JP2014070915A - Liquid sucking method, and solid phase washing method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid sucking method that enables with a simple configuration the remaining quantity of liquid to be effectively reduced and a washing method using the sucking method.SOLUTION: By a method of sucking liquid within a container while bringing down a sucking nozzle, the sucking nozzle goes down in contact with the liquid surface as far as to the bottom of the container while sucking the liquid and ambient gas at such a speed as allows the state of contact to be maintained. By a solid phase washing method, after causing the solid phase component and the liquid phase component to react with each other in the container, the liquid phase is discharged from the container and, after liquid is injected into the container to wash the solid phase, the liquid is sucked by the sucking method described above.

Description

  The present invention relates to a liquid suction method and a solid phase cleaning method using the same.

  Removal of reaction solution after solid-liquid reaction in immunoassay for analyzing components in a sample using solid-liquid reaction, that is, heterogeneous reaction in which a reactant in a solid phase reacts with a reactant in a liquid phase In addition, B / F (Bound / Free) separation or B / F washing for separating and washing the solid phase carrier plays an important role in basic performance such as sensitivity and reproducibility of measurement results. For example, the solid phase reagent and the sample are injected into the upper opening container and reacted, and then the first B / F washing is performed. Further, the labeling reagent is injected into the container and reacted, and then the second B / F washing is performed. In the enzyme immunoassay where the substrate reagent is added and the signal due to the sample is measured, if the cleaning solution used for B / F cleaning remains in the container and the residual amount varies from measurement to measurement, the cleaning efficiency Or the dilution or inhibition of the reagent added after washing leads to a decrease in the accuracy of the measurement value or a variation in the measurement value.

  Patent Documents 1 and 2 describe that when a liquid in an open container having a flat bottom surface is sucked by the suction nozzle, the suction nozzle is horizontally moved near the bottom surface so as not to leave the liquid in the corner of the container. Has been. Patent Document 3 discloses that a suction nozzle portion is made porous and matched to the internal shape of the reaction vessel, and the nozzle portion corresponding to the magnetic collection location of the solid phase fine particles is retracted to widen the gap with the reaction vessel. Therefore, the inhalation of fine particles is prevented and the amount of residual liquid is minimized. Further, Patent Document 4 discloses a technique for minimizing the residual liquid at the corner of the container by using a plurality of brush-like suction tentacles. Patent Document 5 discloses a method of sucking liquid with a constant suction amount larger than the volume change speed obtained by multiplying the maximum cross-sectional area inside the container parallel to the water surface by the lowering speed of the suction nozzle. According to this method, the adhesion of the liquid to the outer peripheral surface of the suction nozzle can be minimized.

Japanese Patent Laid-Open No. 7-83939 JP 2003-190899 A JP-A-8-62215 JP 2003-42912 A JP 2008-76273 A

  In the above prior art documents, the techniques of Patent Documents 1 and 2 require an operation control mechanism for operating the suction nozzle in the horizontal direction, and it takes extra time for suction. The techniques of Patent Documents 3 and 4 are not economically preferable because the suction nozzle requires a component having a complicated shape. According to the technique of Patent Document 5, the suction nozzle is always above the liquid surface, and the outer peripheral surface of the suction nozzle does not contact the liquid. The present invention has been made in view of such circumstances, and based on a simple configuration, a rapid liquid suction method capable of effectively reducing the residual amount of liquid and cleaning of a solid phase using the same. It aims to provide a method.

As a result of intensive studies on the above problems, the present inventor has reached the present invention. That is, the present invention is as follows.
(1) A method of sucking the liquid in the container while lowering the suction nozzle, wherein the suction nozzle is in contact with the liquid surface and sucks the liquid and the atmospheric gas while maintaining the contact state. A liquid suction method, wherein the liquid is lowered to the bottom.
(2) In the method described in (1) above, the suction method described in (1) is performed a plurality of times, and the speed at which the suction nozzle descends is the highest in the last round compared to the previous round. A method of sucking a liquid to make it smaller.
(3) After reacting the solid phase component and the liquid phase component in the container, the liquid phase is discharged from the container, then the liquid is injected into the container to wash the solid phase, and then the method according to (1) A method of washing a solid phase, wherein the liquid is sucked by the method.
(4) In the method described in (3) above, after the liquid is injected into the container and the solid phase is washed, the step of sucking the liquid by the method described in (1) is performed a plurality of times, and the last round In the solid phase washing method, the speed at which the suction nozzle descends is minimized as compared with the previous time.

  Hereinafter, the present invention will be described in more detail.

  In the present invention, the liquid in the container is sucked while lowering the suction nozzle, but the nozzle and the liquid level are substantially reduced while sucking the liquid and the atmospheric gas while maintaining the state where the suction nozzle is in contact with the liquid level. Will descend at the same speed. In this case, the degree of decompression in the suction pipe from the initial stage to the final stage of suction is almost constant without drifting, although there is a slight fluctuation due to air contamination, and the liquid from the liquid surface part gradually falls in the final stage of suction. Aspirate. For this reason, it is presumed that the liquid molecular group attached to the corner of the container also moves smoothly to the center of the liquid surface and is sucked by the nozzle.

  On the other hand, when the liquid is sucked at a position lower than the liquid level by the suction nozzle, the suction of the atmospheric gas does not occur compared to when the liquid is sucked in contact with the liquid level. Increases and suction speed increases. For this reason, the liquid adhering to the corner of the container at the end of the suction is not allowed to move to the suction nozzle, and only the liquid near the nozzle is sucked so as to be torn off, and the suction process is completed. It is inferred that the unsucked droplets remain.

  Further, according to the present invention, the suction nozzle sucks the liquid in contact with the liquid surface and does not immerse deeply below the liquid surface, so that adhesion of the liquid to the outer surface of the nozzle can be avoided. preferable.

  In the present invention, the suction nozzle descends at a speed that maintains the state in contact with the liquid surface. In other words, the nozzle lowering speed depends on the balance with the liquid suction speed.

  The shape of the suction nozzle used in the present invention is not particularly limited. The material is not limited to a specific material as long as it is a stable material that does not corrode, deform, break, or the like when in contact with the liquid or container to be used. As an example, a stainless steel tube that hardly changes in quality even when it is in contact with liquid for a long period of time and a plastic (for example, polyetheretherketone) tube that has excellent chemical resistance can be given. A slit as shown in FIG. 2 is preferably formed at the tip of the suction nozzle so that the nozzle is not blocked by the bottom of the container. Depending on the height of the slit, a large amount of atmospheric gas is mixed when the liquid is sucked out at the final stage, the degree of decompression in the suction pipe is weakened, the liquid suction is incomplete, and the remaining liquid increases. For this reason, when a stainless steel tube with an inner diameter of about 1 mm is used as the suction nozzle, it is preferable to provide slits at two diameter positions on the circular edge of the nozzle, and if one or three slits are provided, the remaining liquid increases. There was a tendency to.

  In the present invention, the container can contain a liquid whose upper part is open or can be opened, and examples thereof include devices called wells, cups, cells, test tubes, reaction vessels, and the like. The container may be a single container, or may be a multiple container or a microtiter plate container in which two, three, and side walls are integrally formed adjacent to each other.

  In the present invention, after performing suction by the above suction method, a liquid may be added to the container, and suction by the above suction method may be performed again. As described above, when the suction method of the present invention is repeatedly performed a plurality of times, it is preferable that the last suction time has the lowest speed at which the suction nozzle descends compared to the previous time. This means that the liquid suction speed is minimized. As a result, the lowering speed of the suction nozzles other than the last time is set larger than the last time to shorten the suction time, and in the last time, the lowering speed of the suction nozzle is compared with the previous times. By making it the smallest, that is, making the liquid suction speed the smallest, it becomes possible to effectively reduce the remaining liquid amount.

  In the suction method of the present invention, after the solid phase component and the liquid phase component are reacted in the container, the liquid phase is discharged from the container, and then the liquid is injected into the container to wash the solid phase, and then the liquid is sucked. Can also be used when

  The solid phase component is not particularly limited. For example, in immunoassay for analyzing a component in a sample, a reagent component having specific affinity such as an antibody or an antigen is physically / chemically coupled to a solid phase carrier. Or a reaction complex in which an analysis component in a sample solution and / or a label component in a labeling reagent is directly or indirectly bound to the solid phase conjugate. Examples of the liquid phase component include an analysis component in a sample solution or a label component in a labeling reagent that binds to the solid phase conjugate or the reaction complex.

  In the present invention, in the cleaning method described above, the step of sucking the liquid by the suction method (1) described above after injecting the liquid and cleaning the solid phase may be repeated a plurality of times. In that case, it is preferable to make the speed at which the suction nozzle descends the smallest in the last round compared to the previous round. This means that the liquid suction speed is minimized. As a result, the lowering speed of the suction nozzles other than the last time is set larger than the last time to shorten the suction time, and in the last time, the lowering speed of the suction nozzle is compared with the previous times. By making it the smallest, that is, making the liquid suction speed the smallest, it becomes possible to effectively reduce the remaining liquid amount.

  Thus, the cleaning method of the present invention can be used as B / F separation or B / F cleaning when component analysis in a sample is performed.

  The case where the present invention is used for immunoassay for analyzing components in a sample will be described below as an example.

  The solid phase carrier constituting the solid phase component can be accommodated in the reaction vessel, and there is no particular limitation on any of the material, size, and shape, but magnetic force, gravity, etc. for B / F separation. What is suitable for localizing in the bottom corner part or side wall in a reaction container by external force is preferable. In addition, the inner wall of the reaction vessel itself can be used as a solid phase carrier. For example, for a cup-shaped container having a volume of about 0.5 mL to 1 mL, a solid phase carrier having a size ranging from fine particles having a particle size of about 0.01 μm to beads having a particle size of about 1 mm can be used. Among them, use is made of 0.1 to 10 μm magnetic fine particles suitable for increasing the reaction efficiency between the solid phase component and the liquid phase component and for collecting on the side wall of the container by the magnetic force from the outside of the container at the time of B / F separation. Is preferred.

  The washing solution is not particularly limited, but the unreacted sample and the labeling reagent can be effectively used without dissociating the solid-phase conjugate, the reaction complex, or the reaction complex further bound with the labeling component. What is necessary is just to select the electrolyte concentration, pH, type and concentration of the surfactant, etc. that can be cleaned cleanly.

  According to the present invention, when the liquid is sucked, the amount of the remaining liquid can be effectively reduced. Moreover, since the suction nozzle does not immerse deeply below the liquid surface, it is possible to suppress the adhesion of the liquid to the outer surface of the nozzle.

It is a figure which shows BF washing | cleaning apparatus and reaction container. It is a figure which shows the suction pipe front-end | tip shape.

  FIG. 1 shows a cleaning apparatus and reaction in which a suction nozzle 1 and a discharge nozzle 2 suitable for carrying out the present invention are integrated with a support portion 5 connected to at least a drive means (not shown) capable of moving up and down. The container 6 is shown. The suction nozzle 1 is connected to a suction pipe 3 whose base end is connected to a vacuum pump (not shown) via a waste liquid means (not shown). The discharge nozzle 2 is connected to a controllable liquid feeding means such as a syringe pump from a supply source of the cleaning water via a discharge pipe 4. The raising and lowering drive means is preferably programmed and controlled by a stepping motor, and in the suction process, the suction nozzle tip is lowered until it contacts the bottom of the container in order to suck the liquid in the container as much as possible. For this reason, it is desirable to provide a buffer means when the tip of the suction nozzle 1 collides with the bottom surface in the container 6.

  Table 1 shows an example of a B / F washing method in the case where a two-step immunoassay using magnetic fine particles as a solid phase is performed with an automatic apparatus. In the table, the first B / F cleaning refers to a cleaning step performed to separate and clean an unreacted sample after injecting and reacting a solid phase reagent and the sample into a reaction vessel. The second B / F washing is a washing step performed to separate and wash the unreacted labeling reagent after adding the labeling reagent to the solid phase component after the first B / F washing and causing the reaction. In the example of Table 1, as the second B / F separation, separation and washing of the labeling reagent-solid phase component complex (Bound) and the unreacted labeling reagent (Free) are performed in three steps. In order to thoroughly clean the labeling reagent that directly affects the detection sensitivity, the magnetic fine particles are completely dispersed in the reaction vessel by discharging the cleaning liquid after releasing the magnetically collected state. This is because it is necessary to secure a long time for the magnetic flux collecting operation after the liquid is dispersed over the entire liquid, while the cycle time of the automatic device for sequence control is not excessively increased.

  In the first B / F cleaning, the reaction solution of the solid phase reagent and the sample is sucked over 0.4 seconds and discarded through the suction pipe while lowering the suction nozzle at a speed of 90 mm / s. Here, the lowering speed of the suction nozzle of 90 mm / s is a speed lower than about 100 mm / s at which the liquid level descends when the suction nozzle is immersed in the cleaning liquid in the same shape container to suck the liquid. This means that the nozzle comes into contact with the liquid surface and descends to the bottom of the container at a speed that maintains the contact state while sucking the liquid and the atmospheric gas. The container used in this example is a polypropylene flat bottom cylindrical container having a bottom diameter of about 5 mm and a height of about 20 mm.

  After this suction step, a magnet is brought close to the outer surface of the reaction vessel to collect magnetic fine particles on the inner surface of the vessel. However, when the cleaning liquid is discharged for the second time in the second B / F cleaning, the magnet is moved away from the container to release the magnetism collecting state in order to perform the above complete dispersion.

  After sucking the reaction liquid in the first B / F cleaning as described above, the suction nozzle is raised. At this time, it is preferable that the suction nozzle is raised to a position higher than the liquid level after the maximum 400 μL of the cleaning liquid is discharged in the subsequent process. Next, at this position, 250 μL of the cleaning liquid is discharged from the discharge nozzle. After a predetermined time (several seconds), as in the case of the above-mentioned reaction liquid suction, while the suction nozzle is lowered at a speed of 90 mm / s, the mixed liquid of the remaining reaction liquid and the cleaning liquid is sucked over 0.4 seconds and discarded through the suction pipe. . Next, the suction nozzle is raised, and 400 μL of the cleaning liquid is discharged from the discharge nozzle. Next, as the final suction step of the first B / F cleaning, the cleaning liquid is sucked over 0.5 seconds while the suction nozzle is lowered at a speed of 45 mm / s.

  Also in the second B / F cleaning, the operations of the suction nozzle and the discharge nozzle are basically the same as those in the first B / F cleaning. In the first second B / F washing, as in the case of the first B / F washing reaction liquid suction, it takes 0.4 seconds by the suction nozzle that lowers the unreacted reaction reagent (labeling reagent) at a speed of 90 mm / s. Suction and discard through suction pipe. Next, the suction nozzle / discharge nozzle is raised, and 100 μL of the cleaning liquid is discharged from the discharge nozzle. The reason why the discharge amount is 100 μL and less than 250 μL is to minimize contamination due to adsorption of the labeling reagent to the container. After discharging 100 μL of the cleaning solution, the mixed solution of the residual reaction reagent and the cleaning solution is aspirated and discarded over 0.4 seconds. Further, after the process of discharging 400 μL of the cleaning liquid and sucking it for 0.5 seconds was repeated twice, when the cleaning liquid was discharged by 400 μL, the first second cleaning was completed.

  In the next cycle of the automatic apparatus (cycle time 65 seconds), the second B / F cleaning is performed for the second time. After 400 μL of the cleaning liquid discharged at the end of the second B / F cleaning is sucked for 0.5 seconds, the magnetic flux collection state is canceled and 150 μL of cleaning liquid is discharged to clean the magnetic fine particle component. After a predetermined time (about 30 seconds) has elapsed since the start of the magnetic flux collection operation, 150 μL of the cleaning liquid is sucked in over 0.4 seconds, the magnetic flux collection state is released again, and when the cleaning liquid is discharged by 150 μL, the second cleaning is completed. To do.

  Further, in the next cycle of the automatic apparatus, the second B / F cleaning is performed for the third time. After 150 μL of the cleaning liquid discharged at the end of the second B / F cleaning is sucked for 0.4 seconds, 400 μL of the cleaning liquid is discharged and then sucked again for 0.5 seconds, and then the second B / F cleaning is finished. . In this apparatus, at the time of the last suction of the second B / F cleaning, the lowering speed of the suction nozzle can be lowered at 90 mm / s or a half speed of 45 mm / s. It is shown that it is preferable to be 45 mm / s.

[Example 1]
Using the B / F cleaning apparatus shown in FIG. 1, B / F cleaning suction and discharge operations were performed under the conditions shown in Table 1, and the remaining amount of the cleaning liquid was weighed. Here, in order to compare the remaining amount of the cleaning liquid, an empty container without magnetic fine particles was used, and the suction / discharge operation of the cleaning liquid was performed without adding any sample or labeling reagent. The suction nozzle 1 was a stainless steel tube having an inner diameter of 0.92 mm, and the suction pipe 3 was a polytetrafluoroethylene tube having an inner diameter of 1.5 mm. As the washing solution, Tris buffer (pH 8) containing sodium chloride and a surfactant was used.

  Table 2 shows the results. If the lowering speed of the suction nozzle in the suction step to be executed last is 90 mm / s, the average value of the remaining amount of the cleaning liquid 10 times is 0.0033 g, whereas if the lowering speed is 45 mm / s, it is 0. 0005 g, decreased to 1/6 or less.

  In the above experiment, the width of the rectangular slit at the tip of the suction nozzle 1 shown in FIG. 2 was 0.30 mm, and the height of the slit was 0.55 mm. When the slit height was 1.0 mm and the lowering speed of the suction nozzle in the last suction step was 45 mm / s, the residual amount of cleaning liquid (average value of 10 measurements) was measured to be 0.0012 g Thus, the residual amount was more than twice as long as the slit height of 0.55 mm. Table 3 shows the results.

[Example 2]
An enzyme immunoassay for TSH (thyroid stimulating hormone) was performed under the same apparatus and conditions as in Example 1. That is, using the B / F cleaning apparatus shown in FIG. 1, the suction / discharge operation of B / F cleaning was performed under the conditions shown in Table 1, enzyme immunoassay was performed, and the reproducibility of the measured values was compared. Samples were prepared by diluting TSH standards to a concentration of about 10 μIU / mL. The measurement is carried out in accordance with a conventional method. After reacting a sample with a solid phase reagent in which anti-TSH monoclonal antibody is immobilized on magnetic fine particles having a particle size of about 2.5 μm, the 1B / F washing is performed, and an alkaline phosphatase-labeled anti-TSH monoclonal antibody is obtained. After the reaction, the second B / F was washed, and a dioxetane luminescent substrate was allowed to act to measure the chemiluminescence intensity.

  As a result, when the lowering speed of the suction nozzle in the last suction step to be executed is 90 mm / s, the coefficient of variation (CV) based on the 10 measurements is 2.65%, while the lowering speed is 45 mm. / S, CV decreased to 1.46%.

  From the above, according to the method of the present invention, it becomes possible to effectively reduce the remaining amount of the cleaning liquid, and in particular, the lowering speed (45 mm / s) of the last suction nozzle can be reduced. By setting it lower than the lowering speed of the suction nozzle (90 mm / s), it becomes possible to more effectively reduce the remaining amount of the cleaning liquid in the last suction process, and this is the enzyme immunoassay. It was found to improve the reproducibility of.

1: Suction nozzle 2: Discharge nozzle 3: Suction pipe 4: Discharge pipe 5: Support part 6: Reaction vessel 7: Slit nozzle tip slit part

Claims (4)

A method of sucking the liquid in the container while lowering the suction nozzle, and the suction nozzle descends to the bottom of the container at a speed that maintains the contact state while contacting the liquid surface and sucking the liquid and atmospheric gas. A method for sucking a liquid. The method according to claim 1, wherein the suction method according to claim 1 is performed a plurality of times, and at the last time, the speed at which the suction nozzle descends is minimized as compared with the previous time. Suction method. After the solid phase component and the liquid phase component are reacted in the container, the liquid phase is discharged from the container, then the liquid is injected into the container to wash the solid phase, and then the liquid is discharged by the method according to claim 1. A method for washing a solid phase, comprising sucking. 4. The method according to claim 3, wherein the step of injecting the liquid into the container to wash the solid phase and then sucking the liquid by the method according to claim 1 is performed a plurality of times. The solid phase cleaning method minimizes the speed at which the suction nozzle descends compared to the first time.

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