205 is composed of a first suction port 205 a and a second suction port 205 b, which are arranged side by side on the front surface of the main body 201.

  In the main body 201, a filter 207, an ion generator 208, a blower 209, and an on-off valve 210 are provided.

  The filter 207 passes the air sucked from the outside and cleans it, and is provided in the first compartment 203. The filter 207 includes a dust collection filter and a deodorizing filter. The dust collection filter is for removing dust contained in the air, and the deodorization filter is for removing odor components in the air.

  The filter 207 is provided inside the main body 201 so that only air sucked from any one of the suction ports passes. In particular, in the present embodiment, only air sucked from the first suction port 205a is passed. It is provided just behind (leeward side) the first suction port 205a so as to pass through. That is, the filter 207 is not disposed behind the second suction port 205b. Thereby, as a path | route of the air suck | inhaled inside the main body 201, the path | route (henceforth the 1st path | route A) inhaled from the 1st inlet 205a and passing the filter 207, and the 2nd inlet 205b Thus, there are two paths, a path that is sucked in through the filter 207 and does not pass through the filter 207 (hereinafter referred to as a second path B).

  The ion generator 208 includes a discharge electrode and an induction electrode that are arranged to face each other via a dielectric, and generates ions by discharging between these electrodes. It is provided in the vicinity of 206. The ion generator 208 can generate at least one of positive and negative ions. If both positive and negative ions are generated from the ion generator 208, these ions can inactivate airborne bacteria in the air and remove harmful substances in the air.

  The air blowing means 209 discharges the air sucked from outside through the ion generator 208, and has a fan 209a and a motor 209b that rotates the fan 209a. The fan 209 a is provided in the second compartment 4, and the motor 209 b is provided across the first compartment 3 and the second compartment 4.

  The on-off valve 210 is provided immediately behind the second suction port 205b, and opens and closes the second suction port 205b by its own opening / closing operation.

  The sample collection and protein separation / extraction / concentration apparatus 300 is provided at a predetermined location including the suction port 205b, and a conventionally proposed allergen detection microchannel device 500 is inserted under the suction port 205b, for example. An insertion port 205c is provided. When the allergen detection microchannel device 500 is inserted from the insertion port 205c, the allergen detection microchip 500 and the sample collection, protein separation extraction and concentration apparatus 300 of the present invention are connected. This connected state is the same as in the sixth embodiment.

  When the air cleaner is activated, air that does not pass through the filter 207 is sent to the specimen collection and protein separation extraction concentration apparatus 300. As described in Embodiment 3 and Embodiment 6, pollen is collected, protein is extracted, protein is concentrated, and the amount of protein is detected.

  Depending on the amount of protein detected, the rotation amount (fan amount) of the fan 209a and the ion generation amount of the ion generator are controlled.

  Here, the allergen-detecting microchannel device 500 is used a plurality of times after use by performing a regeneration process. The allergen-detecting microchannel device 500 with reduced sensitivity is taken out from the insertion port 205c, and a new allergen-detecting microchannel device 500 is inserted.

  In the present embodiment, the application example of the sample collection and protein separation extraction concentration detection device of the present invention has been described by taking an air cleaner as an example, but it can be applied to an air conditioner such as an air conditioner, a humidifier, and a dehumidifier. Needless to say.

(Example 1)
A protein separation extraction concentration device 100 having a shape as shown in FIG. The apparatus 100 includes a container 101 and a lid 102, and the material of the container 101 and the lid 102 is acrylic resin.

  The first space 1 has a volume of 1 ml, and the second space 2 has a volume of 70 μl. The first space 1 and the second space 2 are partitioned by a partition 3 made of a filter having a hole diameter of about 1 μm fixed to the fixing jig 16. The first space 1 and the second space 2 are provided with electrodes 4 and 5 arranged to face each other, and this is connected to the constant voltage generator 6 through the wiring 7.

  In the second space 2, a discharge channel 8 and an injection channel 9 are provided, and valves 11 and 12 are provided in both channels. The first space 1 is provided with an extraction liquid injection channel 13, and a valve 14 is also provided in this channel.

  First, the valve 11 is closed, the valve 12 and the valve 14 are opened, the casein solution is injected into the container 100 from the tube 109 using the syringe 116, and the air in the container is removed through the extract injection channel 13. Then, the inside of the container 100 is washed by continuing to flow the liquid, and a nonspecific adsorption preventing film is formed inside the container.

  The valve 12 is closed, the valve 11 and the valve 14 are opened, and Tris hydrochloric acid buffer solution (pH about 6) and ammonium hydrogen carbonate solution (total 1.5 ml) containing 10 mg of cedar pollen are passed through the extract injection channel 13. , Put in the first space 1. At this time, the second space 2 is also filled with the solution through the filter.

  Next, the valve 11 and the valve 14 were closed, and a voltage of 7 kV was applied using the constant voltage generator 6 so that the electrode 5 became a positive electrode and the electrode 4 became a negative electrode. The application was continued for 1 minute and then intermittently applied for 20 minutes (the total application time was 10 minutes).

  Next, the valve 11 and the valve 12 were opened, and the solution in the second space 2 was extracted from the discharge channel 8 by flowing a buffer solution from the injection channel 9.

  About 0.1 ml of the concentrated solution was treated with sodium dodecyl sulfate (SDS), then subjected to electrophoresis using acrylamide gel, and the gel after electrophoresis was stained with Coomassie Brilliant Blue (CBB). A dark spot could be confirmed at the position of Cry-J1, which is the allergen.

  From this, it can be seen that the target protein can be efficiently concentrated by using the apparatus according to this example.

  As described above, according to the present invention, a protein concentrator capable of efficiently concentrating a target protein can be realized. In addition, operations from collection of a sample contained in an external space such as pollen to protein extraction and protein concentration can be efficiently performed with one apparatus. Therefore, its industrial significance is great.

FIG. 1 is a schematic diagram for explaining the outline of the protein concentrating device according to the first embodiment. FIG. 2 is a schematic diagram for explaining the outline of the protein extraction and concentration apparatus according to the third embodiment. FIG. 3 is a partially cutaway perspective view for explaining the outline of the protein extraction and concentration apparatus according to the third embodiment. FIG. 4 is a schematic diagram showing a configuration when the second space is a microchannel. It is a block diagram which shows operation control of the protein extraction concentration apparatus concerning Embodiment 3. FIG. FIG. 6 is a schematic diagram for explaining how to use the protein extraction and concentration apparatus according to the third embodiment. FIG. 7 is a schematic diagram for explaining the outline of the protein extraction and concentration apparatus according to the fourth embodiment. FIG. 8 is a schematic diagram illustrating a modification of the protein extraction and concentration apparatus. FIG. 9 is a schematic diagram for explaining the outline of the protein extraction and concentration apparatus according to the fifth embodiment. FIG. 10 is a schematic diagram for explaining a method of using the quality extraction and concentration apparatus according to the fifth embodiment. FIG. 11 is a schematic diagram for explaining the outline of the protein measuring apparatus according to the sixth embodiment. FIG. 12 is a schematic diagram for explaining the outline of the air cleaner according to the seventh embodiment. 13A and 13B are schematic diagrams for explaining an overview of a conventional allergen detection microchip. FIG. 13A is a plan view, and FIG. 13B is a cross-sectional view taken along line XY in FIG. .

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st space 2 2nd space 3 Partition 4 Electrode 5 Electrode 6 Constant voltage generator 7 Wiring 8 Discharge flow path 9 Injection flow path 10 Carrier 11 Valve 12 Valve 13 Protein solution injection flow path (extract liquid injection flow path)
14 Valve 15 Liquid container 16 Partition fixing jig 18 Buffer solution injection channel 19 Valve 20 Pollen (specimen)
21 Pollen adhesion part 22a Marker generator 22b Marker detector 24 Protein (allergen)
25 Adsorbing plate 26 Concentration container 27 Channel 28 Stand 29 Valve 30 Valve 31 First container 32 Second container 33 Motor 34 Input unit 35 Operation control unit 36 Voltage application unit 37 Valve drive unit 38 Liquid feed pump drive unit 39 Marker formation unit 40 Marker detection unit 41 First container drive unit 42 Carrier drive unit 43 Air introduction pump drive unit 44 Air introduction pump 45 Drive motor 46 Liquid feed pump 48 Support body 49 Pollen removal jig 50 Sample (pollen) collection unit 60 Extraction / concentration unit 70 Moving means 80 Extraction unit 90 Concentration unit 100 Protein concentration device 101 Device main body 102 Lid 201 Main body 202 Partition plate 205a First suction port 205b Second suction port 206 Outlet 207 Filter 208 Ion generator 209a Fan 209b Motor 210 On-off valve 300 Protein extraction and concentration device 500 Protein detection microchannel device 501 substrate 502 antibody with the beads 503 reaction part 504 flow path 505 flow path 504A introducing hole 505A discharge hole 508 damming portion 507 connecting the pad 509 interconnect 510 lid 511 Joint

Claims (31)

An apparatus for concentrating proteins in a test solution,
A first space with a large volume;
A second space having a smaller volume than the first space;
A partition that separates the first space and the second space and allows protein to pass through;
Electric field generating means for generating a DC electric field from the first space toward the second space;
A discharge port for discharging the liquid contained in the second space;
A protein concentrating device comprising: The protein concentrator according to claim 1,
The electric field generating means has a pair of electrodes arranged in a state of facing the first space and the second space.
A protein concentrator characterized by that. The protein concentrator according to claim 1,
The volume ratio of the second space to the first space is 1/20 to 1/1000000.
A protein concentrator characterized by that. The protein concentrator according to claim 1,
The volume of the first space is 10 to 1000 ml;
The volume of the second space is 1 to 500 μl;
A protein concentrator characterized by that. The protein extraction and concentration apparatus according to claim 1,
The partition has a plurality of holes having a hole diameter of 0.002 to 60 μm.
A protein extraction and concentration device characterized by that. The protein concentrator according to claim 1,
The second space is composed of a micro flow channel having a cross-sectional area perpendicular to the flow direction of 400 to 250,000 μm ² .
A protein concentrator characterized by that. The protein concentrator according to any one of claims 1 to 6,
The first space has a function of extracting a protein by immersing a specimen in an extraction solution.
A protein concentrator characterized by that. The protein concentrator according to claim 7,
The partition has a filter function that does not allow the specimen to pass through,
A protein concentrator characterized by that. A movable carrier carrying a sample collected from an external space;
An extraction and concentration unit for extracting the protein from the specimen carried on the carrier into a solution and concentrating the protein extraction solution;
Moving means for moving the carrier to the extraction and concentration unit;
A protein concentrating device comprising:
The extraction and concentration unit includes:
A first space with a large volume;
A second space having a smaller volume than the first space;
A partition that separates the first space and the second space and allows protein to pass through;
Electric field generating means for generating a DC electric field from the first space toward the second space;
A discharge port for discharging the liquid contained in the second space;
Having
Protein concentrator. The protein concentrator according to claim 8,
The extraction and concentration unit includes:
An extraction unit for extracting the protein contained in the specimen into a solution;
A concentration unit for concentrating the protein extraction solution extracted by the extraction unit;
A flow path connecting the extraction unit and the concentration unit;
A protein concentrating device comprising: The protein concentrator according to claim 8 or 9,
The movably supported carrier also serves as the partition;
A protein concentrator characterized by that. The protein concentrator according to claim 9,
The extraction and concentration unit has a first container having an opening and a second container having an opening and an openable / closable discharge port, and at least one of the first container and the second container is movable, It is a structure in which the first space and the second space are formed by integrating the openings of both containers by facing each other in a state where the carrier is interposed therebetween, and
The partition is provided in the second container;
A protein concentrator characterized by that. The protein concentrator according to claim 9,
The extraction and concentration unit has a first container having an opening and a second container having an opening and an openable / closable discharge port, and at least one of the first container and the second container is movable, It is a structure in which the first space and the second space are formed by being integrated by contacting the openings of both containers,
The partition is provided in the second container;
A protein concentrator characterized by that. The protein concentrator according to claim 11,
The extraction and concentration unit has a first container having an opening and a second container having an opening and an openable / closable discharge port, and at least one of the first container and the second container is movable, It is a structure in which the first space and the second space are formed by integrating the openings of the two containers facing each other in a state where the carrier is interposed,
A carrier interposed between the first container and the second container also serves as the partition;
A protein concentrator characterized by that. The protein concentrator according to claim 10,
Each of the extraction units has an opening, and at least one of the extraction parts has a movable first container and a second container, and the openings of the first container and the second container are the above-mentioned carriers. It is a structure that faces and abuts with each other with the holding body interposed therebetween, and is integrated.
A protein concentrator characterized by that. The protein concentrator according to claim 10 or 15,
The extraction unit includes:
A cutting part for cutting the carrier;
An adsorbing plate for adsorbing the cut carrier;
Vibration means for vibrating the suction plate;
A protein concentrating device comprising: The protein concentrator according to claim 10 or 15,
The extraction unit includes an ultrasonic generator.
A protein concentrator characterized by that. The protein concentrator according to claim 10 or 15,
The extraction unit includes a micro heater.
A protein concentrator characterized by that. The protein concentrator according to claim 10 or 15,
The protein concentrating device further includes pH adjusting means for adjusting a solution pH in the concentrating unit by injecting a pH adjusting solution into a flow path connecting the extracting unit and the concentrating unit.
A protein concentrator characterized by that. The protein concentrator according to claim 10 or 15,
The protein concentrating device further includes an electric field increasing substance adding means for adding a charge increasing substance for increasing the charge of the protein in a flow path connecting the extraction section and the concentration section.
A protein concentrator characterized by that. The protein concentrating device according to claim 12, 13, 14 or 15,
The carrier is strip-shaped,
The moving means includes a roller for advancing the belt-shaped carrier,
A motor for driving the roller,
A protein concentrator characterized by that. The protein concentrator according to claim 21,
The protein concentrator is
Marking means for marking the belt-shaped carrier for specifying the specimen-carrying region;
Mark detection means for detecting the marking;
Further comprising
A protein concentrator characterized by that. The protein concentrator according to claim 22,
The protein concentrator is
A container drive unit that drives the first container and / or the second container, and further includes an operation control unit that controls operations of the container drive unit and the moving unit, the operation control unit including the mark detection unit; When the marking is detected by the means, the operation of the moving means is stopped, and the first container and / or the second container are driven to integrate the first container and the second container,
A protein concentrator characterized by that. The protein concentrator according to claim 9, 10 or 11,
The electric field generating means includes a pair of electrodes arranged in a state facing the first space and the second space,
A protein concentrator characterized by that. The protein concentrating device according to claim 12, 13, 14 or 15,
The volume ratio of the second space to the first space is 1/20 to 1/1000000.
A protein concentrator characterized by that. The protein concentrating device according to claim 12, 13, 14 or 15,
The volume of the first space is 10 to 1000 ml;
The volume of the second space is 1 to 500 μl.
A protein concentrator characterized by that. The protein concentrator according to claim 9, 10 or 11,
The partition has a plurality of holes having a hole diameter of 0.001 to 100 μm,
A protein concentrator. The protein concentrator according to claim 9, 10 or 11,
The second space is a microchannel having a cross-sectional area perpendicular to the flow direction of 400 to 250,000 μm ² .
A protein concentrator characterized by that. A protein concentrator according to any one of claims 9 to 28;
A microchannel device for measuring the type or amount of protein concentrated by the protein concentrator,
The microchannel device is:
A reaction channel;
A reactant fixed in the reaction channel;
A detection unit for detecting the amount of protein;
A protein measuring apparatus comprising: A protein measuring apparatus according to claim 29;
A blowing means for sending outside air outside the apparatus into the apparatus;
Blower control means for increasing or decreasing the blast volume of the blast means according to the amount of protein contained in the outside air measured by the protein measuring device;
Air conditioner equipped with. A protein measuring apparatus according to claim 29;
A protein-containing substance removing means for removing the protein-containing substance;
A removal operation control means for controlling the operation of the protein-containing substance removal means according to the amount of protein measured by the protein measuring device;
Air conditioner equipped with.