JP2008241250A - Stirring rod using specifically binding reaction, and analyzing method and device using stirring rod thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analyzing method to collect polarity-labeled molecules without using a container wall, dispense with sucking and cleaning operations of solution and improve measurement accuracy by improving the diffuser efficiency of target molecules. <P>SOLUTION: A stirring rod is provided which is used for the analyzing method by specifically binding reaction of a biomolecule and with which a magnetic force is given arbitrarily. The stirring rod is composed of a magnet rod made of an electromagnet rod or a magnet rod made of a permanent magnet, and a nonmagnetic sheath into and from which the magnet rod can be inserted and drawn arbitrarily. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an analysis method based on a specific binding reaction of biomolecules.

  In the measurement of biomolecules using specific binding reactions, a method of selectively separating reactants using magnetic force by labeling the reaction molecules with a magnetic substance has been performed. In general, a specimen containing a target molecule is first injected into a container and reacted with a reactive molecule labeled with a magnetic substance. As a result, the target molecule is magnetically labeled via the reactive molecule. Next, a magnet is pressed against the outer wall of the container, the magnetically labeled target molecule is immobilized on the wall surface of the container, and the solution in the container is aspirated in this state. Next, a washing liquid is poured, and the reaction product is washed by stirring with the magnet removed. Next, the reaction product is again fixed to the wall surface of the container with a magnet, the cleaning liquid is sucked, and then a reaction solution for a desired test reaction is injected.

  Patent Document 1 discloses B / F separation cleaning in which a liquid is sucked by a nozzle in a state where magnetically labeled molecules are attracted to a container wall by a magnetic force. In Patent Document 1, after the second reagent is injected, the particles are redispersed by stirring with a stick.

  In Patent Document 2, after immobilizing magnetically labeled molecules on the container wall, the mass of the magnetically labeled molecules on the wall is moved by further moving the magnet to promote cleaning inside the particle mass and redispersion No need to spread with a stick for

However, in the method as described above, since all the reactions are performed in the same container, there is a problem that when the container itself is contaminated, the influence remains until the final process. In addition, since all reactions are performed in the same container, there is a problem that it takes time to inject and suck each solution. Furthermore, equipment such as a pipetter and a probe is required for injecting and aspirating the solution, and equipment such as a vibration device is required for stirring the molecules in the solution. Furthermore, since the magnetically labeled molecules are collected at one point on the wall surface, there is a problem that the efficiency of stirring and washing the molecules is poor.
Japanese Patent Laid-Open No. 9-257796 JP 2001-91521 A

  In view of the above problems, the present invention can collect magnetically labeled molecules without using a container wall, eliminates the need for solution aspiration and washing operations, and improves target molecule diffusion efficiency. Thus, an object of the present invention is to provide an analysis method with improved measurement accuracy.

  According to the present invention, there is provided a stirrer bar that can be arbitrarily supplied with magnetic force, which is used in an analysis method based on a specific binding reaction of biomolecules. The stirring bar is preferably a magnet bar made of an electromagnet. Or it is preferable that this stirring rod is comprised from the magnet rod which consists of a permanent magnet, and the nonmagnetic sheath which can insert and extract this magnet rod arbitrarily.

  The stirring bar may have a shape having a protrusion. Or you may manufacture from a porous body. Alternatively, it may have a twisted shape. Alternatively, it may have a shape having wings for promoting stirring of the solution. Since the wing portion has a high resistance to flow from the solution during stirring, the non-magnetic material is used except for the wing portion so that the reagent substance magnetically labeled is preferentially adsorbed to the wing portion. It is preferable. By doing so, the reagent substance once adsorbed can be easily diffused into the solution in a state where the magnetic force is released.

  From another aspect of the present invention, the above stirring rod is used, and (a) a reaction solution containing a magnetically labeled reagent substance and a target substance to be detected is added to the stirring bar in a state where no magnetic force is supplied. (B) supplying a magnetic force to the stirring bar to adsorb the magnetically labeled reagent substance and the target substance, and stirring the reaction bar. And (c) immersing the stirring rod in the cleaning solution, stopping the supply of magnetic force to the stirring rod, and diffusing the adsorbed reactant, d) stirring the washing solution with the stirring rod in a state where no magnetic force is supplied, and washing the reactant; and (e) supplying magnetic force to the stirring rod to adsorb the washed reactant, Withdrawing the rod from the washing solution; and (f) immersing the stirring rod in the detection solution. And stopping the supply of magnetic force to the stirring rod to diffuse the adsorbed reactant, and (g) detecting the reactant in the solution by withdrawing the stirring rod from the detection solution. And a biomolecule analysis method comprising: Here, the reaction molecules may be washed by repeating the steps (c) to (e).

  According to another aspect of the present invention, there is provided an analyzer comprising the stirring bar, a moving unit for moving the stirring bar, and a driving unit for rotating, vibrating, or ultrasonically oscillating the stirring bar. Is done. The analyzer preferably includes a plurality of stirring bars.

  According to the present invention, magnetically labeled molecules can be collected without using a container wall, solution aspiration and washing operations are unnecessary, and further, by improving the diffusion efficiency of target molecules, An analysis method with improved measurement accuracy can be provided.

  The present invention provides a stirring bar used in a method for analyzing a target substance utilizing a specific binding reaction of biomolecules. The analysis method using a specific binding reaction of a biomolecule is an analysis method using, for example, an antigen-antibody reaction, and includes a nucleic acid chain binding reaction, for example, an analysis method by hybridization. Here, the biomolecule refers to a molecule such as a nucleic acid, an antigen, or an antibody, and further includes a molecule similar to these artificially synthesized molecules.

  The stirring rod of the present invention has a configuration that can arbitrarily supply magnetic force. Such a configuration can be achieved, for example, by using a magnet bar made of an electromagnet. Or it can achieve by comprising the magnet bar which consists of a permanent magnet, and the nonmagnetic sheath which can insert and extract this magnet bar arbitrarily.

  The stirring rod of the present invention is immersed in a solution and agitates the solution by rotating, vibrating, or ultrasonically oscillating to promote biomolecule reaction and washing. In addition, by supplying a magnetic force, molecules labeled with magnetic particles present in the solution are adsorbed. Here, supplying magnetic force to the stirring bar means that, in the case of a magnet bar made of an electromagnet, current or voltage is applied to generate magnetic force. In the case of a stirring bar composed of a permanent magnet magnet bar and a non-magnetic sheath, magnetic force is generated by inserting the magnet rod into the sheath, and conversely, the magnet rod is withdrawn from the sheath. To stop the supply of magnetic force.

  The stirring bar of the present invention may have a bar shape as shown in FIG. Moreover, the shape provided with protrusion as shown in FIG.1 (b) may be sufficient. Moreover, as shown in FIG.1 (c), it manufactures with a porous body and can also raise the adsorption | suction efficiency of a target substance. Furthermore, as shown in FIG.1 (d), it can also have a twist shape. Moreover, as shown in FIG.1 (e), it can also have a wing | blade for accelerating | stimulating stirring of a solution. As shown in FIG. 1, the stirring efficiency of the solution can be improved by changing the shape of the rod. In addition, minute magnetically labeled molecules can be captured efficiently.

  Next, an analysis method using the stirring rod of the present invention will be described with reference to FIG. Here, description will be given by taking as an example a stirring bar 1 composed of a magnet bar 3 made of a permanent magnet and a nonmagnetic sheath 2 having wings.

  First, a reagent solution containing the magnetically labeled reagent substance 5 and a sample solution containing the target substance 7 to be detected are injected into the container. The reaction solution 4 in the container is stirred with the stirring rod 1 in a state where no magnetic force is supplied, and a reaction process for binding the reagent substance 5 and the target substance 7 is performed. In this reaction step, when the target substance 7 reacts with the reagent substance 5, the target substance 7 is magnetically labeled. Hereinafter, the target substance magnetically labeled by the reaction is referred to as a reactant.

  Next, a magnetic force is supplied to the stirring bar to adsorb the magnetically labeled reactant on the outer peripheral surface of the stirring bar, and then the stirring bar is withdrawn from the reaction solution.

  Next, the cleaning solution 8 is poured into another container in advance, and the stirring rod 1 on which the reactant is adsorbed is immersed in the cleaning solution 8. Thereafter, the supply of magnetic force to the stirring bar 1 is stopped, and the stirring bar 1 is rotated, vibrated, or ultrasonically oscillated to diffuse the reactant adsorbed on the outer peripheral surface of the stirring bar. The washing solution 8 is stirred with the stirring rod 1 in a state where the magnetic force is not supplied, and the reaction product is washed.

  Next, the magnetic force is again supplied to the stirring bar to adsorb the reactant diffused in the cleaning liquid, and then the stirring bar is withdrawn from the cleaning solution.

  A detection solution 9 suitable for detection of a reactant is poured into another container in advance, and a stirring rod on which the reactant is adsorbed is immersed in the solution. Thereafter, the supply of magnetic force to the stirring rod is stopped, and the adsorbed reactant is diffused by rotating, vibrating, or ultrasonically oscillating the stirring rod. The detection solution is stirred with the stirring rod in a state where no magnetic force is supplied, and the adsorbed reactant is completely diffused, and then the stirring rod is withdrawn. Thereafter, the reactant in the detection solution is detected by an appropriate method. Note that the washing step may be repeated to more thoroughly wash the reactants.

  The detection of the reactant in the detection solution may be performed using a well-known method, and may be selected depending on the target substance. For example, detection may be performed using a fluorescent dye, or detection may be performed using a dye that develops color when a substrate is added. Further, a detection method using an intercalator may be used.

  In the analysis method using the stirring rod of the present invention described above, the reaction product can be easily dispersed and stirred, and the reaction product can be prevented from being adsorbed on the container wall, so that the reaction efficiency can be improved. Further, by changing the container for each process, the influence of the container contamination can be minimized. Furthermore, since the solution can be injected into the container in advance, the operation of injecting and sucking the solution is not necessary. Therefore, the inspection time can be shortened, and further, equipment for sucking the solution, for example, a flow path system such as a pipetter, a tube, a pump, and a tank is not necessary, and analysis can be performed easily and at low cost. Furthermore, since the stirring and the B / F separation step can be performed continuously, the processing capacity can be improved.

  In a further aspect of the present invention, the above-mentioned stirring bar 1, moving means 20 for moving the stirring bar 1, and stirring bar driving means 21 for rotating, vibrating, or ultrasonically oscillating the stirring bar 1 are provided. An analysis device is provided (FIG. 4). This analyzer can also include a plurality of stirring rods 1. By providing a plurality of stirring bars, a large number of samples can be analyzed simultaneously. An example is shown in FIG.

  In another aspect, the analyzer further includes a magnet bar driving means 22 for inserting and extracting the magnet bar from the sheath (FIG. 5).

  A titer plate 12 having a plurality of cells is used, and a stirring rod 10 equal to the number of specimens is used. The reaction solution is injected into the cells 13 in the first row of the titer plate 12, and a stirring bar is immersed to perform stirring and adsorption. A washing solution is injected into the cells in the second and subsequent rows of the titer plate 12, and the stirring rod is continuously moved from the cells 14 in the second row to be washed. The last row of cells 16 is filled with a detection solution, and a stir bar can be immersed to diffuse the reactants for detection. In this way, if a titer plate or the like is prepared in advance, a large number of specimens can be analyzed simultaneously with ease by simply moving the stirring rod in order and repeating stirring, adsorption, and diffusion. .

The figure which shows the Example of a stirring rod. The conceptual diagram which shows the procedure of the analysis method using a stirring rod. The schematic diagram which shows one Embodiment of the analyzer provided with the stirring rod. The conceptual diagram of the analysis method which processes many samples with a some stirring rod. The schematic diagram which shows other embodiment of the analyzer provided with the stirring rod.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Stirring bar, 2 ... Sheath, 3 ... Magnet bar, 4 ... Reaction solution, 5 ... Reagent molecule made into magnetic particles, 6 ... Pipette, 7 ... Target molecule, 8 ... Washing solution, 9 ... Detection solution, 20 ... Moving means, 21 ... stirring bar driving means, 22 ... magnet bar driving means.

Claims (12)

  A stir bar used for an analysis method based on a specific binding reaction of biomolecules, wherein magnetic force can be arbitrarily supplied.   The stirring bar according to claim 1, wherein the stirring bar is a magnet bar made of an electromagnet.   The stirring bar according to claim 1, comprising a magnet bar made of a permanent magnet or an electromagnet, and a nonmagnetic sheath capable of arbitrarily inserting and extracting the magnet bar.   The stirring bar according to any one of claims 1 to 3, wherein the stirring bar has a shape having a protrusion.   The stirring bar according to claim 3, wherein the stirring bar is manufactured from a porous body.   The stirring bar according to claim 1, wherein the stirring bar has a twisted shape.   The stirring bar according to any one of claims 1 to 3, wherein the stirring bar has wings for promoting stirring of the solution. An analysis method by a specific binding reaction of a biomolecule, using the stirring rod according to any one of claims 1 to 7,
(A) A reaction step in which a reaction solution containing a magnetically labeled reagent substance and a target substance to be detected is stirred with the stirring rod in a state where no magnetic force is supplied, and the reagent substance and the target substance are bound to each other. When,
(B) supplying a magnetic force to the stirring bar, adsorbing a reaction product of the magnetically labeled reagent substance and the target substance, and allowing the stirring bar to exit from the reaction solution;
(C) immersing the stirring bar in a cleaning solution, stopping the supply of magnetic force to the stirring bar, and diffusing the adsorbed reactant;
(D) stirring the cleaning solution with the stirring rod in a state where no magnetic force is supplied, and cleaning the reaction product;
(E) supplying a magnetic force to the stirring bar to adsorb the washed reactant, and causing the stirring bar to exit the cleaning solution;
(F) immersing the stirring bar in a detection solution, stopping the supply of magnetic force to the stirring bar, and diffusing the adsorbed reactant;
(G) withdrawing the stirring bar from the detection solution to detect a reactant in the solution;
A method for analyzing biomolecules.   9. The analysis method according to claim 8, wherein the reaction molecules are washed by repeating the steps (c) to (e).   A stirring bar according to any one of claims 1 to 7, a moving means for moving the stirring bar, and a stirring bar driving means for rotating, vibrating, or ultrasonically oscillating the stirring bar. An analyzer using a magnetic particle-immobilized stirring bar.   The analyzer according to claim 10, further comprising magnet bar driving means for inserting and extracting the magnet bar from the sheath.   The analyzer according to claim 10, comprising a plurality of the stirring bars.

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