JPH0623767B2 - Method and apparatus for analyzing liquid sample - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method and an apparatus for analyzing a liquid sample, and more specifically, to a reaction of a liquid sample and a reactive substance (reagent) on a disk and the properties of the reaction product. The present invention relates to an analysis method and an apparatus thereof, which can significantly simplify equipment and operation by performing measurement and obtain highly accurate measurement results.

[Problems to be Solved by Prior Art and Invention]

In recent years, for example, liquid samples such as serum in blood are reacted with various reagents, and hormones, vitamins, new viruses or immunological substances contained in trace amounts are detected, and various diseases, especially early stages of cancer and AIDS are detected. It is hoped that it will contribute to discovery. However, the conventional method for measuring immunological substances using the antigen-antibody reaction is to impregnate a paper (film) with a reagent, apply a liquid sample (blood, urine, etc.) to this to react, and judge from the change in its color. Although the dry film method is a simple measurement method, it has a drawback that it cannot perform highly accurate analysis. On the other hand, as an analysis method using a large-scale and complicated device, for example, there is a method using an automatic chemical device described in JP-A-61-193072, in which a fluid restraining means is provided in the radial direction of a rotatable disk. A groove-shaped reaction zone having the above is formed, and a reagent is attached to the reaction zone in advance, and a liquid sample is supplied to the disk and moved in the radial direction by centrifugal force to react with the reagent, and the reaction product is appropriately reacted. It is taken out with a probe and subjected to other treatment such as gel electrophoresis to analyze necessary properties such as DNA sequencing.

Also, Japanese Patent Publication No. 54-36879 discloses a method and an apparatus for analyzing a liquid material. In this method, a plurality of cavities are provided in a radial direction of a rotatable disc, where a liquid sample and a reagent are reacted, and the obtained reaction product is further moved to a chromatographic column having a liquid phase separation medium to separate it. The reaction product that has passed through this column is received by a tube, and the tube is removed to measure the radiation dose of the contents, etc. to analyze the necessary properties.

In these analytical methods, since the reaction between the liquid sample and the reagent is performed using a rotatable disk, the reaction is efficiently performed, but the property of the reaction product is measured by measuring the reaction product on the disk. Since it is taken out of the apparatus and is used by another measuring means, it is inevitable that the apparatus becomes large-scaled and complicated, and the measuring method becomes complicated, and it takes a long time for analysis.

Further, Japanese Unexamined Patent Publication (Kokai) No. 59-193359 discloses an immunological automatic analyzer, and the measuring method by this device is to dispense a sample into a U-shaped tube and cause an antigen-antibody reaction in it. A beaded carrier on which an antigen (antibody) is fixed is charged and reacted, and the reaction solution is taken out and measured by a colorimeter or the like. Therefore, the analysis method by this method also requires a complicated mechanism such as carrier movement, washing, and separation, and the procedure is very complicated.

[Means for Solving the Problems]

In order to solve the above-mentioned problems, the inventors of the present invention have made earnest researches, and as a result, it is possible to carry out a reaction between a liquid sample and a reagent on a disk and to measure the properties of the obtained reaction product. By using the device, it was found that the equipment and operation can be remarkably simplified and the measurement can be performed with high accuracy, and the present invention has been completed based on this finding.

That is, according to the present invention, among a plurality of channels formed in the radial direction on a rotatable disk, a reactive substance is fixed to the outer peripheral portion of at least one channel, and a liquid sample is placed on the inner peripheral portion of the channel. Introducing, rotating the disk to make the liquid sample flow by centrifugal force to react with the reactive substance, and then measure the properties of the obtained reaction product on the disk. (A) A disc having a plurality of channels formed in the radial direction on the upper surface and having a reactive substance fixed to the outer peripheral portion of at least one of the channels, (B) a rotating means for the disk, (C) a means for supplying a liquid sample to the inner peripheral portion of the flow path, (D) a means for measuring the property of a reaction product on the disk, and (E) these Liquid test characterized by comprising control means And it provides a analytical device.

Hereinafter, the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram showing one mode of an apparatus suitable for carrying out the present invention.

In the figure, reference numeral 1 is a disk, and a plurality of channels 2 are provided in a groove shape in the radial direction on the upper surface thereof as shown in FIG. Various cross-sectional shapes (radial cross-sections) of the flow path 2 are conceivable, and for example, the shapes shown in FIGS. 3A to 3E are used. 3 (a) to (e) are partial cutouts of the disk showing the cross-sectional shape of the flow path 2, and the center of the disk is on the left side of the drawing. FIG. 3 (a) shows a simple groove-like flow path, FIG. 3 (b) shows one having a liquid sample dropping portion, and FIG. 3 (c) shows deep groove portions provided at predetermined intervals. FIG. 3 (d) shows the deep groove portion of FIG. 3 (c) with a step, and FIG. 3 (e) shows the groove portion having a convex portion in the middle thereof. Shows. Among these, the shape shown in FIG. 3A is particularly preferable.

Further, in FIG. 2 (a), a straight flow path is shown as the radial flow path 2, but a bent flow path or curved flow path as shown in FIG. 2 (b) (a), (b) is used. It may be a road. By doing so, the flow (movement) of the liquid sample or the washing liquid due to the centrifugal force is facilitated, and the mixing of the liquid between the channels can be prevented. Further, the flow path 2 may be as shown in FIG. 2 (b) (c).

The width and length of each of these flow paths 2 are not particularly limited, but for serum analysis and the like, the width is usually 1 to 10 mm and the length is 50 to 1 mm.
Those with a depth of 00 mm and a depth of 0.1 to 2 mm are used. In addition,
The material of this disk is not particularly limited, but for example, an injection molded product of resin such as polycarbonate, acryl, polystyrene or the like is suitable, and a surface-treated product may be used depending on the type of sample and reagent. These plural flow paths 2
The reactive substance 3 is fixed to the outer peripheral portion of at least one of the flow paths, that is, the portion close to the outer periphery of the disk.

Here, as the reactive substance 3, for example, an immunoactive substance is used, and this immunoactive substance reacts with a liquid sample described later. Usually, in order to facilitate the measurement of the properties of the reaction product, the labeled substance (second reagent) together with the reactive substance 3 (first reagent) is added to the inner peripheral portion of the channel 2, that is, in the disc. Apply to the part close to the circumference. Here, it is preferable to use the labeled antigen labeled with a known fluorescent substance such as fluorescein and rhodamines. When such a labeled antigen (second reagent) is used, an antibody that specifically reacts with the liquid sample and the labeled antigen (second reagent) is used as the reactive substance 3 (first reagent).

The reactive substance 3 can be fixed on the upper surface of the disk in the channel 2 by various methods. For example, the reactive substance 3 can be directly applied to the channel 2 or the channel 2 can be coated with an adsorbent. After that, it can be carried out by a method of supplying the reactive substance 3 for adsorption.

Further, preferably, a waste liquid treatment means 4 is provided outside the outer peripheral portion of the disc 1. As the drainage processing means 4,
For example, a peripheral weir may be provided outside the outer peripheral portion of the disk 1, and a combination of this and an appropriate drainage storage portion may be used.

Next, reference numeral 5 in the drawing is a rotating means of the disc 1,
A known disk rotating device can be used. As the rotating means 5 for this disk, one that can rotate stably up to about 3,000 rpm is preferable.

Further, in the present invention, the means 6 for supplying the liquid sample to the inner peripheral portion of the flow path 2 is provided.

The liquid sample supply means 6 may be any device that can supply a required amount (μ unit) of a liquid sample, a reagent and the like to a predetermined position of the flow path, and a dispenser with a normal position control mechanism is used.

In the present invention, various kinds of liquid samples to be analyzed can be mentioned, and it is particularly effective for analysis of liquid samples such as whole blood, blood, serum, urine and body fluid. When performing analysis using whole blood, a membrane filter (not shown) may be provided on the disc 1, blood cells and serum may be separated using this membrane filter, and serum may be used. Alternatively, serum obtained by previously removing blood cells using a centrifuge may be used.

Furthermore, in the present invention, means 7 for measuring the properties of the reaction product on the disk 1 is provided. As the measuring means 7 for measuring the properties of the reaction product, known means may be appropriately used. For example, when a fluoroimmunoassay method using a labeled antigen labeled with a fluorescent substance as the second reagent is applied, the obtained reaction product is quantified by fluorescence analysis, and therefore, a known fluorescence analysis measurement device is used. You can use it.

Further, the reaction product may be quantified by the change in concentration using a colorimeter.

The reaction product measuring means 7 is provided on the outer peripheral portion of the disk 1, but may have a movable structure if necessary.

Finally, in the present invention, the control means 8 is used in order to automate the above-mentioned means and perform the operations from reaction to measurement quickly and accurately. Particularly, when there are many flow paths on the disk, precise control means is essential. Such control means is not particularly limited as long as it can organically perform the above-mentioned operation, and a known control device may be appropriately used.

Next, the analysis method of the present invention will be described. First, the reactive component 3 is fixed to the outer peripheral portion of at least one channel 2. Here, as described above, the reaction product can be easily measured by using the labeled antigen (second reagent) together with the reactive substance 3 (first reagent). Therefore, it is preferable to apply the labeled antigen to the inner peripheral portion of the channel 2. As this labeled antigen, one labeled with a radioisotope, an enzyme, a fluorescent substance, or the like is used.

On the other hand, the liquid sample is supplied to the inner peripheral portion of the channel 2 by using the liquid sample supply means 6. When the labeled antigen (second reagent) is used, the liquid sample is supplied to the inner circumference from the place where the labeled antigen is applied. Then disk 1,
The disc sample is rotated by using the disc rotating means 5, the liquid sample is caused to flow by centrifugal force, and when the labeled antigen is applied, it is dissolved and mixed with the labeled antigen. If the mixture is sufficiently mixed, the rotation speed of the disc 1 is increased and the dissolved mixture is moved to the outer peripheral portion of the disc 1 to react with the reactive substance 3 (immobilized antibody) immobilized on the outer peripheral portion and the antigen-antibody reaction. Do.

An immunoassay suitable for the determination of trace components in blood, urine, or body fluids uses an antigen-antibody reaction that occurs between a specific antigen and an antibody, and is usually labeled with a radioactive isotope, enzyme, or fluorescence. Although a substance or the like is used, there is also a method of optically reading and quantifying the concentration change due to the reaction.

After the reaction as described above, a cleaning liquid is flowed if necessary, and a part of the reaction liquid containing unreacted substances and the like is removed by using the drainage means 4 provided outside the outer peripheral portion of the disc. Specifically, the number of revolutions of the disc 1 may be further increased to feed unnecessary liquid over the weir at the end of the disc flow path and into the drainage storage unit.

Then, with respect to the remaining reaction product, required properties are measured on the disk 1 by using the measuring means 7.
For example, when a labeled antigen labeled with a fluorescent substance is used, the fluorescence intensity of the remaining reaction product can be measured to calculate the amount of antigen in the liquid sample.

When the above-mentioned labeled antigen is not used, it may be quantified by a change in concentration due to the reaction using a measuring means such as a colorimeter.

If the liquid sample contains an antibody, it can be measured by using the labeled antigen (second reagent) as the labeled antibody and the immobilized antibody (first reagent) as the immobilized antigen. Is.

〔Example〕

 Next, the present invention will be described in detail with reference to Examples.

Example 1 A liquid sample was analyzed using the analyzer shown in FIG.

The disc 1 in this analyzer is made of polycarbonate with a diameter of 200 mm and a thickness of 2 mm, and a channel 2 (a groove depth of 1.0 mm and a width of 4 m) as shown in FIG.
m, and the length of the groove was 80 mm) were used, which had 18 pieces at equal intervals on the circumference.

A schematic diagram of this reaction is shown in FIG.

First, carcinoembryonic antigen (CEA) fluorescently labeled with Rhodamine B is applied to the inner peripheral part of the channel 2 (hereinafter referred to as zone I), and the outer peripheral part of the channel 2 (hereinafter referred to as zone II).
Say. ) Was immobilized on the channel by the physical adsorption method with the reactive substance 3 (the antigen of the liquid sample and the antibody that specifically reacts with the labeled antigen).

First, 0.2 ml of serum as a liquid sample was placed on the innermost side of Zone I.
(10% aqueous solution) is dropped, the disk 1 is rotated,
The serum and the fluorescently labeled antigen applied to Zone I were dissolved and mixed. Then, the number of revolutions of the disk was increased and the dissolved mixture in zone I was moved to zone II, and in zone II, the reactive substance 3 immobilized on the flow channel was mixed and the antigen-antibody reaction was carried out. After the reaction is completed, the cleaning liquid (pure water) is introduced into zone I, the disk is rotated again, and zone I and zone I
II was washed and unreacted antigen was released. Then Zone II
The remaining antigen, which reacted with the antibody, was quantified by a fluorometer and the CEA was determined to be 50 × 10 ⁻⁹ g / ml serum.

[Effects of the Invention] According to the method of the present invention, since the reaction of the liquid sample and the reagent on the disk and the measurement after the reaction are performed, the operability is excellent and the accuracy is good, so a small amount of the sample is sufficient
Easy to automate and continuous.

Further, according to the apparatus of the present invention, the equipment can be downsized and the operability can be improved.

Further, various inspections and analyzes can be performed by changing the shape of the (channel) of the groove of the disk, and various analyzes can be simultaneously performed by increasing the number of grooves (channel).

[Brief description of drawings]

FIG. 1 is a schematic view showing one mode of the analyzer according to the present invention. 2 (a) and 2 (b) are cross-sectional views showing an example of the shape of the flow path on the disk. 3 (a) to (e) are partially cutaway views of the disk showing the cross-sectional shape of the flow path. FIG. 4 is a schematic diagram of the reaction in the example. 1 ... Tisk, 2 ... Flow path, 3 ... Reactive substance, 4 ... Drainage means, 5 ... Disk rotation means, 6 ... Liquid sample supply means, 7 ... Reaction product measuring means , 8 ... Control means

Claims (2)

[Claims] 1. A reactive material is fixed to the outer peripheral portion of at least one of the plurality of flow channels formed in the radial direction on a rotatable disk, and a liquid sample is applied to the inner peripheral portion of the flow channel. Introducing, rotating the disk to make the liquid sample flow by centrifugal force to react with the reactive substance, and then measure the properties of the obtained reaction product on the disk. Analysis method. 2. (A) A disc having a plurality of flow paths formed in the radial direction on the upper surface, and a reactive substance being fixed to the outer peripheral portion of at least one of the flow paths,
(B) rotating means for the disk, (C) means for supplying a liquid sample to the inner peripheral portion of the flow path, (D) on the disk,
A liquid sample analyzer characterized by comprising means for measuring properties of a reaction product and (E) these controlling means.