JP4111505B2 - Biochemical treatment apparatus and biochemical treatment method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique for analyzing a cell, a microorganism, a chromosome, a nucleic acid, or the like in a specimen using a biochemical reaction such as an antigen-antibody reaction or a nucleic acid hybridization reaction.
[0002]
[Prior art]
Many analyzers for specimens such as blood use an immunological method utilizing an antigen-antibody reaction or a method utilizing nucleic acid hybridization. Examples of such analysis methods include antibodies or antigens that specifically bind to a substance to be detected, or single-stranded nucleic acids as probes that are immobilized on a solid phase surface such as fine particles, beads, or glass plates to be detected. The substance is allowed to undergo antigen-antibody reaction or nucleic acid hybridization. Using a labeled substance having a specific interaction carrying a labeling substance having high detection sensitivity such as an enzyme, a fluorescent substance, or a luminescent substance, such as a labeled antibody, a labeled antigen, or a labeled nucleic acid, The antibody compound or double-stranded nucleic acid is detected to detect the presence or absence of the test substance or to quantify the test substance.
[0003]
As a development of these technologies, US Pat. No. 5,445,934 discloses a so-called DNA array in which a large number of DNA (deoxyribonucleic acid) probes having different base sequences are arranged in an array on a substrate. is there. Anal. Biochem., 270 (1), 103-111, 1999 discloses a method for preparing a protein array having a structure such as a DNA array by arranging many types of proteins on a membrane filter. With the use of DNA arrays, protein arrays, etc., it has become possible to inspect a very large number of items at once.
[0004]
We also propose disposable biochemical reaction cartridges that perform necessary reactions internally for the purpose of reducing sample contamination, improving reaction efficiency, miniaturizing equipment, and simplifying work in various sample analysis methods. In JP-T-11-509094, there are a plurality of chambers in a biochemical reaction cartridge including a DNA array, the solution is moved by differential pressure, and DNA is extracted or amplified from the specimen inside, or A biochemical reaction cartridge that enables reactions such as hybridization is disclosed.
[0005]
And as a method of flowing the solution from the outside into the biochemical reaction cartridge as described above, there is a method using an external syringe pump or vacuum pump. Also, as a method of moving the solution inside the biochemical reaction cartridge, Patent No. 2832117 can be used as a micropump that utilizes gravity, capillary action, electrophoresis, or a micro pump that can be disposed inside the biochemical reaction cartridge because of its small size. Japanese Patent No. 2000-274375 discloses a diaphragm using a heat generating element, Japanese Patent Application Laid-Open No. 11-509094 discloses a diaphragm pump.
[0006]
[Patent Document 1]
US Patent No. 5,445,934 [Patent Document 2]
Special table hei 11-509094 [patent document 3]
Patent No. 2832117 [Patent Document 4]
JP 2000-274375 [Non-Patent Document 1]
Anal. Biochem., 270 (1), 103-111, 1999.
[0007]
[Problems to be solved by the invention]
However, in order to prevent secondary infection and contamination (contamination), it is preferable to use a disposable cartridge containing a necessary solution from the viewpoint of ease of use. However, a cartridge containing a pump is expensive.
[0008]
The present invention has been made in view of the above-described problems. When a biochemical reaction is performed using a disposable cartridge, the user automatically injects a sample after injecting a specimen while using an external pump. An object of the present invention is to provide an apparatus and method for advancing biochemical reactions.
[0009]
[Means for Solving the Problems]
In order to solve the above-described problems and achieve the object, each aspect of the present invention is listed below.
[0010]
[Aspect 1]
A cartridge having a plurality of chambers for holding a solution for biochemical treatment of the specimen, a nozzle portion connected to each of the communication passages communicating with each chamber of the cartridge, and a flow communicating with the plurality of chambers And a controller that controls the air pressure in the cartridge via the nozzle , and the controller moves the solution in the cartridge back and forth in the flow path or the chamber of the cartridge. The air pressure is controlled as follows.
[0011]
[Aspect 2]
In the first aspect, the control unit controls air pressure for the plurality of cartridges simultaneously.
[0012]
[Aspect 3]
In the first aspect, a unit having a plurality of nozzle portions moves to the cartridge side, and each of the nozzle portions is simultaneously connected to all the communication paths.
[0013]
[Aspect 4]
In the first aspect, the control unit controls changes with time in pressurization and decompression.
[0014]
[Aspect 5]
In the above aspect 4, the temporal change is controlled by shifting the pressurization and depressurization timings.
[0015]
[Aspect 6]
In the said aspect 4 or 5, the said control part controls the said time-dependent change using a syringe pump.
[0016]
[Aspect 7]
In the above aspect 1, the nozzle portion is disposed on two corresponding side surfaces of the cartridge.
[Aspect 8 ]
A cartridge having a plurality of chambers for holding a solution for biochemical treatment of a specimen is set, and a nozzle portion is connected to each of the communication passages that communicate with the chambers of the cartridge, and communicates with the plurality of chambers. The solution is moved to the flow path, and a biochemical reaction is generated by controlling the air pressure in the cartridge so that the solution in the cartridge moves back and forth in the flow path or the chamber of the cartridge.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
[0018]
FIG. 1 shows an external view of a biochemical reaction cartridge according to this embodiment. In the biochemical reaction cartridge of this embodiment, when a liquid sample such as blood is injected and set in a processing apparatus to be described later, DNA is extracted and amplified inside the biochemical reaction cartridge, and the amplified sample DNA and the living sample are amplified. Steps such as hybridization with a DNA probe on a DNA microarray inside the chemical reaction cartridge, and washing of the fluorescently labeled sample DNA and fluorescent label that have not been hybridized are performed.
[0019]
The main body of the biochemical reaction cartridge 1 is made of a transparent or translucent plastic such as polymethyl methacrylate (PMMA), acrylonitrile-butadiene-styrene (ABS), polystyrene, polycarbonate, polyester, polyvinyl chloride or the like. If optical measurement of the reactant in the biochemical reaction cartridge 1 is not required, the material may not be a transparent material. On the upper part of the biochemical reaction cartridge 1, a rubber cap is fixed to a portion of the sample inlet 2 when a sample such as blood is injected using a syringe. In addition, a plurality of nozzle inlets 3 are formed on the side surface to insert and pressurize or depressurize the nozzle in order to move the solution in the biochemical reaction cartridge 1, and a rubber cap is fixed to the opposite side surface. Has the same structure.
[0020]
FIG. 2 is a plan sectional view of the biochemical reaction cartridge of FIG. There are ten nozzle inlets 3a to 3j on one side, and ten nozzle inlets 3k to 3t on the opposite side. Each nozzle inlet is connected to a chamber 5 which is a place where a solution is stored or where a reaction occurs, via an air flow path 4 through which air flows. However, since the nozzle inlets 3n, 3p, 3q, and 3s are not used in the process of this embodiment, they are not connected to the chamber and are reserved. Eventually, the nozzle inlets 3a to 3j are connected to the chambers 5a to 5j via the flow paths 4a to 4j, and the nozzle inlets 3k, 3l, 3m, 3o, 3r, and 3t on the opposite side are respectively connected to the flow paths 4k, 4l, 4m, It is connected to chambers 5k, 5l, 5m, 5o, 5r, and 5t via 4o, 4r, and 4t.
[0021]
The chambers 5a, 5b, 5c and 5k are connected to the chamber 7, the chambers 5g and 5o are connected to the chamber 8, and the chambers 5h, 5i, 5j, 5r and 5t are connected to the chamber 9. Further, the chamber 7 is connected to the chamber 8 via the flow path 10, and the chamber 8 is connected to the chamber 9 via the flow path 11. Chambers 5d, 5e, 5f, 5l, and 5m are connected to the flow path 10 through flow paths 6d, 6e, 6f, 6l, and 6m, respectively. In addition, a square hole is formed in the bottom surface of the chamber 9, and a DNA microarray 12 made of a glass substrate is attached to the bottom surface of the chamber 9 so that the probe surface is on the bottom, thereby forming the chamber 9.
[0022]
The DNA microarray 12 is formed by arranging tens to hundreds of thousands of different types of DNA probes on a solid surface such as a glass plate of about 1 inch square. By performing a hybridization reaction with the sample DNA using this DNA microarray 12, it is possible to test many genes at once. In addition, these DNA probes are regularly arranged in a matrix, and the address of each DNA probe (for example, the number of rows and columns) can be easily taken out as information. Examples of genes to be examined include infectious disease viruses / bacteria, disease-related genes, and individual polymorphisms.
[0023]
In the chambers 5a and 5b, a first hemolytic agent containing EDTA and a second hemolytic agent containing a protein denaturant such as a surfactant are accumulated. Silica-coated magnetic particles that adsorb DNA are accumulated in the chamber 5c, and the first and second extraction detergents used for purifying the DNA at the time of DNA extraction in the chambers 5l and 5m. Is accumulated. The chamber 5d contains an eluate composed of a low-concentration salt buffer that elutes DNA from magnetic particles, and the chamber 5g contains primers necessary for PCR, polymerase, dNTP solution, buffer, Cy-3 dUTP containing a fluorescent agent, and the like. The mixture is filled. In the chambers 5h and 5j, a cleaning agent containing a detergent for washing the fluorescently labeled sample DNA and the fluorescent label, and in the chamber 5i, the chamber 9 including the DNA microarray 12 is dried. Of alcohol has accumulated.
[0024]
The chamber 5e is a chamber for collecting dust other than blood DNA, the chamber 5f is a chamber for collecting waste liquids of the first and second extraction detergents in the chambers 5l and 5m, and the chamber 5r is for the first and second detergents. Chambers 5k, 5o, and 5t are blank chambers provided to prevent the solution from flowing into the nozzle inlet.
[0025]
FIG. 3 shows a processing apparatus for controlling the movement of the solution and various reactions in the biochemical reaction cartridge 1. The table 13 is a place where the biochemical reaction cartridge 1 is set. On the table 13, an electromagnet 14 that is activated when DNA or the like from the specimen is extracted in the biochemical reaction cartridge 1, and DNA (DNA) from the specimen is PCR (Polymerase). Peltier element 15 for temperature control when amplifying by a method such as Chain Reaction, hybridization of amplified sample DNA and DNA probe on DNA microarray in biochemical reaction cartridge, sample DNA not hybridized A Peltier element 16 for controlling the temperature at the time of cleaning is disposed, and these are connected to a control unit 17 for controlling the entire processing apparatus. Pumps having electric syringe pumps 18 and 19 on both sides (upper and lower sides of FIG. 3) and a plurality of pump nozzles 20 and 21 each having 10 outlets and outlets for discharging or sucking air by these pumps on both sides of the table 13 Blocks 22 and 23 are arranged. A plurality of well-known electric switching valves (not shown) are arranged between the electric syringe pumps 18 and 19 and the pump nozzles 20 and 21, and are connected to the control unit 17 together with the electric syringe pumps 18 and 19. The control unit 17 can perform control such that one of the ten pump nozzles on one side is selectively opened with respect to the electric syringe pumps 18 and 19 or all the pump nozzles are closed. Moreover, the control part 17 is connected to the input part 24 which an inspector inputs.
[0026]
In this embodiment, blood is used as a sample, and when the examiner injects blood through the rubber cap of the sample inlet 2 using a syringe, the blood flows into the chamber 7. Thereafter, when the examiner places the biochemical reaction cartridge 1 on the table 13 and operates a lever (not shown), the pump blocks 22 and 23 are moved in the direction of the arrow in FIG. The rubber cap is inserted through the nozzle inlet 3.
[0027]
As described with reference to FIG. 2, since the nozzle inlets are concentrated in two areas of the biochemical reaction cartridge, the shape and arrangement of the electric syringe pump, the electric switching valve, and the pump blocks 22 and 23 incorporating the pump nozzle are simple. become. Furthermore, since a plurality of nozzle inlets are provided concentrated on the side surfaces on both sides, the biochemical reaction cartridge is sandwiched simultaneously by the pump blocks 22 and 23 while securing the necessary chambers and flow paths. The pump nozzles 20 and 21 can be inserted, and the configuration of the pump blocks 22 and 23 is simplified. If the nozzle inlets are all arranged at the same height, that is, linearly, the heights of the flow paths connected to the nozzle inlets are all the same, and the flow paths can be easily manufactured.
[0028]
Further, in the processing apparatus of FIG. 3, if the pump blocks 22 and 23 are made n times longer for a plurality (n) of biochemical reaction cartridges, n biochemical reaction cartridges are arranged in series and necessary. This process can be performed simultaneously on n biochemical reaction cartridges, and biochemical reactions can be performed on a large number of biochemical reaction cartridges with a very simple configuration.
[0029]
When the inspector inputs a process start command at the input unit 24, the process starts. FIG. 4 is a flowchart for explaining a processing procedure in the processing apparatus of this embodiment. First, in step S1, the control unit 17 opens only the nozzle inlets 3a and 3k, discharges air from the electric syringe pump 18, sucks air from the electric syringe pump 19, and removes the first hemolytic agent in the chamber 5a from the blood. Pour into the chamber 7 inside. At this time, depending on the viscosity of the hemolytic agent and the resistance of the flow path, if the suction of the air from the electric syringe pump 19 is controlled to start 10 to 200 mS after the discharge of the air from the electric syringe pump 18 starts, the flowing solution The solution flows smoothly without jumping out at the top. In this way, the control of air supply is shifted to control the pressurization and depressurization so that the solution flows smoothly, and the suction of air from the electric syringe pump 19 By performing fine control, such as controlling to increase linearly from the start of discharge, it is possible to flow more smoothly. The same applies to the movement of the following solutions. The control of the air supply can be easily realized by using a syringe pump. Then, only the nozzle inlets 3a and 3o are opened, and the electric syringe pumps 18 and 19 alternately repeat the discharge and suction of air, and the operation of flowing the solution in the chamber 7 through the flow path 10 and returning it thereafter is repeated. Alternatively, air may be continuously discharged from the electric syringe pump 19 to generate bubbles and stir.
[0030]
FIG. 5 is a cross-sectional view through the chamber 5a, the chamber 7 and the chamber 5k of FIG. 2. The pump nozzle 20 is inserted into the nozzle inlet 3a and pressurized, and the pump nozzle 21 is inserted into the nozzle inlet 3k and depressurized. , The first hemolytic agent in the chamber 5a flows into the chamber 7 containing blood.
[0031]
Returning to FIG. 4, next, in step S2, only the nozzle inlets 3b and 3k are opened, and the second hemolytic agent in the chamber 5b is poured into the chamber 7 in the same manner. In step S3, only the nozzle inlets 3c and 3k are opened. Similarly, the magnetic particles in the chamber 5 c are poured into the chamber 7. Steps S2 and S3 are stirred in the same manner as S1. In step S3, the DNA that has come out of the cells dissolved in steps S1 and S2 adheres to the magnetic particles.
[0032]
In step S4, the electromagnet 14 is turned on, only the nozzle inlets 3e and 3k are opened, the air is discharged from the electric syringe pump 20, the air is sucked from the electric syringe pump 19, and the solution in the chamber 7 is moved to the chamber 5e. To do. During this movement, magnetic particles and DNA are captured on the electromagnet 14 of the flow path 10. The suction and discharge of the electric syringe pumps 18 and 19 are alternately repeated to reciprocate the solution twice between the chambers 7 and 5e, thereby increasing the DNA capture efficiency. Increasing the number of times can further increase the capture efficiency, but it takes additional processing time.
[0033]
As described above, DNA is captured in a flowing state on a small channel having a width of about 1 to 2 mm and a height of about 0.2 to 1 mm by using magnetic particles, so that it can be captured extremely efficiently. . The same applies when the capture target substance is RNA or protein.
[0034]
Next, in step S5, the electromagnet 14 is turned off, only the nozzle inlets 3f and 3l are opened, air is discharged from the electric syringe pump 19, and air is sucked from the electric syringe pump 17 to extract the first extraction cleaning liquid in the chamber 5l. Is moved to the chamber 5f. At this time, the magnetic particles and DNA captured in step S4 move together with the extraction cleaning liquid, and cleaning is performed. After reciprocating twice in the same manner as in step S4, the electromagnet 14 is turned on and reciprocating twice in the same manner to collect the magnetic particles and DNA on the electromagnet 14 in the flow path 10 and return the solution to the chamber 5l. Keep it.
[0035]
In step S6, the second extraction washing liquid in the chamber 5m is further washed by performing the same process as in step S5 using the nozzle inlets 3e and 3m. In step S7, with the electromagnet 14 turned on, only the nozzle inlets 3f and 3l are opened, the air is discharged from the electric syringe pump 18, the air is sucked from the electric syringe pump 19, and the eluate from the chamber 5d is put into the chamber 8. Moving. At this time, the magnetic particles and DNA are separated by the action of the eluate, and only the DNA moves to the chamber 8 together with the eluate, and the magnetic particles remain in the flow path 10. DNA extraction and purification are performed as described above. Since a chamber containing the extraction washing liquid and a chamber containing the washing waste liquid were prepared, it became possible to extract and purify DNA in the biochemical reaction cartridge 1.
[0036]
Next, in step S8, only the nozzle inlets 3g and 3o are opened, air is discharged from the electric syringe pump 17, the air is sucked from the electric syringe pump 19, and the PCR drug in the chamber 5i is poured into the chamber 8. Further, only the nozzle inlets 3g and 3t are opened, and the electric syringe pumps 18 and 19 alternately repeat the discharge and suction of air, and the operation of flowing the solution in the chamber 7 into the flow path 11 and returning it thereafter is repeated. After controlling the Peltier element 15 and keeping the solution in the chamber 8 at a temperature of 96 ° C. for 10 minutes, the process of 96 ° C. for 10 seconds, 55 ° C. for 10 seconds, and 72 ° C. for 1 minute is repeated 30 times to be eluted. The obtained DNA is amplified by PCR.
[0037]
In step S9, only the nozzle inlets 3g and 3t are opened, air is discharged from the electric syringe pump 18, the air is sucked from the electric syringe pump 19, and the solution in the chamber 8 is moved to the chamber 9. Further, the Peltier element 16 is controlled to perform hybridization by keeping the solution in the chamber 9 at 45 ° C. for 2 hours. At this time, the electric syringe pumps 18 and 19 alternately discharge and suck air, move the solution in the chamber 9 to the flow path 6t, and then repeat the returning operation to proceed with hybridization while stirring.
[0038]
Next, in step S10, while maintaining the same 45 ° C., this time, only the nozzle inlets 3h and 3r are opened, air is discharged from the electric syringe pump 18, air is sucked from the electric syringe pump 19, and the inside of the chamber 9 is discharged. The first cleaning solution in the chamber 5h is poured into the chamber 5r through the chamber 9 while moving the solution in the chamber 5r. The suction and discharge of the electric syringe pumps 18 and 19 are alternately repeated to reciprocate the solution twice between the chambers 5h, 9, and 5r, and finally return to the chamber 5h. In this way, the fluorescently labeled sample DNA and the fluorescent label that have not been hybridized are washed.
[0039]
FIG. 6 is a cross-sectional view passing through the chamber 5h, chamber 9, and chamber 5r of FIG. 2. The pump nozzle 20 is inserted into the nozzle inlet 3h and pressurized, and the pump nozzle 21 is inserted into the nozzle inlet 3r and depressurized. , A state in which the first cleaning liquid in the chamber 5h flows into the chamber 5r through the chamber 9 is shown.
[0040]
Returning to FIG. 4, in step S11, the second cleaning liquid in the chamber 5j is further cleaned by using the nozzle inlets 3j and 3r while maintaining the same 45 ° C., and further cleaned by performing the same process as in step S10. Return to chamber 5j. Thus, since the chambers 5h and 5j containing the cleaning liquid and the chamber 5r containing the waste liquid after the cleaning are prepared, the DNA microarray 12 can be cleaned in the biochemical reaction cartridge 1.
[0041]
In step S12, only the nozzle inlets 3i and 3r are opened, air is discharged from the electric syringe pump 18, the air is sucked from the electric syringe pump 19, and the alcohol in the chamber 5i is moved to the chamber 5r through the chamber 9. Thereafter, in step S13, only the nozzle inlets 3i and 3t are opened, air is discharged from the electric syringe pump 18, and air is sucked from the electric syringe pump 19 to dry the inside of the chamber 9.
[0042]
Thereafter, when the inspector operates a lever (not shown), the pump blocks 22 and 23 move away from the biochemical reaction cartridge 1 by a mechanism (not shown), and the pump nozzles 20 and 21 enter the nozzle inlet of the biochemical reaction cartridge 1. Deviate from 3. Then, the examiner inserts this biochemical reaction cartridge into a well-known scanner (not shown) such as a scanner for DNA microarray to perform measurement and analysis.
【The invention's effect】
As described above, according to the present invention, the air pressure in the cartridge is controlled by the pump on the apparatus side, and the solution in the cartridge is moved only in the cartridge to cause a necessary biochemical reaction. A disposable cartridge that does not discharge the solution to the outside is possible, and there is no risk of secondary infection or contamination. Further, since the cartridge contains a necessary solution, it is not necessary to prepare a reagent / cleaning solution on the apparatus side, and there is an effect that labor and labor of the reagent are eliminated.
[Brief description of the drawings]
FIG. 1 is an external view of a biochemical reaction cartridge according to an embodiment of the present invention.
2 is a plan sectional view of the biochemical reaction cartridge of FIG. 1. FIG.
FIG. 3 is a processing apparatus for controlling the movement of a solution and various reactions in the biochemical reaction cartridge of the present embodiment.
FIG. 4 is a flowchart illustrating a processing procedure in the processing apparatus according to the embodiment.
5 is a sectional view through the chamber 5a, the chamber 7, and the chamber 5k of FIG.
6 is a cross-sectional view through the chamber 5h, the chamber 9, and the chamber 5r of FIG.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Biochemical reaction cartridge 2 Specimen inlet 3 Nozzle inlet 4 Air flow path 5, 7-9 Chamber 6, 10, 11 Flow path 12 DNA microarray 13 Table 17 Control part 18, 19 Electric syringe pump 20, 21 Pump nozzle 22, 23 Pump block

Claims (8)

A cartridge having a plurality of chambers for holding a solution for biochemical treatment of the specimen;
A nozzle portion connected to each of the communication passages communicating with the chambers of the cartridge;
A flow path communicating with the plurality of chambers;
A control unit for controlling the air pressure in the cartridge through the nozzle unit,
The biochemical treatment apparatus, wherein the control unit controls the air pressure so that the solution in the cartridge moves back and forth through the flow path or the chamber of the cartridge.   The biochemical processing apparatus according to claim 1, wherein the control unit controls air pressure simultaneously with respect to the plurality of cartridges.   The biochemical processing apparatus according to claim 1, wherein a unit having a plurality of nozzle portions moves toward the cartridge, and each of the nozzle portions is connected to all the communication paths at the same time.   The biochemical treatment apparatus according to claim 1, wherein the control unit controls changes with time in pressurization and decompression.   The biochemical treatment apparatus according to claim 4, wherein the time-dependent change is controlled by shifting the pressurization and depressurization timings.   The biochemical treatment apparatus according to claim 4 or 5, wherein the control unit controls the temporal change using a syringe pump. The biochemical treatment apparatus according to claim 1, wherein the nozzle portion is disposed on two corresponding side surfaces of the cartridge. Set a cartridge having a plurality of chambers for holding a solution for biochemical processing of the specimen;
A nozzle portion is connected to each of the communication paths leading to the chambers of the cartridge;
Moving the solution to a flow path communicating with the plurality of chambers;
A biochemical treatment method, wherein a biochemical reaction is generated by controlling air pressure in the cartridge so that the solution in the cartridge moves back and forth through the flow path or the chamber of the cartridge.

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