JP2009209094A - Microchip for protein extraction, protein extraction apparatus, protein measurement apparatus, protein extraction method using them, and air conditioner - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for simply and quickly extracting a protein from a specimen. <P>SOLUTION: The protein extraction apparatus comprises a microchip for extraction having a weir for retaining a specimen in the inside of the chip and a transportation mechanism that transports the specimen and an extractant to the extraction tank of the microchip, wherein the transportation mechanism includes at least a specimen solution receptacle that holds the specimen and the extractant and a transportation pump that transports the specimen and the extractant from the receptacle to the extraction tank inside the microchip. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein extraction apparatus that extracts a protein from a specimen containing a specific protein, generates a high-concentration protein solution, and separates the generated protein solution from the specimen, and a protein extraction method and air using the same. It relates to the adjusting machine.

  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 recent years, attempts have been made to measure harmful substances in the air environment and the health of human beings on a chip. These techniques are referred to as μ-TAS (micro total analysis systems), MEMS (micro electro mechanical systems), etc., and in particular, biotechnology for analyzing a measurement object using a specific reaction of molecules contained in a living body. Sensor chips are attracting attention. In particular, there is an increasing expectation for a so-called Lab-on-a-chip technique that performs all analysis operations such as protein extraction, separation, recognition, and detection on a chip without performing pre-processing in a macro system.

  Many people suffer from allergic symptoms caused by airborne pollutants, mites, fungi, volatile organic compounds, and other airborne micro- and chemical substances. Therefore, in order to measure the amount of allergen and other proteins that cause allergies, development of microchannel devices to which the above-described μTAS and MEMS technologies are applied is extensive. A microchannel device is a microchannel (mainly a channel with a diameter of several μm to several hundreds of μm) that passes through a fluid such as a liquid or gas, and performs separation, chemical reaction or analysis / measurement. It is a system to do.

  Since the microchannel device has a fine structure, it does not require a large amount of fluid for transporting the sample and can be analyzed and measured with a small amount of sample. Furthermore, there is an advantage that the analytical measurement apparatus itself can be downsized and the analytical measurement time can be increased.

  However, the microchannel device has a limit when the concentration of the target substance in the sample is too low. When a protein is extracted from a specimen for immunoassay, a large amount of extraction solution is used, so that only a protein having a low protein concentration can be obtained. For this reason, when this extract was injected into the microchannel device and the amount of protein in the test solution was measured, the protein concentration was too low, resulting in a problem that accurate measurement could not be performed.

  The shape of the microchannel device is not limited as long as the device has a microchannel. Examples of the microchannel device include a microchip, a capillary, and a tube.

  Patent Document 1 proposes a technique for applying a micro-channel device in which micro-order channels are formed on a substrate to immunoassay. Patent Document 1 discloses a microchannel reaction tank section, a microchannel separation section having a longitudinal cross-sectional area smaller than the diameter of the solid microparticles, and an introduction section or microchannel that separately introduces an antigen and a labeled antibody to the reaction tank section. A microchannel device having an inflow portion is disclosed. However, Patent Document 1 does not describe extracting a protein using a microchannel device. In Patent Document 1, a thermal lens is used for detection. In this case, the apparatus becomes large.

  On the other hand, Patent Document 2 proposes a technique using an electrochemical detection method instead of the thermal lens method. The technology of Patent Document 2 shows a micro-channel device that integrally constitutes a micro-channel device and a method for manufacturing the micro-channel device, does not require a joining technique, and is not restricted by measurement due to light absorption or fluorescence of a resin. It is about.

  As a form of the micro-channel device, generally, a form in which a reaction part (a part that actually performs reaction / analysis and the like) is in the form of a microchip, a tube is connected thereto, and an external pump is used is used. This form has the advantage that the intended work can be easily switched by simply replacing the microchip portion.

  On the other hand, in order to perform immunoassay, it is necessary to efficiently collect a specimen containing a target antigen or antibody. In the prior art, the method for detecting pollen collection is to collect pollen on a slide glass with petrolatum thinly and evenly applied on the surface or on an adhesive tape wound around a drum. Furthermore, after the collected pollen was put into a buffer solution and an antigen was extracted from the pollen, the pollen was separated by centrifugation, and a buffer solution (supernatant) was taken out as a specimen containing the antigen and used as an analysis sample.

Inventions relating to pollen collection and analysis have also been proposed. A technique related to an automatic pollen collecting apparatus is proposed in Patent Document 3. 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

  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, so accurate measurement is possible. There are cases where it is not possible.

  In order to solve this problem, a method of injecting a large amount of protein extract into the microchannel device in order to increase the absolute amount of protein to be extracted is also conceivable. However, a microchannel device is usually used at a flow rate of about 6 μl / min. Therefore, if a protein extract of about 10 to 1000 ml is allowed to flow through the microchannel, it will take about 30 to 2800 hours to finish flowing. It will take. Therefore, this method is not practical.

  Therefore, there is a need for a technique for concentrating a low concentration protein extract easily and in a short time.

  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 protein extraction and measurement.

  The present invention is a protein extraction microchip, a protein extraction apparatus, a protein measurement apparatus, and an air conditioner that can automatically perform a series of steps from sample collection to antigen protein extraction and measurement. The purpose is to provide a machine.

  According to a first aspect of the present invention, there is provided an extraction tank section for extracting a target component from a specimen, at least one injection channel connected to the extraction tank section, and the extraction tank section. In addition, the microchip for extraction includes at least one discharge channel.

  One aspect of the extraction microchip according to the present invention is that a damming portion having a curved chip plan view shape is provided between the injection channel and the discharge channel in the extraction tank unit. It is characterized by.

  If it is the said structure, it will become possible to perform extraction of the protein which is the target component from a test substance, and isolation | separation of a test substance and the extracted protein within a microchip. Moreover, since protein extraction can be performed with a small amount of extraction liquid, a high-concentration protein extraction liquid can be generated, and a complicated concentration step conventionally required is not required. Further, by using this high-concentration protein extract as a test solution for protein detection, it becomes possible to measure the amount of protein with high accuracy.

  In the microchip of the above aspect, the damming portion has a curved shape in plan view of the chip. Here, the chip plan view shape means a shape when the extraction microchip is placed on a plane and seen through from above. According to this configuration, it is possible to increase the damming area, increase the damming effect, and suppress a decrease in the liquid feeding speed due to the specimen clogging the damming part. Although various curved shapes can be designed, it is preferable that the curved shape is convex toward the discharge channel.

  In the microchip of the above aspect, the shape of the extraction tank portion in a plan view of the chip is preferably a circular shape or an elliptical shape because excellent movement of the specimen within the chip is excellent.

  In the microchip of the above aspect, in the first invention, the damming portion may have a slit structure. According to this configuration, it becomes possible to make the depth of the extraction tank part and the damming part the same, and the manufacture of the microchip becomes easy.

  Another aspect of the microchip for extraction according to the present invention is such that the damming portion is provided in a shape connecting both end faces in a direction perpendicular to the inflow / outflow direction of the extraction tank section, and the slit width in the vicinity of both end faces is the center. It is characterized by being wider than the slit width in the region. According to this configuration, it is possible to prevent the sample from concentrating on the central portion, and to suppress a decrease in the liquid feeding speed due to the sample being clogged with the damming portion. The specimen can be collected efficiently.

In another aspect of the extraction microchip according to the present invention, at least two rows of damming portions having a slit structure are provided between the injection flow channel and the discharge flow channel in the extraction tank. It is characterized by being. According to this configuration, the damming area can be increased, and the damming effect can be increased. Further, by changing the slit width at the front and rear, the specimen can be separated depending on the size of the specimen, and there is an effect of making aggregation less likely to occur.

  According to still another aspect of the extraction microchip of the present invention, a plurality of damming portions having a slit structure are disposed between the injection channel and the discharge channel in the extraction tank, The parts are arranged in a multistage manner in the flow direction. According to this configuration, the damming area can be increased, and the damming effect can be increased. In addition, since the specimen is retained at a plurality of locations, there is an effect that aggregation is unlikely to occur.

  According to still another aspect of the extraction microchip of the present invention, the extraction tank is connected to at least two discharge channels, and a damming portion is provided between each discharge channel and the injection channel. It is characterized by being.

  If it is the said structure, by providing two or more discharge flow paths, and providing a damming part in each discharge flow path, a sample will be disperse | distributed to each damming part and a sample will be clogged by a damming part. It is possible to suppress a decrease in the liquid feeding speed. In addition, even when one flow path is clogged, it is possible to continue liquid feeding because there are other discharge flow paths. Furthermore, by dispersing the specimen, protein extraction from the specimen and collection of the specimen after extraction can be performed efficiently.

  In the microchip for extraction of each aspect described above, the volume of the extraction tank in the microchip can be 5 to 50 μl. According to this configuration, since protein is extracted in a small amount of extraction liquid, a high concentration protein solution can be obtained.

  The microchip of the present invention is suitable for protein extraction processes such as pollen, but can also be used for extraction of components other than proteins (for example, amino acids or nucleic acids). Furthermore, any process other than the extraction process can be applied as long as the solid is retained in the damming portion and the fluid is allowed to flow there. Examples of other processes include a process or a reaction process in which the solid inside is completely dissolved in the inflowing liquid.

  A second invention for solving the above-described problems includes the extraction microchip according to the present invention, and a liquid feeding mechanism section for feeding the specimen and the extraction liquid to the extraction tank section of the extraction microchip. The liquid feeding mechanism part feeds the sample liquid storage container for storing the specimen and the extraction liquid, and the specimen and the extraction liquid from the sample liquid storage container to the extraction tank part in the extraction microchip. A protein extraction apparatus comprising at least a liquid feed pump.

  According to the present invention, since protein is extracted in a small amount of extraction liquid, a high-concentration protein extract can be generated, and a complicated concentration step that has been conventionally required is unnecessary. Therefore, the apparatus can be miniaturized and automation is facilitated. Further, by using this high-concentration protein extract as a test solution for protein detection, it becomes possible to measure the amount of protein with high accuracy.

  In the above-described protein extraction apparatus of the present invention, the extraction liquid can be sent in an amount of 5 to 100 μl. According to this configuration, since protein is extracted in a small amount of extraction liquid, a high concentration protein solution can be obtained.

  In the above-described protein extraction apparatus of the present invention, the microchip can be provided with means for vibrating the extraction tank section. Due to the vibration, a stirring effect is generated in the microchip, so that the protein is easily extracted from the specimen, and the extraction efficiency can be increased.

  As the vibration device, an ultrasonic generator is preferable. Thereby, the extraction efficiency of the protein from the specimen can be further increased by the fine vibration by the ultrasonic wave.

  In the above-described protein extraction apparatus of the present invention, a micro heater can be provided on the microchip. As a result, when the temperature in the microchip rises, the solubility of the protein generally increases, so that the effect of increasing the amount of protein dissolved in the extraction liquid can be obtained.

  In the above-described protein extraction apparatus, the liquid feeding mechanism unit may be further provided with a suction pump.

  According to this configuration, a method for extracting proteins by operating a liquid feeding pump and a suction pump and alternately swinging the extraction liquid in the extraction tank section in the flow direction and the reverse direction becomes possible.

  In addition, after the protein is extracted from the specimen, a washing process can be performed in which the specimen and the extraction liquid remaining in the extraction tank are discharged from the extraction tank using the above-described suction pump. Thus, it becomes possible to repeatedly extract the target protein.

  In the present invention, in the above-described protein extraction apparatus, there is a method for extracting a protein by feeding a transport liquid mixed with a specimen with a liquid feed pump, and feeding the specimen into the microchip together with the transport liquid. Further provided. According to this method, it is possible to easily send the specimen to a minute part in the microchip. Furthermore, it becomes easy to send all the specimens to the extraction tank provided in the microchip.

  In the above protein extraction method, various specimens can be extracted as the specimen in addition to biological substances such as pollen.

  The transport liquid is preferably a poor protein solvent in which the protein extracted from the specimen is difficult to dissolve. When the sample is pollen, pure water is preferred. As a result, the protein in the sample is prevented from being discharged together with the transport liquid during transport, and thus the protein extraction efficiency can be increased.

  It is preferable that the amount of the liquid for transportation described above is 0.5 to 10 ml. If it is this range, it will become possible to send a sample easily to the extraction tank part provided in the microchip in a short time.

  In the above protein extraction method, when the specimen is pollen, the extraction solution is preferably a phosphate buffer or a Tris buffer as the extract.

  In the protein extraction apparatus according to the second invention group, a collection unit for collecting a sample from an external space is further provided, and the sample collected by the collection unit is sent to an extraction tank unit of an extraction microchip. It can be set as the structure to do. According to this configuration, it is possible to automatically perform protein extraction and separation operations with a single device from collection of a sample from the air. In addition, since protein is extracted in a small amount of extract, a high concentration protein extract can be produced, and a complicated concentration step that has been conventionally required is not required, and the apparatus can be miniaturized. . Furthermore, by using this high concentration protein extract as a test solution for protein detection, it is possible to measure the amount of protein with high accuracy.

  3rd invention for solving the said subject is related with the protein measuring apparatus incorporating the protein extraction apparatus concerning the above-mentioned 2nd invention group.

  The protein measuring apparatus of the present invention includes a protein extraction apparatus having any one of the above-described configurations of the second invention group, a microchannel device for detecting the type and amount of the protein extracted by the protein extraction apparatus, And the microchannel device has a reaction part containing a reactive substance and a detection part for detecting the amount of protein.

  With such a configuration, protein extraction and measurement can be performed continuously. In particular, in the case of a protein measuring apparatus equipped with the above-described collection unit, operations from sample collection from external space to protein extraction and protein detection can be performed with one apparatus. Work efficiency of identification and content measurement is greatly improved.

  As the microchannel device, for example, a device including a reaction channel, a reactive substance fixed in the reaction channel, and a detection unit (electrode) as shown in FIG. 14 can be used.

  4th invention for solving the said subject is related with the air conditioner incorporating the protein measuring apparatus concerning above-mentioned 3rd invention.

  The air conditioner according to the present invention is configured to control the amount of air blown from the air blowing unit according to the amount of protein contained in the outside air measured by the protein measuring device, the air blowing unit, and the protein measuring device according to the third invention. And a ventilation control means for increasing and decreasing.

  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.

  As another aspect of the air conditioner according to the present invention, the protein measuring device according to the third invention, the protein-containing material removing means for removing the protein-containing material, and the amount of protein measured by the protein measuring device. Correspondingly, there is provided one provided with a removal operation control means for controlling the operation of the protein-containing substance removal means.

  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 means includes, for example, an ion generation section or plasma generation section and an adsorption section that adsorbs a specimen charged by ions or plasma. When the protein measuring apparatus detects a protein, ions or plasma are generated, whereby the protein-containing substance is charged and adsorbed and removed by the adsorption unit.

  As described above, according to the protein extraction apparatus of the present invention, operations from the collection of the specimen from the external space to the extraction and separation of the protein can be efficiently performed in a short time. In addition, the protein extraction apparatus of the present invention does not require a complicated concentration step that has been necessary in the past, and the apparatus can be miniaturized, facilitating automation. Such a protein extraction 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, 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.

[First Embodiment]
A protein extraction microchip (hereinafter referred to as an extraction chip or simply a chip) according to this embodiment will be described with reference to FIG. FIG. 1 shows an embodiment of the extraction chip of the present invention. (A) is a top view, (b) is sectional drawing in the XX 'line of (a). FIG. 6C is a perspective view before the chip substrate 22 and the lid equipment 23 are attached.

  As shown in each drawing of FIG. 1, the extraction chip 1 includes an extraction tank unit 16 that extracts proteins from a specimen, an injection port 17, a discharge port 18, an injection channel 19, and a discharge channel 20. A damming portion 21 for preventing the specimen from being discharged from the discharge port 18 is formed in the extraction tank portion 16.

  As can be seen from FIG. 1C, the extraction chip of the present invention has a structure in which a substrate 22 and a lid base material 23 are attached. A recess is formed in the substrate 22, and the recess becomes a space between the substrate 22 and the lid base 23 after being attached to the lid base 23. Two holes are made in the lid base material 23 perpendicular to the substrate surface so as to communicate with the gap. One hole is a liquid inlet 17 and the other hole is an outlet 18. As viewed from above the substrate surface, the shape of the gap is narrow in the vicinity of the inlet 17 and outlet 18 to form a flow path (19, 20), and wide in the vicinity of the center of the substrate. The portion where the width is wide is referred to as an extraction tank portion 16.

  This extraction tank part needs to be a space having a sufficient width with respect to the width of the extraction chip. This is because if the space of the extraction tank is narrow, the possibility that the specimen aggregates increases and the contact area between the specimen and the extraction liquid becomes small, so that extraction cannot be performed efficiently.

  In the chip of FIG. 1, the injection port 17 and the discharge port 18 are perforated perpendicularly to the substrate surface, but other modes may be used. For example, the case where the inlet 17 and the outlet 18 are linearly connected to the inlet channel 19 and the outlet channel 20 is also possible.

  The extraction tank in the microchip is provided with a damming portion having a shape that prevents the specimen from passing therethrough. By this damming portion, the sample can be held in the extraction tank during extraction, and the protein solution after extraction and the sample can be separated.

  Any structure can be used for the damming part as long as the specimen cannot pass therethrough. For example, a convex portion having a size that does not allow the specimen to pass through the gap between the extraction tanks may be provided, but a configuration having a slit structure with a width that the specimen cannot pass is preferable (see c in FIG. 1). According to this structure, it becomes possible to make the depth of an extraction tank part and a damming part the same, and manufacture of the microchip of this invention becomes easy.

  On the surface of the substrate 22 of the extraction chip 1, an extraction tank portion 16, an injection flow channel 19, a discharge flow channel 20, and a damming portion 21 are formed, and their depth is about 50 to 200 μm. When shallower than this, it is necessary to increase the area of the extraction tank section 16, and the size of the extraction chip 1 is increased. If it becomes deeper than this, the volume of the injection flow path 19 and the discharge flow path 20 will increase, and it will be necessary to increase the amount of the extraction liquid. Moreover, the width | variety of the injection | throwing flow path 19 and the discharge flow path 20 is about 50-200 micrometers.

  As for the volume of the extraction tank part 16, about 5-50 microliters is preferable. Within this range, a high concentration protein solution can be obtained by extraction.

  The lid base 23 is formed with an inlet 17 and an outlet 18 to which a liquid feeding tube is connected.

  As the material of the substrate 22 and the lid base material 23, a resin such as silicon, glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), COP (cyclic olefin polymer) or COC (cyclic olefin copolymer) is used. Can do. In particular, in the case of a resin, pattern formation and bonding are easy, and the mass productivity is excellent.

  When an extraction tank is provided in the device as in the present invention, a microchannel device is manufactured by forming a channel pattern on a substrate and then attaching a lid base material to complete the device. The method is preferred. As a method for forming the flow path pattern, anisotropic dry etching, wet etching using hydrofluoric acid, or the like is used in the case of silicon, glass, or the like. Examples of the resin material pattern forming method include a soft lithography method, an optical nanoimprint method, and a hot embossing method.

  The damming unit 21 has a structure that dams the sample in the extraction tank unit 16. For example, a slit-like pattern having the same depth as the extraction tank section 16 and a width through which the specimen cannot pass is used. In the case of the slit structure, the depth of the extraction tank portion and the damming portion can be made the same, and the manufacture of the microchip becomes easy. When the specimen is cedar pollen, cedar pollen having a particle size of about 30 μm can be blocked by using a slit having a width of 10 μm. A pattern other than a slit such as a dam shape or a pillar shape may be used as long as the sample can be prevented from passing therethrough.

[Second Embodiment]
A protein extraction apparatus according to this embodiment will be described with reference to FIGS. As shown in FIG. 2, the protein extraction apparatus includes an extraction chip 1 that extracts a protein from a sample, and a liquid feeding mechanism unit 111 that sends the sample and the extraction liquid to the extraction unit.

  The liquid feeding mechanism section includes liquid containers 2, 3, 4 for storing liquid, pumps 5, 6 for feeding the specimen and liquid, valves 7, 8, 9, 10, 11, and liquid feeding tube 12. The sample is sent to the extraction chip 1, the extraction liquid is filled into the extraction chip 1, and the extracted protein is sent to the detection unit 13. The specific contents of the apparatus will be described below.

(Transportation of specimen to microchip)
First, the sample and the transport liquid are placed in the recovery liquid container 2. As the specimen, for example, cedar pollen collected from the air is used. A protein poor solvent in which the protein extracted from the sample is difficult to dissolve is used for the transport liquid. As a result, the protein in the sample is prevented from being discharged together with the transport liquid during transport, and thus the protein extraction efficiency can be increased. When the specimen is cedar pollen, pure water can be used as the transport liquid.

  The amount of the liquid for conveyance is preferably about 0.5 to 10 ml. If the amount is less than this, it becomes difficult to send the sample into the transport liquid. If the amount is more than this, it takes a long time to send the sample to the extraction tank, and the liquid container needs to be enlarged, resulting in a large apparatus.

  In the second embodiment, cedar pollen will be described as an example of the sample, but the present invention can be applied to other pollen such as cypress and ragweed, and a sample other than pollen (floating matter in the air such as tick). The liquid for transport is not limited to pure water, and can be selected according to the protein extracted from the specimen.

  Next, the mixed liquid 14 of the specimen and the transport liquid in the collection liquid container 2 is sent into the extraction tank section 16 in the extraction chip 1 using the pump 5. The liquid feed flow rate is preferably about 0.5 to 10 ml / min in consideration of the liquid feed time and the durability of the extraction tip 1.

  In addition, it is desirable to stir the specimen and the transport liquid in the recovery liquid container 2 when the mixed liquid 14 of the specimen and the transport liquid is fed into the extraction tank section 16. By stirring, it becomes easy to send all the specimens from the collection liquid container 2 to the extraction tank section 16. As a stirring method, methods such as applying vibration to the container, mechanically stirring, or stirring by feeding air can be used.

  The shape of the collection liquid container 2 is desirably a shape that makes it difficult for the specimen to remain in the container, and the bottom surface portion preferably has a sharp tip, for example, so that the bottom surface area is reduced as much as possible.

  In addition, in order to prevent the protein extracted from the specimen from adsorbing to the extraction tank section 16 and the discharge flow path 20, a non-specific adsorption prevention process for proteins is performed in advance on the surfaces of the extraction tank section 16 and the discharge flow path 20. You may give it. For example, a blocking agent such as a casein solution or a BSA (bovine serum albumin) solution may be injected into the extraction tank section 16 before the mixed liquid 14 of the specimen and the transport liquid is fed into the extraction tank section 16. Absent. By covering the surfaces of the extraction tank section 16 and the discharge flow path 20 with a blocking agent, protein adsorption to the extraction tank section 16 and the discharge flow path 20 can be suppressed, and protein can be extracted efficiently. .

  The specimen sent to the extraction chip 1 remains in the extraction tank section 16 by the damming section 21, and only the transport liquid passes through the discharge channel 20 and is discharged from the discharge port 18 to the discharge liquid container 4. .

  In this way, by mixing the sample with the transport liquid and sending it, it is possible to easily send the sample to a minute portion in the chip.

  The discharged transport liquid can be reused when it is continuously extracted. By doing so, the amount of waste liquid can be reduced, and the apparatus can be further downsized.

(Filling with extraction liquid)
Next, the extraction liquid 15 in the extraction liquid container 3 is filled into the extraction tank section 16 in the extraction chip 1 using the pump 6.

  As the extraction solution 15, it is preferable to use a solution having molecules and pH that are excellent in the extraction efficiency of the protein in the specimen. In the case of cedar pollen, a phosphate buffered saline buffer solution or a Tris buffer solution can be used as the extraction solution.

  The amount of the extraction liquid 15 is preferably about 5 to 100 μl. If it is this range, a highly concentrated protein solution can be obtained. When the amount of liquid increases, the concentration of the protein extract decreases, making it difficult to measure the amount of protein in the detection unit, and concentration is required.

  The flow rate and flow rate of the transport liquid and the extract liquid can be controlled by the pressures of the pumps 5 and 6 and the inner diameter of the liquid feed tube 12. Moreover, you may provide the mechanism for controlling a flow velocity and a flow volume separately.

  When three or more flow paths are connected to the extraction chip, an injection port, an injection flow path, a discharge flow path, and a discharge port may be provided separately for the transfer liquid and the extraction liquid. According to this configuration, it is possible to independently adjust the flow velocity and the flow rate for each liquid, and the flow rate and the liquid amount can be easily controlled.

  Further, a part of the valve for liquid feeding may be provided in the extraction chip 1. By providing a microvalve in the microchip, the dead volume of the extract (a small amount of liquid remaining in the container) can be reduced, and the liquid volume can be easily controlled. Further, the number of parts is reduced, and the apparatus can be made smaller.

(Protein extraction process and detection process)
Next, the protein in the specimen is extracted into the extraction liquid in a state where the specimen and the extraction liquid 15 are filled in the extraction tank section 16. The extraction time is preferably about 1 to 60 minutes.

  A means for vibrating the extraction tank section 16 may be provided in the extraction chip 1. According to this configuration, the protein can be easily extracted from the specimen, and the extraction efficiency can be increased.

  Further, the extraction chip 1 may be provided with an ultrasonic generator. By extracting ultrasonic waves in the extraction tank section 16 at the time of protein extraction, it becomes possible to further increase the protein extraction efficiency from the specimen.

  Further, the extraction chip 1 may be provided with a micro heater. At the time of protein extraction, the extraction tank portion 16 is warmed by a microheater, whereby the protein extraction efficiency from the specimen can be increased.

  Further, the extraction liquid 15 in the extraction chip 1 may be swung during protein extraction. For example, by providing a swing mechanism unit that controls repetition of suction and discharge to the extraction tank unit 16 by a pump, pressurization and depressurization to the mechanical extraction tank unit 16, It can be swung. By swinging the extraction liquid 15, the protein extraction efficiency from the specimen can be increased.

(Detection of protein amount)
Finally, the protein solution from which the protein has been extracted in the extraction liquid is sent from the discharge channel 20 to the detection unit 13 through the discharge port 18, and the amount of the extracted protein is detected. When the protein solution is discharged from the discharge channel 20, the sample from which the protein is extracted from the sample is blocked by the blocking unit 21 and is not discharged from the discharge channel 20, so that the protein solution and the sample can be easily separated. Can do.

  The detection unit 13 can be widely selected from means suitable for protein detection. In addition to the electrochemical detection type micro-channel device described later, spectroscopic measurement methods such as IR, liquid chromatography, gas chromatography, and the like can be used.

  As described above, according to this embodiment, since protein is extracted in a small amount of extract, a high-concentration protein extract can be generated, and a complicated concentration step that has been conventionally required is not necessary. . Therefore, the apparatus can be miniaturized and automation is facilitated. Further, by using this high-concentration protein extract as a test solution for protein detection, it becomes possible to measure the amount of protein with high accuracy.

  In the present embodiment, the specimen is mixed with the transport liquid and injected into the extraction tank section 16 in the extraction chip 1. However, the specimen may be directly injected into the extraction tank section 16 using a syringe or the like. Absent. By extracting the sample in the extraction tank section 16 and performing extraction, the protein is extracted in a small amount of the extract, and thus it is possible to generate a protein extract with a high concentration.

  Next, the present invention will be specifically described based on examples. In addition, this invention is not limited to each following Example, In the range which does not change the summary of this invention, it can change suitably and can implement.

Example 1
Example 1 is an example according to the protein extraction apparatus described in the second embodiment. The protein extraction apparatus according to this example will be described with reference to FIGS.

  In the protein extraction apparatus of this example, the extraction chip 1 shown in FIG. 1 and the liquid feeding mechanism 111 shown in FIG. 2 were used. Using airborne cedar pollen as a specimen, the allergen protein Cry-J1 contained in the cedar pollen was extracted with a protein extraction device, and the amount of the extracted Cry-J1 was detected by the detection unit.

  As shown in FIG. 1, the damming portion 21 of the extraction chip 1 has a curved shape that is convex in the flow direction in order to increase the damming area.

  The recovery liquid container 2, the extraction liquid container 3, and the discharge liquid container 4 are TOP 15ml centrifuge tubes, the pumps 5 and 6 are motor-type pumps (CM-50-12 manufactured by Enomoto Micropump Manufacture), valves Electromagnetic valves were used for 7, 8, 9, 10 and 11, and a Teflon (registered trademark) tube having an inner diameter of 0.75 μm was used for the liquid feeding tube 12.

  First, the casein solution was injected into the extraction tank section 16 before the mixed liquid 14 of the specimen and the transport liquid was fed into the extraction tank section 16. By covering the surfaces of the extraction tank section 16 and the discharge flow path 20 with the casein solution, protein adsorption to the extraction tank section 16 and the discharge flow path 20 is suppressed, and the protein can be efficiently extracted.

  Next, cedar pollen collected from the air and 5 ml of pure water are placed in the collection liquid container 2 and sufficiently stirred. Then, the valves 7, 9, and 10 are opened, the pump 5 is turned on, and cedar pollen is turned on. And pure water were fed into the extraction tank 16 in the extraction chip 1 at a liquid feed flow rate of about 2 ml / min.

  The cedar pollen sent to the extraction chip 1 remains in the extraction tank section 16 by the damming section 21, and only pure water passes through the discharge channel 20 and is discharged from the discharge port 18 to the discharge liquid container 4. . In this way, by mixing cedar pollen with pure water and sending it, it was possible to easily send the sample into a minute part in the chip.

  The extraction chip 1 has the structure shown in FIG. 1, and a silicon substrate was used for the substrate 22 and PDMS was used for the lid base material. The depths of the extraction tank section 16, the injection flow path 19, the discharge flow path 20, and the damming section 21 were each 200 μm, and the widths of the injection flow path 19 and the discharge flow path 20 were each 200 μm. Moreover, the volume of the extraction tank part 16 was about 10 microliters.

  The damming part 21 formed a slit-like pattern having the same depth as the extraction tank part 16. It was possible to dampen cedar pollen having a slit width of about 10 μm and a particle size of about 30 μm.

  Next, the valves 8, 9, 10 were opened, the pump 6 was turned on, and the extraction liquid 15 in the extraction liquid container 3 was filled in the extraction tank section 16 in the extraction chip 1. Quantification of the liquid feeding amount of the extraction liquid 15 was performed by making the valve opening / closing time constant by microcomputer control. As the extraction solution 15, a phosphate buffered saline buffer solution was used, and the amount of solution fed was about 50 μl.

  Next, Cry-J1 of the allergen protein contained in the cedar pollen was extracted with the valves 9, 10, and 11 closed and the extraction tank 15 filled in the extraction tank 16. The extraction time was about 15 minutes, and the extraction tank 16 was vibrated by a vibration motor provided in the extraction chip 1 during extraction.

  Finally, the extract from which Cry-J1 was extracted was sent from the discharge channel 20 through the discharge port 18 to the detection unit 13, and the amount of the extracted Cry-J1 was detected. When the extract was discharged from the discharge channel 20, the pollen shell was blocked by the blocking unit 21 and was not discharged from the discharge channel 20, so that the extract and the pollen shell could be easily separated.

  It was confirmed that the extracted amount of Cry-J1 can be detected by a conventional electrochemical detection type microchannel device. For this reason, when the apparatus according to this example is used, it is possible to extract the target protein at a high concentration, which eliminates the need for a complicated concentration step that has been necessary in the past, and the apparatus can be downsized and automated. It turns out that it becomes easy. In addition, by using a high concentration protein extract as a test solution for protein detection, it is possible to measure the amount of protein with high accuracy.

(Example 2) (Cleaning of microchip)
In Example 2, a process of washing the inside of the extraction chip after performing the extraction process with the protein extraction apparatus described in Example 1 will be described. The protein extraction apparatus according to this example will be described with reference to FIGS. 1 and 3.

  In the protein extraction apparatus of this example, the liquid feeding mechanism unit having the suction pump 25 shown in FIG. 3 and the extraction chip 1 shown in FIG. 1 were used. In the same manner as in Example 1, using a cedar pollen in the air as a specimen, the allergen protein Cry-J1 contained in the cedar pollen was extracted by a protein extraction device, and the amount of the extracted Cry-J1 was detected by the detection unit.

  After performing the extraction process in the same manner as in Example 1, the valves 9 and 27 are opened, the suction pump 25 is turned on, and the transport liquid 28 in the transport liquid container 24 is discharged from the discharge port 18 into the extraction chip 1. The pollen shell remaining in the extraction tank 16 was recovered from the inlet 17 into the recovery liquid container 2. Pure water was used as the transport liquid. Thereafter, the valve 26 was opened, the pump 5 was turned on, and pure water and pollen shells in the collection liquid container 2 were discharged into the discharge liquid container 4. If necessary, the inside of the extraction chip 1 was cleaned by repeating this cleaning step.

  By performing this washing step, it became possible to repeatedly extract the target protein from the specimen. Further, when the specimen is sent into the extraction chip, the amount of waste liquid can be reduced by reusing the discharged transport liquid, and the apparatus can be further downsized.

(Example 3)
The extraction process was performed in the same manner as in Example 1 except that the extraction chip was the chip 101 of FIG. In the extraction chip 101, the depth of the extraction tank section 16, the injection flow path 19, the discharge flow path 20 and the damming section 21 was 200 μm, and the width of the injection flow path 19 and the discharge flow path 20 was 200 μm. Moreover, the volume of the extraction tank part 16 was about 10 microliters.

  The damming unit 21 formed a slit-like pattern having the same depth as the extraction tank unit 16. The slit width was narrowest in the vicinity of the center and increased with increasing distance from the center. The slit width varied in the range of about 5 μm to about 20 μm, and it was possible to dampen cedar pollen having a particle size of about 30 μm.

  In this chip, by making the slit width of the damming portion 21 wider in the peripheral portion than in the central portion, it is possible to prevent the sample from concentrating on the central portion, and to reduce the liquid feeding speed due to the sample being clogged in the damming portion. Can be suppressed.

  Similar to the first embodiment, the sample is introduced into the extraction tank 16, the extraction liquid 15 is injected, the protein is extracted from the sample, and the extract is extracted from the Cry-J1. By the method. By making the slit width of the damming portion 102 wider at the peripheral portion than at the central portion, it was possible to prevent the sample from concentrating on the central portion and to efficiently extract the protein from the sample.

  As in Example 1, it was confirmed that the extracted Cry-J1 amount could be detected by a conventional electrochemical detection type microchannel device.

  Furthermore, the collection | recovery of the pollen shell which remained in the extraction tank part 16, and the washing | cleaning of the extraction tank part were performed by the method similar to Example 2. FIG.

Example 4
The extraction process was performed in the same manner as in Example 1 except that the extraction chip was the chip 102 in FIG. In the extraction chip 102, the depth of the extraction tank section 16, the injection flow path 19, the discharge flow path 20, and the damming section 21 was 200 μm, and the width of the injection flow path 19 and the discharge flow path 20 was 200 μm. Moreover, the volume of the extraction tank part 16 was about 10 microliters.

  The damming portion 21 of the extraction chip 102 formed a slit-like pattern having the same depth as the extraction tank portion 16. There are two rows of slits, the slit interval is about 10 to 30 μm, and the front (inlet side) slit interval is wider than the rear (outlet side) slit interval. With this structure, it is possible to disperse the specimens in the respective rows based on the size, and it is possible to suppress a decrease in the liquid feeding speed due to the specimen clogging the damming portion.

  Moreover, the area of the damming portion could be increased by increasing the number of slit rows of the damming portion 21 to increase the protein extraction effect.

  Similar to the first embodiment, the sample is introduced into the extraction tank 16, the extraction liquid 15 is injected, the protein is extracted from the sample, and the extract is extracted from the Cry-J1. By the method.

  By using a plurality of slit rows in the damming unit 21, the specimen was dispersed, and protein could be extracted efficiently from the specimen.

  As in Example 1, it was confirmed that the extracted Cry-J1 amount could be detected by a conventional electrochemical detection type microchannel device.

  Furthermore, the pollen shells remaining in the extraction tank unit 16 were collected by the same method as in Example 2.

(Example 5)
The extraction process was performed in the same manner as in Example 1 except that the extraction chip was changed to the chip 103 in FIG. In the extraction chip 103, the depth of the extraction tank section 16, the injection flow path 19, the discharge flow path 20, and the damming section 21 was 200 μm, and the width of the injection flow path 19 and the discharge flow path 20 was 200 μm. Moreover, the volume of the extraction tank part 16 was about 10 microliters.

  The damming unit 21 formed a slit-like pattern having the same depth as the extraction tank unit 16. It was possible to dampen cedar pollen having a slit width of about 10 μm and a particle size of about 30 μm.

  The damming portion 21 of this chip is composed of a slit row having the same width as that of the extraction tank 16 and a plurality of slit rows shorter than the width of the extraction tank 16 disposed on the inlet side from the slit row. It was.

  Dispersing and providing a plurality of damming units in the extraction tank unit 16 prevents the sample from concentrating on a specific portion, and suppresses a decrease in the liquid feeding speed due to the sample clogging the damming unit. it can.

  The sample is fed into the extraction tank unit 16, the extraction liquid 15 is injected, the protein is extracted from the sample, and the solution is fed to the detection unit 13 of the extracted solution from which Cry-J1 is extracted. By the method.

  By providing a plurality of damming portions dispersed in the extraction tank portion 16, it was possible to prevent the sample from concentrating on a specific portion and to efficiently extract proteins from the sample.

  As in Example 1, it was confirmed that the extracted Cry-J1 amount could be detected by a conventional electrochemical detection type microchannel device.

(Example 6)
In this embodiment, the extraction chip is the chip 104 in FIG. 7A and the chip 105 in FIG. 7B. Corresponding to the fact that there are two discharge ports, a slight design change such as connecting a tube to the discharge port was made to the liquid feeding mechanism 111. Otherwise, the extraction process was performed in the same manner as in Example 1.

  The extraction chip 104 shown in FIG. 7A is provided with one injection channel 19 and one injection port 17 and two discharge channels 20 and eight discharge ports 18. The depth of the extraction tank section 16, the injection flow path 19, the discharge flow path 20, and the damming section 21 was 200 μm, and the width of the injection flow path 19 and the discharge flow path 20 was 200 μm. Moreover, the volume of the extraction tank part 16 was about 10 microliters.

  The damming portion 21 of this chip was provided independently with respect to the two discharge ports 18, and formed a slit-like pattern having the same depth as the extraction tank portion 16. It was possible to dampen cedar pollen having a slit width of about 10 μm and a particle size of about 30 μm.

  By providing the damming portions 21 at two locations, the sample is dispersed in the two damming portions, and protein can be efficiently extracted from the sample. Further, by providing two discharge ports, it is possible to suppress a decrease in the liquid feeding speed due to the specimen being dispersed in the two damming portions and the specimen being clogged by the damming portions. Further, even when one of the discharge channels 20 is clogged, liquid can be fed by the other discharge channel 20. This prevents interruption of the extraction process due to clogging, and further increases the durability of the chip because the internal pressure is less likely to increase due to clogging.

  The sample is fed into the extraction tank unit 16, the extraction liquid 15 is injected, the protein is extracted from the sample, and the solution is fed to the detection unit 13 of the extracted solution from which Cry-J1 is extracted. By the method.

  It was confirmed that the extracted amount of Cry-J1 can be detected by a conventional electrochemical detection type microchannel device.

  Further, the pollen shell remaining in the extraction tank section 16 was collected by the same method as in Example 1. In this example, by providing the damming portions 21 at two locations, the specimen was dispersed in the two damming portions, and the pollen shells could be collected efficiently.

  Further, the extraction chip used in the present embodiment is not limited to the structure of the extraction chip 104 shown in FIG. Two or more discharge flow paths 20 and discharge ports 18 may be provided, and a damming portion may be provided independently for each discharge port 18. For example, for extraction shown in FIG. A structure like the chip 105 may be used. In this case, the two damming portions 21 are provided opposite to each other and the liquid is discharged in the opposite direction, so that the specimen is more easily dispersed and extraction and recovery can be performed more efficiently.

(Example 7)
Example 7 is yet another example of the protein extraction apparatus described in the first embodiment. The protein extraction apparatus according to this example will be described with reference to FIGS.

  The protein extraction apparatus of the present embodiment is obtained by introducing the extraction chip 106 shown in FIG. 9A into the extraction chip 1 portion of the liquid feeding mechanism shown in FIG. Using airborne cedar pollen as a specimen, the allergen protein Cry-J1 contained in the cedar pollen was extracted with a protein extraction device, and the amount of the extracted Cry-J1 was detected by the detection unit.

  The containers, pumps, valves and tubes used were the same as in Example 1.

  First, the casein solution was injected into the extraction tank section 16 before the mixed liquid 14 of the specimen and the transport liquid was fed into the extraction tank section 16. By covering the surfaces of the extraction tank section 16 and the discharge flow path 20 with the casein solution, protein adsorption to the extraction tank section 16 and the discharge flow path 20 is suppressed, and the protein can be efficiently extracted.

  Next, cedar pollen collected from the air and 5 ml of pure water are placed in the collection liquid container 2 and sufficiently stirred. Then, the valves 9, 401 and 27 are opened, the pump 5 is turned on, and cedar pollen and pure water are collected. The water mixture was fed into the extraction tank section 16 in the extraction chip 106 at a liquid feed flow rate of about 2 ml / min.

  As shown in FIG. 9A, the extraction chip 106 has a structure in which an injection port, an injection flow channel, a discharge flow channel, and a discharge port are separately provided for the transfer liquid and the extraction liquid. The depths of the extraction tank section 16, the transfer liquid injection flow path 32, the extraction liquid injection flow path 33, the transfer liquid discharge flow path 34, the extraction liquid discharge flow path 35, and the damming section 21 of the extraction chip 106 are determined. The width of the transport liquid injection channel 32 and the transport liquid discharge channel 34 was 200 μm, and the width of the extraction liquid injection channel 33 and the extraction liquid discharge channel 35 was 100 μm. Moreover, the volume of the extraction tank part 16 was about 10 microliters. The mixed liquid of cedar pollen and pure water was injected into the extraction tank section 16 from the transfer liquid injection port 30 through the transfer liquid injection flow path 32.

  The cedar pollen sent to the extraction chip 106 remains in the extraction tank unit 16 by the damming unit 21, and only pure water passes through the transfer liquid discharge channel 34 and is transferred from the transfer liquid discharge port 36. It was discharged into the container 24. In this way, by mixing cedar pollen with pure water and sending it, it was possible to easily send the sample into a minute part in the chip.

  The damming unit 21 formed a slit-like pattern having the same depth as the extraction tank unit 16. The cedar pollen having a slit width of about 10 μm and a particle size of about 30 μm can be dammed up.

  Next, the valve 8 was opened, the pump 25 was turned on, and the extraction liquid 15 in the extraction liquid container 3 was sent into the recovery liquid container 2. Thereafter, the valves 402, 403, and 10 were opened, the pump 5 was turned on, and the extraction liquid 15 in the recovery liquid container 2 was fed into the extraction tank section 16 in the extraction chip 106. The amount of the extraction liquid 15 fed was quantified by making the valve opening and closing time constant under microcomputer control. As the extraction solution 15, a phosphate buffered saline buffer solution was used, and the amount of solution fed was about 50 μl. The extraction liquid 15 was injected from the extraction liquid injection port 31 through the extraction liquid injection flow path 33 into the extraction tank section 16.

  Next, Cry-J1 of the allergen protein contained in the cedar pollen was extracted in the state where the extraction solution 15 was filled in the extraction tank section 16. The extraction time was about 15 minutes.

  During extraction, the valves 402, 403, and 10 are opened, and the pump 5 and the pump 25 are repeatedly switched on and off by microcomputer control every short time, whereby the extraction liquid 15 in the extraction tank section 16 flows in the flow direction and It was swung in the reverse direction. As a result, the protein was easily extracted from the specimen, and the extraction efficiency was improved compared to the stationary state.

  Next, the extraction liquid from which Cry-J1 has been extracted is sent from the extraction liquid discharge channel 35 to the detection unit 13 through the extraction liquid discharge port 37, and the amount of the extracted Cry-J1 is detected. I did it. When the extraction liquid is discharged from the extraction liquid discharge port 37, the pollen shell is blocked by the blocking unit 21 and is not discharged from the extraction liquid discharge port 37. Therefore, the extraction liquid and the pollen shell can be easily separated. It was.

  As in Example 1, it was confirmed that the extracted Cry-J1 amount could be detected by a conventional electrochemical detection type microchannel device.

  In the present embodiment, the transfer liquid and the extraction liquid are separately provided with the inlet, the injection flow path, the discharge flow path, and the discharge opening of the extraction chip, so that the flow velocity and flow rate for each liquid are set. Can be adjusted independently, and the flow rate and liquid volume can be easily controlled.

  Further, the extraction chip used in the present embodiment is not limited to the structure of the extraction chip 106 shown in FIG. The extraction liquid 107 may have a structure in which an inlet, an injection channel, a discharge channel, and a discharge port of the extraction chip are separately provided for the transport liquid and the extraction liquid. For example, the extraction chip 107 illustrated in FIG. A structure such as

  If the configuration of FIG. 9B, the feeding direction of the transport liquid and the extraction liquid is different, so when injecting the extract liquid, it is difficult to be affected by the pollen dammed in the damming portion 21, More stable liquid feeding becomes possible. Furthermore, when rocking the extraction liquid 15 in the extraction tank section 16 during extraction, the damming sections 21 are provided on both sides of the extraction tank section 16 on the extraction liquid inlet 31 side and the extraction liquid outlet 37 side. Therefore, the specimen can be prevented from going out of the extraction tank section 16, and the extraction efficiency can be further increased.

  Further, the configuration of FIG. 9B has an advantage that the sample aggregated in the damming portion at the time of carry-in can be extracted while eliminating the aggregation by flowing the extraction liquid from the vertical direction.

  Furthermore, in this embodiment, the liquid feeding liquid and the extraction liquid are fed using the same pump, but different pumps may be used. For example, the configuration of the transport mechanism shown in FIG. 10 may be used. In this case, the extraction liquid can be quantified more accurately by using a pump for supplying a small amount of liquid such as a syringe pump as the pumps 404 and 405 for supplying the extraction liquid.

[Third Embodiment]
In the present embodiment, the protein extraction apparatus includes a collection mechanism unit that collects a specimen from the air.

  As shown in FIG. 11, the protein extraction apparatus of this embodiment includes a collection mechanism unit 40 that collects a sample from the air, a liquid feeding mechanism unit that sends the sample to the extraction unit, and protein extraction from the sample. It consists of a chip 1 for extraction.

  FIG. 12 shows the structure of the collection mechanism 40. (A) is a front view, (b) is a cross-sectional view taken along the line YY ′ of (a), and (c) is a perspective view of the collection mechanism 40. In the collection mechanism unit 40, a sample in the air is sucked by a pump or a fan and collected in the collection container 42 of the collection mechanism unit 40.

  A carrier 43 is attached in the collection container 42, and an air sample is sucked from the suction port 45 by an inhalation fan 44 provided on the back surface of the carrier 43, thereby allowing an air sample to be contained in the carrier 43. Capture on top. In this embodiment, a fan is used for inhalation, but other mechanisms such as a pump may be used.

  The carrier 43 is preferably a member that can be sucked from the back surface, can be captured on the surface without passing through the target sample, and can easily collect the sample captured on the surface. As the carrier 43, for example, a filter paper, a film, a filter, or the like can be used. Stainless steel, copper, nickel, nylon, polyester, carbon, etc. can be used as the material of the filter. Among them, a metal filter is particularly preferable because the collected specimen can be easily collected.

  Further, an antistatic coating may be applied to the surface of the carrier 43. By doing so, adsorption of the specimen due to static electricity is prevented, and the specimen captured on the carrier can be more easily recovered from the carrier.

  The collection container 42 is preferably a member that hardly adsorbs the specimen to the inner wall portion. For example, it is possible to use a metal material such as stainless steel or aluminum, or a plastic material such as polycarbonate or acrylic having a surface coated with an antistatic coating.

  The suction port 45 may be provided with a dust filter for preventing entry of dust having a diameter larger than that of the specimen. The dust filter is preferably a filter whose hole is larger than the sample and through which the sample can easily pass, and the sample is difficult to adsorb on the filter. For example, a metal filter can be used.

  For example, when a flat woven wire mesh filter having a filtration accuracy of 100 μm is provided as a dust filter, it is possible to prevent the intrusion of dust of 100 μm or more, and to collect and extract cedar pollen more stably. It becomes.

  Next, after suctioning air from the suction port 45 for a certain period of time, the shutter 46 provided in the collection container 42 is closed, and the collection container 42 is sealed, and a suction pump or the like is used from the recovery port 47 for a certain period of time. By aspirating, the specimen captured on the carrier 43 is sent into the recovery liquid container 2.

  By injecting the transport liquid into the collection liquid container 2 in advance and feeding the specimen into the transport liquid together with air, the specimen and the transport liquid are agitated. Therefore, it is not necessary to provide a separate stirring means, and the apparatus can be downsized.

  When collecting the specimen on the carrier 43, the carrier 43 may be provided with a vibration mechanism 48, and suction may be performed while vibrating the carrier 43. By vibrating the carrier 43, the specimen on the carrier 43 can be taken more easily, and the collection efficiency of the specimen from the air can be increased.

  Further, when collecting the specimen on the carrier 43, suction may be performed while hitting a part of the back surface of the carrier 43. By applying an impact to the carrier 43, the specimen on the carrier 43 can be taken more easily, and the collection efficiency of the specimen from the air can be increased.

  Further, when the specimen on the carrier 43 is collected, suction may be performed while blowing air from the back surface or the front surface of the carrier 43 with a fan or the like. By applying wind pressure to the carrier 43, the specimen on the carrier 43 can be taken more easily, and the collection efficiency of the specimen from the air can be increased.

  Further, when collecting the specimen on the carrier 43, suction may be performed while scraping the specimen on the carrier 43 with a metal brush or the like. By directly scraping the specimen, the specimen on the carrier 43 can be taken more easily, and the collection efficiency of the specimen from the air can be increased.

  Further, when the specimen on the carrier 43 is collected, suction may be performed on the surface of the carrier 43 while moving the suction nozzle by making a suction mechanism such as a pump or a fan movable. By directly aspirating the specimen, the specimen on the carrier 43 can be taken more easily, and the collection efficiency of the specimen from the air can be increased.

  Further, when the specimen on the carrier 43 is collected, suction may be performed while flowing the transport liquid into the collection container 42. By washing away with the transfer liquid, the sample in the collection container 42 can be more easily collected, and the collection efficiency of the sample from the air can be increased.

  The sample collected in the collection liquid container 2 can be subjected to protein extraction by the same method as in the first embodiment.

  As described above, according to the present embodiment, it is possible to automatically perform operations from the collection of a sample from the air to the extraction and separation of proteins using a single device. By collecting the specimen by once collecting it on the carrier 43 and collecting it, it becomes easy to increase the suction speed at the time of capture, and the specimen collection time can be shortened. In addition, since protein is extracted in a small amount of extract, a high concentration protein extract can be produced, and a complicated concentration step that has been conventionally required is not required, and the apparatus can be miniaturized. . Furthermore, by using this high concentration protein extract as a test solution for protein detection, it is possible to measure the amount of protein with high accuracy.

  In the present embodiment, in the collection of the specimen from the air, a method is used in which the sample is once collected on the carrier 43 and then collected. However, the sample is aspirated by a pump or the like without using the carrier 43 and is collected. You may collect directly in 2. Compared with the case where the carrier 43 is used, the restriction on the suction speed becomes stricter, but the adsorption to the carrier 43 and the like is reduced, and it becomes easy to increase the collection efficiency.

  In this embodiment, the filter is used for separating the dust and the specimen, but the present invention is not limited to this. For example, a method of separating using a cyclone structure using a difference in centrifugal force, a method of separating using a difference in gravity, or the like may be used.

  In this embodiment, the sample is mixed with the transport solution and injected into the extraction tank 16 in the extraction chip 1. However, the sample may be directly injected into the extraction tank 16. By extracting the sample in the extraction tank section 16 and performing extraction, the protein is extracted in a small amount of the extract, and thus it is possible to generate a protein extract with a high concentration.

(Example 8)
An example of the protein extraction apparatus according to the third embodiment will be described in this example.

  In the protein extraction apparatus of this example, the collection mechanism 40 shown in FIG. 12, the liquid feeding mechanism 111 shown in FIG. 11, and the extraction chip 1 shown in FIG. 1 were used. Using airborne cedar pollen as a specimen, the allergen protein Cry-J1 contained in the cedar pollen was extracted with a protein extraction device, and the amount of the extracted Cry-J1 was detected by the detection unit.

  The containers, pumps, valves and tubes used were the same as in Example 1.

  FIG. 12 shows the structure of the collection mechanism 40. In the collection container 42, a wire mesh filter is attached as a carrier 43, and air cedar pollen is collected by sucking air from the suction port 45 by a suction fan 44 provided on the back surface of the wire mesh filter. Captured on a wire mesh filter.

  As the wire mesh filter, a twilled woven wire mesh filter having a size of 120 mm × 120 mm and a filtration accuracy of 20 μm was used. The flow rate through the wire mesh filter by the suction fan was 0.6 m / s.

  The collection container 42 was made of stainless steel in order to prevent cedar pollen from adsorbing to the inner wall.

  While the air is being sucked from the suction port 45, the valves 9 and 27 are opened, the pump 25 is turned on, and 5 ml of pure water as the transport liquid is injected from the transport liquid container 24 into the recovery liquid container. did.

  Next, after inhaling air from the inlet 45 for 5 minutes, the shutter 46 provided in the collection container 42 is closed, the collection container 42 is closed, the valve 38 is opened, the pump 25 is turned on, and the collection is performed. The cedar pollen captured by the wire mesh filter as the carrier 43 was fed into the collection liquid container 2 from the opening 47.

  When collecting the cedar pollen on the wire mesh filter, it was collected while vibrating the wire mesh filter by a vibration motor attached to the wire mesh filter. By vibrating the wire mesh filter, the cedar pollen on the wire mesh filter was more easily removed, and the collection efficiency of cedar pollen from the air could be increased.

  The cedar pollen and pure water were easily stirred by feeding the cedar pollen together with air into the pure water previously poured into the collection liquid container 2. Therefore, it is not necessary to separately provide a stirring means, and the apparatus can be downsized.

  Before the mixed solution of cedar pollen and pure water was fed into the extraction tank section 16, the casein solution was injected into the extraction tank section 16. By covering the surfaces of the extraction tank section 16 and the discharge flow path 20 with the casein solution, protein adsorption to the extraction tank section 16 and the discharge flow path 20 can be suppressed, and protein can be extracted efficiently.

  Thereafter, in the same manner as in Example 1, the sample transport process and the extraction process in the collection container liquid container 2 were performed. It was confirmed that the extracted amount of Cry-J1 can be detected by a conventional electrochemical detection type microchannel device.

  As described above, according to this example, it was possible to automatically perform operations from the collection of cedar pollen from the air to the extraction and separation of proteins by a single device. In addition, by collecting cedar pollen by once collecting it on a wire mesh filter and collecting it, it becomes easy to increase the suction speed at the time of capture, and it is possible to shorten the collection time of cedar pollen. It was.

[Fourth Embodiment]
In the present embodiment, a protein measurement apparatus in which the protein extraction apparatus according to the third embodiment and a protein detection microchip are integrated will be described.

  FIG. 13 is a schematic diagram of a protein measuring apparatus according to the present embodiment. The protein measuring apparatus has a configuration in which a protein extracting apparatus 300 and a protein detecting microchip 500 are connected by a pipe. This protein extraction apparatus 300 is the same as that of the third embodiment.

  The protein detection microchip 500 includes a substrate 501 with a flow path 504 formed thereon, an inlet 504A, an outlet 505A, and a lid substrate 510 with an electrochemical detection electrode 506 formed thereon.

  The channel 504 is filled with fine particles 502 to which an antibody as a reactant is fixed. In the fine particle 502, an antibody that causes an antigen-antibody reaction with the extracted protein is immobilized. A pipe connected from the protein extraction device 300 is fixed to the inlet 504A by a joint 511.

  In addition, a pipe 302 connected to the cleaning solution, the labeled antibody solution, and the substrate solution storage unit 301 is connected to the pipe via a switching valve 303.

  In this apparatus, by first switching the switching valve 303, the protein extract extracted in the same manner as in the second embodiment is injected from the inlet 504A of the microchannel device 500. Thereby, the allergen contained in the protein extract and the antibody of the microparticles 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 port 504A of the microchannel device 500. As a result, the enzyme-labeled antibody reacts with the antibody-antigen complex immobilized on the fine particles 502, and the enzyme-labeled antibody is captured.

  Thereafter, the cleaning liquid is flowed to clean the flow path 504, and then the substrate solution is injected from the injection port 504A of the micro flow path 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 protein extraction 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.

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

  In the present embodiment, the electrochemical detection method is used as the protein detection microchip detection method, but the present invention is not limited to this. A micro-channel device using another detection method such as a thermal lens method or an SPR method may be used. Also in this case, it is possible to perform operations from sample collection to detection of the amount of allergen with one apparatus.

[Fifth Embodiment]
In the present embodiment, a protein measuring apparatus according to the fourth embodiment that is incorporated in an air cleaner will be described.

  FIG. 15 shows a schematic diagram of an air cleaner according to this embodiment. (A) is a front view of an air cleaner, (b) is sectional drawing when cut | disconnecting by the ZZ 'line of (a).

  As shown in FIG. 15B, the inside of the main body 601 is partitioned forward and backward by a partition plate 602. As a result, a first compartment 603 is formed in front of the main body 601 (upward from the partition plate 602), and a second compartment 604 is formed in the rear (downward from the partition plate 602). . The first compartment 603 and the second compartment 604 communicate with each other via a single or a plurality of openings provided in the partition plate 602. In addition, a suction port 605 for air from the outside is provided in front of the main body 601, and a blower outlet 606 for discharging the sucked air to the outside is provided on the rear upper surface of the main body 601.

  The suction port 605 is composed of a plurality of suction ports. In the present embodiment, the suction port 605 includes a first suction port 605a and a second suction port 605b, and these are arranged in parallel up and down on the front surface of the main body 601.

  Further, a filter 607, an ion generator 608, and a blower 609 are provided in the main body 601.

  The filter 607 passes the air sucked from the outside and cleans it, and is provided in the first compartment 603. The filter 607 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 607 is provided inside the main body 601 so that only air sucked from one of the suction ports passes. In particular, in this embodiment, only air sucked from the first suction port 605a is provided. It is provided just behind (leeward side) the first suction port 605a so as to pass through. That is, the filter 607 is not disposed behind the second suction port 605b. Thereby, as a path | route of the air suck | inhaled inside the main body 601, the path | route (henceforth the 1st path | route A) inhaled from the 1st inlet 605a and passing the filter 607, and inhaled from the 2nd inlet 605b Thus, there are two routes, a route that does not pass through the filter 607 (hereinafter referred to as the second route B).

  The ion generator 608 includes a discharge electrode and an induction electrode that are arranged to face each other via a dielectric, and generates ions by discharging between the two electrodes. In the second compartment 604, the air outlet 606 is provided. It is provided in the vicinity. The ion generator 608 can generate at least one of positive and negative ions. If positive and negative ions are generated from the ion generator 608, airborne bacteria in the air can be inactivated by these ions, and harmful substances in the air can be removed.

  The air blowing means 609 discharges air sucked from outside through the ion generator 608, and has a fan 609a and a motor 609b that rotates the fan 609a. The fan 609a is provided in the second compartment 604, and the motor 609b is provided across the first compartment 603 and the second compartment 604.

  The shutter 610 is provided immediately behind the second suction port 605b, and opens and closes the second suction port 605b by its own opening / closing operation.

  The protein extraction apparatus 300 is provided at a predetermined place where the suction port 605b is inserted, and an insertion port 611 for inserting, for example, a conventionally proposed allergen detection microchip 500 is provided below the suction port 605b. ing. When the allergen detection microchip 500 is inserted from the insertion port 611, the allergen detection microchip 500 and the protein extraction apparatus 300 of the present invention are connected. This connected state is the same as in the third embodiment.

  When the air cleaner is activated, air that does not pass through the filter 607 is sent to the protein extraction apparatus 300. As described in the second and third embodiments, pollen is collected, protein is extracted, and the amount of protein is detected.

  An electrical signal corresponding to the detected amount of protein is sent from the microchip 500 to the air purifier controller. In the air cleaner control unit, the rotation amount (fan amount) of the fan 609a and the ion generation amount of the ion generator are controlled by microcomputer control or the like.

  Here, the allergen detection microchip 500 is used a plurality of times after performing a regeneration process after use. The allergen detection microchip 500 with reduced sensitivity is taken out from the insertion slot 611, and a new allergen detection microchip 500 is inserted.

  In the present embodiment, an application example of the protein measuring apparatus of the present invention has been described by taking an air cleaner as an example, but the present invention can also be applied to other air conditioners such as an air conditioner, a humidifier, and a dehumidifier.

  As described above, according to the present invention, a protein extraction apparatus that can efficiently extract a target protein can be realized. By connecting this apparatus to a measuring instrument, a protein measuring apparatus can be realized. Thereby, operations from collection of a sample contained in an external space such as pollen to protein extraction, concentration, separation, and measurement can be efficiently performed with one apparatus. Therefore, its industrial significance is great.

It is the schematic which shows the chip | tip structure for extraction concerning 1st Embodiment. It is the schematic explaining the outline | summary of the protein extraction apparatus concerning 2nd Embodiment. It is the schematic explaining the outline | summary of the protein extraction apparatus concerning Example 2. FIG. It is the schematic which shows the chip | tip structure for extraction concerning Example 3. FIG. It is the schematic which shows the chip | tip structure for extraction concerning Example 4. FIG. It is the schematic which shows the chip | tip structure for extraction concerning Example 5. FIG. It is the schematic which shows the chip | tip structure for extraction concerning Example 6. FIG. It is the schematic explaining the outline | summary of the protein extraction apparatus concerning Example 7. FIG. It is the schematic which shows the chip | tip structure for extraction concerning Example 7. FIG. It is the schematic explaining the outline | summary of another protein extraction apparatus concerning Example 7. FIG. It is the schematic explaining the outline | summary of the protein extraction apparatus concerning 3rd Embodiment. It is the schematic which shows the structure of the collection mechanism part concerning 3rd Embodiment. It is the schematic explaining the outline | summary of the protein measuring apparatus concerning 4th Embodiment. It is the schematic explaining the outline | summary of the microchip for an allergen detection used in 4th embodiment. It is the schematic explaining the outline | summary of the air cleaner concerning 5th Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Extraction chip 2 Collection liquid container 3 Extraction liquid container 4 Discharge liquid container 5, 6 Liquid feed pump 7, 8, 9, 10, 11 Valve 12 Liquid feed tube 13 Detection part 14 Sample and transport liquid Liquid mixture 15 extraction liquid 16 extraction tank part 17 inlet 18 discharge port 19 injection flow path 20 discharge flow path 21 damming part 22 substrate 23 lid base material 24 liquid container for transport 25 suction pumps 26, 27 valve 28 for transport Liquid 30 Transport liquid inlet 31 Extraction liquid inlet 32 Transport liquid injection path 33 Extraction liquid injection path 34 Transport liquid discharge path 35 Extraction liquid discharge path 36 Transport liquid discharge port 37 Extraction Liquid discharge port 38 Valve 40 Collection mechanism 41 Recovery tube 42 Collection container 43 Carrier 44 Suction fan 45 Suction port 46 Shutter 47 Recovery port 48 Vibration mechanism 101, 102, 103, 104, 105, 106, 107 Extraction chip 111 Liquid feeding mechanism unit 300 Protein extraction device 301 Solution storage unit 302 Pipe 303 Switching valve 401, 402, 403 Valve 404, 405 Pump 500 Microchip for protein detection 501 Substrate 502 Fine particle with antibody 503 Reaction tank unit 504, 505 Flow path 504A Inlet 505A Discharge port 506 Electrode 507 Connection pad 508 Damping portion 509 Wiring 510 Lid substrate 511 Joint 601 Air conditioner body 602 Partition plate 603 First partition chamber 604 Second partition chamber 605a First suction port 605b First 2 Suction port 606 Blowing port 607 Filter 608 Ion generator 609a Fan 609b Motor 610 Shutter 611 Insertion port

Claims (26)

An extraction tank for extracting a target component from a specimen;
At least one injection flow channel connected to the extraction vessel;
At least one discharge channel connected to the extraction tank section;
In an extraction microchip comprising:
Between the injection channel and the discharge channel in the extraction tank unit, a damming unit having a curved chip plan view shape is provided,
An extraction microchip characterized by that. The extraction microchip according to claim 1,
The plan view shape of the extraction tank is a circular shape or an elliptical shape,
An extraction microchip characterized by that. In the extraction microchip according to claim 1 or 2,
The damming portion has a curved shape that is convex toward the discharge channel side.
Extraction microchip characterized by that In the extraction microchip according to any one of claims 1 to 3,
The damming portion has a slit structure.
An extraction microchip characterized by that. An extraction tank for extracting a target component from a specimen;
At least one injection flow channel connected to the extraction vessel;
At least one discharge channel connected to the extraction tank section;
In an extraction microchip comprising:
The damming portion is provided in a shape connecting both end faces in a direction perpendicular to the inflow / outflow direction of the extraction tank section, and the slit width in the vicinity of both end faces is wider than the slit width in the central region,
An extraction microchip characterized by that. An extraction tank for extracting a target component from a specimen;
At least one injection flow channel connected to the extraction vessel;
At least one discharge channel connected to the extraction tank section;
In an extraction microchip comprising:
Between the injection flow path and the discharge flow path in the extraction tank, at least two rows of damming portions having a slit structure are provided in the flow direction.
An extraction microchip characterized by that. An extraction tank for extracting a target component from a specimen;
At least one injection flow channel connected to the extraction vessel;
At least one discharge channel connected to the extraction tank section;
In an extraction microchip comprising:
Between the injection channel and the discharge channel in the extraction tank, a plurality of damming portions having a slit structure are arranged, and the plurality of damming portions are arranged in multiple stages in the flow direction. A microchip for extraction, characterized by An extraction tank for extracting a target component from a specimen;
At least one injection flow channel connected to the extraction vessel;
At least two discharge channels connected to the extraction tank section;
In an extraction microchip comprising:
In the extraction tank part, a damming part having a slit structure is provided between each discharge channel and injection channel,
An extraction microchip characterized by that. The extraction microchip according to any one of claims 1 to 8,
The volume of the extraction tank in the microchip is 5 to 50 μl.
An extraction microchip characterized by that. The extraction microchip according to any one of claims 1 to 9,
A liquid feeding mechanism section for feeding the specimen and the extraction liquid to the extraction tank of the microchip for extraction;
Have
The liquid feeding mechanism section feeds the sample liquid storage container for storing the specimen and the extraction liquid, and the specimen and the extraction liquid from the sample liquid storage container to the extraction tank section in the extraction microchip. A liquid feed pump, comprising at least
The protein extraction apparatus characterized by the above-mentioned. The protein extraction apparatus according to claim 10, wherein
The amount of extraction liquid fed is 5 to 100 μl.
The protein extraction apparatus characterized by the above-mentioned. The protein extraction apparatus according to claim 10 to 11,
A means for vibrating the extraction tank section is provided in the microchip.
The protein extraction apparatus characterized by the above-mentioned. The protein extraction apparatus according to claim 10 to 11,
An ultrasonic generator is provided on the microchip.
The protein extraction apparatus characterized by the above-mentioned. The protein extraction apparatus according to any one of claims 10 to 13,
Provide a micro heater on the microchip,
The protein extraction apparatus characterized by the above-mentioned. The protein extraction device according to any one of claims 10 to 14,
A suction pump is further provided in the liquid feeding mechanism.
The protein extraction apparatus characterized by the above-mentioned. The protein extraction apparatus according to any one of claims 10 to 15,
Provide a collection mechanism to collect the sample from the external space,
A protein extraction apparatus characterized by that. A protein extraction device according to any one of claims 10 to 16,
A microchannel device for detecting the type and amount of protein extracted by the protein extraction device,
The microchannel device has a reaction part containing a reactive substance that reacts with the protein;
A detection unit for detecting the amount of the protein;
Having
A protein measuring apparatus. A protein measuring device according to claim 17,
Air blowing means for moving the specimen together with air;
A blast 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,
An air conditioner characterized by that. A protein measuring device according to claim 17,
A protein-containing substance removing means for removing the protein-containing substance;
Removing operation control means for controlling the operation of the protein-containing substance removing means according to the amount of protein measured by the protein measuring device,
An air conditioner characterized by that. An extraction tank unit that extracts a target component from a specimen, at least one injection channel connected to the extraction tank unit, and at least one discharge channel connected to the extraction tank unit An extraction microchip, characterized in that a damming portion is provided between the injection channel and the discharge channel in the extraction tank unit,
A liquid feeding mechanism section for feeding the specimen and the extraction liquid to the extraction tank of the microchip for extraction;
Have
A sample solution storage container in which the liquid feeding mechanism unit stores the sample and the extraction liquid;
A liquid feed pump for feeding the specimen and the extraction liquid from the sample liquid storage container to the extraction tank in the microchip for extraction;
Using a protein extraction apparatus comprising at least
A protein extraction method for extracting protein from a specimen,
The transport liquid mixed with the specimen is fed by the liquid feed pump, and the specimen is transported together with the transport liquid into the microchip.
A protein extraction method characterized by the above. The extraction microchip according to any one of claims 1 to 9,
A liquid feeding mechanism section for feeding the specimen and the extraction liquid to the extraction tank of the microchip for extraction;
Have
A sample solution storage container in which the liquid feeding mechanism unit stores the sample and the extraction liquid;
A liquid feed pump for feeding the specimen and the extraction liquid from the sample liquid storage container to the extraction tank in the microchip for extraction;
Using a protein extraction apparatus comprising at least
A protein extraction method for extracting protein from a specimen,
The transport liquid mixed with the specimen is fed by the liquid feed pump, and the specimen is transported together with the transport liquid into the microchip.
A protein extraction method characterized by the above. The protein extraction method according to claim 20 to 21, wherein
The extraction liquid is alternately fed and reversely fed by the liquid feeding pump and the suction pump, and the protein is extracted from the specimen by swinging the extraction liquid.
A protein extraction method characterized by the above. The protein extraction method according to any one of claims 20 to 22,
As the transport liquid, a poor protein solvent that does not dissolve the extraction target protein contained in the specimen is used.
A protein extraction method characterized by the above. The protein extraction method according to any one of claims 20 to 23,
The specimen is pollen,
A protein extraction method characterized by the above. The protein extraction method according to any one of claims 20 to 24,
The transport liquid is pure water;
A protein extraction method characterized by the above. In the protein extraction method in any one of Claim 20 thru | or 25,
The extraction solution is a phosphate buffer;
A protein extraction method characterized by the above.