JP2007187587A - Electrophoretic device, and plate for electrophoresis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compact, lightweight, and inexpensive electrophoretic device, enabling setting of a different temperature for each of a plurality of flow paths formed on a plate, dispensing with complicated preparation work, and providing an accurate result in a short period of time. <P>SOLUTION: A flow path temperature adjustment part 45 with a heating/cooling device 33 having a voltage application means 30 built therein is disposed on an upper part of a tray 22 for thereon mounting the plate 10. The adjustment part 45 is provided with blowoff opening parts 46 as many as the flow paths 110 in order to blow a temperature-controlled air flow from the adjustment part 45 into each of the plurality of flow paths for electrophoresis on the plate 10. An inner wall of each blowoff openings 46 is provided with a heater 48 for heating air cooled in the adjustment part 45 to a desired temperature to temperature-control the respective flow paths on the plate 10 on different temperature settings. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an electrophoresis apparatus for electrophoresis of DNA, protein and other biological samples in a buffer, and an electrophoresis plate used for the electrophoresis apparatus. The present invention relates to an electrophoresis apparatus capable of electrophoresis and an electrophoresis plate.

  When a general biological sample is considered, DNA and protein exist largely. In recent years, with the rapid development of molecular biology, the involvement of genes in various diseases has been understood fairly accurately, and attention has been focused on medical treatments targeting genes. Regarding DNA, SNPs (abbreviation of single nucleotide polymorphism, which is generally translated as “single nucleotide polymorphism” and is a general term for a difference of one code (one base) in a gene) has been attracting attention. The reason for this is that the classification of SNPs can predict the prevalence of many diseases and the effects and susceptibility of each individual to drugs, and there are absolutely no humans on the planet who have the same SNPs, even if they are parents and siblings. This is because it is considered that the individual can be completely identified from not doing so.

  Currently, as a method for examining SNPs, sequencing (determination of base sequence) in which the base sequence of DNA is directly read from the end is most commonly used. There are several reports on the sequencing method, but the most common method is the dideoxy method (Sanger method). Sequencing is based on a technique that separates and identifies the difference in length of one base length by denaturing polyacrylamide gel electrophoresis with high resolution or capillary electrophoresis in any method including the Sanger method. This is true.

Another method is affinity ligand capillary electrophoresis.
Affinity ligand capillary electrophoresis uses intermolecular affinity, particularly specific affinity in the ecosystem (enzyme and substrate, antigen and antibody affinity, etc.) to give specificity to separation. Specifically, When an affinity ligand that specifically recognizes the base sequence is added to the electrophoresis solution in the capillary tube and the sample is electrophoresed, only the molecular species that interact in the sample mixture will change in the moving speed. The analysis is performed with attention (for example, see Patent Document 1).

  On the other hand, proteins exist in cells, tissues, and biological fluids, regulate biological activities, supply energy to cells, synthesize important substances, maintain biological structures, and communicate between cells and intracellular information. Involved in transmission. Currently, it has become clear that proteins have multiple functions depending on the various environments, the presence of other interacting proteins, and the degree and type of modification that the protein has undergone.

  Proteins are made by connecting 20 types of amino acids in order according to gene instructions (sequence information), and the types are said to be tens of millions. If you know the sequence of the gene, what amino acid is what? You can get information on whether they are connected in order. A complete set of proteins made from the genes (genomes) of organisms is called a proteome, and now the analysis of the proteome has been actively promoted after the nucleotide sequence of the human genome has been deciphered.

  Such functional analysis of proteins requires not only identification and characterization, but also biochemical assays, protein-protein interaction studies, protein networks, and intracellular and extracellular signaling elucidation. In this protein function research, various techniques are used and there are enzyme assay, yeast two-hybrid assay, chromatographic purification, information tool and database, etc. Especially, protein discrimination by electrophoresis is important. It is a technique. Then, as in electrophoresis, the transport reaction obtained when moving liquids such as samples, analytes, buffers, and reagents in capillary tubes is detected, and analysis, discrimination, determination, etc. of the samples are performed. There are various reports regarding the transportation and orientation of the liquid when it is performed (for example, Patent Documents 2 to 5).

  In recent years, in addition to the capillary electrophoresis apparatus as described above, an electrophoresis apparatus using a biological sample discriminating plate as disclosed in, for example, Patent Document 6 has also been reported.

  FIG. 12 is a diagram illustrating a configuration of a conventional electrophoresis apparatus disclosed in Patent Document 6. In the electrophoresis apparatus 400, a resin-made biological sample discrimination plate 10 (hereinafter simply referred to as “plate”) is used. The one in which a fine channel is formed is used. When the determination is made, the plate 10 in which the DNA sample and the DNA conjugate for separation are injected into the flow path of the plate 10 is mounted on the tray 422, and the plate 10 is rotated at high speed by the high-speed rotation motor 421. Then, after filling a part of the flow path with the separation DNA conjugate, a predetermined position of the flow path is pressurized by the pressurizing unit 424 and rotated at a high speed again by the high-speed rotation motor 421 to fill the flow path. A DNA sample is quantitatively added to the separated DNA conjugate. Then, the plate 10 is lifted by the lifting stage 450 moved up and down by the motor 451, the plate 10 is positioned with respect to the optical detection unit 440 by the fitting pin 434, and the plate 10 is fixedly held by the clamper 436. The heater 433 having a size covering all the flow paths of the plate 10 and the sensor 455 are controlled so that the flow path becomes a predetermined temperature, and the voltage applying means 432a and 432b are inserted at predetermined positions of the flow path of the plate 10. In this state, a predetermined potential gradient is applied to the separation DNA conjugate by the voltage application means 432a and 432b, and a DNA sample is electrophoresed in the separation DNA conjugate, and at the same time, the low-speed motor 431 is rotated. While the plate 10 is rotated, the fluorescent material applied to the sample to be electrophoresed in the flow path of the plate 10 by the optical detection unit 440 is irradiated with light by a laser or LED, and the fluorescent material emits light. The fluorescence intensity distribution is detected.

Therefore, according to the conventional electrophoresis apparatus 400, if a user injects a DNA sample and a separation DNA conjugate into the plate 10 and sets the plate 10 in the apparatus 400, the apparatus 400 side performs all processing, Since the filling process of the DNA sample and the DNA conjugate for separation into the flow path of the plate 10, the electrophoresis process of the DNA sample, and the optical detection process are automatically performed, a complicated preparation work is not performed in a short time. Accurate detection results can be obtained.
JP 7-311198 A Special Table 2000-513813 JP-T-2001-523341 JP 2000-514928 gazette JP 2003-28883 A International Publication No. 2005/064339 Pamphlet

  However, the above-described conventional electrophoresis apparatus 400 shown in FIG. 12 has a configuration in which a heater 433 having a size covering the entire flow path of the plate 10 is in contact with the plate 10 to heat or cool the flow path. However, since all the channels on the plate are heated or cooled at the same temperature, when a plurality of channels are formed on the plate 10, the channel 10 is different for each channel. The temperature could not be controlled by setting the temperature. Therefore, the conventional apparatus 400 has a disadvantage in that it cannot cope with samples or the like having different electrophoresis conditions or SNPs separation conditions on the same plate 10 at the same time.

  The present invention has been made to solve the above-described conventional problems, and can be set at different temperatures for each of a plurality of flow paths formed on a plate, is easy to handle, is small and lightweight. It is an object of the present invention to provide an inexpensive electrophoresis apparatus using an electrophoresis plate and an electrophoresis plate.

In order to solve the above problems, the electrophoresis apparatus of the present invention is a electrophoresis apparatus in which a sample is quantitatively added to a buffering agent filled in a flow path, and the sample is electrophoresed by voltage application. Each of a plurality of plates formed on the plate, a tray for mounting the plate, a voltage applying means for applying a voltage to a buffer in the flow path of the plate, and a plurality of flow paths formed on the plate A plurality of blowing openings for blowing out the temperature-controlled air flow, and blowing the air flow blown out from each of the blowing openings to each of the flow paths, so that each of the plurality of flow paths has a predetermined temperature. And a flow path temperature adjusting unit to be adjusted.
This makes it possible to control at different temperatures for each of a plurality of flow paths formed on the plate. Therefore, if the biological sample is discriminated by the apparatus, the electrophoresis conditions and SNPs can be obtained with one plate. A plurality of samples having different separation conditions and the like can be electrophoresed simultaneously, and an accurate detection result can be obtained in a short time.

Moreover, the electrophoresis apparatus of this invention is provided with the heater means which heats the said air flow to each predetermined temperature in each of the said several blowing opening part of the said flow-path temperature control part.
Thereby, since the air flow controlled by predetermined temperature for every flow path can be sprayed, it can implement | achieve control by different temperature for every some flow path. Further, by providing the heater in the opening, it is possible to realize a small and simple apparatus.

Furthermore, in the electrophoresis apparatus of the present invention, the flow path temperature adjusting unit includes at least one cooling unit and a common chamber cooled by the cooling unit, and the air cooled to a predetermined temperature in the common chamber. The flow is heated to the respective predetermined temperatures by the heater means provided in each of the plurality of blow-off openings, and the air flow temperature-controlled at the predetermined temperatures is blown to the respective flow paths of the plates. Is.
Thereby, since the mechanism for temperature control can be realized with a simple configuration, the electrophoresis apparatus can be made smaller, lighter and less expensive.

Furthermore, the electrophoresis apparatus of the present invention has a light irradiation means for irradiating light to the flow path, and a fluorescence detection means for detecting fluorescence generated from the sample by the light irradiation, An optical detection unit that optically detects the sample that is electrophoresed, and a voltage application contact / separation unit that brings the voltage application unit into contact with or away from the flow path formed in the plate. The voltage application contact / separation means brings the voltage application means into contact with the flow path, and the sample is electrophoresed by voltage application, and then the voltage application contact / separation means causes the voltage application means. Is separated from the flow path, and optical detection is performed by the optical detection unit.
Thereby, since it is not necessary to drive the voltage application means and the plate integrally, the apparatus can be made small and simple.

Furthermore, in the electrophoretic device of the present invention, the voltage applying means is disposed inside the flow path temperature adjusting unit disposed on the tray, and the blowing opening of the flow path temperature adjusting unit A voltage is applied to the buffer in the flow path of the plate.
Thereby, in the said apparatus, since the said electrode application means can be comprised integrally with a flow-path temperature control part, the said whole apparatus can be reduced in size.

Furthermore, in the electrophoresis apparatus of the present invention, the plurality of blowing openings have an area that substantially covers each flow path of the plate.
This makes it possible to control the temperature by reliably blowing an air flow controlled to a predetermined temperature for each flow path formed in the plate.

Further, in the electrophoresis apparatus of the present invention, the outlet shape of the blowing opening is cut obliquely so that the air blown out from the blowing opening flows in the direction from the center of the plate to the outside. is there.
Thereby, the airflow blown out from one blowout opening hardly flows to the adjacent flow path, and it is possible to reduce the influence on the temperature control of other flow paths.

In addition, the electrophoresis plate of the present invention includes a plurality of the flow paths used in an electrophoresis apparatus in which a sample is quantitatively added to a buffer filled in the flow path and the sample is electrophoresed by applying a voltage. In the prepared electrophoresis plate, a slit is provided between the formed adjacent flow paths of the electrophoresis plate.
Thereby, in all the flow paths formed in the plate, heat conduction between the flow paths hardly occurs, so that temperature control for each flow path can be reliably performed.

Further, in the electrophoresis plate of the present invention, a heat insulating material is fitted in the slit provided between the flow paths.
Thereby, temperature control for every flow path can be reliably performed in all flow paths formed in the plate while ensuring the strength of the plate.

Further, in the electrophoresis plate of the present invention, the heat insulating material is made of a material having a higher heat insulating effect than a material constituting the electrophoresis plate.
As a result, heat conduction between the flow paths hardly occurs, and temperature control for each flow path can be reliably performed.

In addition, the electrophoresis plate of the present invention includes a plurality of the flow paths used in an electrophoresis apparatus in which a sample is quantitatively added to a buffer filled in the flow path and the sample is electrophoresed by applying a voltage. In the prepared electrophoresis plate, a region where the thickness of the plate is increased is provided between the formed adjacent flow paths of the electrophoresis plate.
Thereby, while ensuring the strength of the plate, in all the flow paths formed in the plate, heat conduction between the flow paths is less likely to occur, and temperature control for each flow path can be reliably performed. it can.

  According to the electrophoresis apparatus of the present invention, each of the plurality of flow paths on the plate on which a plurality of flow paths for electrophoresis of the sample quantitatively added in the filled buffer are formed is provided for each of the plurality of flow paths. Since a flow path temperature adjusting unit that blows out a temperature-controlled air flow for each flow path is provided, it is possible to perform different temperature adjustments for each flow path of the plate. The temperature at the time of electrophoresis can be set according to the DNA sample to be migrated. As a result, a plurality of samples having different separation conditions can be determined accurately and in a short time with one plate.

  According to the electrophoresis apparatus of the present invention, the flow path temperature adjusting unit is disposed on the tray on which the plate is mounted, and voltage application for applying a voltage to the buffering agent filled in each flow path of the plate is performed. Since the means is disposed inside the flow path temperature adjusting unit, and the voltage application means is further provided with voltage application contact / separation means for bringing the voltage application means into contact with or separating from the plate, The configuration can be further downsized.

  According to the electrophoresis plate of the present invention, a slit penetrating the plate is provided between adjacent flow paths formed on the plate to prevent the influence of heat conduction between the adjacent flow paths. Therefore, it is possible to obtain accurate temperature control performance of each flow path, and as a result, it is possible to obtain highly reliable electrophoresis and SNP separation and light detection results.

(Embodiment 1)
Hereinafter, the electrophoresis apparatus 100 according to the first embodiment will be described.
The present invention relates to an electrophoretic apparatus for performing biological, enzymatic, immunological, and chemical assays by moving a biological sample in a buffer, and a plurality of electrophoretic conditions and separation conditions for SNPs are different. It is possible to simultaneously determine the biological sample.

  In the first embodiment, for the sake of concrete explanation, the biological sample is a DNA sample, and the buffering agent contains a separation DNA conjugate and a DNA binding control agent (hereinafter simply referred to as “separation”). The electrophoresis apparatus 100 adds the quantified DNA sample to the separation DNA conjugate packed in the flow path, causes the electrophoresis, and the flow is performed. The presence or absence of SNPs (single nucleotide polymorphisms) in the DNA sample is determined by detecting the fluorescence intensity or absorbance in the path.

  First, the configuration of the electrophoresis apparatus 100 according to the present embodiment will be described with reference to FIGS. 1 and 2. FIG. 1 is a configuration diagram of an electrophoresis apparatus 100 according to the present embodiment.

  The electrophoresis apparatus 100 according to this embodiment includes a plate 10 in which a separation DNA conjugate is filled, and a flow path 110 is formed in which the DNA sample is quantified and added to the filled separation DNA conjugate. The tray 22 to be mounted, the tray driving means 20 for rotating the plate 10 together with the tray 22, the voltage applying means 30 for applying a voltage to the separation DNA conjugate in the flow path 110, and the movement by the voltage application In order to detect the movement state of the DNA sample in the separating DNA conjugate, an optical detection unit 40 that irradiates the plate 10 with light and detects the fluorescence intensity or absorbance of the DNA sample with the objective lens 106 is provided. .

  On the tray 22, a flow path temperature adjustment unit 45 capable of individually controlling the temperature of each flow path 110 of the plate 10 is provided. The flow path temperature adjustment unit 45 protrudes toward the plate 10 side. The same number of blowing openings 46 having a shape as the number of flow paths 110a for electrophoresis are provided.

  Thus, by providing the flow path temperature adjusting unit 45 on the tray 22, the plate 10 having a plurality of concentric flow paths 110 as shown in FIG. An air flow heated or cooled with respect to the ambient temperature is individually blown to the region including the electrophoresis channel 110a (part a in FIG. 4) of each channel 110, and the electrophoresis It becomes possible to control the temperature for each flow path 110a.

  Hereinafter, the configuration of the flow path temperature adjusting unit 45 will be described in detail with reference to FIGS. 2, 5, and 6. FIG. 2 is a diagram showing the configuration of the flow channel temperature adjusting unit 45 in the first embodiment, and FIG. 6 is a cross-sectional view showing an example of the shape of the blowout opening 46 of the flow channel temperature adjusting unit 45. FIG. 5 is a diagram showing the flow direction of the temperature-adjusted air flow blown out from the blowing opening 46.

  As shown in FIGS. 1 and 2, the flow path temperature adjusting unit 45 includes a common chamber 39 in which the temperature is controlled to be lower than the ambient temperature, and each of the plates 10 from the flow path temperature adjusting unit 45. The plurality of blowout openings 46 sprayed onto the flow path 110.

  The common chamber 39 includes a heating / cooling device 33 that uses a Beltier element or the like capable of controlling both heating and cooling, and a fan 34 that agitates air whose temperature is locally controlled by the heating / cooling device 33. And a heater 48 for controlling the temperature of the air cooled in the common chamber 39 to a predetermined temperature higher than the temperature of each opening 46. A thermistor 49 for monitoring the temperature of the air blown from the opening 46 is provided.

  In addition, the shape of each blowing opening 46 is such that the temperature-controlled air flow blown from one blowing opening 46 is blown only to one flow path 110 on the plate 10. Or the shape and arrangement of the flow paths formed in the plate 10.

  For example, in the first embodiment, the flow paths 110 of a plurality of flow paths are concentrically formed on the plate 10 at positions that are concentric and adjacent to each other. As shown in FIG. 2, it is necessary to adopt a configuration in which the air escapes from the area where the air is blown to the outer edge of the plate 10.

  Therefore, the shape of each blowing opening 46 in the first embodiment has an area that substantially covers one flow path 110 formed in the plate 10, and the outlet shape of the opening 46 is As shown in FIG. 6 (a), the plate is cut obliquely from the plate center side (x side in the figure) to the plate outer edge side (y side in the figure), or FIG. 6 (b). As shown in FIG. 4, the plate is cut obliquely from the plate center side (x side in the figure) to the outer edge side of the plate (y side in the figure), and the blowing angle β of the blown air to the plate 10 is 90 degrees or less. Thus, a valve for changing the air flow is formed on the center side of the plate (x side in the figure).

  In this way, the air cooled to a certain temperature in the common chamber 39 of the flow path temperature adjusting unit 45 is supplied to the heater 48 provided on the inner wall of the opening 46 for each of the blowing openings 46. The air heated to a desired temperature can be blown from the radial center side to the outer edge side with respect to each of the plurality of the vicinity of the electrophoretic flow path 110a. The blown air flow hardly flows into the adjacent flow path, and it becomes possible to set different temperatures for each flow path 110 on the plate 10. In the above description, each blowing opening 46 has an area that substantially covers one flow path 110 formed in the plate 10, but the region of the flow path 110 in which electrophoresis is performed. Since the temperature only needs to be a predetermined temperature, each blowing opening 46 only needs to have an area covering at least a region (a portion in FIG. 4) of each channel 110 where the DNA sample is electrophoresed.

  Furthermore, in the first embodiment, in order to further improve the temperature control performance of each flow path 110 in the plate 10, as shown in FIGS. 4 (a) and 4 (b), the adjacent flow formed on the plate 10 is used. A slit 47 penetrating the plate 10 is provided between the paths 110. 4A and 4B are diagrams showing the configuration of the plate according to the first embodiment. FIG. 4A is a diagram showing a flow path forming surface, FIG. 4B is a sectional view of the plate, and FIG. FIG. 4 is a diagram showing in detail a flow path formed in the plate. The detailed configuration of the flow path 110 formed in the plate 10 of the first embodiment will be described later with reference to FIG.

  As shown in FIG. 4A, the slit 47 has a long axis in the radial direction of the plate 10, and the length of the slit 47 is a radius on the plate 10 of the flow path 110a for electrophoresis for temperature control. Make it sufficiently longer than the length in the direction. Thereby, it can suppress as much as possible that the precision of the temperature control of each flow path 110 is influenced by the heat conduction between the adjacent flow paths formed in the plate 10.

  By the way, when the slits 47 are provided in the plate 10 as described above, the number of slits increases as the number of flow paths formed in the plate 10 increases, so that the strength of the plate 10 becomes a problem.

  In such a case, for example, as shown in FIG. 7A, the slit 47 is made of a material different from the constituent material (for example, acrylic) of the plate 10 as the slit heat insulating material 151, and the plate 10. A material having a lower thermal conductivity (for example, PVC) than the constituent material is filled. In this way, the strength of the plate 10 can be obtained, and heat conduction to the adjacent flow path can be reduced as compared with the case where the plate 10 is made of, for example, an acrylic simple substance.

  Further, instead of the slit 47, as shown in FIGS. 7B and 7C, a plate thickness adjusting portion 150 that is a region where the thickness of the plate 10 is increased may be provided between adjacent flow paths. . In this way, the time until the thermal equilibrium between the adjacent flow paths is reached due to heat conduction can be increased, so that the temperature-controlled air is applied to the region (the flow path 110a for electrophoresis) where temperature control is desired. While blowing a flow, it is difficult to conduct heat to the adjacent flow path. It should be noted that the thickness and width of the plate thickness adjusting portion 150 provided between the flow paths of the plates 10 may be in a range that does not hinder the apparatus configuration.

  The tray driving means 20 of the first embodiment includes a motor 51 as a drive source, a high-speed rotation switching gear 21a for driving the tray 22 to rotate at a high speed, and a low speed for driving the tray 22 at a low speed. The rotation switching gear 21b is provided in a switchable state, thereby enabling high-speed rotation and low-speed rotation of the tray 22 by the motor 51.

  The voltage applying means 30 includes a plurality of electrode probes 32a to 32i for applying a voltage, an electrode probe substrate 27 for electrically connecting and fixing the plurality of electrode probes 32a to 32i, and the electrode probes A plurality of electrode probes 32a to 32i fixed to the substrate 27 are provided with voltage application contact / separation means 80 for bringing the electrode 10 into contact with or separating from the plate 10. The voltage application contact / separation means 80 includes a drive motor 81 such as a DC motor for moving the electrode probe substrate 27 up and down with respect to the plate 10, and an electrode probe substrate 27 moved by the drive motor 81. An electrode height detection sensor 82 for detecting a position, whereby the electrode probe substrate 27 is moved up and down between predetermined positions to bring the electrode probe 32 into contact with or away from the plate 10. Making it possible. Each electrode probe 32 is provided with a compression spring at the tip thereof in order to adjust the contact pressure when the electrode probe 32 is brought into contact with the plate 10 by the electricity applying means contact / separation mechanism 80. A built-in spring 85 is provided.

  FIG. 8 is a diagram showing a state where the electrode probe is moved up and down by the voltage application contact / separation means in the first embodiment. When a voltage is applied to the plate 10 by the electrode probe 32, first, as shown in FIG. 8A, the electrode probe substrate 27 positioned at the “a” position of the electrode height detection sensor 82 is used. Then, the drive motor 81 is driven to move to the “b” position of the electrode height detection sensor 82 shown in FIG. Thereby, it becomes possible to make each said electrode probe 32 electrically contact each electrode part (not shown) of the plate 10, and to apply a voltage to the buffering agent with which the flow path was filled.

  After the voltage application by the voltage application means 30 is completed, the drive motor 81 is driven again, and the electrode probe substrate 27 is moved from the “b” position of the electrode height detection sensor 82 to the “a” position. . Thereby, each electrode probe 32 that has been in contact with each electrode portion (not shown) of the plate 10 can be separated from the plate 10.

  Thus, if the voltage application unit 30 is provided with the voltage application contact / separation means 80 for bringing the electrode probes 32 a to 32 i into contact with or away from the plate 10, the electrode application means 30 is integrated with the plate 10. Since a rotating mechanism is not necessary, the device 100 can be made small and simple.

  The casing of the electrophoresis apparatus 100 according to the first embodiment includes a main body upper chassis 101 and a main body lower chassis 102, and all the above-described constituent elements are arranged inside the casing. Specifically, the tray 22 and the optical detection unit 40 and the tray driving unit 20 are disposed in the lower chassis 102 of the main body, and the voltage application is applied to the upper chassis 101 of the main body. By adopting a configuration in which the flow path temperature adjusting unit 45 in which the means 30 is incorporated is disposed in a state of being locked through the guide shaft 26, the entire apparatus is downsized.

Hereinafter, the positional relationship between the flow path temperature adjusting unit 45 and the positional relationship between the main body chassis 101 will be described.
The flow path temperature adjusting unit 45 and the main body upper chassis 101 are connected via the guide shaft 26 as described above, and the guide shaft 26 has one end connected to the main body chassis 101 and the other end. It is connected to the upper surface of the flow path temperature adjusting unit 45. The guide shaft 26 is provided with a compression spring 25 as an elastic member coaxially with the shaft 26, and the length of the guide shaft 26 is such that the flow path temperature adjusting unit is closed when the main body chassis 101 is closed. A part of the lower wall of 45 is set to be slightly overstroked relative to the positioning pin locking portion 211, and the entire flow path temperature adjusting portion 45 is elastic with respect to the chassis 101 on the main body. It is locked to.

  Therefore, when the main body upper chassis 101 is closed, the flow path temperature adjusting unit 45 moves together with the main body upper chassis 101 and is disposed on the tray 22, and is provided in the flow path temperature adjusting unit 45. The compression spring 25 of the guide shaft 26 is compressed until the positioning pin 210 contacts the positioning pin locking portion 211 of the lower chassis 102, and the positioning pin 210 and the positioning pin locking portion 211 come into contact with each other. At that time, the flow path temperature adjusting unit 45 stops and the position of the flow path temperature adjusting unit 45 is determined. On the other hand, when the operation of opening the main body chassis 101 of the apparatus 100 is performed, as shown in FIG. 3, the flow path temperature adjusting unit 45 locked to the main body chassis 101 is moved together with the main body chassis 101 to the tray 22. Since the tray 22 is exposed to a position sufficiently separated from the tray 22, the plate 10 can be mounted on the tray 22 or the measured plate 10 can be removed from the tray 22.

  By the way, as described above, when the main body upper chassis 101 is closed, the flow path temperature adjusting portion 45 is pressed against the lower main body chassis 102 by the compression spring 25 of the guide shaft 26. It is in a state of being biased in a direction away from 10. In the first embodiment, the electrode probes 32a to 32i are lowered by the voltage application contact / separation means 80 in this state, and the electrode probes 32 are pressed against the plate 10 via the built-in springs 85 to make contact. Voltage application. Therefore, at the time of applying an electrode, each electrode probe 32 is separated from the plate 10 in two stages by two elastic bodies, that is, a built-in spring 85 provided at the tip thereof and a compression spring 25 of the guide shaft 26. It is energized in the direction to do.

  At this time, if the elastic force of the built-in spring 85 of the electrode probe 32 is larger than the elastic force of the compression spring 25 of the guide shaft 26, the positioning pin 210 provided on the voltage applying means base plate 28 will move the lower body chassis. As a result, the entire voltage applying means 30 is biased in a direction away from the plate 10 to the electrode portion provided on the plate 10 of the electrode probe 32. The pressure of the electrode probe 32 is reduced, the contact resistance between the electrode probe 32 and the electrode of the plate 10 is increased, or the contact is poor, so that an accurate voltage during electrophoresis cannot be applied. appear.

  Therefore, in the first embodiment, the total elastic force when the built-in spring 85 is subjected to a predetermined amount of bending is the same as that of the compression spring 25 locking the voltage applying means 30. It should be set so that it is smaller than the total elastic force generated.

  In addition to the above-described configuration, the electrophoresis apparatus 100 includes a plate confirmation sensor 23 for confirming the position of the plate 10 on the tray 22 and the voltage application means 30 just below the tray 22 as in the conventional apparatus. A high-voltage power supply 66 connected to the electrode probes 32a to 32i, a control board 68 for controlling the operation of the entire apparatus 100, an apparatus power supply (not shown), and a power switch (not shown) for switching the on / off state of the apparatus. , An LED (not shown) that lights when the power switch is ON, a cooling fan 64 that cools the inside of the apparatus 100, and a rubber leg 65 that can protect the apparatus 100 from vibration and can be adjusted in height. Yes.

Here, an example of the flow path 110 formed in the plate 10 used in the first embodiment will be described with reference to FIG.
As shown in FIG. 4C, the flow path 110 is a fine flow path formed by grooves having a very small width and depth. In the first embodiment, the flow of the same shape is formed on the plate 10. Eight concentric circles 110 are provided. The flow channel 110 is provided with a conjugate injection port 306 and a sample injection port 308. The separation DNA conjugate injected from the conjugate injection port 306 is temporarily held by the conjugate injection unit 307, and The DNA sample injected from the sample injection port 308 is temporarily held by the sample injection unit 309. Reference numeral 312 denotes a positive electrode portion into which a positive electrode is inserted, and reference numeral 313 denotes a negative electrode portion into which a negative electrode is inserted. These are connected via the flow path 314 and further connected to the conjugate injection part 307 via the flow paths 310 and 311, respectively. In addition, a conductive film 12 (not shown) is attached to the positive electrode portion 312 and the negative electrode portion 313, and when the electrode probe 32 contacts the film 12 and a voltage is applied to the film 12. A voltage is applied to the DNA conjugate for separation filled in the electrode portions 312 and 313. Reference numerals 316, 318, and 321 denote a chamber part, a sample holding part, and a buffer part, respectively, the chamber part 316 is connected to the sample injection part 309 via a flow path 315, and the sample holding part 320 is The buffer section 321 is connected to the chamber section 316 and the sample holding section 320 through flow paths 317, 318, and 319 through flow paths 317 and 318. Further, the buffer section 321 can be vented by the flow path 322. Reference numeral 323 denotes a sample quantification unit, which is provided at a joint portion between the flow channel 314 and the flow channel 319, and the DNA sample is quantified at this portion. Then, electrophoresis of the DNA sample is performed in the electrophoresis channel 110a connecting the electrode portions 312 and 313.

  Next, using FIG. 9 to FIG. 11, a DNA sample is sampled by the electrophoresis apparatus 100 of the first embodiment using the plate 10 in which a plurality of flow paths 110 as described above are formed concentrically. A series of operations when the SNPs are determined will be described.

  First, the main body chassis 101 of the apparatus 100 is opened, and the plate 10 into which the separation DNA conjugate and the DNA sample are injected into each flow path 110 is set on the tray 22. At this time, the fitting pin 31 of the tray 22 is inserted into the fitting hole 11 of the plate 10, and the plate 10 is positioned with respect to the tray 22.

  Then, by closing the main body chassis 101 of the apparatus 100, the flow path temperature adjusting unit 45 locked to the main body upper chassis 101 by the guide shaft 26 is disposed on the tray 22, and the flow path temperature adjusting unit 45 Until the positioning pin 210 provided and the positioning pin locking portion 211 of the lower chassis 102 come into contact with each other, the flow path temperature adjusting portion 45 is moved by the compression spring 25 provided on the guide shaft 26. Then, it is pressed against the positioning pin locking portion 211.

Next, the tray 22 is rotated at a high speed by the tray driving means 20 so that the separation DNA conjugate added to the plate 10 is filled into the electrophoresis channel 110a. Is quantitatively added to the packed DNA conjugate for separation. Specifically, in the first embodiment, the tray 22 is rotated at a high speed for a certain period of time by the motor 51 of the tray driving means 20, and then suddenly stopped, and then rotated again at a medium speed for several seconds. FIG. 10 shows the movement state of the DNA conjugate for separation and the DNA sample injected into the plate 10 used in the first embodiment, and FIG. 10 (a) shows the state after a certain time has elapsed from the start of high-speed rotation. (B) shows a state immediately after the rapid rotation of the high-speed rotation, and (c) shows a state after the medium-speed rotation.
By such a filling process, the DNA sample remaining in the sample quantification unit 323 becomes the final sample for determining the SNPs.

  After that, air whose temperature is controlled to a predetermined temperature is blown from the flow path temperature adjusting unit 45 to the electrophoresis flow path 110a of each flow path 110, and the temperature is controlled for each electrophoresis flow path 110a. Thereafter, as shown in FIG. 9, the electrode probes 32 a to 32 i of the voltage application means 30 are brought into contact with the film 12 on the electrode portions 312 and 313 by the voltage application contact / separation means 80 to apply a voltage. Then, the DNA sample is electrophoresed. FIG. 9 is a diagram illustrating a positional relationship among the flow path temperature adjustment unit 45, the electrode probe, the plate, and the objective lens during the voltage application operation in the first embodiment.

  In the first embodiment, the voltage application contact / separation unit 80 performs the sequence control to alternately perform the voltage application operation by the voltage application unit 30 and the light detection operation by the optical detection unit 40. After the probes 32 a to 32 i are separated from the plate 10, the movement state of the DNA sample to be electrophoresed in the electrophoresis channel 110 a is detected by the optical detection unit 40.

  Specifically, the voltage application contact / separation means 80 raises the electrode probe 32 to a predetermined position (“a” position in FIG. 8), and in that state, the tray 22 is moved to the motor 51 of the tray driving means 20. While rotating at a low speed, the excitation channel 110a is irradiated with excitation light, and the fluorescence intensity from the electrophoresis channel 110a is read.

  As described above, in the first embodiment, the electrophoresis operation and the light detection operation are not performed simultaneously, and the light detection operation by the light detection unit 40 is stopped during the voltage application operation by the voltage application unit 30. During the light detection operation by the light detection unit 40, the voltage application operation by the voltage application unit 30 is stopped, so that a mechanism for rotating the voltage application unit 30 and the tray 22 integrally becomes unnecessary. The configuration of the device 100 can be simplified. Further, as described above, since the operation for supplying the high voltage for electrophoresis from the voltage applying means 30 to the plate 10 side and the operation for rotating the plate 10 are performed separately, the power supply mechanism can be simplified. Can also be obtained.

  In the first embodiment, the electrophoresis operation is performed by bringing the electrode probe 32 into contact with the positive electrode portion 312 and the negative electrode portion 313 in the flow path 110 and applying a voltage of several hundred volts. As a result, the DNA sample in the plate 10 is electrophoresed in the separation DNA conjugate filled in the electrophoresis channel 110a, and moves in the direction A in the electrophoresis channel 110a. During this movement, the DNA sample is electrophoresed while repeating the binding with the separating DNA conjugate. At this time, normal DNA in the DNA sample has a strong binding force with the separation DNA conjugate, and thus the migration speed is slow. On the other hand, the mutant DNA in the DNA sample is separated from the separation DNA conjugate. Since the binding force is weak, the migration speed is faster than that of the normal DNA. That is, when both the normal DNA and the mutant DNA exist in the DNA sample, the normal DNA and the mutant DNA are separated, and the SNPs can be discriminated.

  In addition, the above-described light detection operation was performed, for example, by exciting a DNA modified with a fluorescent label (for example, FITC) with light of 470 nm and detecting light near 520 nm. FIG. 11 is a graph showing the movement state of the DNA sample obtained at this time. The horizontal axis represents the position on the electrophoresis channel 110a, which is an area for light detection, and the DNA sample migrates from left to right. . That is, the left side is the sample quantification unit 323 and the right side is the positive electrode unit 312 side. The vertical axis represents the fluorescence intensity, and represents a waveform that changes in time every minute from the top. It can be seen from FIG. 11 that the two mountains are gradually separated. The mountain on the right side of the graph shows a mutant DNA because the migration speed is fast, and the mountain on the left side shows a normal DNA because the swimming speed is slow. That is, in this case, it was determined that the same amount of normal DNA and mutant DNA were present in the DNA sample. In addition, you may perform the said detection using the light absorbency of 260 nm.

  Then, after the light detection operation by the optical detection unit 40 is completed, the measured plate 10 is taken out from the tray 22. In this case, the main body chassis 101 of the apparatus 100 is opened, and the flow path temperature adjusting unit 45 locked to the main body chassis 101 is retracted to a position sufficiently separated from the tray 22, thereby exposing the above-described exposed portion. The plate 10 is removed from the tray 22.

  As described above, according to the first embodiment, a plurality of blowout openings for blowing a temperature-controlled air flow for each flow path 110 with respect to the plurality of flow paths 110 formed in the plate 10. A flow path temperature adjusting section 45 including a section 46 is disposed on the tray 22 on which the plate 10 is mounted, and heating and cooling provided inside the flow path temperature adjusting section 45 is provided on the inner wall of each blowing opening 46. Since the heater 48 that heats the air cooled by the device 33 to a desired temperature is provided, different temperature adjustment is performed for each electrophoresis channel 110 a of the plate 10 by the air blown from the blowing openings 46. In each electrophoresis channel 110a, it is possible to set the temperature during electrophoresis according to the DNA sample to be electrophoresed. As a result, one plate , It is possible to determine a plurality of samples having different SNP of separation conditions.

  Further, according to the first embodiment, the slit 47 penetrating the plate 10 is provided between the adjacent flow paths on the plate 10 to prevent the influence of heat conduction between the adjacent flow paths. Therefore, it is possible to obtain accurate temperature control performance of each electrophoresis channel 110a, and as a result, highly reliable electrophoresis, SNP separation, and light detection results can be obtained.

  Further, the slit 47 provided between the adjacent flow paths on the plate 10 is fitted with a heat insulating material 151 having a higher heat insulating effect than the material constituting the plate 10, or the area where the thickness of the plate is increased instead of the slit 47. If 150 is provided, the influence by heat conduction can be prevented and the plate 10 can be given strength.

  In the first embodiment, a filling operation for quantifying the fine flow path 110 formed in the plate 10 by filling the DNA sample and the separating DNA conjugate is performed, and the plate 10 is driven by a plate such as the motor 51. Although the description has been made on the assumption that the means 20 is used for centrifugal filling and quantification, the present invention is not limited to this. Needless to say, the present invention is also applied to a reaction type apparatus.

  The electrophoretic device of the present invention is useful as a device that enables a biological sample such as a DNA sample to be discriminated at a low cost.

It is a figure which shows the structure of the electrophoresis apparatus in Embodiment 1 of this invention. It is sectional drawing which shows the structural example of the flow-path temperature control part of the electrophoresis apparatus in Embodiment 1 of this invention. It is a figure which shows the state which opened the main body upper chassis of the electrophoresis apparatus in Embodiment 1 of this invention. It is a figure which shows the structural example of the plate in Embodiment 1 of this invention, A figure (a) is a flow-path formation surface of a plate, A figure (b) is a cross section of a plate, A figure (c) is a plate. It is a figure which shows the formed flow path in detail. It is a figure which shows the flow direction of the temperature-controlled air flow in Embodiment 1 of this invention. It is sectional drawing which shows the structural example of the blowing opening part of the electrophoresis apparatus in Embodiment 1 of this invention. It is sectional drawing which shows another structural example of the plate in Embodiment 1 of this invention, A figure (a) shows the structural example which provided the heat insulating material in the slit, A figure (b), (c) is a plate The structural example which provided the thickness adjustment part is shown. It is a figure which shows the up-and-down movement state of the electrode probe board | substrate by the drive means in Embodiment 1 of this invention, A figure (a) shows the state which separated the electrode probe from the said plate, A figure (b) shows an electrode probe. Shows a state in contact with the plate. It is a figure which shows the positional relationship of the flow path on a plate, the blowing opening part, and an electrode probe in Embodiment 1 of this invention. The figure which shows the state by which the DNA conjugate for a separation is filled in the flow path formed in the plate in Embodiment 1 of this invention, and the state by which a DNA sample is quantitatively added to this DNA conjugate for separation. is there. It is a figure which shows the fluorescence intensity distribution of a mode that it moves in the separation DNA conjugate with which the DNA sample was filled in the flow path formed in the plate in Embodiment 1 of this invention. It is the figure which showed one Example of the conventional electrophoresis apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Plate 11 Mating pin hole 12 Film 20 Tray drive means 21a High speed rotation switching gear 21b Low speed rotation switching gear 22 Tray 23 Plate confirmation sensor 25 Compression spring 26 Guide shaft 27 Electrode board unit 30 Voltage application means 32a-32i Electrode probe 33 Heating Cooling device 34 Fan 39 Common room 40 Optical detection unit 45 Flow path temperature adjustment unit 46 Outlet opening 47 Slit 48 Heater 49 Thermistor 51 Motor 64, 464 Cooling fan 65 Rubber leg 66 High voltage power supply 68 Control board 80 Voltage application contact / separation means 81 Drive motor 82 Electrode height detection sensor 85 Built-in spring 100, 400 Electrophoresis device 101 Upper body chassis 102 Lower body chassis 106 Objective lens 110 Flow path 110a Electrophoresis flow path 150 Play Thickness adjusting part 151 Slit insulation 210 Positioning pin 211 Positioning pin locking part 306 Conjugate injection port 307 Conjugate injection unit 308 Sample injection port 309 Sample injection unit 310, 311, 314, 315, 317, 318, 319, 322 flow Path 312 Positive electrode part 313 Negative electrode part 316 Chamber part 320 Sample holding part 321 Buffer part 323 Sample quantification part 421 High speed rotation motor 424 Pressurization part 431 Low speed rotation motor 432a, b Voltage application means 433 Heater 434 Fitting pin 436 Clamper 450 Lifting stage 451 Motor 455 Sensor A Migration direction

Claims (11)

In an electrophoresis apparatus in which a sample is quantitatively added to a buffer filled in a flow path, and the sample is electrophoresed by applying a voltage,
A plate in which a plurality of the flow paths are formed;
A tray for mounting the plate;
Voltage application means for applying a voltage to the buffer in the flow path of the plate;
Each of the plurality of flow paths formed in the plate has a plurality of blowing openings for blowing a temperature-controlled air flow, and blows an air flow blown from each blowing opening to each flow path, A flow path temperature adjusting unit that adjusts each of the plurality of flow paths to each predetermined temperature,
An electrophoresis apparatus characterized by that. The electrophoresis apparatus according to claim 1,
Heater means for heating the air flow to each predetermined temperature is provided in each of the plurality of blowing openings of the flow path temperature adjusting unit,
An electrophoresis apparatus characterized by that. The electrophoresis apparatus according to claim 2,
The flow path temperature adjusting unit has at least one cooling means and a common chamber cooled by the cooling means,
The air flow cooled to a predetermined temperature in the common chamber is heated to each predetermined temperature by the heater means provided in each of the plurality of blowing openings, and the air flow is temperature-controlled to each predetermined temperature. Spray each channel of the plate,
An electrophoresis apparatus characterized by that. The electrophoresis apparatus according to claim 1,
A light irradiating means for irradiating light to the flow path; and a fluorescence detecting means for detecting fluorescence generated from the sample by the light irradiation; and optical detection of the sample that is electrophoresed in the flow path. An optical detector to perform;
A voltage application contact / separation means for bringing the voltage application means into contact with or separating from the flow path formed in the plate;
After the voltage application contact / separation means brings the voltage application means into contact with the flow path and the sample is electrophoresed by voltage application, the voltage application contact / separation means causes the voltage application means to Separated from the flow path, optical detection is performed by the optical detection unit,
An electrophoresis apparatus characterized by that. The electrophoresis apparatus according to claim 1,
The voltage applying means is disposed inside the flow path temperature adjusting unit disposed on the tray, and is provided in the flow path of the plate through the blowing opening of the flow path temperature adjusting unit. Applying a voltage to the buffer,
An electrophoresis apparatus characterized by that. The electrophoresis apparatus according to claim 1,
The plurality of blowing openings have an area substantially covering each flow path of the plate,
An electrophoresis apparatus characterized by that. The electrophoresis apparatus according to claim 6, wherein
The outlet shape of the blowing opening is cut obliquely so that the air blown out from the blowing opening flows in a direction from the center of the plate toward the outside.
An electrophoresis apparatus characterized by that. In an electrophoresis plate in which a plurality of the flow paths are formed, which is used in an electrophoresis apparatus in which a sample is quantitatively added to a buffering agent filled in the flow path, and the sample is electrophoresed by applying a voltage.
A slit is provided between the formed adjacent flow paths of the electrophoresis plate.
Electrophoresis plate characterized by the above. The electrophoresis plate according to claim 8,
A heat insulating material is fitted in the slit provided between the flow paths,
Electrophoresis plate characterized by the above. The electrophoresis plate according to claim 9,
The heat insulating material is made of a material having a higher heat insulating effect than the material constituting the electrophoresis plate,
Electrophoresis plate characterized by the above. In an electrophoresis plate in which a plurality of the flow paths are formed, which is used in an electrophoresis apparatus in which a sample is quantitatively added to a buffering agent filled in the flow path, and the sample is electrophoresed by applying a voltage.
A region where the thickness of the plate is increased is provided between the formed adjacent flow paths of the electrophoresis plate.
Electrophoresis plate characterized by the above.

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