JP5599266B2 - Reaction plate suction and cleaning devices - Google Patents

Description

  The present invention relates to a device for aspirating fluid from a reaction plate having a plurality of reaction cells. The present invention also relates to a reaction plate cleaning device and a cleaning apparatus provided with the suction device.

  In the field of analytical chemistry such as genetic analysis and immunoassay, it is possible to fix nucleic acids and proteins to be quantified on magnetic beads and wash them with the beads left in the reaction cell in order to remove unreacted substances. This is a frequently performed process. Especially when the target molecule is washed and used for repeated analysis, and especially when samples that have undergone an amplification process such as polymerase chain reaction (PCR) are repeatedly analyzed, the substance remaining in the sample solution should be sufficient. Needed to be removed and needed manual work. When the number of reaction cells is increased, labor is required and automation is required. However, there are many inspections that do not allow a small amount of residual material, and a new cleaning device or apparatus is desired.

  Conventionally, an apparatus that automatically executes the process of washing such magnetic beads has a single dispensing mechanism corresponding to one reaction cell, and an automatic transfer mechanism is used for many reaction cells. It corresponded. Even when multiple dispensing mechanisms were included, few (up to about 8) were present.

  However, it is not suitable for cleaning the interior of many reaction cells at low cost and quickly, and the automatic moving mechanism is expensive because it contains moving parts, and there are also problems such as failure. .

  On the other hand, in order to wash many tens or more of reaction cells at once, it is often performed by inserting a suction capillary into each of the plurality of reaction cells and sucking with a pump. In many cases, an extreme variation in speed occurs and a suction residue inevitably occurs (Patent Documents 1 and 2).

  As an improvement measure, there is a method of preparing as many suction cylinders as there are reaction cells (Patent Document 3). However, in this method, it is necessary to separately provide a drive system for simultaneously controlling and driving a plurality of syringes and a means for discharging the solution in the sucked syringe after suction in the cylinder. Cost will rise.

  In addition, there is known a method in which the reagent contained in the cell is pushed out to the dispensing means by the suction means by pressurizing the cell. In this method, the suction speed of the reagent is stabilized by the pressurization, but it is added to the cell. A new means for pressing is required, and the apparatus configuration is complicated (Patent Document 4).

JP 2002-1092 A US Patent Application Publication 2001/0055545 A1 Patent No.4431276 US Patent Application Publication 2001/0053337 A1

  In order to solve the above problems, an inexpensive and easy-to-use device or apparatus that can easily and repeatedly wash many reaction cells is desired. The object of the present invention is to provide such a device or apparatus.

  As a result of intensive studies to solve the above problems, the present inventor has established that each reaction cell is sealed and includes at least two tubes (for leaks) as a means for reliably and inexpensively washing all reaction cells. It has been found that the above-mentioned problem can be solved by making the structure in contact with the outside through a thin tube and a suction thin tube, and making the conductance of the leak thin tube smaller than that of the suction thin tube. The present invention is based on such findings and includes the following.

[1] A device for aspirating fluid in a plurality of reaction cells of a reaction plate,
A sealing material for making the pressure inside the plurality of reaction cells of the reaction plate independent,
At least one suction tubule and at least one leakage tubule penetrating the sealant corresponding to each reaction cell;
Means for joining the suction capillary tube outside the reaction cell, and the leak capillary tube applies a pressure difference across the suction capillary tube when the fluid in the reaction cell is suctioned from the suction capillary tube A suction device characterized by being.
[2] When the conductance to the air flowing through the two thin tubes is compared by applying a pressure difference between the two ends of the suction thin tube and the leak thin tube, the conductance of the leak thin tube is smaller than the conductance of the suction thin tube The suction device according to [1], which is a device.
[3] The suction device according to [1] or [2], wherein a ratio of an inner diameter of the suction capillary and the leaking capillary is 5: 1 to 2: 1.
[4] The suction device according to any one of [1] to [3], wherein a distance between a lower end of the suction thin tube and a bottom portion of the reaction cell is smaller than an inner radius of the suction thin tube.
[5] The suction device according to any one of [1] to [4], wherein the suction thin tube includes a check valve for preventing backflow of the sucked fluid into the reaction cell.
[6] The suction device according to any one of [1] to [5], further comprising means for sucking air inside the means for joining the suction thin tubes outside the reaction cell.

[7] A device for cleaning a plurality of reaction cells of a reaction plate,
A suction device according to any one of [1] to [6];
A cleaning device, comprising: a cleaning liquid injecting thin tube penetrating the sealing material corresponding to each reaction cell.
[8] The cleaning device according to [7], further comprising means for joining the cleaning liquid injection thin tubes.
[9] In the above [7] or [8], the distance between the cleaning liquid injection capillary and the inner wall of the reaction cell is smaller than the distance between the suction capillary and the cleaning liquid injection capillary The cleaning device described.
[10] The apparatus further includes a magnet for collecting magnetic beads on a part of the inner wall of the reaction cell, and the cleaning liquid injection capillary is arranged to correspond to the position of the magnet. [9] The cleaning device according to any of [9].
[11] An apparatus for cleaning a plurality of reaction cells of a reaction plate,
[7] to [10] The cleaning device according to any one of
A cleaning liquid supply unit;
A cleaning device comprising an exhaust and a waste liquid section.

  The present invention provides a device for aspirating fluid in a plurality of reaction cells of a reaction plate. This suction device can efficiently and evenly suck fluid in a plurality of reaction cells on the reaction plate. The device is also useful for cleaning the reaction cell as a cleaning device and a cleaning device for the reaction plate. The device and apparatus do not require an expensive operating device or a complicated configuration, and a single suction pump can collectively suck and discard the fluid.

The device structural example for attracting | sucking a solution from the reaction cell in a comparative example is shown. The device structural example for attracting | sucking a solution from the reaction cell in this invention is shown. It is the figure which showed the pressure difference concerning the both ends of the comparative example and the attraction | suction thin tube in this invention. The example of a data of the pressure difference concerning the both ends of the suction thin tube in a comparative example, and the suction speed is shown. The example of data of the pressure difference concerning the both ends of the thin tube for suction in this invention and suction speed is shown. It is a block diagram of the suction device corresponding to Example 1, the cleaning device for the reaction plate, and the cleaning apparatus. It is device sectional drawing when a sealing material isolate | separates into plurality. (A) And (b) is the figure which looked at the suction device from the top, and shows the shape of the sealing material in the case of a round reaction cell (a) and the case of a polygonal reaction cell (b). It is device sectional drawing at the time of attaching a taper-shaped disposable chip | tip to the front-end | tip of the thin tube for suction. It is device sectional drawing in the case of including the mechanism in which the lower end of the thin tube for suction is automatically adjusted to the optimal position. FIG. 6 is a configuration diagram of a suction device and a cleaning device when a suction manifold is disposed between a cleaning liquid injection manifold and a seal material. FIG. 5 is a configuration diagram of a cleaning apparatus when a cleaning liquid injection manifold is not used. It is an apparatus block diagram in the case of performing only solution aspiration independently. It is an apparatus block diagram when performing only washing | cleaning liquid injection | pouring independently.

  The present invention relates to a device for aspirating a fluid from a reaction plate having a plurality of reaction cells, and a reaction plate cleaning device and a cleaning apparatus.

  As described above, conventionally, in devices that realize suction of a solution from a plurality of reaction cells without using robotics, it has been desired to improve the point that suction variation occurs or suction efficiency is lowered.

  As a comparative example, FIG. 1 shows a typical configuration example of a device for simultaneously sucking a solution from a plurality of reaction cells. A suction capillary 4 for aspirating the solution 3 in the plurality of reaction cells 2 on the plate 1 is inserted in the center of the reaction cell 2. The vacuum manifold 5 (means for sucking air) causes the suction manifold 6 (means for joining all of the plurality of suction capillaries 4 outside the reaction cell 2) to become negative pressure, so that the upper end of the suction capillaries 4 is The pressure is lower than the lower end, and the solution in the reaction cell is sucked. At this time, in order to operate the pump effectively, a solution separation container 7 for separating the solution is often inserted in the middle of the flow path, but this is not essential.

  In such a configuration, the suction of the solution may be promoted by operating the vacuum pump. However, since an equal amount of the solution is not necessarily dispensed in the reaction cell 2, the empty reaction cell Problems arise when 2 occurs.

  In the comparative example of FIG. 1, the three containers (B, C, D) other than the leftmost container (A) of the reaction cell 2 are empty. Even if the vacuum pump 5 is operated in this state to suck the solution 3, a large amount of air flows into the suction manifold 6 from the suction thin tube 4 inserted into the empty reaction cell 2, so that the inside of the manifold The pressure is lower than when all the reaction cells contain the solution. This means that the pressure difference applied to both ends of the suction capillary 6 is reduced as it is, so that the solution suction speed is reduced. As the number of empty containers increases, the decrease in the suction speed of the solution due to this phenomenon increases, and eventually it may be hardly sucked. Therefore, there remains a reaction cell that cannot be sucked even if suction is continued for a very long time.

  A pressure diagram illustrating this is shown in FIG. The pressure diagram in the case where the solution remains in all the reaction cells 2 is shown as Comparative Example 1 in FIG. The uppermost dotted line indicates the atmospheric pressure, and the negative pressure Pe in the suction manifold 6 sucked by the pump increases (indicated by a downward arrow in the figure). Since the viscosity of the solution is two orders of magnitude higher than that of air, the pressure drop hardly occurs, and the pressure directly reflecting the performance of the vacuum pump 5 becomes the pressure Pe of the suction manifold 6. However, when the containers B, C, and D are empty, as shown in the pressure diagram of Comparative Example 2 in FIG. 3, the pressure difference generated at both ends of the suction thin tube 4 exceeds the suction speed of the pump. Air is sucked from the suction thin tubes 4 for B, C, and D, and the pressure Pe in the suction manifold 6 decreases.

  The pressure drop is then evaluated under relatively practical conditions. When using a suction capillary 4 with an inner diameter of 0.5 mm and a length of 30 mm and a pump of 5 L / min, assuming that a 96-well plate was used and 95 reaction cells were empty, the pressure applied to both ends of the capillary Is 6.9Pa. As shown in the diagram of FIG. 3, the pressure difference applied to both ends of the capillary tube sucking air is the same as the pressure difference applied to the capillary tube where the solution 3 remains, so the suction speed is greatly reduced to 0.23 μL / sec. (Comparative Example 2).

  As a device for overcoming the above-described problems and reliably and reliably aspirating the fluid in all the reaction cells, in the present invention, each reaction cell is sealed, and has at least two tubes (air leaking tubule and The structure is in contact with the outside through a solution suction capillary, and the problem is overcome by making the conductance of the leak capillary smaller than that of the suction capillary.

FIG. 2 shows the basic configuration of the device of the present invention. As shown in FIG. 2, each of the plurality of reaction cells 2 is sealed with a sealing material 8 and arranged so that the suction thin tube 4 and the leaking thin tube 9 penetrate the sealing material 8. The lower end of the suction capillary 4 is arranged near the bottom of the reaction cell 2 so that the solution 3 in the reaction cell 2 can be sucked without leaving as much as possible. The upper end of the suction capillary is connected to the suction manifold 6 so that negative pressure can be applied. The suction manifold 6 is made to have a sufficient negative pressure by using a vacuum pump 5 having a sufficient displacement. At this time, it is possible to reduce the conductance of the leaking capillary 9 and apply a sufficient pressure difference to both ends of the suction capillary where the solution remains. The pressure diagram in the present invention is shown on the right side of FIG. When the B, C, and D containers are emptied, the internal pressure of the suction manifold 6 and the pressure difference ΔP _air inside the B, C, and D containers become small values as in Comparative Example 2. At this time, assuming that the flow rate of air flowing through the narrow tube is S _air , in the configuration of the present invention, the air flows also at the B, C, D leaking narrow tube 9 at the speed of S _air. A pressure difference of ΔP _{leak — B, C, D} = S _air / C _leak occurs between the atmospheric pressures, with the conductance of the leaking narrow tube 9 as C _leak . At this time, ΔP _leak can be sufficiently increased if C _leak is sufficiently small. On the other hand, for the container A in which the solution 3 remains, the velocity S _sol of the solution flowing through the suction capillary 4 is smaller than S _air by two orders of magnitude or more due to the difference in characteristics as the fluid. _{Therefore, ΔP leak_A = S sol / C} leak ( Equation 1) is _{ΔP leak_B, C,} reduced more than two orders of magnitude as compared with _D. On the other hand, since the pressure difference applied to both ends of the suction _thin tube of the container A is ΔP _sol = ΔP _air + ΔP _{leak_B, C, D} -ΔP _{leak_A} , ΔP _{leak_B, C, D} -ΔP _{leak_A ≈ΔP leak_B, C, D} The pressure difference can be greatly increased by this amount. Therefore, it is possible to continue to apply a sufficiently large pressure difference to both ends of the suction thin tube where the solution 3 remains, no matter how many empty containers are generated, and the solution 3 can be continuously sucked at a high speed.

  FIG. 4 shows the pressure difference at both ends of the suction capillary and the suction speed when the diameter of the suction capillary in the comparative example is changed. At this time, the pump suction speed was 5 L / min, and the ultimate vacuum was 1 Pa or less. The precondition is that the length of the narrow tube is 3 cm, the viscosity of air is 18 μPasec, the viscosity of the solution to be sucked is 2 mPasec, and 95 of 96 containers are empty. As can be seen from FIG. 4, the pressure difference decreases as the diameter of the suction tube increases. For this reason, the suction speed is also reduced. Further, when the inner diameter of the suction thin tube is 0.4 mm or less, it decreases to 1 kPa or less. In such a case, the influence of external factors increases, and the pressure difference may further decrease. On the other hand, when the inner diameter of the suction thin tube is reduced, the applied pressure becomes sufficiently large, but this time, the flow velocity rapidly decreases. Therefore, there is an optimal suction capillary inner diameter, but the suction speed is not always sufficient.

On the other hand, FIG. 5 shows the pressure difference and suction speed in the present invention under the same conditions as in FIG. However, the diameter of the capillary tube for suction is fixed at 0.5 mm, and the diameter of the capillary tube for leak is changed. As can be seen from FIG. 5, when the diameter of the leaking capillary is about 1/3 of that of the suction capillary, a pressure difference can be applied to both ends of the suction capillary with the performance of the vacuum pump being sufficiently drawn. The suction speed can be suctioned at a suction speed corresponding to the suction speed of the pump. Also, the conductance when a fluid such as air is sucked through the narrow tube (in the case of laminar flow) is proportional to the fourth power of the inner diameter of the narrow tube and inversely proportional to the length of the narrow tube. As described above, the _{reason why} the solution suction speed of the present invention is higher than that of the comparative example is that the pressure difference applied to both ends of the suction capillary is larger by ΔP _{leak_A} = S _sol / C _leak. The effective pressure difference is effective when the conductance of the leaking thin tube 9 is small. Considering that the small conductance is a relative value with respect to the conductance of the leak suction capillary, it can be considered that it can be generalized from FIG. Therefore, it can be seen that the effect of the present invention is great when the conductance of the leaking capillary is smaller than the conductance of the suction capillary. From the above, it can be seen that, when the conditions of the present invention are satisfied, suction can be performed quickly and without variation for all the reaction cells 2.

Therefore, a device for aspirating fluid in a plurality of reaction cells of a reaction plate according to the present invention (also referred to as “suction device of the present invention”)
A sealing material for making the pressure inside the plurality of reaction cells of the reaction plate independent,
At least one suction tubule and at least one leakage tubule penetrating the sealant corresponding to each reaction cell;
Means for joining the suction capillary tube outside the reaction cell, and the leak capillary tube applies a pressure difference across the suction capillary tube when the fluid in the reaction cell is suctioned from the suction capillary tube It is.

  In the suction device of the present invention, in order to efficiently suck fluid from a plurality of reaction cells at the same time using one pump, the reaction cell is sealed with a sealing material, and a leaking thin tube is added. The pressure drop at both ends of the capillary tube was suppressed.

  The sealing material can be of any shape, size and material as long as the pressure inside the plurality of reaction cells can be made independent. Note that “independent pressure” means that the pressure applied to the inside of each reaction cell is separated from the pressure of other reaction cells and is not affected. For example, the sealing material may be a single plate-like structure having the same size as the reaction plate (for example, FIG. 6) or a plurality of cap-like structures corresponding to individual reaction cells. (For example, FIG. 7). The material is not limited, but resin (for example, polyester resin, polystyrene, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene Resin, phenol resin, melamine resin, epoxy resin and vinyl chloride resin), metal (eg, gold, silver, copper, aluminum, tungsten, molybdenum, chromium, platinum, titanium, nickel), alloy (eg, stainless steel, hastelloy, inconel) , Monel, duralumin etc.), glass (eg glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite and photosensitive glass etc.), silicon, rubber (eg natural and synthetic rubber) etc. Specifically, a peak material (PEEK: polyetheretherketone) or the like can be used.

  The suction thin tube and the leak thin tube can be of any shape as long as they have a shape, size and material through which the fluid can pass. For example, the shape of the thin tube is not limited to a circular shape in general in the art, but may be an elliptical shape, a square shape, a triangular shape, or the like. The material of the thin tube can be made of resin, glass, metal, etc. For example, Teflon, peak, stainless steel (SUS), etc. can be used. It is preferable that the inside of the narrow tube is subjected to a water repellent treatment or is made of a water repellent material because the solution attached to the narrow tube can be reduced. The size of the narrow tube can be appropriately set according to the size of the reaction cell to be used, the reaction volume, and the like.

  The suction thin tube is for sucking a fluid such as a reaction solution or air in the reaction cell. The suction capillary is preferably provided with a check valve for preventing the backflow of the sucked fluid into the reaction cell. On the other hand, the leaking capillary tube communicates the inside and outside air of the reaction cell, and applies a pressure difference to both ends of the suction capillary tube when sucking fluid from the suction capillary tube.

  The suction thin tube and the leak thin tube are disposed so as to penetrate the sealing material, but the penetrating position can be appropriately determined.

In the suction device of the present invention, when the conductance to the air flowing through the two capillaries is compared by applying a pressure difference between both ends of the suction capillaries and the leak capillaries, the conductance of the leak capillaries is more than the conductance of the suction capillaries. Is preferably small. This can be achieved, for example, by setting the ratio of the inner diameters of the suction and leaking capillaries to 5: 1 to 2: 1, preferably about 3: 1.

  Moreover, the lower end of the suction capillary is arranged near the bottom of the reaction cell so that the solution inside the reaction cell can be sucked without leaving as much as possible. Here, the distance between the lower end of the suction capillary and the bottom of the reaction cell is preferably smaller than the inner radius of the suction capillary.

  The means for joining the suction tubules outside the reaction cell (also referred to as “suction manifold” in the present specification) has an arbitrary shape and size in which an appropriate amount of air can exist. The material can be any material such as resin, metal, glass, rubber, etc. as long as the shape and size can be maintained during suction. The suction manifold is connected to a means (for example, a vacuum pump) for sucking air inside the suction manifold in order to apply a negative pressure to one end (upper end) of the suction capillary.

  In the suction device of the present invention, when the fluid is sucked from the suction capillary, the conductance of the leak capillary is reduced, so that even when the reaction cell is empty, the pressure difference applied to both ends of the leak capillary is Covers the pressure drop across the suction tube of the reaction cell where there is residual pressure, so that a sufficient pressure difference can be applied across the suction tube of the reaction cell to aspirate the solution in the reaction cell almost completely. It is.

  The reaction plate used with the suction device of the present invention can be a reaction plate known in the field related to the reaction to be performed. Specifically, it is preferably a solid plane that is insoluble in water and does not melt during heat denaturation. Examples of the material include metals, alloys, silicon, glass materials, plastics such as resins. The shape of the reaction plate is a plane on which the reaction cell is partitioned, for example, a titer plate, a porous or a pore array, and the like.

  The “fluid” is a reagent, a sample, a washing liquid and other additives used for the reaction performed on the reaction plate, and can take the form of a solution, a suspension, a colloidal liquid (sol), or the like.

  Therefore, the suction device of the present invention can be used in various methods in which it is desired to suck a fluid such as a reaction liquid and a cleaning liquid from a reaction plate having a plurality of reaction cells. For example, in reaction such as chemical reaction, biochemical reaction, nucleic acid amplification reaction, nucleic acid hybridization reaction, immunoassay, especially sandwich assay, ELISA (Enzyme-Linked ImmunoSorbent Assay), the reaction solution and washing solution are aspirated during and after the reaction. Therefore, the suction device of the present invention can be used.

A device for cleaning a plurality of reaction cells of a reaction plate according to the present invention (also referred to as “cleaning device of the present invention”) is as follows.
The suction device of the present invention described above;
A cleaning liquid injection thin tube penetrating the sealing material corresponding to each reaction cell is provided.

  The cleaning liquid is injected into the individual reaction cells from the cleaning liquid injection means through the cleaning liquid injection thin tube. The cleaning liquid injection capillary can be of any shape as long as it has a shape, size, and material through which a fluid can pass, like the suction capillary and the leak capillary.

  The cleaning device of the present invention preferably further comprises means for joining the cleaning liquid injection capillaries (also referred to herein as “a cleaning liquid injection manifold”). By this means, it becomes possible to inject the cleaning liquid into the reaction cell through the cleaning liquid injection thin tube. The manifold for injecting the cleaning liquid can have an arbitrary shape and size in which an appropriate amount of the cleaning liquid can exist, and the material is not particularly limited, and resin, metal, glass, rubber Any material can be used. The cleaning liquid injection manifold is connected to a portion for supplying the cleaning liquid (cleaning liquid supply unit).

  In the cleaning device of the present invention, it is preferable that the distance between the cleaning liquid injection capillary and the inner wall of the reaction cell is smaller than the distance between the suction capillary and the cleaning liquid injection capillary. This is to prevent the injected cleaning liquid from forming droplets between the suction thin tube and the cleaning liquid injection thin tube, so that a part of the cleaning liquid is not supplied to the solution at the bottom of the reaction cell.

  The cleaning device of the present invention may further include a magnet for collecting magnetic beads on a part of the inner wall of the reaction cell. This embodiment is useful when a sample (nucleic acid, protein, etc.) is immobilized on a magnetic bead for the reaction. In that case, it is preferable that the cleaning liquid injection capillary is arranged so as to correspond to the position of the magnet. Specifically, the magnetic beads can be efficiently cleaned in the reaction cell by arranging a magnet directly under the tip of the cleaning liquid injection capillary.

Further, according to the present invention, an apparatus for cleaning a plurality of reaction cells of a reaction plate (also referred to as “the cleaning apparatus of the present invention”)
The above-described cleaning device of the present invention;
A cleaning liquid supply unit;
An exhaust and a waste liquid part are provided. The cleaning liquid supply unit is connected to the cleaning liquid injection capillary or the cleaning liquid injection manifold in the cleaning device of the present invention, and supplies the cleaning liquid to the reaction cell. On the other hand, the exhaust and waste liquid parts are connected to the suction manifold in the suction device of the present invention to exhaust and waste the fluid (solution and air) sucked from the reaction cell, and are also connected to the cleaning device to obtain excess cleaning liquid. Waste liquid. As the cleaning liquid supply section and the exhaust and waste liquid section, those conventionally used in this technical field can be used.

  The cleaning device and the cleaning apparatus of the present invention can be used in various methods in which it is desired to clean a reaction plate having a plurality of reaction cells. For example, in a reaction such as a chemical reaction, biochemical reaction, nucleic acid amplification reaction, nucleic acid hybridization reaction, immunoassay, especially sandwich assay, ELISA (Enzyme-Linked ImmunoSorbent Assay), the reaction solution is aspirated, and the washing solution is injected and aspirated. Thus, when cleaning the reaction cell, the cleaning device and the cleaning apparatus of the present invention can be used. In particular, it can be preferably used in a reaction that is repeatedly used while washing a sample.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples do not limit the present invention.

[Example 1]
In this example, a cDNA library obtained from one cell is repeatedly used for an application to precisely analyze the expression level of various genes. MRNA in one cell is obtained as cDNA on magnetic beads (for example, the method described in JP 2007-319028 A). Since the amount of cDNA to be analyzed is small and gene amplification technology (PCR) is used for measurement, it is necessary to completely remove the reagents and reaction solution used in the previous measurement when one measurement is completed and the next measurement is started. There is. Since the measurement proceeds simultaneously in parallel for many cells, a titer plate having 96 reaction cells or a titer plate having more reaction cells is often used. Here, an example using a titer plate having 96 reaction cells will be described.

  In actual operation, first, a reaction solution containing cDNA captured on magnetic beads is placed in a reaction cell, and target DNA is quantitatively analyzed by quantitative PCR. The titer plate is then removed, washed thoroughly, and repeated with quantitative PCR to quantify the expression level of another gene. The present invention relates to a cleaning device or apparatus for use in this process, which will be described in detail.

  The configuration of the cleaning apparatus is shown in FIG. When the plate to be cleaned is a 96-hole plate 61, these reaction cells are arranged in a 12 × 8 square lattice at 9 mm pitch. In FIG. 6, only four reaction cells arranged in the horizontal direction are shown in order to avoid overcomplicating the figure.

  The cleaning apparatus includes a cleaning device section 12, a cleaning liquid supply section 13, and an exhaust / waste liquid section 14. A 96-well titer plate 61 in which the sample solution 3 is dispensed into the reaction cell 2 is inserted into the cleaning device section 12. In the lowermost layer of the cleaning device section 12, a magnet holding plate 15 is disposed on the side wall of the reaction cell 2 of the 96-well plate 61 to collect magnetic bead samples and separate them from the solution. On the plate 61, a sealing material 8 having means capable of individually sealing the 96 reaction cells 2 is adhered. An O-ring 18 is attached to a protrusion 17 (in the shape of an inverted truncated cone) at the bottom of the sealing material 8, and each reaction cell 2 is sealed. A suction tubule 4 and a washing liquid injection tubule 10 corresponding to each reaction cell 2 are attached to the seal material 8 so as to penetrate the seal material 8. The lower end of the suction thin tube 4 is almost in close contact with the bottom of the reaction cell 2, and is designed so that the cleaning liquid can be sucked and removed without leaving any excess. Therefore, the tip of the suction thin tube 4 is made of a plastic thin tube so that it does not break even if it contacts the bottom. On the other hand, the outlet side of the suction thin tube 4 is joined outside the reaction cell and connected to an exhaust pump. The means for joining is a suction manifold 6. In addition, the cleaning liquid enters the reaction cell 2 from the cleaning liquid injection thin tube 10, but when a certain amount of cleaning liquid needs to be put into the reaction cell, a leakage thin tube 9 having a lower end at that level is provided. At this time, a cleaning liquid injection manifold 11 is provided for distributing the cleaning liquid to a plurality of cleaning liquid injection capillaries 10, and the pressure is supplied here, and when the reagent is aspirated from the reaction cell, the cleaning liquid is quickly injected into the reaction cell. In the case where it is desired, the structure can be set to a positive pressure.

  In the apparatus shown in this embodiment, the following process for washing the magnetic beads on which the DNA sample is fixed can be performed.

  That is, the magnetic bead sample suspended in the solution 3 is dispensed into all or part of the 96-well plate 61, and the magnet holding plate 15 is overlaid on the plate 61 as shown in FIG. By this operation, the magnetic beads are attracted to the periphery of the magnet 19 on the side surface inside the reaction cell 2 within a few seconds and become a sample bead lump 16. This operation also makes it possible to suck an unnecessary solution without damaging the target DNA. Next, with the magnet holding plate 15 and the 96-hole plate 61 being stacked, the magnet holding plate 15 and the sealing material 8 having the suction thin tubes 4 are stacked.

  Next, in a state where the cleaning liquid is not injected into the cleaning liquid injection manifold 11, the vacuum pump 5 is driven and the suction control valve 20 is opened, so that the suction manifold 6 is set to a negative pressure (pressure below atmospheric pressure). As a result, a pressure difference is generated between the upper end and the lower end of the suction thin tube 4, and thereby the solution 3 is sucked. At this time, some of the reaction cells are empty with no solution dispensed, and there is a variation in the initial suction speed for some reason, and even if an empty reaction cell occurs during the suction process, as described above. In addition, it is possible to prevent the pressure difference between both ends of the suction capillary 4 from being lowered and suck the solution.

  In the configuration of this embodiment, the leaking narrow tube 9 and the cleaning liquid injection thin tube 10 function as the leaking narrow tube 9. In this embodiment, the inside diameter of the leaking narrow tube 9 and the cleaning liquid injection thin tube 10 is about 50 μm, and the lengths thereof are 22 mm and 11 mm, respectively. Therefore, the conductance to the air flow when the two thin tubes are arranged in parallel is sufficient. A decrease in pressure (increase in the pressure of the suction manifold 6) applied to both ends of the suction capillary 4 inserted in the small reaction cell 2 in which the solution remains is suppressed to a minimum. That is, this allows the solution 3 in the reaction cell 2 to be sucked efficiently without changing the suction speed even when the solution is contained in only a part of the 96 reaction cells 2. The sucked solution flows through the bottom surface of the suction manifold 6 and is collected in the solution separation container 7.

  When the apparatus shown in this example is used, if the solution dispensed in the reaction cell 2 is about several tens of μL, all the solution is sucked within 30 seconds. Therefore, after the suction is started, the suction control valve 20 is closed 30 seconds, the cleaning liquid control valve 21 is opened for 5 seconds, and the cleaning liquid is injected from the cleaning liquid tank 22 into the cleaning liquid injection manifold 11 by gravity and pressure difference. At this time, when an excessive cleaning liquid is injected into the cleaning liquid injection manifold 11, the cleaning liquid is recovered in the excess cleaning liquid recovery container 27.

  Next, the pressure control valves 1 (23) and 2 (24) are closed to prevent the positive pressure from escaping, and then the positive pressure control valve 25 is opened to control the compressed air controlled to 2 atm. The compressed air from the cylinder 26 is injected into the cleaning liquid manifold 11. By this operation, 50 μL or more of the cleaning solution can be injected into the reaction cell 2 in about 10 seconds. At this time, the cleaning liquid may remain in the cleaning liquid injection manifold 11, but this is discharged through the reaction cell 2 in the next suction process.

  In order to return to the initial suction process, the positive pressure control valve 25 is closed, the pressure control valves 1 (23) and 2 (24) are opened, the suction control valve 20 is opened, and the suction of the cleaning liquid is executed in the same manner as described above. .

  Highly efficient cleaning can be realized by repeatedly injecting and discharging the cleaning liquid about three times.

Since the cleaning efficiency in the reaction cell 2 varies depending on the degree of adsorption of the chemical substance to be cleaned / removed on the bead surface and the inner wall of the reaction cell 2, it is difficult to obtain general efficiency. Therefore, the cleaning efficiency is evaluated by the solution exchange efficiency. During suction remaining when using the apparatus since was below 0.2 [mu] L, the initial solution volume and 20 [mu] L, three injections 100μL of washing liquid, when the time was aspirated, solution exchange efficiency is 10 ⁹ times or more Is calculated. This indicates that the apparatus shown in this example can potentially achieve a very high cleaning capacity.

  Next, the structure of the cleaning device and apparatus and the specifications of each part will be described. As the plate 61, a resin-made 96-hole plate (9 mm pitch between reaction cells) that is used as a standard product in the market is used, but it is also possible to handle a 384-hole plate or other number of containers.

  A magnet 19 made of neodymium having a diameter of 2 mm and a thickness of 1 mm was attached to the magnet holding plate 15 so as to be in contact with the side surface of each reaction cell 2. The bead washing effect can be enhanced by setting the position where the magnet 19 is attached directly below the tip of the washing liquid injection thin tube 10.

  In addition, since the height at which the magnet 19 is attached also depends on the amount of the solution at the beginning, the magnet holding plate 15 having an optimum height according to the amount of the solution was prepared and used. Further, the number of magnets 19 is not provided one for each reaction cell 2, but one magnet 19 may be used for four reaction cells 2. In this case, since the relative positions of the sample bead mass 16 formed by the magnet 19 in the reaction cell 2 are different from each other, the position of the tip of the cleaning liquid injection capillary 10 is set to the position where the sample bead mass 16 is formed. The effect similar to the above can be obtained by changing every four in total.

  Next, the sealing material 8, the cleaning liquid injection manifold 11, and the suction manifold 6 are formed by cutting the peak material, and as shown in FIG. 6, the external dimensions of these three stacked states are 104 mm long, 140 mm wide, and 76 mm high. . In the example shown in FIG. 6, the sealing material 8 has a single plate-like structure, but any structure may be used as long as there is a structure that covers the upper part of the reaction cell 2. As shown in FIG. The same number of cap-like structures as the number of cells 2 may be used. Further, depending on the shape of the reaction cell, for example, the shape and structure of the sealing material can be changed according to the round reaction cell shown in FIG. 7A or the polygonal reaction cell shown in FIG. Here, the sealing material 8 is fixed to the cleaning liquid injection manifold 11. On the other hand, also in this case, the suction thin tube 4, the cleaning liquid injection thin tube 10, and the leak thin tube 9 penetrate the seal material 8.

  The inner diameter, outer diameter, and length of the suction thin tube 4 were 0.681 mm, 0.91 mm, and 60 mm, respectively. The internal size of the suction manifold 6 was 70 mm long, 110 mm wide, maximum height 38 mm, and minimum 33 mm. The pressure at the upper end of the suction thin tube 4 is large enough not to depend on the location. Further, the vertical and horizontal inside the cleaning liquid injection manifold 11 are the same size as the suction manifold 6 and the height is 13 mm.

  A check valve was provided at the tip of the suction thin tube 4. This is because when the pump is stopped and the suction control valve 20 is opened, the pressure difference applied to both ends of the suction capillary 4 is eliminated or reduced, and the solution remaining in the suction capillary 4 is caused by the difference in gravity and atmospheric pressure. , Provided to avoid backflowing toward reaction cell 2. The check valve used was a 3.5 mm diameter SUS ball, spring and O-ring. Other structures may be used for the check valve structure.

  The sucked solution flows from the solution discharge port 28 toward the bottom surface of the suction manifold 6. The check valve is always installed above the bottom surface of the suction manifold 6, and the solution discharged from the solution discharge port 28 flows into the bottom as water droplets. It has a structure that does not come into contact with the solution staying inside. In addition, the inside of the suction capillary 4 is made water-repellent, and the vicinity of the discharge port 28 and the outside of the suction capillary 4 are hydrophilically processed to reduce the amount of residual solution. Can be reached.

  Further, the bottom surface of the suction manifold 6 is inclined so that it flows toward the suction port 29 by the action of gravity and is discharged into the waste liquid container by the action of the pump.

  A piston-free pump was used as the suction pump (vacuum pump 5), and the maximum exhaust speed was 25L / min and the degree of vacuum reached -46.7kPa. However, as to the type and performance of the pump, any type and performance may be used as long as a sufficient pressure difference (for example, 1 kPa or more) is obtained at both ends of the suction thin tube 4.

  A combination of a compressed air cylinder and a pressure regulator was used as a means for applying a positive pressure inside the cleaning liquid injection manifold 11, but a line gas may be used, and the type of gas is not air, but nitrogen or Carbon dioxide gas may be used.

  A silicon tube with an outer diameter of 3 mm is used between the cleaning device section 12 and the cleaning liquid supply section 13 and between the cleaning device section 12 and the exhaust / waste liquid section 14, and a pinch valve is used as the control valve. Controlled.

  Three narrow tubes are passed through the sealing material 8. A suction capillary 4, a leak capillary 9, and a cleaning liquid injection capillary 10. In order to improve the cleaning efficiency, it is preferable that the suction thin tube 4 has reached the vicinity of the bottom surface of the reaction cell 2 in order to reduce the remaining amount of the solution during suction. The extent to which the suction capillary 4 should be closer to the bottom surface of the reaction cell 2 depends on the water repellency and water immersion of the inner wall of the reaction cell 2, but empirically the bottom surface of the reaction cell 2 and the suction cell The distance between the lower ends of the thin tubes 4 is preferably smaller than the inner radius of the suction thin tube 4.

  In order to discharge the solution without leaving the reaction cell 2, the position of the tip of the suction capillary 4 is important. However, controlling the distance between the lower end of the suction thin tube 4 and the bottom surface of the reaction cell 2 for all 96 is difficult in terms of processing and assembly accuracy. Therefore, as shown in FIG. 8, a pressing spring 32 is provided to press the suction capillary 4 against the bottom of the reaction cell 2, and the lower end of the suction capillary 4 is cut at an angle 33 so that the solution flows. Is effective. This mechanism effectively controls the position of the lower end of the suction thin tube 4.

  The shape of the lower end of the suction thin tube 4 may be a shape in which a portion of the suction thin tube 4 is not in contact with the bottom of the reaction cell 2, and may be an obliquely cut shape or a V-shape. .

  If the outer diameter of the suction capillary 4 is large when the amount of solution is small, the suction capillary 4 may come into contact with the sample bead lump 16 and may be accidentally suctioned. In order to avoid this, as shown in FIG. 9, in order to prevent the beads from being accidentally sucked by thinning the tip of the suction capillary, the taper disposable tip 31 made of resin is attached to the suction capillary 4. It is also possible to deal with it by attaching it to the tip of the. The disposable part may be only the chip, or may be a part or the whole of the cleaning device.

In addition, although a commercially available catalan needle is used as the suction thin tube 4, a SUS thin tube, a glass thin tube, or a resin thin tube such as PEEK or Teflon may be used.
In particular, when a resin thin tube is used, it is often easy to make the inside of the thin tube water-repellent.

  Further, although glass capillaries having an inner diameter of 50 μm and an outer diameter of 350 μm are used for the cleaning liquid injection thin tube 10 and the leak thin tube 9, other metal, glass, or resin thin tubes may be used.

  In this embodiment, it is assumed that the check valve and the suction thin tube 4 are disposable for each sample. Therefore, the suction capillary 4 is sealed so that the solution does not leak with two O-rings, but can be removed.

  In addition, when performing a reaction in which it is desirable to avoid mixing of solutions between samples, the entire cleaning device unit can be made disposable.

  In addition, since it is more efficient to avoid the leaking capillary 9 from coming into contact with the solution, the height in the reaction cell 2 at the lower end of the three capillaries is higher from the higher side. Preferably, the injection capillary 10 and the suction capillary 4 are used. When the cleaning liquid is injected, the pressure difference between the inside of the reaction cell 2 and the cleaning liquid injection manifold is not large, so the maximum height of the liquid level is limited by the height of the lower end of the cleaning liquid injection thin tube.

[Example 2]
In the present embodiment, a configuration example is shown in which the suction capillary 4 is made as short as possible so that the solution does not remain in the suction capillary 4 without a check valve and the solution can be efficiently suctioned.

FIG. 10 shows a configuration diagram of the cleaning apparatus and the cleaning device.
In this embodiment, as shown in FIG. 10, the suction manifold 6 is disposed between the cleaning liquid injection manifold 11 and the sealing material 8 in order to shorten the length of the suction thin tube 4. As a result, the length of the suction thin tube 4 can be reduced to 12 mm and the inner diameter can be reduced to 0.4 mm. As a result, the internal volume of the suction capillary 4 can be reduced to 1.5 μl, and the amount of residual solution at the end of the suction process can be reduced. At the same time, the probability that no residual solution remains at the end of the suction process can be improved. In addition, the suction tube 4 is made of resin, and ultraviolet ozone treatment is applied so that the inner wall of the capillary tube is a water-repellent surface and the outer wall is a hydrophilic surface. It will be sucked up quickly. As a result, when reaching the upper end of the suction thin tube 4, the solution is quickly conveyed to the bottom of the suction manifold 6 along the hydrophilic outer wall. For this reason, in this example, it was found that the average solution remaining amount could be reduced to substantially the same as in Example 1 without a check valve, and the bead sample could be washed with high efficiency.

  In this embodiment, the washing liquid injection thin tube 10 and the leak thin tube 9 were capillaries having an inner diameter of 75 μm and an outer diameter of 350 μm, and the lengths thereof were 50 mm and 65 mm, respectively. In this case as well, even if there are many empty reaction cells 2, the pressure at the upper end of the suction capillary 4 does not increase (the pressure applied to both ends of the suction capillary 4 with respect to the reaction cell 2 where the solution remains) does not occur. It is possible to aspirate the solution at a sufficient rate.

  Further, as shown in FIG. 10, the leaking narrow tube 9 may be 40 mm long so as not to reach the cleaning liquid injection manifold 11 and to prevent the suction solution from flowing backward. At this time, a pressure difference is generated at both ends of the cleaning liquid introducing capillary 10 due to the air flow during solution suction, and the leaking capillary 9 functions to relieve the pressure in the reaction cell when introducing the cleaning liquid.

[Example 3]
In the present embodiment, a case in which a reaction liquid is directly injected into the reaction cell without using the cleaning liquid injection manifold 11 is shown.

  FIG. 11 shows the apparatus configuration. A distributor 40 is disposed in the cleaning liquid supply unit 13 instead of the cleaning liquid injection manifold 11 of the cleaning device unit 12. When the cleaning liquid is injected into the reaction cell 2, the cleaning liquid stored in the cleaning liquid tank 22 opens the cleaning liquid injection control valve 1 (35), closes the cleaning liquid injection control valve 2 (36), pulls the syringe 37, and supplies the cleaning liquid. After the suction, the opening and closing of the control valve is reversed, and the syringe 37 is pushed to supply the cleaning liquid through the distributor 40 and the cleaning liquid injection thin tube 10. In this embodiment, air is not contained in the thin tube into which the cleaning liquid is injected. Therefore, when the solution is sucked from the reaction cell 2, the pressure in the reaction cell 2 is adjusted using only the leak thin tube 9. Also in this example, a capillary having an inner diameter of 75 μm and an outer diameter of 350 μm was used as the leaking thin tube 9. Other configurations are the same as those of the second embodiment.

[Example 4]
In the present embodiment, the apparatus configuration in the case where the suction of the solution and the injection of the cleaning liquid are performed by different apparatuses is shown. FIG. 12 shows an apparatus configuration diagram when performing only solution suction independently, and FIG. 13 shows an apparatus configuration diagram when only cleaning liquid injection is performed independently.

  As a method of use, a sample solution containing magnetic beads is dispensed on a 96-well plate 61, and a solution other than the magnetic beads is sucked with the apparatus shown in FIG. Thereafter, the 96-hole plate 61 is removed, the 96-hole plate 61 is set in the cleaning liquid injection apparatus shown in FIG. 13, and after the dispensing of the cleaning liquid, the cleaning liquid is sucked again using the apparatus of FIG. repeat. The detailed structure of the device is the same as that described in Examples 1-3.

  The present invention provides a device for aspirating fluid in a plurality of reaction cells of a reaction plate. This suction device can efficiently and evenly suck fluid in a plurality of reaction cells on the reaction plate. Moreover, this device can also be used for the washing | cleaning in a reaction cell as a washing | cleaning device and washing | cleaning apparatus of a reaction plate. Therefore, this invention is useful in the field | area which reacts in the several cell on a reaction plate, for example, fields, such as a chemistry, biochemistry, biotechnology, a pharmaceutical.

1 plate
2 reaction cells
3 Solution in reaction cell
4 Suction tube
5 Vacuum pump
6 Manifold for suction
7 Solution separation container
8 Sealing material
9 Leak capillary
10 Washing liquid injection tube
11 Manifold for injecting cleaning solution
12 Cleaning device section
13 Cleaning liquid supply unit
14 Exhaust and waste liquid section
15 Magnet holding plate
16 Sample bead mass
17 Sealing material protrusion
18 O-ring
19 Magnet
20 Suction control valve
21 Cleaning fluid control valve
22 Cleaning liquid tank
23 Pressure control valve 1
24 Pressure control valve 2
25 Positive pressure control valve
26 Compressed air cylinder
27 Surplus cleaning liquid collection container
28 Solution outlet
29 Suction port
31 Tapered disposable tip made of resin
32 Pressing spring
33 Tip of suction tube cut diagonally
35 Cleaning fluid injection control valve 1
36 Cleaning fluid injection control valve 2
37 syringe
61 96-well plate

Claims (10)

A device for aspirating fluid in a plurality of reaction cells of a reaction plate,
A sealing material for making the pressure inside the plurality of reaction cells of the reaction plate independent,
At least one suction tubule and at least one leakage tubule penetrating the sealant corresponding to each reaction cell;
Means for joining the suction capillary tube outside the reaction cell, and the leak capillary tube applies a pressure difference across the suction capillary tube when the fluid in the reaction cell is suctioned from the suction capillary tube der is,
When the conductance for the air flowing through the two capillaries is compared by applying a pressure difference between both ends of the suction capillaries and the leak capillaries, the conductance of the leak capillaries is smaller than the conductance of the suction capillaries. A suction device characterized by that. The suction device according to claim 1, wherein a ratio of an inner diameter of the suction capillary and the leaking capillary is 5: 1 to 2: 1. The suction device according to claim 1 or 2 , wherein the distance between the lower end of the suction capillary and the bottom of the reaction cell is smaller than the inner radius of the suction capillary. The suction device according to any one of claims 1 to 3 , wherein the suction thin tube includes a check valve for preventing a backflow of the sucked fluid into the reaction cell. The suction device according to any one of claims 1 to 4 , further comprising means for sucking air inside the means for joining the suction thin tubes outside the reaction cell. A device for cleaning a plurality of reaction cells of a reaction plate,
The suction device according to any one of claims 1 to 5 ,
A cleaning device, comprising: a cleaning liquid injecting thin tube penetrating the sealing material corresponding to each reaction cell. The cleaning device according to claim 6 , further comprising means for joining the cleaning liquid injection thin tubes. The cleaning device according to claim 6 or 7 , wherein the distance between the cleaning liquid injection capillary and the inner wall of the reaction cell is smaller than the distance between the suction capillary and the cleaning liquid injection capillary. Wherein the portion of the inner wall of the reaction cell further comprising a magnet for collecting the magnetic beads, the cleaning liquid injection capillary is disposed so as to correspond to the position of the magnet, any one of claims 6-8 2. A cleaning device according to item 1. An apparatus for washing a plurality of reaction cells of a reaction plate,
The cleaning device according to any one of claims 6 to 9 ,
A cleaning liquid supply unit;
A cleaning device comprising an exhaust and a waste liquid section.

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