JP2006003349A - DNA microarray processing equipment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To operate easily and efficiently when conducting a large amount of analysis using a DNA microarray having a structure in which a polymer gel with a probe immobilized is filled in a plurality of holes penetrating front and back An apparatus capable of well performing hybridization and washing is provided.
An apparatus for performing hybridization and washing treatment of a DNA microarray having a structure in which a polymer gel on which a probe is immobilized is held in a plurality of holes penetrating front and back,
A DNA microarray processing apparatus including a plurality of individually sealable compartments in which each of a plurality of DNA microarrays is accommodated, and a hybridization / washing plate in which slots are formed.
[Selection] Figure 4

Description

  The present invention relates to a hybridization / washing apparatus for DNA microarrays.

  There is an analysis method called a DNA microarray method (DNA chip method) as a method for performing batch expression analysis of a large number of genes. In this method, a large number of DNA fragments are aligned and fixed on a flat substrate piece such as a slide glass or silicon called a microarray at high density. This is a method for nucleic acid detection and quantification based on a hybridization reaction.

  As a method of using the microarray method, for example, a sample solution containing a sample in which a gene expressed in a cell to be studied is labeled with a fluorescent dye is applied to the microarray, hybridization is performed, and complementary nucleic acids are bound to each other. A method of detecting and quantifying nucleic acid by reading a section where a hybrid is formed at high speed with a high-resolution analyzer can be mentioned.

  In addition to the above-described planar base piece, for example, a polymer gel in which a probe that functions as a probe for a substance to be inspected is immobilized has a microarray, and a probe is placed in each of a plurality of holes penetrating the front and back. A DNA microarray having a structure in which a fixed polymer gel is held is known. As an example, there is known a DNA microarray (fiber type DNA microarray) in which holes are formed of hollow fibers (Patent Document 1).

  Patent Document 1 discloses the production of a fiber-type DNA microarray including sequentially performing the following steps (1) to (4).

  (1) A plurality of hollow fibers are prepared, and these hollow fibers are bundled.

  (2) A hollow fiber array in which the bundle is fixed with resin or the like is created.

  (3) From one end portion of the array, a polymerizable monomer solution such as acrylamide containing a capture probe is introduced into the hollow portion of each hollow fiber, and a polymerization reaction is performed in the hollow portion.

  (4) The cutting of the resin-fixed portion of the hollow fiber array is repeated in a direction intersecting with the longitudinal direction of the hollow fiber.

  In the fiber-type DNA microarray, a polymer gel having a capture probe immobilized on the surface of the microarray is exposed. Therefore, it is necessary to handle with great care so as not to damage the delicate gel part exposed on the surface in the hybridization, washing process and analysis process. Therefore, it is preferable to protect the microarray by using, for example, a holder as shown in FIG.

  On the other hand, with regard to hybridization and washing treatment of DNA microarrays, a microarray (hereinafter referred to as glass DNA microarray) in which a large number of DNA fragments are aligned and fixed on a slide glass substrate piece at high density is disclosed in, for example, Patent Document 2 These operations can be carried out using the containers that are provided.

  When processing a large amount of microarrays, it is important that the operation is simple and efficient. Therefore, the glass DNA microarray can be implemented using the apparatus disclosed in Patent Document 3.

However, there is no disclosure of an apparatus that can be handled easily and efficiently in a fiber-type DNA microarray. In addition, since the apparatus disclosed in Patent Document 3 processes glass DNA microarrays in a horizontal state, the number of microarrays that can be simultaneously processed per area occupied by the apparatus is small. Therefore, it is difficult to say that the apparatus is capable of efficient processing. That is, in order to process a plurality of microarrays, it has been desired to develop a hybridization / washing apparatus for DNA microarrays that has a large number of simultaneous processes per area occupied by the apparatus.
JP 2001-133453 A Japanese Patent Laid-Open No. 2003-125751 JP 2003-57257 A

  Therefore, an object of the present invention is to perform a large amount of analysis using a DNA microarray having a structure in which a polymer gel having a probe fixed therein is held in each of a plurality of holes penetrating the front and back. An object of the present invention is to provide an apparatus that is simple in operation and capable of efficient hybridization and washing treatment.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found the following apparatus capable of hybridization / washing a DNA microarray, and have completed the present invention.

That is, the present invention
A device for performing hybridization and washing treatment of a DNA microarray having a structure in which a polymer gel with a probe fixed is held in each of a plurality of holes penetrating the front and back,
A hybridization / washing plate having a plurality of compartments each housing a plurality of DNA microarrays;
A temperature adjustment mechanism capable of holding the liquid held in the compartment at a set temperature;
A cleaning liquid injection nozzle for injecting the cleaning liquid;
A cleaning liquid suction nozzle for discharging the cleaning liquid;
A liquid feed pump for feeding the cleaning liquid to the cleaning liquid injection nozzle;
A switching valve capable of switching at least one type of cleaning liquid fed to the cleaning liquid injection nozzle; and
A suction pump for sucking and transferring the cleaning liquid from the cleaning liquid suction nozzle;
A liquid bottle for storing cleaning liquid and cleaning waste liquid;
A moving mechanism for moving a nozzle block in which the cleaning liquid injection nozzle and the cleaning liquid suction nozzle are fixed horizontally and vertically with respect to the hybridization / cleaning plate;
A DNA microarray processing apparatus.

  The DNA microarray processing apparatus is characterized by having a mechanism capable of heating / cooling and supplying the cleaning liquid injected into the compartment from the cleaning liquid injection nozzle. Precise and efficient DNA microarray can be obtained by heating / cooling the washing liquid to a predetermined temperature, for example, the same temperature as the temperature at which the hybridization / washing plate is kept, and then injecting the DNA microarray into the compartment where the DNA microarray is inserted and housed. Can be cleaned.

  Further, the DNA microarray processing apparatus is characterized in that a cleaning liquid heating / cooling mechanism is incorporated in a nozzle block equipped with a cleaning liquid injection nozzle. By adopting such a structure, the cleaning liquid is adjusted to a predetermined temperature immediately before being injected into the compartment in which the DNA microarray is inserted and housed. It becomes possible to control the temperature of the cleaning liquid.

  According to the present invention, there is provided a DNA microarray processing apparatus that is simple in operation and capable of efficiently performing a hybridization / washing process on a plurality of DNA microarrays.

  Hereinafter, the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a schematic diagram of a DNA microarray main body (10) having a structure in which a polymer gel having a probe fixed therein is held in each of a plurality of hole portions penetrating front and back. An example of a detailed manufacturing method of the DNA microarray main body (10) is described in Patent Document 1.

  In the DNA microarray main body (10), a polymer gel having probes immobilized thereon is exposed. Here, the probe refers to, for example, a probe used for searching for a target substance in a specimen. As the probe, a nucleic acid, peptide, protein, lipid, peptide nucleic acid or the like can be used. In hybridization, washing treatment, and analysis treatment, care must be taken not to damage the delicate polymer gel exposed on the surface. Therefore, as exemplified in FIG. 2, the DNA microarray body (10) is preferably used in a state of being inserted and protected by the holder (20).

  The holder (20) has a concave marker portion (21) for the purpose of preventing the microarray from being inserted in the wrong direction. On the other side into which the holder (20) is inserted, that is, on the microarray body, a convex marker portion that fits into the concave marker portion (21) is provided. Thereby, when the holder (20) is inserted in the wrong direction, the holder (20) is pushed up by the convex marker portion, and it can be easily determined that the holder (20) is inserted in the wrong direction.

  FIG. 3A shows a hybridization in which a plurality of DNA microarrays are vertically accommodated in individually sealable compartments, and a polymer gel portion on which a probe is immobilized is immersed in a hybridization solution. / It is the upper surface of the plate for washing (30). The cross-sectional views are shown in FIGS. 3B and 3C.

  A plurality of DNA microarrays can be individually sealed in a compartment (31) in which the DNA microarrays are accommodated, and a slot (32) for immersing a polymer gel portion on which a probe is immobilized in a hybridization solution Is formed.

  The compartment (31) is an opening (31a) in which only the upper part is opened before sealing. The DNA microarray is inserted into the slot (32) in the partition (31) from this opening (31a), and the hybridization solution is injected. Moreover, after these work is complete | finished, each division can also be sealed by plugging this opening part (31a).

As a method of sealing each compartment, a cap having a structure that exactly fits each other may be fitted. For example, a method of using a commercially available adhesive film or heat-sealing film and closing all the openings (31a) of the hybridization / washing plate (30) at once is simple, reliable and preferable.
Furthermore, if the said adhesive film and a heat sealing | fusion film are pressed down with a pressing plate, more reliable sealing will be attained. In addition, a heater may be provided in the holding plate for keeping the temperature of each section.

  On the bottom of the slot (32), there is formed a wrong direction insertion preventing convex portion (33) that fits into the wrong direction insertion preventing concave portion (21) of the holder (20) described above. When the holder (20) is inserted in the wrong direction and the wrong direction insertion preventing concave portion (21) and the wrong direction insertion preventing convex portion (33) are not fitted, the holder (20) becomes the wrong direction insertion preventing convex portion. The holder (20) is pushed upward by (33) and protrudes from the opening (31a). Thereby, it can be easily determined that the card has been inserted in the wrong direction.

  The material constituting the hybridization / washing plate is not particularly limited as long as it does not contain a substance that inhibits a detection reaction such as hybridization or an antigen-antibody reaction. For example, a thermoplastic resin material such as polypropylene, polyethylene, polymethyl methacrylate, and polycarbonate can be used and can be manufactured in large quantities at low cost by injection molding or the like.

  FIG. 4A is a top view of the DNA microarray processing apparatus (40). FIG. 4B is a side view of the processing apparatus (40). Inside the upper cover (41), a hybridization / washing plate (30) is mounted in a plate mounting groove (42). The upper cover (41) is opened when the hybridization / washing plate (30) is attached / detached from the plate mounting groove (42), and is closed during the hybridization process or the washing process to protect against disturbances and ambient temperature. Stabilize.

  The plate mounting groove (42) includes a temperature adjustment mechanism for holding the mounted hybridization / washing plate (30) at a set temperature. As an example of the temperature adjustment mechanism, a method of covering the plate mounting groove (42) with an electric heater when the temperature adjustment is only heating and the natural cooling is sufficient for cooling. Further, when cooling is also necessary, a method may be mentioned in which a jacket structure is provided around the plate mounting groove (42) and a heat medium heated or cooled to a predetermined temperature is circulated.

  When a Peltier element that can be easily reversed by reversing the polarity of the power supply is attached to the back wall surface of the plate mounting groove (42) and used for temperature adjustment, the heat capacity of the mechanism is small and rapid temperature increase / decrease is possible. Is also particularly preferred.

  Further, as a means for heating / cooling the cleaning liquid injected into the compartment of the hybridization / washing plate (30) from the cleaning liquid injection nozzle to a predetermined temperature, for example, the hybridization / washing plate is kept on the hybridization / washing plate. In the case of heating / cooling to the same temperature as the existing temperature, if a part of the temperature adjustment mechanism covering the periphery of the plate mounting groove (42) is used as a liquid heat exchange unit, the cleaning liquid is brought to a predetermined temperature. The heating / cooling mechanism can be simplified.

  The DNA microarray processing apparatus (40) includes a cleaning solution bottle A (43) and a cleaning solution bottle B (44) filled with a cleaning solution supplied to the hybridization / washing plate (30), and a hybridization / washing plate. A waste liquid bottle (45) for storing the sucked waste liquid is connected.

  The cleaning liquid bottle A (43) and the cleaning liquid bottle B (44) contain cleaning solutions having different compositions. They can switch the type of cleaning liquid fed to the injection nozzle by a switching valve (not shown). Here, two types of cleaning liquid are illustrated, but one type or two or more types may be used without any particular limitation.

  On the upper surface of the DNA microarray processing apparatus (40), the heating / cooling temperature, temperature holding time, temperature switching, washing time, and section of the hybridization / washing plate (30) mounted in the plate mounting groove (42) are divided into one section. There is a sequence input unit (46) for inputting a sequence such as the amount of injected liquid, the standing time after injection of the cleaning liquid, and the amount of movement of the nozzle arm. The sequence input unit includes a key (46a) for inputting a sequence, and a display panel (46b) for displaying the input sequence and the progress of the sequence.

  5A and 5B are detailed views of the hybridization / washing plate mounting portion of the DNA microarray processing apparatus. Above the hybridization / washing plate (30), there is a nozzle block (47) to which a washing liquid injection nozzle (47a) and a washing liquid suction nozzle (47b) are fixed. The nozzle block (47) is a nozzle arm ( 48).

  The nozzle arm (48) can be arbitrarily moved in the horizontal and vertical directions with respect to the hybridization / washing plate (30) by an actuator such as a screw feed, a belt feed, or a linear motor. For vertical movement, an electromagnetic cylinder or the like may be used as long as it moves up and down at regular intervals.

  Next, a cleaning method using the apparatus of FIG. 5 will be described.

First, the DNA microarray is inserted into the compartment (31) of the hybridization / washing plate (30). Subsequently, after injecting the hybridization solution into the opening (31a), each compartment is sealed with an adhesive film or a heat-sealing film (50) in order to prevent evaporation and contamination of the internal liquid. Hybridization is performed under predetermined conditions (temperature, time, etc.), and then the nozzle block (47) is lowered at a predetermined position in each section, and the cleaning liquid injection nozzle (47a) and the cleaning liquid suction nozzle (47b) are used. Perform a washing operation. Here, for example, the tip of the cleaning liquid injection nozzle (47a) and the cleaning liquid suction nozzle (47b) are sharpened like an injection needle, thereby penetrating the film (50) and injecting the cleaning liquid from the nozzle tip into the inside. The cleaning waste liquid can be sucked.
Further, if a hole corresponding to the partition (31) is provided in the pressing plate, the cleaning operation can be performed while the above-described film (50) is pressed by the pressing plate.

  When a heat exchange unit for heating / cooling the cleaning liquid is built in the nozzle block (47), the cleaning liquid is adjusted to a predetermined temperature immediately before being injected into the compartment in which the DNA microarray is inserted and housed. Therefore, compared with the case where a heat exchange unit for heating / cooling the cleaning liquid is installed at a location away from the nozzle block (47), it is preferable that the temperature of the cleaning liquid is not easily changed due to heat dissipation, heat absorption or the like in the liquid feed pump or the tube.

  As an example of the temperature adjustment mechanism of the nozzle block (47), as described above, the same mechanism as the temperature adjustment of the hybridization / washing plate (30) can be adopted, but if heating / cooling is required, the nozzle block In order to summarize (47) compactly, it is particularly preferable to use a Peltier element for temperature adjustment.

  When the cleaning solution is heated / cooled and supplied to the same temperature as the temperature at which the hybridization / washing plate (30) is maintained, the temperature adjusting mechanism covering the plate mounting groove (42) described above is used. Using both a method that uses a part as a liquid heat exchange unit and a mechanism that incorporates a heat exchange unit that heats / cools the cleaning liquid in the nozzle block (47) can increase the heat exchange area and heat exchange time. Further, it is more preferable in suppressing the liquid temperature fluctuation in the cleaning cycle and improving the temperature control accuracy.

  Here, a method of using a part of the temperature adjustment mechanism of the plate mounting groove (42) as a liquid heat exchange unit, a method of incorporating a heat exchange unit for heating / cooling the cleaning liquid in the nozzle block, and a method of using both of them together In these heat exchange units, the cleaning liquid in the heat exchange unit whose temperature has already been adjusted is piston-flowed by the cleaning liquid that is supplied from the cleaning liquid bottle to the heat exchange unit by a supply pump or the like and is not adjusted in temperature. For example, the flow path in the heat exchange unit is divided into one narrow continuous flow path or one narrow flow path into a plurality of narrower flow paths, and converges to a single flow path It is preferable to have a structure or the like.

  With such a structure, the cleaning liquid that has not been temperature-adjusted supplied from the cleaning liquid bottle to the heat exchange unit by a supply pump or the like, and the cleaning liquid that has been temperature-controlled in the heat exchange unit are agitated and mixed. This can prevent the temperature fluctuation of the injected cleaning liquid by mixing cleaning liquids with different temperatures, improve the accuracy of temperature control, and when the type of cleaning liquid is changed, there is less liquid retention in the heat exchange unit, and the cleaning liquid It is particularly preferred because it can be replaced quickly.

It is a schematic diagram of a DNA microarray main body. It is a perspective view which shows the holder of the state accommodated in the DNA microarray main body. FIG. 4 is a structural diagram of a hybridization / washing plate that houses a plurality of holders. It is a general-view figure of a DNA microarray processing apparatus. It is a detailed view of a hybridization / washing plate mounting portion of the DNA microarray processing apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... DNA microarray main body 11 ... Polymer gel 20 ... Holder 21 ... Concave marker part 30 ... Hybridization / washing plate 31 ... ··· Division 31a ··· Opening 32 ··· Slot 33 · · · Protrusion portion for preventing incorrect insertion 40 · · · DNA microarray processing device 41 · · · Upper cover 42 ··· ... Plate mounting groove 43 ... Cleaning liquid bottle A
44 ... Cleaning fluid bottle B
45 ...... Waste liquid bottle 46 ... Sequence input part 46a ... Key 46b ... Display panel 47 ... Nozzle block 47a ... Cleaning liquid injection nozzle 47b ... ..Nozzle for cleaning liquid suction 48 ... Nozzle arm 50 ... Film

Claims (3)

A device for performing hybridization and washing treatment of a DNA microarray having a structure in which a polymer gel with a probe fixed is held in each of a plurality of holes penetrating the front and back,
A hybridization / washing plate having a plurality of compartments each housing a plurality of DNA microarrays;
A temperature adjustment mechanism capable of holding the liquid held in the compartment at a set temperature;
A cleaning liquid injection nozzle for injecting the cleaning liquid;
A cleaning liquid suction nozzle for discharging the cleaning liquid;
A liquid feed pump for feeding the cleaning liquid to the cleaning liquid injection nozzle;
A switching valve capable of switching at least one type of cleaning liquid to be fed to the cleaning liquid injection nozzle; and
A suction pump for sucking and transferring the cleaning liquid from the cleaning liquid suction nozzle;
A liquid bottle for storing cleaning liquid and cleaning waste liquid;
A moving mechanism for moving a nozzle block in which the cleaning liquid injection nozzle and the cleaning liquid suction nozzle are fixed horizontally and vertically with respect to the hybridization / cleaning plate;
A DNA microarray processing apparatus.   The processing apparatus according to claim 1, further comprising a mechanism capable of heating and cooling the cleaning liquid injected into the compartment from the cleaning liquid injection nozzle to a predetermined temperature and supplying the cleaning liquid. The processing apparatus according to claim 2, wherein the cleaning liquid is heated and cooled by a heating and cooling mechanism incorporated in a nozzle block equipped with a cleaning liquid injection nozzle.

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