JP2017085951A - JIG, PROCESSING DEVICE, PROCESSING METHOD, AND cDNA SYNTHESIS METHOD - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a jig that does not require labor for recovering magnetic substance particles 5 with a sample after processing.SOLUTION: A jig 100 comprises a substrate 1 that has a first region 2 holding processing droplets for magnetic substance particles which move by magnetic field and magnetic substance particles 5 with a sample containing a sample immobilized on the magnetic substance particles, and a second region 3 having a larger water contact angle than the first region 2 on the entire surface. In this case, a jig that does not require labor for recovering magnetic substance particles 5 with a sample after processing can be provided.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a jig, a processing apparatus, and a processing method, and more particularly, a jig for processing a sample or the like, a processing apparatus including the jig, a processing method using the processing apparatus, and a cDNA synthesis method. About.

  Patent Document 1 discloses a jig for holding a droplet for processing with respect to a magnetic particle with a sample including a magnetic particle moving by a magnetic field and a sample fixed to the magnetic particle.

  However, in the jig disclosed in Patent Document 1, it takes time to collect the magnetic particles with the sample after the treatment.

  The present invention includes a first region for holding a droplet for processing with respect to a magnetic particle with a sample including a magnetic particle moving by a magnetic field and a sample fixed to the magnetic particle, and the first region And a substrate having a second region having a large water contact angle on one side.

  According to the present invention, it does not take time to recover the magnetic particles with the sample after the treatment.

It is the top view which looked at the jig | tool with which the processing apparatus of one Embodiment is provided from the downward direction. It is a figure which shows the relationship between a water contact angle and surface tension. FIGS. 3A to 3D are diagrams (No. 1 to No. 4) for explaining the processing method of the first embodiment, respectively. FIGS. 4A to 4D are diagrams (No. 1 to No. 4) for explaining the processing method of the second embodiment. FIG. 5A to FIG. 5D are diagrams (No. 1 to No. 4) for explaining the processing method of the third embodiment, respectively.

  Hereinafter, an embodiment will be described.

  The processing apparatus according to one embodiment includes a jig 100 for processing a sample such as a micro liquid or a microorganism used for molecular biological operations in genetic analysis, genetic testing, and the like.

  Specifically, the jig 100 is a jig that enables sample processing for gene expression analysis such as RT (Reverse Transcription) -PCR, quantitative PCR, microarray, and next-generation sequencer.

  The jig 100 can also be used for antibody development using a system for rapidly and rapidly isolating monoclonal antibodies specific for the target antigen from antibody-producing cells derived from various animal species. is there.

  Furthermore, the jig 100 can also be applied to large-scale and trace continuous reactions of nucleic acids, proteins, lipids, carbohydrates, complex carbohydrates, and chemical substances.

  The jig 100 can also be used for immunostaining of immobilized cells.

[Processing equipment]
FIG. 1 shows a plan view of the jig 100 of the processing apparatus as viewed from below.

  As shown in FIG. 1 as an example, the jig 100 includes a substrate 1 disposed substantially parallel to a horizontal plane, and a tray 8 (not shown in FIG. 1, FIGS. 3A to 5 (not shown in FIG. 1). d)). Note that the jig 100 only needs to include at least the substrate 1. That is, the tray 8 may not be a component of the jig 100.

  The substrate 1 includes a first region 2 which is a majority region on the lower surface of a plurality of droplets for processing with respect to magnetic particles 5 with a sample including magnetic particles moving by a magnetic field and a sample fixed to the magnetic particles. Hold on. Examples of the material of the substrate 1 include resins such as polyester, polyethylene, polystyrene, polypropylene, Teflon (registered trademark), and polycarbonate, glass, ceramic, and silicone.

  In addition to the jig 100, the processing apparatus 200 includes a plurality of magnets 4 (not shown in FIG. 1, see FIGS. 3A to 3D) and the plurality of magnets arranged on the upper surface side of the substrate 1. 4 includes a magnet unit including a holding member (not shown) for holding 4.

  The magnet 4 is a small neodymium magnet, and is movable along the substrate 1 while applying a magnetic force (attractive force here) to magnetic particles (magnetic beads) located on the lower surface side of the substrate 1. The magnet 4 may be a permanent magnet other than a neodymium magnet such as a ferrite magnet or an alnico magnet, or a temporary magnet such as an electromagnet or soft iron. However, in the case of a temporary magnet, it is necessary to generate an external magnetic field.

  Therefore, by moving the magnet 4 along the substrate 10, the magnetic particles on which the sample is immobilized can be moved between a plurality of droplets.

  Here, a plurality of (for example, 72) droplets are held in the first region 2 so as to form a matrix arrangement of 6 rows × 12 columns. Note that the number and arrangement of the plurality of droplets are merely examples, and can be changed as appropriate. Further, the lower surface of the substrate 1 further has a second region 3 adjacent to the first region 2. Here, the first region 2 is set larger than the second region 3, but the size of the first region 2 may be equal to or smaller than the size of the second region 3.

  The magnet unit is held by the holding member so that a plurality of (for example, twelve) magnets 4 corresponding to a plurality of (for example, twelve) magnetic particles are arranged at substantially equal intervals in the column direction of the matrix arrangement. Along the row direction of the matrix array. The magnet unit can be moved manually or by an actuator (drive device). As this actuator, for example, a linear actuator including a stepping motor can be used.

  Although a single magnet 4 can be used instead of the magnet unit, it is not efficient to cover the entire range of the matrix arrangement with one magnet 4.

  Here, the shape of the water droplet on the solid is determined by the lateral balance of the surface tension γL of water, the surface tension γS of the solid, and the interfacial tension γSL between the water and the solid.

  As shown in FIG. 2, these three forces are balanced at a contact point where they intersect at one point. The balance equation is called Young's equation and is represented by γS = γSL + γL cos θ. Here, θ is an angle formed by a tangent to the surface of the water droplet drawn from the contact point and the solid surface, and is a water contact angle defined by the angle on the water droplet side. In general, the greater the water contact angle, the higher the water repellency.

  The second region 3 has a larger water contact angle and higher water repellency than the first region 2. In the present specification, “water contact angle” means a contact angle with respect to pure water.

  Specifically, the first region 2 is a region having a water contact angle of less than 150 ° (hereinafter also referred to as “droplet holding region 2”), and the second region 3 has a water contact angle of 150 °. This is the above region (hereinafter also referred to as “super water-repellent region 3”).

  Here, the substrate 1 and the tray 8 form a processing space on the droplet holding region 2 side as shown in FIGS. 3A, 4A, and 5A, for example. A particle collection space communicating with the use space in the vicinity of the substrate 1 is formed on the super-water-repellent region 3 side. A container is installed below the particle recovery space.

  In the droplet holding region 2 and the super water-repellent region 3, for example, a thin film (for example, a film) whose water contact angle is adjusted to a desired value may be attached on the base material of the substrate 1, or the base material itself may be attached. You may form by processing.

  In particular, resin or nanoparticles may be applied to form the super water-repellent region 3. The lower surface of the substrate 1 is preferably flat within a range in which the magnetic particles can move, but an uneven structure or fractal structure of several nm to several μm may be formed. The fractal structure may be a structure in which a small uneven structure is formed in a large uneven structure.

  Further, in order to form the super water-repellent region 3, it is particularly important to increase the surface area. Therefore, a fluororesin, an AKD (alkyl ketene dimer) wax, etc. can be applied, or nanoparticles, carbon nanotubes, self-organization can be applied. A monomolecular film, a needle-like structure or the like may be formed.

  In the droplet holding region 2, droplets of the same or different types of solutions are held. The solution held as droplets is not particularly limited, and can be, for example, a reaction solution or a cleaning solution. The kind of the reaction liquid and the cleaning liquid can be appropriately selected depending on the purpose of use.

  A surfactant may be added to the reaction solution or the cleaning solution in order to facilitate the penetration of the magnetic particles into the droplets. Examples of the surfactant include anionic surfactant lithium dodecyl sulfate and a nonionic surfactant TritonX100.

  The magnetic particles positioned on the lower surface side of the substrate 1 are sequentially moved from the A row droplets to the F row droplets in the droplet holding region 2 while being attracted to the lower surfaces by the corresponding magnets 4. Move to the water region 3 side. By allowing the magnetic particles to enter each droplet, the sample is reacted and washed.

  Then, when the super water-repellent region 3 is reached, the magnetic particles wet with the droplets fall from the substrate 1. A container is installed below the super water-repellent region 3, and the magnetic particles carrying the sample after reaction and / or washing can be collected by the container.

  The magnetic particles carrying the sample collected in this container are used in the next analysis step for each container.

[Processing method]
The processing method of this embodiment can be implemented using the processing apparatus 200.
First, a substance to be treated by the treatment method of the present embodiment is immobilized on the surface of magnetic particles. The substance to be treated is not particularly limited, and examples thereof include nucleic acids (DNA, RNA, etc.), peptides, proteins, saccharides, lipids, complex glycolipids and the like.

  The treatment method of the present embodiment can be applied to a large-scale / trace continuous reaction of nucleic acids, proteins, lipids, carbohydrates, and chemical substances.

  A cDNA synthesis method can be given as an example of the processing method of the present embodiment. In this cDNA synthesis method, at least one cell lysis solution, mRNA / magnetic particle cleaning solution, reverse transcription reaction cleaning solution, and reverse transcription reaction solution droplets are arranged in this order in at least one row direction of the jig 100. Each of the mRNAs (samples) held and immobilized on the magnetic particles is sequentially moved using a magnet 4 disposed on the opposite side of the substrate 1 from the droplet. Thereby, cDNA immobilized on the magnetic particles can be obtained.

  The solution for cell lysis can be, for example, a 3 μl total solution containing 100 mM Tris HCl (pH 7.5), 500 mM LiCl, 1% lithium dodecyl sulfate 5 mM dithiothreitol (DTT). The mRNA washing solution can be a 3 μl total solution containing 10 mM Tris HCl (pH 7.5), 0.15 M LiCl, 0.1% lithium dodecyl sulfate. The washing solution for reverse transcription reaction is a 3 μl total solution containing 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.1% Triton X-100, 0.5 mM dNTP, 5 mM DTT, and 2 unit RNase inhibitor. be able to. The reverse transcription reaction solution is 3 μl in total volume containing 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.1% Triton X-100, 0.5 mM dNTP, 5 mM DTT, 2 unit RNase inhibitor, 8 unit SuperScript III Reverse transcriptase. Solution.

  However, these are examples and are not limited to these solutions.

  There is no restriction | limiting in particular in the kind of mRNA, length, etc. mRNA from various organisms can be used. As the magnetic particles, for example, particles having a particle diameter of 2.8 μm and oligo (dT) 25 covalently bonded to the surface can be used.

  As the magnet 4, for example, a small neodymium magnet can be used. In each droplet, magnetic particles on which the sample is immobilized are retained for a time required for reaction or washing. The time required for the reaction or washing varies depending on the reaction conditions and washing conditions, but can be, for example, in the range of 1 second to 1 hour.

  The reaction and washing can be performed at room temperature (room temperature), but the temperature can be adjusted as necessary. Moreover, in order to adjust humidity, the wet filter paper 10 may be put in the tray 8 (refer FIG. 3 (a), FIG. 4 (a), FIG. 5 (a)).

  The mRNA immobilized on the magnetic particles can be obtained by sequentially retaining and passing the mRNA immobilized on the magnetic particles in the droplets. The obtained cDNA can be used in the next step as it is. Further, by using a plurality of different magnetic particles in parallel, a plurality of different types of cDNA can be obtained simultaneously.

  It is also possible to automatically synthesize antibody cDNA using oligo dT magnetic particles.

  Examples of containers used in the present embodiment include petri dishes (petri dishes), tubes, biochips, microarrays, PCR microwell plates, and the like. The tube may be made of generally used polypropylene or polystyrene, but may be a nucleic acid low adsorption tube made of special low adhesion polypropylene. A tube that can be inverted may be used. Alternatively, tubes may be arranged in a rack and a plurality of samples may be processed in parallel.

  The PCR microwell plate can be either a 98 well plate or a 384 well plate. When twelve magnets 4 are used at the same time, for example, by sequentially moving from the even-numbered column to the odd-numbered column, the sample can be put into the 384 well plate at high speed.

  Conventionally, a sample is collected manually by a pipette after cDNA synthesis and put back into the container for the next analysis device. However, by using the jig 100 of this embodiment, the sample can be easily moved to the next container. Therefore, contamination can be suppressed.

  Next, the present embodiment will be described more specifically by way of examples. However, the present embodiment is not limited to the following examples.

<Example 1>
Hereinafter, Example 1 will be described with reference to FIGS. 3 (a) to 3 (d). In FIG. 3A to FIG. 3D, the jig is denoted by reference numeral 100-1.
(Production of substrate)
A PET film was placed on the silicon rubber, a parafilm was further stacked, and a stainless steel mold having a concavo-convex structure of about 1 μm in a part of the region was placed and fixed from above. Heated in a drying cabinet set at 50 ° C. for 5 minutes. The mold was taken out from the drying cabinet, and a molding die was pressure-bonded to the PET film from above. After removing the mold and cooling, the water-repellent fluorine coating agent Fluorosurf (manufactured by Fluoro Technology) was applied only to the portion where the irregularities were formed on the parafilm and dried.

Automatic contact angle measurement OCA20 (Eihiro Seiki Co., Ltd.) was used to measure the water contact angle. The measurement conditions were dropping liquid H ₂ O, dropping liquid volume 3.0 μL, flow rate 10 μL / s, and measurement was performed 9 seconds after dropping. When the region where the uneven structure was not formed on the parafilm was measured three times, the water contact angle was an average of 108 °. When the region where the concavo-convex structure was formed on the parafilm and the water-repellent fluororesin was applied was measured three times, the water contact angle was 153 ° on average.

(Magnetic particle manipulation on the jig)
3 μL of the magnetic particle dispersion was dispensed into a region having a water contact angle of less than 150 ° (droplet holding region 2). Next, 3 μL of UHRR (Universal Human Reference RNA) aqueous solution and 3 μL of mRNA-magnetic particle washing solution were dispensed in the same row. A filter paper (wet filter paper 10) moistened with ion-exchanged water is placed in the lower container 9 of the tray, the substrate 1 dispensed in the tray 8 is set and fixed, and then the tray 8 is inverted to place the droplets in a suspended state. . Next, the petri dish 7 was placed below the region (super water-repellent region 3) having a water contact angle of 150 ° or more on the parafilm. In this state, the magnet 4 was moved from the opposite side of the substrate 1 onto the magnetic particle dispersion liquid droplet 11 (see FIG. 3A). After the magnetic particles in the magnetic particle dispersion liquid droplet 11 gathered on the substrate 1 side, the magnet 4 was operated to move the magnetic particle to the next UHRR aqueous solution droplet 12 (FIG. 3B). reference). After fixing the magnet 4 on the UHRR aqueous solution droplet 12 for 1 minute, the magnet 4 was operated to move the magnetic particles to the next mRNA-magnetic particle cleaning liquid droplet 13 (see FIG. 3C). After fixing for 1 minute, the magnetic particles were moved to a region where the water contact angle on the parafilm was 150 ° or more. Immediately after entering the region where the water contact angle was 150 ° or more, it was dropped by its own weight and could be put into the petri dish 7 that was placed below (see FIG. 3 (d)).

<Example 2>
The second embodiment will be described below with reference to FIGS. 4 (a) to 4 (d). In FIGS. 4A to 4D, reference numeral 100-2 is attached to the jig.
(Magnetic particle manipulation on the jig)
The same substrate as in Example 1 was used. Droplets were placed in the following order in a region with a water contact angle of less than 150 °.

  Line A: Magnetic particle dispersion liquid droplet 11, Line B: UHRR aqueous solution liquid droplet 12, Line C: mRNA-magnetic material particle cleaning liquid droplet 13, Line D: Reverse transcription reaction cleaning liquid droplet 15, Line E: Reverse transcription reaction Solution droplet 16, line F: PCR reaction solution 17.

  As the magnetic particles, Dynabeads Oligo (dT) 25 (manufactured by Thermo Fisher) was used. As the mRNA-magnetic particle washing solution, a total volume of 3 μl containing 10 mM Tris HCl (pH 7.5), 0.15 M LiCl, 0.1% lithium dodecyl sulfate was used. As the washing solution for reverse transcription reaction, a total volume of 3 μl containing 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.1% Triton X-100, 0.5 mM dNTP, 5 mM DTT, 2 unit RNase inhibitor was used. . The reverse transcription reaction solution is 3 μl in total volume containing 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.1% Triton X-100, 0.5 mM dNTP, 5 mM DTT, 2 unit RNase inhibitor, 8 unit SuperScript III Reverse transcriptase. The solution of was used. A PCR reaction solution was prepared by mixing PrimeStar GC Buffer (TaKaRa Clontech) and TE Buffer (Thermo Fisher).

  After dispensing, the substrate 1 was set on the tray 8 and reversed by the same procedure as in Example 1, and the droplets were suspended. In this state, the magnetic particles were sequentially moved from the A row to the F row by the magnet 4 from the opposite side of the substrate 1 (see FIGS. 4A to 4D). In the droplets of the B and E rows, the magnet 4 was moved up and down to stir the magnetic particles. After moving the magnetic particles to the F row, the magnetic particles were moved to a region of the substrate 1 having a water contact angle of 150 ° or more. Immediately after entering the region with a water contact angle of 150 ° or more, the magnetic particles dropped by their own weight, and could be put into the PCR tube 14 installed below.

  The tube 14 was agitated to eliminate precipitation of magnetic particles, and then set in a Thermo Cycler T100 (manufactured by BIO RAD). After performing the PCR reaction, electrophoresis was performed. From this result, it was confirmed that DNA was amplified by cDNA synthesis and PCR using magnetic particles.

<Example 3>
Example 3 will be described below with reference to FIGS. 5 (a) to 5 (d). In FIGS. 5A to 5D, reference numeral 100-3 is attached to the jig.
(Production of substrate)
A low molecular weight gelling agent (12-hydroxystearic acid) and a polymer material (polystyrene) were dispersed in 1 mL of an organic solvent, and the solution was applied to a portion on a polypropylene substrate and allowed to stand at room temperature and dried. The water contact angle was measured by the same method as in Example 1. When the region where the gelling material was not applied was measured three times, the water contact angle was an average of 82 °. When the region to which the gelling material was applied was measured three times, the water contact angle was an average of 152 °.

(Magnetic particle manipulation on the jig)
In a region having a water contact angle of less than 150 °, 6 rows × 12 columns (= 72) of droplets were arranged by an automatic dispenser in the following order. Row A: Magnetic dispersion liquid droplet 12, Row B: (Cell lysis solution + cell) 19, Row C: mRNA-magnetic particle cleaning droplet 13, Row D: Reverse transcription reaction cleaning droplet 15, Row E: Reverse transcription reaction solution droplet 16, F row: PCR reaction solution 17.

  As the cell lysis solution, a total volume of 3 μL containing 100 mM Tris HCl (pH 7.5), 500 mM LiCl, 1% lithium dodecyl sulfate 5 mM dithiothreitol (DTT) was used. As the cells, dermal fibroblasts (Promo Cell) were used. Except for the above, the same liquid as in Example 2 was used.

  After dispensing, the substrate 1 was set on the tray 8 and reversed by the same procedure as in Example 1, and the droplets were suspended. In this state, the magnetic particles were sequentially moved from the A row to the F row by the twelve magnets 4 simultaneously from the opposite side of the substrate 1 (see FIGS. 5A to 5D). In the droplets of the B and E rows, the magnet 4 was moved up and down to stir the magnetic particles.

  After moving the magnetic particles to the F row, the magnetic particles were moved to a region of the substrate 1 having a water contact angle of 150 ° or more. Immediately after entering the region with a water contact angle of 150 ° or more, the magnetic particles dropped by their own weight, and could be placed in 12 rows simultaneously in the 96-well microwell array 18 placed below.

  This 96-microwell array 18 was set in Thermo Cycler T100 (manufactured by BIO RAD), subjected to PCR reaction, and further analyzed by real-time PCR (manufactured by Life Technologies). As a result, all 12 samples were confirmed to have specific gene expression in dermal fibroblasts without being affected by contamination.

  The jig 100 of the present embodiment described above holds the first droplet for processing the magnetic particles 5 with a sample including the magnetic particles moving by the magnetic field and the sample fixed to the magnetic particles. A substrate 1 having a region 2 and a second region 3 having a water contact angle larger than that of the first region 2 is provided.

  In this case, the sample-attached magnetic particles 5 are treated with droplets in the first region 2 and the treated sample-attached magnetic particles 5 are moved from the first region 2 side to the second region 3 side. The sample-attached magnetic particles 5 can be detached from the substrate 1.

  As a result, it does not take time to recover the treated magnetic particles 5 with the sample.

  In addition, when the first region 2 is a region having a water contact angle of less than 150 ° and the second region 3 is a region having a water contact angle of 150 ° or more, the substrate of the magnetic particle 5 with sample after processing The releasability from 1 can be improved more.

  In addition, when the first region 2 is a region having a water contact angle of less than 90 °, it is possible to improve the retention of liquid droplets in the first region 2. In this case, the second region 3 may have a water contact angle of 90 ° or more and less than 150 °.

  Moreover, when the 1st area | region 2 and the 2nd area | region 3 mutually adjoin, the magnetic substance particle 5 with a sample after a process can be collect | recovered rapidly.

  Note that the first region 2 and the second region 3 do not necessarily have to be adjacent to each other. That is, another region may be interposed between the first region 2 and the second region 3.

  Further, when there are a plurality of droplets for processing on the magnetic particles 5 with a sample and the plurality of droplets include at least one of a droplet of a reaction liquid and a droplet of a cleaning liquid, Thus, a plurality of processes can be performed continuously.

  It should be noted that the number of droplets for processing on the sample-attached magnetic particles 5 may be only one (for example, a reaction liquid or a cleaning liquid).

  In addition, a plurality of liquid droplets correspond to each of the plurality of sample-attached magnetic particles 5, and a droplet row composed of at least two droplets arranged in one direction (the row direction described above) is perpendicular to the one direction. It includes a plurality of droplet arrays arranged in the direction (the column direction).

  In this case, it is possible to perform a plurality of treatments on the sample continuously between the samples in parallel and easily collect the samples after the plurality of treatments in parallel.

  In addition, in order to move the magnetic particle 5 with a sample between droplets in parallel between the magnetic particles 5 with a sample, a plurality of magnets 4 corresponding to the plurality of magnetic particles 5 with a sample are moved in the other direction. It is preferable to use magnet units arranged in the above-described row direction.

  Note that the number of droplet rows may be one.

  Moreover, the processing apparatus of this embodiment is a magnet which can move along the substrate 1 while applying a magnetic force to the magnetic particles located on one surface (for example, the lower surface) side of the substrate 1 on which the jig 100 and the sample are fixed. 4 is provided.

  In this case, the magnetic particles carrying the sample can be attracted to the first region 2 of the substrate 1 and processed with droplets, and the magnetic particles carrying the treated sample can be treated as the second region of the substrate 1. 3 and can be detached from the substrate 1.

  Further, when the magnet 4 is disposed on the other surface side of the substrate 1, the magnet 4 is not contaminated with droplets, and there is no need for cleaning.

  Further, since the jig 100 is used with one surface of the substrate 1 facing downward, the weight of the sample-attached magnetic particles 5 is moved when the processed sample-attached magnetic particles 5 are moved to the second region 3. Can be dropped from the substrate 1. Note that the jig 100 may be used in a state where one surface of the substrate 1 is obliquely downward.

  Further, in the case where a container for collecting the sample-attached magnetic particles 5 disposed below the second region 3 is further provided, the sample-attached magnetic particles 5 dropped from the substrate 1 can be collected, and the sample is attached. The entire container containing the magnetic particles 5 can be used in the next step (for example, analysis step).

  Further, in the case where a driving device (actuator) that can move the magnet 4 along the substrate 1 is further provided so that the magnetic particle 5 with sample moves between droplets, the movement of the magnetic particle 5 with sample is automated. it can.

  The driving timing of the magnet 4 by the driving device is set by inputting and storing in advance a computer (including a storage device) that controls the driving device for the time required for reaction and washing with each droplet. be able to.

  Further, the processing method using the processing apparatus of the present embodiment includes a step of reacting and / or cleaning by moving the magnetic particles 5 with a sample between droplets held in the first region 2, and with a sample. Moving the magnetic particles 5 from the first region 2 side to the second region 3 side.

  In this case, the magnetic particles carrying the sample can be attracted to the first region 2 of the substrate 1 and processed with droplets, and the magnetic particles carrying the treated sample can be treated as the second region of the substrate 1. 3 and can be detached from the substrate 1.

  In the cDNA synthesis method using the processing apparatus 200 of the present embodiment, at least the cell lysis solution, the mRNA-magnetic particle cleaning solution, the reverse transcription reaction cleaning solution, and the reverse transcription reaction solution droplets are provided in the first region 2. Are held in this order, and the magnetic particles 5 with a sample containing the magnetic particles and mRNA as a sample immobilized on the magnetic particles are moved between the droplets in the above order to perform reaction or / and washing. And a step of moving the sample-attached magnetic particles 5 from the first region 2 side to the second region 3 side.

  In this case, magnetic particles carrying mRNA (magnetic particles with sample 5) can be attracted to the first region 2 of the substrate 1 and treated with droplets, and magnetic particles carrying the synthesized cDNA. Can be moved to the second region 3 of the substrate 1 to be detached from the substrate 1.

  As can be seen from the above description, in the present embodiment, the sample-attached magnetic particles can be used by using a jig including one substrate without cleaning or replacing the tool every time the sample-attached magnetic particles 5 are moved. A plurality of types of treatments (reactions and washing) can be performed in parallel between the sample-attached magnetic particles 5 and the sample-attached sample magnetic particles 5 are dropped by their own weight. By doing so, the sample-attached magnetic particles 5 can be easily put in a container provided for the next step. Furthermore, contamination can also be suppressed.

  Below, the inventors will explain the thought process that led to the idea of the above embodiment.

  In general, gene-related reactions such as synthesis of cDNA from mRNA and amplification of DNA are often performed in parallel in a small volume. In that case, for example, using a 96-well or 384-well microwell array, prepare an array containing reaction solution or washing solution in each well. When the reaction or washing is completed, move the solution to the next well using a pipette. Generally, it is used for the following treatment. When using a pipette, it is necessary to wash or replace each time and use it for the next processing, and the operation is very complicated.

  Instead of using a pipette, a magnetic bead is used, and a sample immobilized on the magnetic bead is lifted and moved by a magnet (see Japanese Patent Publication No. 2007-504835).

  Further, Patent Document 1 automates the method using the magnetic beads, and performs a plurality of types of reactions and washing operations in parallel without washing or exchanging an instrument used for moving a solution or the like for each operation. A suspended droplet array magnetic bead reaction method is disclosed. In this method, the reaction solution used for the reaction is dispensed onto the reaction plate, and the reaction can be performed automatically by moving the magnetic beads between the droplets using a magnet from the opposite side of the plate. is there. However, although the reaction is automated, it is necessary to manually collect a sample from the last line where the reaction has been completed and place it in a container for the next analysis device by a pipette.

  Therefore, the inventors have devised the above embodiment after intensive studies to solve this problem.

  DESCRIPTION OF SYMBOLS 1 ... Board | substrate (a part of jig | tool), 2 ... 1st area | region, 3 ... 2nd area | region, 4 ... Magnet, 5 ... Magnetic body particle | grains with a sample, 6 ... Droplet, 7 ... Petri dish (container), 8 ... Tray (part of jig), 11 ... Magnetic particle dispersion liquid droplet, 12 ... UHRR aqueous solution droplet, 13 ... mRNA-magnetic particle washing droplet, 14 ... Tube (container), 15 ... For reverse transcription reaction Cleaning droplet, 16 ... Reverse transcription reaction solution droplet, 18 ... Microwell plate (container), 100 ... Jig (part of processing apparatus).

Japanese Patent No. 5244130

Claims (13)

  A first region for holding a droplet for processing with respect to a magnetic particle with a sample including a magnetic particle moving by a magnetic field and a sample fixed to the magnetic particle, and a water contact angle with respect to the first region A jig having a substrate having a second region having a large area on one side. The first region is a region having a water contact angle of less than 150 °,
The jig according to claim 1, wherein the second region is a region having a water contact angle of 150 ° or more.   The jig according to claim 1 or 2, wherein the first region is a region having a water contact angle of less than 90 °.   The jig according to any one of claims 1 to 3, wherein the first region and the second region are adjacent to each other. There are a plurality of the droplets,
The jig according to any one of claims 1 to 4, wherein the plurality of droplets include a droplet of a reaction liquid and a droplet of a cleaning liquid.   The plurality of droplets are arranged in a direction perpendicular to the one direction, each of which is a plurality of droplet rows composed of at least two droplets arranged in one direction corresponding to the plurality of magnetic particles with the sample. The jig according to claim 5, comprising a droplet array. The jig according to any one of claims 1 to 6,
A processing apparatus comprising: a magnet movable along the substrate while exerting a magnetic force on the sample-attached magnetic particles positioned on the one surface side.   The processing apparatus according to claim 7, wherein the magnet is disposed on the other surface side of the substrate.   The processing apparatus according to claim 7, wherein the jig is used in a state where the one surface is downward or obliquely downward.   The processing apparatus according to claim 9, further comprising a container installed under the second region for collecting the sample-attached magnetic particles.   11. The apparatus according to claim 7, further comprising a driving device capable of moving the magnet along the substrate so that the sample-attached magnetic particles move between the droplets. Processing equipment. A processing method using the processing apparatus according to any one of claims 7 to 11,
Performing reaction or / and washing by moving the magnetic particles with the sample between the droplets held in the first region;
Moving the sample-attached magnetic particles from the first region side to the second region side. A cDNA synthesis method using the processing apparatus according to any one of claims 7 to 11,
Holding at least the cell lysis solution, the mRNA-magnetic particle washing solution, the reverse transcription reaction washing solution and the reverse transcription reaction solution droplets in this order in the first region;
A step of moving the magnetic particles with a sample containing the magnetic particles and mRNA as the sample immobilized on the magnetic particles between the droplets in the order, and performing reaction or / and washing;
Moving the sample-attached magnetic particles from the first region side to the second region side.