JPH06343496A - Analyzing method, analyzing apparatus and reactor - Google Patents

Abstract

PURPOSE:To obtain an analyzing method, an analyzing apparatus and a reactor, capable of automatically determining a nucleic acid in a sample in high sensitivity. CONSTITUTION:A reagent of DNA probe 402 to which fine particles 401 provided with a labeled material are bound and a reagent of DNA probe 404 to which fine particles 403 having particle diameter larger than that of the fine particles 401 are injected into a reacting chamber 341 of a reactor 34a together with a sample. Then, a sample DNA 405 in the sample is hybridized with DNA probes 402 and 404 to form double-stranded chain and unbound excess fluorescence particles-labeled DNA probe 402 is removed using a filter 406 having the middle pore diameter between particle diameters of two kinds of fine particles. Then, a restriction enzyme 407 reacts with the formed double-stranded polynucleotide to cut the double-stranded chain part and isolated fluorescent fine particles 401 are fractionated with a filter 406 and introduced into a flow cell to count particle numbers. Thereby, separation of B/F and cleaning process is extremely simplified and automation of measurement is facilitated.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical method, an analytical apparatus and a reaction container, and more particularly, to an assay for labeling a measurement object with particles and then guiding the particles to a flow cell to measure the concentration of the measurement object. The present invention relates to a method and an analytical device, and a reaction container used for the analytical method.

[0002]

2. Description of the Related Art Detection and identification of pathogenic microorganisms are essential for diagnosing infectious diseases. However, it takes time to detect and identify the causative microorganism of the infectious disease by culture, and it is often inferior to the diagnosis and treatment of the disease.

Therefore, in recent years, it has become possible to identify the pathogen of infectious disease and diagnose before the onset of infectious disease by examining the presence or absence of a specific base sequence in a sample using the technique of nucleic acid hybridization.

That is, it is a method using a DNA probe. This utilizes single-stranded DNA having a sequence complementary to the base sequence of the nucleic acid to be detected (this is called a DNA probe) as a specific reaction reagent. The presence or absence of the target pathogen can be determined by detecting the presence or absence of the base sequence complementary to the base sequence of the DNA probe in the sample.

Various concrete methods have been proposed. For example, Japanese Clinical, 47, 737-754 (198).
9) etc. An example is the method called the dot hybridization method. In this, single-stranded DNA (ss-DNA) obtained by denaturing a sample is bound to a solid phase, and ss-DNA labeled with a radioisotope is allowed to act on this solid phase to form a solid phase ss-DNA. This is a method of measuring the radioactivity of the solid phase by removing unreacted labeled ss-DNA after forming a hybrid.

As a modification of this method, there is a sandwich hybridization method. This method can reduce the background due to adsorption and is particularly effective when an impure sample is used. This method uses at least two DNA fragments derived from the labeled nucleic acid to be identified. One DNA fragment is bound to a solid phase and used as a capture reagent. The other DNA fragment is labeled as a detection reagent and is added to the hybridization solution together with the sample solubilized. When a base sequence homologous to both reagents is present in a sample specimen, the sequence should hybridize to both the capture reagent and the detection reagent. Whether or not they have hybridized can be known through the labeling on the solid phase.

Further, Japanese Patent Publication No. 3-78120 proposes a method using a restriction enzyme. In this method, a single-stranded polynucleotide that is a measurement target, a label is bound and a single-stranded polynucleotide that can react with the single-stranded polynucleotide that is a measurement target to form a double-stranded polynucleotide A double-stranded polynucleotide is formed by contacting the phase with a solution, and the double-stranded polynucleotide that is formed is cleaved by acting a restriction enzyme on the formed double-stranded polynucleotide to give a solution or solid-phase labeled substance. taking measurement.

Similarly, a method using a restriction enzyme or a reagent having a selectable cleavage site introduced therein is disclosed in JP-A-2-92300.

On the other hand, a method of using a reaction container provided with a discharge hole at the bottom of a filter in an immunoassay method, which is not associated with nucleic acid analysis, is disclosed in JP-A-2-228562 and JP-A-2-228563. Has been proposed to. This conventional technique is used as a method for washing reagents on a solid phase and
/ F separation (bound type-bound- and free type-fr
As a method for easily performing the operation (separation from ee-), pressure is applied from above the filter of the reaction container, and the liquid is discharged through the discharge hole provided below the filter.

Although not associated with nucleic acid analysis, JP-A-4-273065 proposes a highly sensitive immunoassay method using fine particles as a label. In this conventional technique, fine particles are used as a label, and the fine particles proportional to the amount of the object to be measured are captured in the reaction solid phase by a specific reaction such as an antigen-antibody reaction, and then released, and the number of fine particles in the free liquid is counted. By doing so, the amount of the measuring object is obtained. The solution to be measured containing educts is introduced into the flow cell, and the pulsed fluorescence generated when the fine particles pass through the laser light flux irradiated from the direction orthogonal to the flow is detected, and the number of pulses is counted to obtain the fine particles. Is counted. In this conventional technique, the labeled substance, ie, the fine particles, once captured on the reaction solid phase is released, and then the measurement is performed. Therefore, the influence of the labeled substance that nonspecifically binds to the solid phase can be avoided. By using fine particles as labels and counting them, highly linear detection is realized even at low concentrations.

[0011]

As described above, a DNA probe labeled with a radioactive isotope is widely used in the conventional nucleic acid detection method. The cost of using isotopes is high, and safety and disposal must be taken into consideration when handling. Further, the conventional dot hybridization method and sandwich hybridization method have a problem when the amount of nucleic acid to be detected is small, and moreover, many work steps are required at the time of measurement, and the measurement is automated. It is hard to say that it is suitable to see.

In the prior art disclosed in Japanese Examined Patent Publication No. 3-78120, a labeled substance attached to a single-stranded polynucleotide is released by a reaction for measurement. However, since the released labeled substance is measured in a form that depends on the concentration of the measurement target substance in the entire reaction solution, the detection sensitivity decreases when the measurement target substance in the sample is extremely small. Also, the shape of the reaction vessel is not mentioned, and when using a normal bottomed reaction vessel, the washing process and B / F separation work become complicated, which is also suitable from the viewpoint of automation of measurement. It is hard to say that

The conventional technique described in Japanese Patent Application Laid-Open No. 2-92300 has the same problem as Japanese Patent Publication No. 3-78120.

JP-A-2-228562 and JP-A-2-2
In the prior art described in 28563, a washing process and a B / F separation operation are performed by applying pressure from the upper part of the filter using a reaction container provided with a discharge hole under the filter and discharging the liquid through the discharge hole. Easy going. However, the released label is measured by bringing the reaction solution remaining on the filter together with the reaction container to the detector. This method requires a special detector that can accommodate the reaction container, and also has a problem in sensitivity because the measurement is performed through the wall of the container.

In the prior art described in Japanese Patent Application Laid-Open No. 4-273065, in order to measure liberated labeled fine particles, a liquid to be measured containing the fine particles is introduced into a flow cell and the number of fine particles is optically measured. However, since this conventional technique also uses a bottomed reaction vessel, the washing process and B / F separation work become complicated, which is also called automation of measurement. From the point of view, it is hard to say that it is suitable.

An object of the present invention is to provide an analytical method, an analytical device, and a reaction container capable of automatically and highly sensitively measuring nucleic acid in a sample.

[0017]

In order to achieve the above object, the analysis method of the present invention comprises: (a) two kinds of fine particles having different particle diameters, one of which is a small particle having a label. The particles, the other fine particles being large particles having a larger particle size than the small particles, are prepared.
Binding fine particles of a kind through an object to be measured in a sample, (c) unbonding by using a filter having a pore size larger than the small particles and smaller than the large particles. Removing small particles, (d) specifically cleaving a bound product of small particles and large particles bound via an object to be measured, (e) separating only the small particles using the filter, (F) It is characterized in that the concentration of the measurement target in the sample is measured by measuring the labeled substance of the small particles.

In order to achieve the above object, the analyzer of the present invention comprises two kinds of fine particles having different particle sizes,
An analyzer for measuring the concentration of an object to be measured in a sample by using one of the microparticles as a small particle with a labeled substance and the other microparticle as a large particle having a larger particle size than the small particle. In (a) at least one reaction chamber, a filter disposed at the bottom of the reaction chamber, having a pore size larger than the particle size of the small particles and smaller than the particle size of the large particles, further below the filter. A liquid flow-down resistance member provided, a reaction container having a small hole for discharging the liquid that has passed through the filter and the liquid flow-down resistance member from the reaction chamber, and (b) a turntable in which a plurality of the reaction containers are arranged, (C) Dispensing and supplying means for injecting the sample, the reagent containing the two kinds of fine particles, the washing liquid, and the reagent that specifically cleaves the binding site into the reaction chamber of the reaction container, (d)
Pressure difference generating means for generating a pressure difference between the reaction chamber and the small hole to discharge the liquid in the reaction chamber from the small hole; and (e) the pressure difference generating means included in the liquid discharged from the small hole. It is characterized in that it is provided with a measuring means for measuring the labeled substance of small particles and measuring the concentration of the measuring object in the sample.

In the above analyzer, the measuring means preferably has a flow cell into which the liquid is introduced through the small holes of the reaction vessel, and measures the optical characteristics of the small particles passing through the flow cell based on the labeled substance. And measure the concentration of the measurement object in the sample. Further, preferably, the small hole of the reaction container is formed in a discharge nozzle provided at a lower end of the reaction container, and the discharge nozzle is directly inserted into an inlet of the injection chamber of the flow cell.

In order to achieve the above object, the reaction container of the present invention comprises two kinds of fine particles having different particle sizes,
An analysis method for measuring the concentration of a measurement target in a sample by using one of the microparticles as a small particle with a labeled substance and the other microparticle as a large particle having a larger particle size than the small particle In the reaction vessel used for the above, at least one reaction chamber, a filter arranged at the bottom of the reaction chamber, having a pore size larger than the particle size of the small particles and smaller than the particle size of the large particles, and It is characterized by further having a liquid flow-down resistance member provided below and a small hole for discharging the liquid that has passed through the filter and the liquid flow-down resistance member from the reaction chamber.

The reaction vessel preferably comprises a first vessel forming the reaction chamber and having the filter arranged therein,
A first container and a second container that are separate bodies, in which the liquid flow-down resistance member is arranged and the small holes are formed;
The first container and the second container can be joined at the bottom of the first container and the top of the second container.

The reaction container may further have a small hole for suction in addition to the small hole for discharging the liquid.
Further, the reaction container may have a plurality of reaction chambers including the reaction chamber. Furthermore, the reaction container preferably further has at least one sample chamber, and the sample chamber contains a reagent containing small particles having the labeled substance together with a storage solution.

[0023]

In the analysis method of the present invention, two different particle sizes are used.
By using two kinds of fine particles and a filter with a pore size intermediate between those particle sizes, two kinds of fine particles are combined with each other through the measurement object in the sample, and then unbound small particles are discharged by passing through the filter. (B / F separation), and the small particles after cutting the bound product of small particles and large particles are also separated by passing through a filter (B / F separation). Further, when the cleaning liquid is injected, the cleaning liquid is also discharged through the filter and the reaction chamber and the filter are cleaned. As described above, since the B / F separation and the washing process are performed by using the two kinds of fine particles and the filter having the pore size between those of the particle diameters, the reaction solution and the washing solution are tilted by using the pipetting nozzle or tilting the container. Operations such as taking out are not necessary, the B / F separation and the washing steps are extremely simple, and the automation of measurement is facilitated. Also,
The separated fluorescent fine particles can be directly introduced into the flow cell, and the operation time can be shortened. Furthermore, since the labeled substance can be measured by the flow cell, highly sensitive measurement is possible.

In the analyzer of the present invention, the sample and the reagent containing the above-mentioned two kinds of fine particles are injected into the reaction chamber of the reaction container by using the dispensing supply means, and the two kinds of fine particles are discharged in the reaction chamber. After binding through the measurement object in the sample, a pressure difference is generated between the reaction chamber and the small hole by using the pressure difference generating means, and the unbound small particles resist the resistance of the liquid flow resistance member. And remove through a filter. At this time, a cleaning liquid is injected as needed using the dispensing supply means to clean the reaction chamber and the filter. Then, a reagent that specifically cuts the bonding site is injected into the reaction chamber by using a dispensing and feeding means, the combined product of the small particles and the large particles is cut, and the small particles after cutting are again used by the pressure difference generating means. Is separated through a filter against the resistance of the liquid flow resistance member. At this time as well, the cleaning liquid is injected by using the dispensing supply means if necessary. Small particles, which are minute particles with labeled substances separated in this way, are measuring means,
Preferably, it is introduced into a flow cell, a small particle label is measured, and the concentration of the measurement target in the sample is measured. Thus, the analysis method of the present invention can be carried out.

By forming a small hole of the reaction container in the discharge nozzle provided at the lower end of the reaction container and inserting the discharge nozzle directly into the inlet of the flow chamber, the non-measurement liquid can be easily transferred to the flow cell. Measurement time is shortened.

Further, the measurement time is further shortened by accommodating a reagent containing small particles with a label together with a preservation solution in at least one sample chamber of the reaction container.

[0027]

First, one embodiment of the analysis method of the present invention will be described with reference to FIG. First, a polynucleotide (DNA probe) 402 bound to a labeled microparticle 401
A first reagent containing γ and microparticles 403 without a label
Bound to the polynucleotide (DNA probe) 40
A second reagent containing 4 is prepared. Here, the polynucleotide (DNA probe) 40 of the first and second reagents
2,404 has a nucleotide sequence complementary to the sample DNA that is the measurement target in the sample,
Those that do not have nucleotide sequences complementary to each other are used. Further, the fine particles 401 of the first reagent are small particles, and the fine particles 403 of the second reagent are large particles having a larger particle size than the fine particles 401 of the first reagent. It is preferable to use fluorescent microparticles as the microparticles 401 having a label.

After preparing the first and second reagents in this manner, the sample and the first reagent are injected into the reaction chamber of the reaction container (FIG. 1), and the sample DNA 405 and the first reagent in the sample are injected. The fluorescent particle-labeled DNA probe 402 therein is hybridized to form a double strand (FIG. 1). Then
The second reagent is injected into the reaction chamber of the reaction container (FIG. 1), and the sample DNA 405 in the sample and the non-fluorescent particle-bound DNA probe 404 in the second reagent are further hybridized to form a double strand. (Figure 1).

In this way, two kinds of fine particles 401,
After binding 403 to the sample DNA 405, the reaction is performed using a filter 406 having a pore size larger than the particle size of the microparticles 401 in the first reagent and smaller than the particle size of the microparticles 403 in the second reagent. The liquid is discharged through a filter 406, the unbound excess fluorescent particle-labeled DNA probe 402 is removed, and the first B / F separation is performed (FIG. 1).
). At this time, a cleaning solution is injected as needed to clean the reaction container and the filter 406. These operations are
The reaction container used up to now may be provided with a mechanism capable of controlling the discharge of the reaction liquid together with the filter 406,
It may be performed using another reaction chamber having a filter 406 and a discharge opening.

Then, a suitable restriction enzyme solution is injected into the reaction chamber to act the restriction enzyme 407 to cleave the double-stranded site formed by hybridization (FIG. 1).
As a result, the fluorescent fine particles 401 trapped in the filter 406 are released, and the released fluorescent fine particles 401 are separated together with the reaction solution through the filter 406 (FIG. 1). In this way the second B
/ F separation is performed. Also at this time, a cleaning liquid is added as needed.

The effluent containing the fluorescent fine particles 401 separated in this way is introduced into the flow cell, the fluorescence due to the fine particles passing through the flow cell is detected, and the number of particles is counted. Based on the counted number of particles, it is possible to calculate the concentration of the sample DNA which is the measurement object in the sample.

According to the above-described analysis method, B / F separation and washing steps are performed using two types of fine particles 401 and 403 having different particle sizes and a filter 406 having a pore size intermediate between those particle sizes. Therefore, it is not necessary to incline the container or take out the reaction liquid or the washing liquid by using the pipetting nozzle, and the B / F separation or the washing process becomes extremely simple. Therefore, automation of measurement is facilitated. Further, it is also possible to directly introduce the separated fluorescent fine particles 401 into the flow cell, and the operation time can be shortened. Further, since the labeled substance is counted using the flow cell, highly sensitive measurement is possible.

In the above embodiments, the fluorescent particle-labeled DN
First reagent containing A probe 402 and non-fluorescent particle binding D
The injection order of the second reagent containing the NA probe 404 may be reversed or may be simultaneous.

Next, FIG. 2 shows an embodiment of an apparatus for carrying out the above-described analysis method and a reaction container used in the analysis method.
~ It demonstrates by FIG.

FIG. 2 is a schematic view showing the overall structure of an automatic analyzer for carrying out the analysis method of the present invention, and FIGS. 3 to 5 are views showing the structure of a reaction container used in the analysis method. .

In FIG. 1, the automatic analyzer of this embodiment has four turntables 1, 2, 3, 35.
On the turntable 1, a plurality of sample containers 33 containing a sample such as blood are arranged in a circle. The turntable 2 has a plurality of reaction vessels 34a arranged in a circle, and the turntable 3 has a plurality of reaction vessels 34b arranged in a circle. Further, the turntable 35 is a reagent table, and various reagent solution containers necessary for the analysis operation are arranged in a circle. That is, the containers 90a, 90b, 90c containing the fluorescent particle-labeled probe liquid, which is the first reagent, corresponding to the analysis items A, B, C whose nucleic acids are to be measured, and the particle binding, which is the second reagent, are bound. A container 36a containing a probe liquid,
36b, 36c, a restriction enzyme solution storage container 38, a cleaning solution and other necessary buffer solution containers and the like are held on the reagent table 35. The particles contained in the particle-bound probe solution which is the second reagent have a larger particle size than the fluorescent particles contained in the fluorescent particle-labeled probe solution which is the first reagent.

The turntables 1, 2 and 35 are pulse motors 91, which are controlled by a controller (not shown).
92 and 93 are rotatably configured to stop a desired sample container 33, reaction container 34a or reagent liquid container at a suction position or a discharge position by the pipetting nozzle 43. The turntable 3 is also configured to be rotatable by a pulse motor 94 controlled by a controller (not shown) so as to stop the desired reaction container 34b at a position aligned with the reaction container 34a on the turntable 2. Also, turntable 2
Is provided with a drive device 95 for moving the turntable 2 up and down.

As shown in FIG. 3, the reaction container 34a includes a plurality of reaction chambers 341 and 342 and a plurality of sample chambers 343 and 34.
5, the reaction chamber 341, which is one of the reaction chambers, has an open bottom, and the filter 346 is arranged on the opened bottom. The reaction container 34b is, as shown in FIG.
It has a cap 347 in the upper part and a discharge nozzle 348a having a small hole 348 in the lower part. The cap 347 can be coupled to the lower opening of the filter 346 of the reaction vessel 34a as shown in FIG. 5, and when there is no pressure difference between the reaction chamber 341 and the small hole 348 when coupled to the lower opening. It has a structure as a liquid flow-down resistance member for holding the liquid in the reaction chamber 341. Specifically, the cap 347 is made of an elastic material, and a cross-shaped cut is made in the material as shown in FIG. 4 (B). When a pressure difference occurs above and below the cap 347, the cut opens and the pressure is increased. It is a structure that closes when there is no difference. In the present embodiment, the pressure difference generated between the reaction chamber 341 and the small hole 348 is the pressure in the pressure chamber 101 connected to the pressure source 102 as shown in FIG.
It is generated by applying pressure from the 41 side.

The filter 346 is larger than the particle size of the fluorescent particles contained in the fluorescent particle-labeled probe solution as the first reagent and smaller than the particle size of the particles contained in the particle-bound probe solution as the second reagent. Has a pore size.

Reference numeral 39 denotes an automatic pipette mechanism, which is attached to the movable arm 42 and has the above-mentioned pipetting nozzle 4
3, a rotation drive unit 45 for horizontally rotating the movable arm 42, and a vertical drive unit 4 for vertically moving the movable arm 42.
4, a syringe pump 40 connected to the nozzle 43 via a tube 41, a cleaning liquid tank 37 also serving as an extruding liquid, and a nozzle cleaning tank 96. As the movable arm 42 moves, the pipetting nozzle 43 moves the sample suction position on the turntable 1, the reagent suction position on the turntable 35,
It is possible to rotate the sample / reagent dispensing position on the turntable 2 and the nozzle cleaning tank 96 as a radius of rotation,
It can descend and rise in each position.

The turntables 1, 2, 3, 35 and the automatic pipette mechanism are arranged in the reaction thermostatic bath 98.

The operation of the above apparatus is as follows. First,
The sample in the sample container 33 is transferred by the pipetting nozzle 43. That is, a fixed amount of the sample is sucked by the pipetting nozzle 43 and transferred to the sample discharge position in the sample chamber 343 in the reaction container 34a.
After being discharged into the reaction chamber and diluted as necessary, it is discharged into the reaction chamber 342 by the pipetting nozzle 43.

The pulse motor 93 rotates the reagent table 35 at a desired angle, and positions the designated reagent container at a required timing so as to stop at the suction position by the nozzle 43. The first reagent is injected by the nozzle 43 into the reaction chamber 342 of the reaction container 34a at the first reagent dispensing position (see FIG. 1). Further, the second reagent is supplied to the reaction chamber 3 of the reaction container 34a at the second reagent dispensing position by the nozzle 43.
It is injected into 42 (see FIG. 1).

Now, when the reaction container 34a reaches the first reagent dispensing position, the nozzle 43 sucks a certain amount of the first reagent into the reaction chamber 342 inside the reaction container 34a which is being transferred to the dispensing position. Dispense. When this operation is completed, the turntable 2 rotates counterclockwise by 360 ° + one pitch (1 cycle) of the reaction container and stops. Now, assuming that the time during which the turntable is rotating and stopping is 20 seconds, the above operation is repeated with 20 seconds as one cycle.
That is, the specific reaction container 34 in which the first reagent is dispensed
With respect to a, as the cycle progresses, the position of the turntable 2 in the stopped state advances counterclockwise by one pitch of the reaction container. When looking at a specific reaction container into which the first reagent has been dispensed, the second reagent is dispensed at a position where the turntable 2 is stopped, for example, a position advanced by one pitch of the reaction container. As a result, the sample, the first reagent, and the second reagent are dispensed into the reaction chamber 342 in the reaction container 34a, and the reaction proceeds.

The hybridization reaction liquid of the sample, the particle-labeled probe, and the fluorescent particle-labeled probe reacted in the reaction chamber 342 of the reaction container 34a is supplied to the reaction container 3 by the pipetting nozzle 43 at the reaction liquid dispensing position.
It is injected into the reaction chamber 341 of 4a. In the filter cleaning position, the pressure device 101 operates and the filter 346 is activated.
The unreacted fluorescent particle probe in the first reagent, which has a smaller pore size than the above, is passed through the filter 346 and discharged to the outside of the reaction container (see FIG. 1). The cleaning liquid is injected into the reaction chamber 341 by the nozzle 43 and the pressurizing device 101 is operated repeatedly to thoroughly clean the filter 346.

When the cleaning of the reaction chamber 341 is completed, as shown in FIG. 6, the turntable 2 is lowered by the operation of the driving device 95 at the container connecting position, and the container 34b on the turntable 3 is connected to the reaction container 34a. After that, drive device 9
5 operates in the opposite direction to raise the turntable 2.
After that, at the restriction enzyme solution dispensing position, the restriction enzyme solution is dispensed from the restriction enzyme solution container 38 by the nozzle 43 (see FIG. 1). After a lapse of a predetermined time, the driving device 95 is operated at the measurement liquid take-out position to process the turntable 2, and the discharge nozzle 348a of the container 34b is moved to the inlet 48 of the injection chamber 48 of the sheath flow cell 47 as shown in FIG.
, Pressurizing device 101 is activated, and the measured solution is filled with small holes 3
It is introduced into the sheath flow cell 47 from 48 (FIG. 1).
reference).

The internal structure of the sheath flow cell 47 is the same as that used in a known flow cytometer, but the reagent injection chamber equivalent to that shown in Japanese Patent Application Laid-Open No. 2-80937 is provided above. Is opened. Therefore, the liquid to be measured can be discharged into the sheath flow cell 47 from the injection chamber inlet 48 via the small hole 348. Then, the sheath liquid in the sheath liquid tank 6 is fed at a constant flow rate by the liquid feed pump 9, flows along the inner wall of the flow cell 47, and is discharged to the waste liquid reservoir 95. The liquid to be measured introduced into the flow cell 47 flows through the center of the sheath liquid flow.

The laser light source 49 has an oscillation wavelength of 488 nm.
Argon laser can be emitted, and the beam width of this laser beam is expanded by the beam expander 50, and then the laser beam is narrowed down by the lens 51 and applied to the sheath flow cell 47 so as to be focused on the flow of the liquid to be measured. . An objective lens 5 for a microscope is used to collect the fluorescence from the flow cell 47.
2 is used. A spatial filter 54 and a wavelength selection filter 55 are provided in front of the photomultiplier 53 as a photoelectric detector to remove scattered light and Raman light. The output of the photomultiplier 53 is amplified by the preamplifier 18 and then by the linear amplifier 19, and the lower limit wave height discriminator 20a is obtained.
And the upper limit wave height discriminator 20b removes noise. After that, the counter 56 integrates the pulses between the two kinds of threshold values.

A high voltage is applied to the photomultiplier 53 via the transformer 96 and the high voltage power source 97. The sample number, counting result, calibration curve, histogram of fluorescence measurement, etc. are output to the display 57, the printer 22, and the floppy disk 58. It can also communicate with a personal computer via the interface 59.

Next, a specific example will be described by taking nucleic acid analysis as an example. Since the reaction process for each measurement target item of the nucleic acid analysis is similar, the analysis operation process in the reaction container will be described here by taking the case of HBV (hepatitis B virus) as an example. The analysis item is specified by the type of particle probe added after the start of the operation.

In the fluorescent particle-labeled probe liquid container 90a, single-stranded HBV-DNA probe type 1 was bound to fluorescent-labeled latex particles (0.1 μm in diameter) containing a coumarin derivative as a fluorescent substance. Prepare a fluorescent labeled latex particle reagent. On the other hand, in the particle-bound probe liquid container 36a, a latex particle reagent (diameter of 0.
9 um) is prepared. Each single-stranded HBV-D
NA probe (single-stranded HBV-DNA probe type 1
The single-stranded HBV-DNA probe type 2) and the single-stranded HBV-DNA probe type 2) used had nucleotide sequences complementary to the nucleic acid component to be measured, but did not have nucleotide sequences complementary to each other.

As the restriction enzyme solution, as a restriction enzyme for cleaving the double-stranded DNA formed by hybridizing the single-stranded HBV-DNA probe type 1 bound to the fluorescent labeled latex particles and the nucleic acid component to be measured, For example, Hae
Prepare III.

When the analysis operation is started, the nozzle 43 sucks the fluorescent-labeled latex particle reagent from the container 60a on the reagent table 35 by the nozzle 43 and discharges it into the reaction chamber 342 in the corresponding reaction container 34a. Specifies that the reaction vessel is for HBV analysis.

The reaction vessel 34a thus prepared
Then, the sample collected by the nozzle 43 from the sample container 33 is added to the reaction chamber 342 of FIG. The HBV-DNA in the sample reacts with the fluorescent-labeled latex particles having the single-stranded HBV-DNA probe type 1 bound thereto.
The reaction was allowed to run for 15 minutes.

Next, a certain amount of the latex particle reagent is sucked from the latex particle reagent liquid container 34a by the nozzle 43 and discharged into the reaction chamber 342 of the corresponding reaction container 34a. The reaction container 34a is kept at a predetermined temperature (37 ° C.) for a certain time (1
Maintain reaction on turntable for 5 minutes. As a result, the single-stranded HBV-
DNA probe type 2 reacts.

At the reaction liquid dispensing position on the turntable 2, the pressurizing device 1 is provided above the reaction chamber 341 of the reaction container 34a.
01 is arranged. When the reaction container 34a is transferred to this position, the reaction liquid is injected into the reaction chamber 341 of the reaction container 34a by the pipetting nozzle 43. As the filter 346, a membrane filter having a pore diameter of 0.6 μm and a diameter of 10 mm was used. At the filter cleaning position, the cleaning liquid is supplied from the nozzle 43 to the reaction chamber 34 in the reaction container 34a.
The operation of injecting No. 1 and activating the pressurizing device 101 was repeated. Then, when the turntable 3 is rotated, the reaction container 43b is turned into a corresponding reaction container 34a on the turntable 2.
After being connected to the reaction chamber 341, the restriction enzyme liquid container 38 is positioned at the suction position by the rotation of the reagent table 35,
A certain amount of the liquid containing the restriction enzyme HaeIII in the container 38 is sucked by the pipetting nozzle 43 and discharged into the reaction chamber 341 of the corresponding reaction container 34a on the turntable 2. In the reaction chamber 341, the double-stranded DNA formed by hybridizing the single-stranded HBV-DNA probe type 1 bound to the fluorescent-labeled latex particles and the nucleic acid component to be measured is cleaved at a predetermined cleavage site. As a result, the fluorescent labeled latex particles float in the liquid in the reaction chamber 341. The solution to be measured containing these educts is discharged to the sheath flow cell 47 at the measurement solution take-out position by the pressurizing device 101 being operated.

Another embodiment of the reaction vessel used in the analytical method of the present invention is shown in FIG. In this example, the reaction vessel 6
0 has an integral structure, unlike that shown in FIGS. That is, the reaction container 60 is integrated with a container part having the filter 346 and a container part having the discharge nozzle 607 a having the cap 355 as the liquid flow-down resistance member and the small hole 607. In this embodiment, since the two container parts are integrated as described above, the turntable 3 is unnecessary.

Further, as another embodiment of the reaction vessel, FIG.
Alternatively, reaction vessels 34a and 34c as shown in FIG. 10 may be used. In this embodiment, the reaction container 34c is provided with a discharge nozzle 351 having a small hole 351 formed on the wall surface of the container. In this case, the automatic analyzer shown in FIG. 2 requires a suction device in addition to the pressure device 101. This suction device is connected to a discharge nozzle 351a having a small hole 351 of the container 34c when the two containers 34a and 34c are connected as shown in FIG. A pressure difference is generated before and after the cap 350, which is a member, so that the reaction liquid in the reaction chamber 341 is transferred to the container 3
Move into 4c.

The operation of the automatic analyzer shown in FIG. 2 using the reaction vessels 34a and 34c of this embodiment is as follows.

First, the sample in the sample container 33 is transferred by the pipetting nozzle 43.
A fixed amount of the sample is sucked in by the pipetting nozzle 43, and is transferred to the sample discharge position.
It is discharged into the sample chamber 343 therein, diluted as necessary, and then discharged into the reaction chamber 342 by the pipetting nozzle 43.

The pulse motor 93 rotates the reagent table 35 at a desired angle, and positions the designated reagent container at a required timing so as to stop at the suction position by the nozzle 43. That is, the first reagent is injected by the nozzle 43 into the reaction chamber 342 of the reaction container 34a at the first reagent dispensing position. Further, the second reagent is injected by the nozzle 43 into the reaction chamber 342 of the reaction container 34a at the second reagent dispensing position.

The hybridization reaction liquid of the sample, the particle-labeled probe, and the fluorescent particle-labeled probe reacted in the reaction chamber 342 of the reaction container 34a is fed to the reaction container 3 by the pipetting nozzle 43 at the reaction liquid dispensing position.
It is injected into the reaction chamber 341 of 4a. In the filter cleaning position, the pressure device 101 operates and the filter 346 is activated.
The fluorescent particle probe in the unreacted first reagent, which is smaller than the pore diameter of the above, is passed through the filter 346 and discharged to the outside of the reaction container. The cleaning liquid is injected into the reaction chamber 341 by the nozzle 43 and the pressurizing device 101 is operated repeatedly to thoroughly clean the filter 346.

When the cleaning of the reaction chamber 341 is completed, as shown in FIG. 6, the turntable 2 is lowered by the operation of the drive device 95 at the container connecting position, and the container 34c on the turntable 3 is connected to the reaction container 34a. After that, drive device 9
5 operates in the opposite direction to raise the turntable 2.
After that, the restriction enzyme solution is dispensed from the restriction enzyme solution container 38 by the nozzle 43 at the restriction enzyme solution dispensing position. After a lapse of a predetermined time, a suction device (not shown) is connected to the discharge nozzle 351a, and suction is performed through the small hole 351 so that the reaction liquid in the reaction chamber 341 is stored in the container 43c.
Move to. After that, the container 34a and the container 34c are separated, and the container 34c is moved to the measurement liquid takeout position.
At the measurement liquid take-out position, the measurement liquid is taken out of the container 34c by the nozzle 43 and introduced into the sheath flow cell 47.

As still another embodiment of the reaction container, FIG.
And a reaction container 34a and a reaction container 34 as shown in FIG.
It may be combined with f. In this case, the reaction container 34f
Is provided with a nozzle 353a having a suction hole 353 and a discharge nozzle 354a having a small hole 354 for discharging, and the measurement liquid overcomes the resistance of the cap 352 due to the suction of the small hole 353, and thus has a small hole. It is taken out from 354 and introduced into the sheath flow cell 47.

Further, the reaction container shown in FIGS. Similar to the case shown in FIG. 8, the reaction container 60 shown in FIG.
Like the above, the container portion having the filter 346, the cap 356 as the liquid flow-down resistance member, and the small holes 608, 60.
The container part having the nozzles 608a and 609a each having 9 may be integrated, and in this case, the turntable 3 is also unnecessary.

Further, the fluorescent particle-labeled probe solution or the particle-bound probe solution may be put together with the preservation solution in one of the sample chambers of the reaction container, for example, the sample chamber 345 of the reaction container 34a. In this case, the container for storing the first reagent or the second reagent is not put in, and the measurement time can be further shortened.

[0067]

According to the present invention, the B / F separation and the washing step are performed by using two kinds of fine particles having different particle diameters and a filter having a pore diameter between those particle diameters, so that the container may be tilted. It is not necessary to use a pipetting nozzle to take out the reaction solution or the washing solution, and the B / F separation and washing steps are extremely simple, which facilitates automation of measurement. Further, the separated fluorescent fine particles can be directly introduced into the flow cell, and the operation time can be shortened. Furthermore, since the labeled substance can be measured by the flow cell, highly sensitive measurement is possible.

Further, according to the present invention, the small hole of the reaction container is formed in the discharge nozzle provided at the lower end of the reaction container, and the discharge nozzle is directly inserted into the inlet of the flow chamber to perform the measurement. You can save time.

Furthermore, since the reagent containing the small particles with the labeling substance is stored in the sample chamber of the reaction container together with the storage solution, the measurement time can be further shortened.

[Brief description of drawings]

FIG. 1 is a diagram illustrating an operation procedure of an analysis method according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing the overall configuration of an analyzer according to an embodiment of the present invention.

3A and 3B are views showing an embodiment of a reaction container used in the apparatus shown in FIG. 2, FIG. 3A being a vertical sectional view and FIG. 3B being a top view.

4A and 4B are views showing an example of a reaction container used in the apparatus shown in FIG. 2, in which FIG. 4A is a vertical sectional view and FIG. 4B is a top view.

5 is a vertical cross-sectional view showing a state in which the container shown in FIG. 3 and the container shown in FIG. 4 are combined.

6A and 6B are views showing an operation procedure for connecting the container shown in FIG. 3 and the container shown in FIG. 4, where FIG. 6A is a state before being combined, FIG. 6B is a state when being combined, and FIG. Indicates the status.

FIG. 7 is a vertical cross-sectional view showing how the discharge nozzle of the reaction container shown in FIGS. 3 and 4 is inserted into a flow cell.

FIG. 8 is a vertical cross-sectional view of a reaction container according to another embodiment of the present invention.

FIG. 9 is a vertical cross-sectional view of a reaction container according to still another embodiment of the present invention.

10 is a cross-sectional view of a lower portion of the reaction container shown in FIG.

FIG. 11 is a vertical cross-sectional view of a reaction container according to still another embodiment of the present invention.

12 is a sectional view of a lower portion of the reaction container shown in FIG.

FIG. 13 is a vertical cross-sectional view of a reaction container according to still another embodiment of the present invention.

[Explanation of symbols]

1,2,3 Turntable 33 Sample container 34a, 34b Reaction container 35 Turntable (reagent table) 36a, 36b, 36c Fluorescent particle labeled probe liquid container 38 Restriction enzyme liquid storage container 42 Movable arm 43 Pipetting nozzle 46 Controller 47 Sheath flow cell 48 Injection chamber inlet 56 Counter 90a, 90b, 90c Particle labeling probe liquid container 94 Nozzle washing tank 102 Compressor 401 Sample DNA 402 Fluorescent particle labeling DNA probe 403 Non-fluorescent particle labeling DNA probe 404 Filter 405 Restriction enzyme

Claims (9)

[Claims] 1. (a) Two kinds of fine particles having different particle diameters, one of which is a small particle having a label, and the other of which is larger than the small particle. Preparing large particles, (b) binding the two types of fine particles through a measurement target in a sample,
(C) removing unbound small particles by using a filter having a pore size larger than the particle size of the small particles and smaller than the particle size of the large particles, and (d) binding through the measurement object. To specifically cleave the bound material of small particles and large particles,
(E) A method of analysis, characterized in that only small particles are separated using the filter, and (f) the concentration of the measurement object in the sample is measured by measuring the labeled substance of the small particles. 2. Two kinds of fine particles having different particle diameters, one fine particle is a small particle with a labeling substance, and the other fine particle is a large particle having a larger particle diameter than the small particle. In the analyzer for measuring the concentration of the measurement object in the sample by using the above, (a) at least one reaction chamber, which is arranged at the bottom of the reaction chamber and is larger than the particle size of the small particles, A filter having a pore size smaller than the particle size of the particles, a liquid flow-down resistance member provided below the filter, and a reaction vessel having small holes for discharging the liquid that has passed through the filter and the liquid flow-down resistance member from the reaction chamber. And (b) a turntable in which a plurality of the reaction vessels are arranged, and (c) a reaction chamber of the reaction vessel, which specifically cuts the sample, a reagent containing the two kinds of fine particles, a washing solution, and a binding site. Try to (D) Dispensing and supplying means for injecting a drug, (d) Pressure difference generating means for generating a pressure difference between the reaction chamber and the small hole, and discharging the liquid in the reaction chamber from the small hole, (e) ) A nucleic acid analyzer, comprising: a measuring unit that measures the labeled substance of the small particles contained in the liquid discharged from the small hole, and measures the concentration of the measurement target in the sample. 3. The analyzer according to claim 2, wherein the measuring means has a flow cell into which a liquid is introduced through a small hole of the reaction container, and an optical system based on a small particle marker passing through the flow cell. An analyzer characterized by measuring characteristics and measuring a concentration of an object to be measured in a sample. 4. The analyzer according to claim 3, wherein the small hole of the reaction container is formed in a discharge nozzle provided at the lower end of the reaction container, and the discharge nozzle is directly inserted into the inlet of the injection chamber of the flow cell. An analyzer characterized by. 5. Two types of microparticles having different particle sizes, one microparticle being a small particle with a label, and the other microparticle being a large particle having a larger particle size than the small particle. In the reaction container used in the analysis method for measuring the concentration of the measurement object in the sample using the above, at least one reaction chamber, arranged at the bottom of the reaction chamber, larger than the particle size of the small particles, A filter having a pore size smaller than the particle size of the large particles, a liquid flow resistance member provided further below the filter, and a small hole for discharging the liquid that has passed through the filter and the liquid flow resistance member from the reaction chamber. And a reaction container comprising: 6. The reaction container according to claim 5, wherein the first container forming the reaction chamber and arranging the filter, and the liquid flow resistance member arranged and the small hole are formed. Having one container and a second container which is a separate body,
A reaction vessel, wherein the first vessel and the second vessel can be joined at the bottom of the first vessel and the top of the second vessel. 7. The reaction container according to claim 5, further comprising a small hole for suction in addition to the small hole for discharging the liquid. 8. The reaction container according to claim 5, having a plurality of reaction chambers including the reaction chamber. 9. The reaction container according to claim 5, further comprising at least one sample chamber, wherein the sample chamber contains a reagent containing small particles having the labeled substance together with a preservative solution. Reaction vessel.