JPH0545359A - Sample measuring apparatus - Google Patents

Abstract

PURPOSE:To separate coagulation of carrier particles to highly accurately measure immunological activation. CONSTITUTION:A wedge-shaped transparent cover member 2 with a recess 2a provided at the center on an inner side is tightly attached on a flat substrate 1 formed of a. transparent member wherein a slit is formed by the recess 2a. Height of the slit between the recess 2a and the substrate 1 is designed to be reduced uniformly. Carrier particles F are sensitized with an immunological active substance, and when a reagent wherein the carrier particles F are dispersed in a liquid medium mainly comprising water is mixed with a sample, reaction occurs so that a plurality of immunological active substances and the carrier particles F form coagulated bodies G. If the reactant solution L is injected into the slit between the substrate 1 and the recess 2a, the reactant solution L enters toward a B direction where a vertical distance is narrow due to surface tension. Since non-coagulated carrier particles F are smaller in diameter, they can move into depth, while the coagulated bodies G are trapped in the midway and suspended depending on their size.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample measuring device for qualitatively or quantitatively detecting a target substance in a sample.

[0002]

2. Description of the Related Art Latex particles, glass particles, ceramic spheres, kaolin, carbon black, animal blood components such as red blood cells, etc. can be used as a method for detecting an immunologically active substance such as an antigen or an antibody in a sample. The carrier particles such as colloidal particles are sensitized with the immunologically active substance, and the carrier particles are reacted with the sample in the liquid medium to observe and confirm the agglutination state of the reaction solution to qualify the immunologically active substance. It is well known how to detect automatically.

[0003]

However, in the above-mentioned conventional example, when the aggregated state is visually judged,
Only detection with poor quantitativeness is possible, and the accuracy and reliability of detection results are lacking.

An object of the present invention is to provide a sample measuring device having a simple structure and capable of qualitative or quantitative detection with high accuracy.

[0005]

The sample measuring device according to the present invention having the above-mentioned structure is provided with a carrier particle carrying a substance that specifically binds to a specific substance and a carrier particle in a reaction solution of the sample. A device for measuring the specific substance in a sample according to the degree of agglomeration, in which the gap decreases uniformly or stepwise from a maximum interval larger than the diameter of the carrier particles, and the reaction solution starts from the maximum interval. Is characterized in that it has a gap that can penetrate.

The analyte measuring device according to the present invention related to the above-mentioned specific invention is characterized by the degree of aggregation of the carrier particles in the reaction solution of the carrier particles carrying the substance that specifically binds to the specific substance and the sample. A device for measuring the specific substance in a sample, wherein a gap decreases uniformly or stepwise from a maximum gap larger than the diameter of the carrier particles, and a gap into which the reaction solution can enter from the maximum gap portion. Part, a detection means for detecting carrier particles in the reaction liquid that has penetrated into the gap, and an operation means for performing a quantitative or qualitative measurement operation of the specific substance based on the output of the detection means. It is characterized by that.

[0007]

In the analyte measuring device having the above-mentioned configuration, when the reaction solution is injected into the gap from the opening of the maximum gap part, carrier particles and aggregates having different sizes are separated due to the difference in spacing, and the degree of aggregation is clearly discriminated. Can be identified.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail based on the illustrated embodiments. FIG. 1 is an external perspective view of the sample table of the first embodiment, and FIG. 2 is a vertical sectional view taken along the line AB of FIG. A wedge-shaped cover member 2 formed of a transparent member and having a recess 2a inside the center thereof is closely adhered onto a flat plate-shaped substrate 1 formed of a transparent member, and a gap is formed by the recess 2a. As shown in FIG. 2, the recess 2a is formed so that the height of the gap between the recess 2a and the substrate 1 is uniformly reduced from the A direction to the B direction, and the vertical spacing DB of the end opening is the carrier used. The diameter is smaller than the diameter R of the particle F, and the vertical gap DA of the opening at the end of the direction is such that the aggregate G can also pass through.
It is set to several times to several hundred times the vertical interval DB.

When the colored carrier particles F are sensitized with an immunologically active substance such as a monoclonal antibody and the carrier particles F are dispersed in a liquid medium mainly composed of water and a specimen such as serum is mixed. When an antigen that specifically reacts with a monoclonal antibody is present in the serum, an antigen-antibody reaction occurs, and a plurality of immunologically active substances and carrier particles F form aggregates G. After sufficiently reacting, as shown in FIG. 3, the reaction liquid L is applied to the gap A between the substrate 1 and the recess 2a.
When injected from the direction, the reaction liquid L intrudes in the direction B with a narrow vertical interval due to the surface tension. Since the unaggregated single carrier particles F have a small diameter, they can move deep in the B direction, but the agglomerates G are trapped and cannot move depending on their size.

The diameter of the aggregate G determined by the number of carrier particles F constituting one aggregate G and the number of the aggregates G trapped in a certain gap are determined by the immunology contained in the reaction solution L. And the number of the chemically active substance, that is, the aggregation state of the aggregate G produced by the reaction. Therefore, when the reaction liquid L flows into such a gap, the amount of the carrier particles F trapped in the gap R equal to the diameter R of the single carrier particle F, the position where the aggregate G is trapped, and the position thereof. The amount can also be easily discriminated and identified visually, and qualitative or quantitative detection of the immunologically active substance can be performed. In practice, a calibration curve is prepared in advance from the reaction solution L that has been reacted with a calibration sample containing a known immunologically active substance, and the calibration curve is compared with that to perform quantification.

Either one of the substrate 1 and the cover member 2 may be an opaque member, and a color tone that facilitates identification depending on the color tone of the colored carrier particles F, for example, if the carrier particles F are a light color system, a member is used. It is also possible to make the identification easy by devising a dark color system. Further, if a substance having a good affinity with the liquid medium of the reaction liquid L is coated on the surface of the gap so that the reaction liquid L easily enters the gap, a better measurement result can be obtained. As the coating material, for example, when the liquid medium is water, a hydrophilic substance, a surfactant, a water-soluble polymer such as methyl cellulose, carboxymethyl cellulose, polyvinyl alcohol, and polyacrylamide are preferable.

Further, if the single particles F emit fluorescence, the measurement can be performed more effectively. The fluorescent carrier particles used are obtained, for example, by the following method.
As the material for the carrier particles, conventionally used materials such as polymer particles, glass particles, ceramic spheres, kaolin and carbon black can be used. Among them, polymer particles are preferable because they are easily made into fluorescent particles. The fluorescent particles are appropriately obtained depending on the material of the carrier particles, the type of the fluorescent substance, etc., but when polymer particles such as polystyrene, polyacrylic acid ester, polyester, polycarbonate and polyamide are taken as an example, fluorescent light will be added to these polymer particles. There are a method of mixing a substance exhibiting properties, for example, a fluorescent dye, and fixing the fluorescent dye by a chemical bond, a physical bond or the like, a method of melting and kneading polymer particles and the fluorescent dye, and the like.

In this embodiment, the substrate 1 is installed horizontally and the reaction solution L is injected in the horizontal direction. However, the measurement is performed with the substrate 1 standing vertically with the direction A in FIG. 1 facing up. It may be performed, and the invasion of the reaction liquid L is promoted by the effect of gravity, and a good measurement result is obtained. As shown in FIG. 4, a gap 3a whose diameter is uniformly reduced in the downward direction may be vertically provided inside the base 3 partially transparent.

FIG. 5 is a block diagram of the sample table of the second embodiment, and FIG. 6 is a vertical sectional view taken along the line AB of FIG. On the flat plate-shaped substrate 1 formed by the transparent member, a flat plate-shaped cover member 2 formed by a transparent member and having a recessed portion 2b in the center is closely attached to the substrate 1 to form a gap. As shown in FIG. 6, the recess 2b is configured such that the height of the gap between the recess 2b and the substrate 1 is reduced from the A direction to the B direction in, for example, four steps, and the vertical spacing of the openings at the B direction ends. DB is smaller than the diameter of the carrier particles F to be used, and the vertical interval DA of the opening at the end in the A direction is set to several times to several hundred times the vertical interval DB so that the aggregate G can pass through. There is.

Also in the case of this embodiment, when the reaction solution L is injected from the direction A, the aggregate G is trapped on the way as shown in FIG. 7, and thus the immunologically active substance is also qualitatively and quantitatively determined. However, for that purpose, the vertical interval of the concave portions 2b needs to be changed in at least three steps.
Also, in the same manner as the first embodiment, the substrate 1 may be vertically used in this embodiment as well, and as shown in FIG. The decreasing gap 3b may be provided vertically.

FIG. 9 is a block diagram of the third embodiment, and FIG.
Reference numeral 0 is a vertical sectional view taken along the line AB of FIG. Similar to the first and second embodiments, the flat plate-shaped cover member 2 formed of the transparent member and having the recess 2c inside the center thereof is provided on the flat-plate-shaped substrate 1 formed of the transparent member. To form a gap. As shown in FIG. 10, the concave portion 2c has a vertical gap DB in which the vertical distance between the concave portion 2c and the substrate 1 is uniformly reduced from the A direction to the B direction and is smaller than the diameter of the carrier particles F used. From the above, the width of the gap is made constant, and the volume of the gap portion SB of the vertical gap DB is substantially equal to the volume of the gap portion SA having a larger vertical gap than the vertical gap DB, or
It is designed to be larger than that. Note that, as in the first embodiment, the vertical distance DA of the opening at the end in the A direction is several times to several hundreds of the vertical distance DB so that the aggregate G can also pass through.
Is doubled.

Also in this embodiment, the reaction liquid L is added to the substrate 1.
When injected from the direction A into the gap between the cover member 2 and
Due to the surface tension, the reaction liquid L intrudes in the direction B having a narrow vertical interval, the carrier particles F and the aggregates G are trapped at positions corresponding to their diameters, and only the liquid mixture of the liquid medium and the specimen has a vertical interval. Move to the gap SB of DB. Since the volume of the gap SB is large, most of the mixed liquid unnecessary for detection flows into this, and in the gap SA having a large vertical gap, only the trapped carrier particles F and aggregates G can be more easily. It can be detected and a good measurement result can be obtained. The vertical gap DB may have any shape as long as the volume condition is satisfied.

Also in this embodiment, it is possible to vertically inject the reaction liquid with the substrate 1 standing upright, and as shown in FIG. The space SB has a diameter that decreases and extends from the position of the diameter DB smaller than the diameter of the carrier particles F at intervals DB in two directions orthogonal to the cross section.
The same effect can be obtained by trapping the carrier particles F in the space SA above the diameter DB and flowing only the mixed liquid of the liquid medium and the sample into the space SB below the space SA. .. Even in this case, the gap portion SB below the diameter DB can have an arbitrary shape even if the gap is equal to or smaller than DB, as long as the above-mentioned volume condition is satisfied.

FIG. 12 is a block diagram of the fourth embodiment, in which the height of the clearance SA in the case of FIG. 9 in the recess 2d of the cover member 2 is set to three levels, and the clearance SB of the clearance DB is rearward. It is provided in.

Also in this embodiment, the same effect as the second and third embodiments can be obtained, and the gap 3d can be changed in the vertical direction by the base 3 as shown in FIG. ..

By the way, in the above third and fourth embodiments, the vertical spacing SB may be set to be slightly larger than the diameter of the carrier particles F, for example, within twice. In this case, the non-aggregated particles, that is, the carrier particles F are not trapped and the gap SB
Therefore, it is possible to more clearly and easily distinguish between aggregation and non-aggregation, and it is possible to obtain a better measurement result.

FIG. 14 is an external perspective view of the fifth embodiment, and FIG. 15 is a vertical sectional view taken along the line AB of FIG. Although the convex lens 4 is formed on the upper surface of the cover member 2 in the first embodiment, this is not limited to the convex lens 4 and may be a Fresnel lens or the like.

In this embodiment, since the convex lens 4 is provided, the inside can be magnified and observed, and the degree of aggregation of the reaction solution can be determined more clearly.

FIG. 16 shows the base 3 of FIG. 4 according to the sixth embodiment.
A convex lens is formed on the base. FIG. 17 is the base 3 of FIG. 5 according to the seventh conventional example, FIG. 18 is the base 3 of FIG. 8 according to the eighth embodiment, and FIG. 19 is the ninth embodiment. Similarly, convex lenses 4 are formed on the base 3 of FIG. 9 so that the state of the reaction solution can be clearly observed through the convex lenses 4.

FIG. 20 is a block diagram of the tenth embodiment for automatically reading the state of the reaction solution, and FIG. 21 is a sectional view of the optical system. The sample table 10 used here is the same as that of the first embodiment shown in FIG.

In order to optically detect the single particles F which emit fluorescence and which are injected into the space of the sample table 10, a fluorescent carrier particle is excited above the sample table 10 through a bandpass filter. An imaging optical system 12 including an imaging lens and a gradient index lens is disposed below the light source 11 and the sample table 10, and a light receiving optical system 13 is provided at the imaging position. The light receiving optical system 13 includes a frame 13
A CCD array 13b in which 2048 photosensitive elements of 14 μm × 14 μm are one-dimensionally arranged is arranged inside a, and the CCD array 13b is protected by a transparent glass protection plate 13c attached to a frame 13a.
The cable 14 outputs the respective photosensitive elements of the CCD array 13b.
Connected to the signal processing device 15 via the signal processing device 1
The output of 5 is connected to the monitor 16.

The internal structure of the signal processor 15 is as shown in FIG. 22, and the output of the CCD array 13b is C
Connected to the CD driver circuit 15a and the arithmetic circuit 15b,
The output of the CCD driver circuit 15a is connected to the arithmetic circuit 15b, the output of the arithmetic circuit 15b is connected to the display circuit,
The output of the display circuit 15c is connected to the monitor 16.

The carrier particles F that emit fluorescence are sensitized with an immunologically active substance such as a monoclonal antibody, and the carrier particles F are sensitized.
When the reagent and the sample dispersed in a liquid medium mainly containing water are mixed, a reaction occurs, and a plurality of immunologically active substances and carrier particles F form aggregates G. After sufficient reaction, as shown in FIG. 23 (a), when this reaction solution L is injected into the gap between the substrate 1 and the recess 2a from the direction A, the reaction solution L has a narrow vertical interval due to surface tension. Infiltrate in the B direction. Since the unaggregated single carrier particles F have a small diameter, B
Although the aggregate G can be moved to the back in the direction, the aggregate G is trapped midway depending on its size and cannot be moved.

The fluorescence image of the reaction liquid L in the recess 2a of the sample table 10 is transferred to the CCD of the light receiving optical system 13 by the image forming optical system 11.
An image is formed on the array 13b, and the CCD driver circuit 15a is formed.
Is photoelectrically converted by and the output voltage value of each photosensitive element is input to the arithmetic circuit 15b.

FIG. 23 (b) shows the output voltage of each photosensitive element corresponding to the separated state image shown in FIG. 23 (a). The output voltage at the portion where they are trapped by the fluorescence emitted by the single particles F and the aggregate G is shown. Becomes larger and its presence is detected.

In practice, a calibration curve is prepared in advance from the reaction solution L that has been reacted with a calibration sample containing a known immunologically active substance, and the calibration curve is compared with that to perform quantification. As the calculation performed by the calculation circuit 15b, for example, the maximum values h1, h2, h3, h4 of the output voltage and the widths d1, d2,
Compare the distribution of d3, d4, etc. with that of the calibration curve. Alternatively, the number of photosensitive elements whose power voltage value is higher than the threshold value Vs may be counted and compared more simply. The processing method is not limited to the above method, and the calculation processing result is displayed on the monitor 16 via the display circuit 15c.

FIG. 24 shows a modification of the tenth embodiment,
A laser scanning optical system 17 is used in place of the light source 9, and the laser light emitted from the laser light source 17a is deflected and scanned by the polygon mirror 17b.
The reaction liquid in the sample table 10 is irradiated by c.

[0033]

As described above, the sample measuring device according to the present invention has a gap that is uniformly larger than the diameter of the carrier particles and has a gap that is reduced in three or more steps from the minimum gap to the minimum gap. It has a simple structure, and when the reaction solution is injected into this gap from the opening with the maximum distance, carrier particles and aggregates that differ in size due to the difference in distance can be separated, and the qualitative analysis of the immunologically active substance in the specimen is possible. Alternatively, quantitative detection can be performed with high accuracy.

[Brief description of drawings]

FIG. 1 is a perspective view of a first embodiment.

FIG. 2 is a vertical sectional view.

FIG. 3 is an explanatory diagram of a measurement principle.

FIG. 4 is a perspective view of a modified example.

FIG. 5 is a perspective view of a second embodiment.

FIG. 6 is a vertical sectional view.

FIG. 7 is an explanatory diagram of a measurement principle.

FIG. 8 is a perspective view of a modified example.

FIG. 9 is a perspective view of a third embodiment.

FIG. 10 is a vertical sectional view.

FIG. 11 is a perspective view of a modified example.

FIG. 12 is a perspective view of a fourth embodiment.

FIG. 13 is a perspective view of a modified example.

FIG. 14 is a perspective view of a fifth embodiment.

FIG. 15 is a vertical sectional view.

FIG. 16 is a perspective view of a sixth embodiment.

FIG. 17 is a perspective view of a seventh embodiment.

FIG. 18 is a perspective view of an eighth embodiment.

FIG. 19 is a perspective view of the ninth embodiment.

FIG. 20 is a configuration diagram of a tenth embodiment.

FIG. 21 is a sectional view of an optical system portion.

FIG. 22 is a configuration diagram of a signal processing device.

FIG. 23 is an explanatory diagram of an optical measurement principle.

FIG. 24 is a perspective view of a modified example using a laser scanning optical system.

[Explanation of symbols]

 1 substrate 2 cover member 2a-2d concave part 3 base 3a-3d gap 4 convex lens 10 sample stand 11 light source 12 imaging optical system 13 light receiving optical system 13b CCD array 14 cable 15 signal processor 15a CCD driver circuit 15b arithmetic circuit 15c display Circuit 16 Monitor 17 Laser scanning optical system

 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Toshikazu Onishi 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Hideto Takayama 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (10)

[Claims] 1. An apparatus for measuring the specific substance in a sample according to the degree of agglomeration of the carrier particle in a reaction liquid of a sample and a carrier particle carrying a substance that specifically binds to the specific substance. In addition, the sample measuring device is characterized in that the gap is uniformly or stepwise reduced from the maximum distance larger than the diameter of the carrier particles, and the reaction solution has a gap into which the reaction liquid can enter. 2. An apparatus for measuring the specific substance in a sample according to the degree of agglomeration of the carrier particle in a reaction liquid of the carrier particle carrying a substance that specifically binds to the specific substance and the sample. Then, the gap decreases uniformly or stepwise from the maximum gap larger than the diameter of the carrier particles, and the gap part into which the reaction liquid can enter from the maximum gap part, and the reaction liquid in the gap part An analyte measuring device comprising: a detection unit that detects carrier particles; and a calculation unit that performs a quantitative or qualitative measurement calculation of the specific substance based on the output of the detection unit. 3. The sample measuring device according to claim 1, wherein the gap portion has openings at two ends of a maximum gap portion and a minimum gap portion. 4. The sample measuring device according to claim 3, wherein a liquid suction means is arranged outside the opening of the minimum gap portion. 5. The sample measuring device according to claim 1, wherein at least one surface surrounding the gap is transparent. 6. The sample measuring device according to claim 5, wherein the transparent surface has a lens function. 7. The sample measuring device according to claim 5, wherein the detecting means optically detects. 8. The sample measuring device according to claim 7, wherein the detection unit has an array of light receiving elements. 9. The sample measuring device according to claim 1, wherein the carrier particles are colored particles or fluorescent particles. 10. The analyte measuring device according to claim 1, wherein the specific substance is an antigen, and a monoclonal antibody that specifically reacts with the antigen is carried on the carrier particles.