JPH05288751A - Subject measuring apparatus - Google Patents

Abstract

PURPOSE:To measure highly accurate immunological activity by separating an agglutinated substance of carrier particles two-dimensionally. CONSTITUTION:A wedge-shaped cover member 3 formed of a transparent member with a recess 3a provided at an inner center is tightly attached on a flat substrate 2 formed of a transparent member, while the recess 3a forms a gap. The recess 3a has its height of the gap between the recess 3a and the substrate 2 reduced uniformly from an A direction to a B direction, a vertical distance DB of an opening at the end of the B direction is smaller than a diameter R of a carrier particle F to be used, and a vertical distance DA of the opening at the end of the A direction is several to several hundreds times the vertical distance DB so that an agglutinated substance G can pass through it. The wedge-shaped cover member 3 comprises wedge members 3b, 3c having electric conductivity and a transparent substrate 3d. In addition the wedge members 3b, 3c are opposite electrodes for forming an electric field in the recess 3a.

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]

A sample measuring device according to the present invention having the above-mentioned structure is provided with a carrier particle carrying a substance which specifically binds to a specific substance and a carrier particle in a reaction solution of the sample. Depending on the degree of aggregation, in the device for measuring the specific substance in the sample, the gap decreases uniformly or stepwise from the maximum interval larger than the diameter of the carrier particles, and the reaction solution enters from the maximum interval part. And a counter electrode for applying an electric field in a direction perpendicular to the inflow direction of the reaction liquid, and a means for applying a voltage to the counter electrode.

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

[0007]

In the analyte measuring device having the above-mentioned structure, when the reaction solution is injected into the gap from the opening of the maximum gap part, carrier particles and agglomerates having different sizes are separated by the gap difference, and the degree of agglutination is clarified. 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 1, and FIG.
FIG. 4 is a vertical cross-sectional view taken along line AB of FIG. On the flat plate-shaped substrate 2 formed of a transparent member, a wedge-shaped cover member 3 formed of a transparent member and having a concave portion 3a inside the center is closely attached, and a gap is formed by the concave portion 3a.
As shown in FIG. 2, the recess 3a is configured such that the height of the gap between the recess 3a and the substrate 2 is uniformly reduced from the A direction to the B direction, and the vertical interval DB of the opening at the B direction end is used. The diameter R is smaller than the diameter R of the carrier particles F, and the vertical distance DA of the opening at the end in the A direction is about several times to several hundred times the vertical distance DB so that the aggregate G can also pass through. The wedge-shaped cover member 3 is composed of wedge-shaped members 3b and 3c having electric conductivity and a transparent substrate 3d, and the recessed portions 3a of the wedge-shaped members 3b and 3c are opposed electrodes 3e and 3f.

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 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, when the reaction liquid L is injected into the gap between the substrate 2 and the concave portion 3b from the A direction, the reaction liquid L intrudes in the B direction with a narrow vertical distance due to surface tension. Do it. Since the non-aggregated single carrier particles F have a small diameter, they can move deep in the B direction,
The aggregate G is trapped midway depending on its size and cannot move, and the aggregate G is separated in the AB direction.

Further, applying a voltage to the counter electrodes 3e, 3f to cause an electric field to act thereon to separate the aggregate G is the same as in the previous embodiment. At this time, if a voltage is applied between the counter electrodes 3e and 3f, the carrier particles F or the aggregates G are separated by the force of the electric field E. Since the electric field E acts perpendicularly to the infiltration direction into the reaction solution, the carrier particles F and the aggregates G are separated two-dimensionally as a result of the separation due to the size of the gap of the recess 3a and the action of the electric field. .. In addition, the aggregate G
The mobility due to ionization depends on the size of the aggregate, the positive / negative of the charge, and the size, and the specific gravity. The positive and negative and magnitude of the charge depend on the trace components contained in the sample, the chemical composition such as ionicity of the carrier particles F and the pH of the reaction solution, and the specific gravity is such as the material composition of the carrier particles F and the specific gravity of the reaction solution. Depends on.

As shown in FIG. 4, when the force due to the electric field E is greater for unaggregated bodies, when the direction of the electric field E is from C to D, they are distributed in the direction from C to D in the descending order of aggregation. As a result, the aggregate G has a two-dimensional distribution. Therefore, it becomes easy to visually observe the trapped positions of the aggregate G and the carrier particles F and the number thereof.

The diameter of the aggregate G is determined by the number of the carrier particles F constituting one aggregate G. The aggregation state of the aggregate G generated by the reaction, that is, the number of carrier particles F forming the aggregate G and the number of the aggregate G in the reaction solution are determined by the concentration of the immunologically active substance contained in the reaction solution L. And depends on the nature. Therefore, when the reaction liquid L flows into the gap,
By visually identifying and identifying the positions where the carrier particles F and the aggregates G are trapped and the number thereof, it is possible to perform qualitative or quantitative detection of the immunologically active substance. 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 target immunologically active substance is quantified by comparison with the calibration curve. ..

As a device for obtaining good measurement results, either the substrate 2 or the cover member 3 may be an opaque member which is colored in contrast with the color tone of the carrier particles F. For example, the carrier particles F are a light color system. In that case, it is possible to make the member easy by identifying the member as a dark color system.

Further, if the surface of the gap is coated with a substance having a good affinity with the liquid medium of the reaction liquid L so that the reaction liquid L can easily enter the gap, a better measurement result can be obtained. As the coating material, for example, when the liquid medium is water,
Hydrophilic substances, surfactants, water-soluble polymers such as methyl cellulose, carboxymethyl cellulose, polyvinyl alcohol, and polyacrylamide are preferred.

Further, in this embodiment, the substrate 2 is installed horizontally and the reaction solution L is injected in the horizontal direction. However, with the direction A in FIG. The measurement may be performed, and the penetration of the reaction liquid L is promoted by the effect of gravity, and a good measurement result is obtained.

Further, when the carrier particles F emit fluorescence, the measurement can be performed optically. The fluorescent carrier particles used are obtained, for example, by the following method. As the material for the carrier particles, polymer particles, glass particles, ceramic spheres, kaolin, carbon black and the like have been conventionally used, but among them, polymer particles are preferable because they are easily made into fluorescent particles. The method of obtaining the fluorescent particles, the material of the carrier particles,
It may be appropriately selected depending on the type of fluorescent substance. Taking polymer particles such as polystyrene, polyacrylic acid ester, polyester, polycarbonate, and polyamide as an example, a substance that emits fluorescence, such as a fluorescent dye, is mixed with these polymer particles, and the fluorescent dye is fixed by a chemical bond or a physical bond. There is a method of melting and kneading polymer particles and a fluorescent dye.

FIG. 5 is a block diagram of the sample table 1 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 2 formed by the transparent member, the flat plate-shaped cover member 3 formed by the transparent member and having the concave portion 3a inside the center is closely attached to the substrate 2 to form a gap. The recess 3a is formed such that the height of the gap between the recess 3a and the substrate 2 decreases from the A direction to the B direction in, for example, four steps, and the vertical spacing DB of the opening at the end in the B direction is the carrier particle to be used. The diameter is smaller than the diameter of F, and the vertical distance DA of the opening at the end in the A direction allows the aggregate G to pass through.
It is set to several times to several hundred times the vertical interval DB. Opposite electrodes 3e and 3f are provided on both side surfaces of the recess 3a, and electrodes 3g and 3h are formed on the side surface of the cover member 3 on the B side by aluminum vapor deposition. The counter electrodes 3e and 3f are electrically connected to the electrodes 3g and 3h, respectively, and serve as extraction electrodes. A conductive paste is used for the adhesive portion.

The operation and effect of the second embodiment are almost the same as those of the first embodiment, and the size of the aggregate G is separated stepwise as shown in FIG.

FIG. 8 is a constitutional view of an embodiment for automatically reading the state of aggregates in a reaction solution which is made to emit fluorescence. The sample table 1 is the same as the sample table 1 of FIG. 1, and in order to optically detect single particles or the like which emit fluorescence and are injected into the gap of the sample table 1, the sample table 1 is provided above the sample table 1. A light source 5 for exciting the fluorescent carrier particles through a band pass filter, and an image forming optical system 6 including an image forming lens, a gradient index lens, and the like are arranged below the sample stage 1, and the image forming position thereof. Is provided with a light receiving optical system 7.
The light receiving optical system 7 includes, for example, 768 × 4 inside the frame 7a.
A CCD array 7b in which 2048 photosensitive elements 93 are two-dimensionally arranged is arranged. The CCD array 7b is a frame 7a.
It is protected by a transparent glass protection plate 7c attached to the. The output of each photosensitive element of the CCD array 7b is connected to the signal processing device 9 via the cable 8, and the output of the signal processing device 9 is connected to the monitor 10. The counter electrodes 3e and 3f provided in the recess 3a are connected to the power supply 11 via the cable 12, and the output of the power supply 11 is controlled by the waveform generator 13.

FIG. 9 shows the internal structure of the signal processing device 9,
The output of the CCD array 7b is connected to the CCD driver circuit 9a and the arithmetic circuit 9b, the output of the CCD driver circuit 9a is connected to the arithmetic circuit 9b, the output of the arithmetic circuit 9b is connected to the display circuit 9c, and the output of the display circuit 9c. Is monitor 10
It is connected to the.

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 a reagent and a sample prepared by dispersing water in a liquid medium mainly containing water are mixed, a plurality of immunologically active substances and carrier particles F are obtained.
And form an aggregate G. When the reaction liquid L is injected into the gap of the sample table 1 after sufficiently reacting, the reaction liquid L intrudes in the depth direction with a small vertical interval due to the surface tension.
Since the unaggregated single carrier particles F have a small diameter, they can move to the inner part, but the agglomerates G are trapped and cannot move depending on the size.

The fluorescent image of the reaction solution L in the sample stage 1 is imaged on the CCD array 7b of the light receiving optical system 7 through the imaging optical system 6, photoelectrically converted by the CCD driver circuit 9a, and the CCD array. The output voltage value of each photosensitive element 7b is input to the arithmetic circuit 9b.

FIG. 10 shows the output voltage of each photosensitive element of the CCD array 7b corresponding to the separated state image of the aggregate G shown in FIG. Due to the fluorescence emitted by the single particles F and the aggregates G, the output voltage increases at the trapped portion, and its presence is detected.

In practice, the calibration is performed in advance according to the two-dimensional position information of the aggregate G in the reaction solution L reacted with the calibration sample containing the known immunologically active substance and the intensity of the output voltage. The target immunologically active substance is quantified by preparing a line and comparing it.

In the present invention, different immunologically active substances are sensitized to the two types of carrier particles F1 and F2, respectively, and the sensitized two types of carrier particles F1 and F2 are used in the sample. It is possible to inspect many items of the trace substances.

FIG. 11 shows two types of carrier particles F having different specific gravities.
9 shows a state in the concave portion 3a when a reaction solution obtained by mixing a reagent containing 1, F2 and a sample and reacting them is measured by the sample measuring device in FIG. If the magnitude of the applied electric field E is properly selected, 2
The aggregate G can be separated in the CD direction depending on the specific gravity of the seed carrier particles F1 and F2. This secondary separation state is detected by the two-dimensional light receiving element, and is displayed on the monitor 10 via the signal processing device 9.

[0027]

As described above, the sample measuring device according to the present invention has a gap portion that is uniformly or stepwise reduced from the maximum interval to the minimum interval that is sufficiently larger than the diameter of the carrier particles, and the reaction solution. Has a structure in which a counter electrode for applying an electric field is provided in a direction perpendicular to the infiltration direction of the carrier particles. Since the aggregates can be separated two-dimensionally, the measurement sensitivity is improved, and the immunologically active substance in the sample can be detected qualitatively and quantitatively with high accuracy. In addition, qualitative and quantitative detection of multiple immunologically active substances can be easily performed at the same time.

[Brief description of drawings]

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

FIG. 2 is a vertical sectional view of a sample table.

FIG. 3 is an explanatory diagram of a separated state of aggregates in a gap portion of a sample table.

FIG. 4 is an explanatory diagram of a separated state of aggregates by an electric field.

FIG. 5 is a perspective view of a sample table according to a second embodiment.

FIG. 6 is a vertical sectional view of a sample table.

FIG. 7 is an explanatory diagram of a separated state of aggregates in a gap portion of a sample table.

FIG. 8 is a configuration diagram of a third embodiment.

FIG. 9 is a configuration diagram of a signal processing device.

FIG. 10 is an explanatory diagram showing, as a model, an output voltage of a photosensitive element corresponding to a separated state of aggregates.

FIG. 11 is an explanatory diagram of a separated state of an aggregate due to an electric field when two types of carrier particles are used.

[Explanation of symbols]

 1 sample table 2 substrate 3 cover member 3a recess 3e to 3h electrode 5 light source 6 imaging optical system 7 light receiving optical system 7b CCD array 8 cable 9 signal processing device 9a CCD driver circuit 9b arithmetic circuit 9c display circuit 10 monitor 11 power supply 13 waveform Generator

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazumi Tanaka 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Ken Ken Miyazaki 3-30-2 Shimomaruko, Ota-ku, Tokyo Kya Non Inc. (72) Inventor Hiroaki Hoshi 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (2)

[Claims] 1. An apparatus for measuring the specific substance in a sample according to the degree of aggregation of the carrier particle in a reaction solution of a carrier particle carrying a substance that specifically binds to a specific substance and a sample, The gap decreases uniformly or stepwise from the maximum spacing larger than the diameter of the carrier particles, and the gap into which the reaction solution can enter from the maximum spacing, and the electric field in the direction perpendicular to the intrusion direction of the reaction solution. An analyte measuring device comprising: a counter electrode for applying a voltage and means for applying a voltage to the counter electrode. 2. An apparatus for measuring the specific substance in a sample according to the degree of agglomeration of the carrier particle in a reaction solution of a carrier particle carrying a substance that specifically binds to a specific substance and a sample, A gap portion in which the gap is uniformly or stepwise reduced from the maximum gap larger than the diameter of the carrier particles, and the reaction liquid can infiltrate from the maximum gap portion, and the carrier particles in the reaction liquid that have penetrated into the gap portion. 2. A sample measuring apparatus, comprising: a detection unit that detects the two-dimensional distribution of the above; and a calculation unit that performs a quantitative or qualitative measurement calculation of the specific substance based on the output from the detection unit.