JPH0436637A - Method and instrument for measuring sample - Google Patents

Abstract

PURPOSE:To take an accurate measurement by providing both a process wherein a carrier on which a specific reaction material for a target material reacts and a sample are mixed and a process wherein the mixture is irradiated with light with an intensity gradient to increase the agglutination reaction efficiency of the carrier. CONSTITUTION:The irradiation luminous flux with the intensity gradient is split by a beam splitter 4; and a photodetector 7 detects the intensity of one beam and the other beam is passed through a liquid-dipped objective 13 to form a beam waist 11 with an intensity gradient at a part to be inspected in a reaction cell 31. Then latex particles are converged on nearby the center of the beam waist 11 by an optical trapping phenomenon to accelerate the agglutination. Consequently, when the concentration of the latex particles is low totally, partial concentration is made high to cause agglutination, thereby enabling high-sensitivity measurement.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to the measurement of a target substance in a specimen by causing an agglutination reaction of a carrier by the action of specific binding such as an antigen-antibody reaction. Regarding the field.

[Prior Art] Conventionally, methods have been known for accurately measuring a target substance in a specimen by utilizing a reaction that specifically binds to the target substance, such as an antigen-antibody reaction. This involves preparing a reagent with a predetermined concentration that contains a substance such as a monoclonal antibody that specifically binds to a specific antigen, which is the target substance, sensitized on the surface of carrier particles such as latex particles, and then applying this reagent to serum etc. The carrier particles are mixed with a specimen to cause binding and aggregation between the carrier particles through an antigen-antibody reaction, and the carrier particles are left at a constant temperature for a sufficient period of time (usually about 20 to 30 minutes) to perform a so-called incubation. Thereafter, the target antigen in the serum is qualitatively or quantitatively measured by mainly measuring the aggregation state of the carrier using an optical method. This is generally called a particle immunoassay method and is widely known.
693, JP 54-108 [i94, JP 54-108695, JP 54-1094]
It is described in detail in JP-A No. 94, JP-A-55-159157, JP-A-62-81567, and the like.

However, in the conventional example described above, during incubation, during the process in which the carriers aggregate to form aggregates, the contact between the carriers is largely due to the Brownian motion of the carriers themselves, which is inefficient, especially when the carrier concentration is low. There was a problem that it took time.

Therefore, the applicant of the present application previously proposed in Japanese Patent Application No. 2-109328 a method of promoting aggregation of carriers by utilizing the optical trapping phenomenon.

The present invention effectively increases the aggregation reaction efficiency with a simple method,
The purpose of this study is to provide a method that can measure the results with even greater precision.

[Means for achieving the object] The outline of the present invention for achieving this object is to mix a carrier sensitized with a substance that specifically reacts with a target substance with a sample containing the target substance to form a mixture. a step of increasing the aggregation reaction efficiency of the carrier by irradiating the mixture with light having an intensity gradient and concentrating the carrier near the light irradiation position by the light pressure; flif a certain state of the carrier in the finished mixture of
It is characterized by having a step of qualitatively or quantitatively measuring the target substance by detecting it as fi information and analyzing the image.

[Example] In explaining the present invention in detail, the basic principle of the present invention will first be explained using FIG. 1.

Generally, as shown in Figure 1(a), when very small particles are dispersed in a liquid, a beam with an intensity gradient, such as a laser beam with a Gaussian distribution, is used in the liquid. It is known that when a waist is formed, as shown in FIG. 1(b), a centripetal force is exerted by the optical pressure of the laser beam, causing floating particles to gather near the center of the beam waist. This is generally called an optical trap, and is a useful method for moving or aggregating particles in a fluid to a specific location. The basic principle of the present invention is to utilize this optical trap phenomenon to concentrate carrier particles at the beam waist S to increase the particle density, thereby promoting aggregation reactions and increasing measurement speed and measurement sensitivity. It is something.

Next, an apparatus according to an embodiment of the present invention will be explained in detail using the drawings. FIG. 2 is a block diagram of a first embodiment of the present invention. In the figure, reference numeral 1 denotes a light source that generates irradiation light having an intensity gradient, and in this embodiment, a YAG laser with a wavelength of 11064 nm is used. 2 is a density filter, 3 is a zoom expander, and 4 is a beam splitter installed obliquely in the optical path.

One of the beams split by the beam splitter 4 is condensed by a condenser lens 6, and the light intensity is detected by a photodetector 7. A control circuit (not shown) monitors the actual output of the light intensity emitted from the light source 1 obtained by the photodetector 7, and controls the amount of light emitted from the light source 1 so that the actual output becomes the set intensity.

The other beam split by the beam splitter 4 reaches a dichroic mirror 5 and an objective lens 13. The objective lens 13 is an immersion type objective lens with a high numerical aperture (NA), and in this embodiment, NA=1.25. The laser beam that has passed through the objective lens 13 is transmitted to the immersion oil 1
2, a beam waist 11 having an intensity gradient is formed in the test area within the reaction cell 31. The reaction cell 31 is made of a transparent material such as glass or plastic, and has a structure in which a reaction sample is sealed inside.

Here, the reaction specimen sealed in the reaction cell 31 has a diameter of 0.
A reagent with a predetermined concentration containing a monoclonal antibody that specifically binds to a specific antigen of interest is prepared on the surface of a large number of latex particles of about 5 μm in size, and a reagent with a predetermined concentration (blood components, urine, etc.) is prepared. It is made by mixing body fluids such as saliva (usually body fluids such as saliva). More details are described in the above publication, so a detailed explanation will be omitted here.

When a beam waist 11 having an intensity gradient is formed in the test area 5 in the reaction cell 31, a large number of latex particles gather near the center of the beam waist due to the optical trap phenomenon, and the density of latex particles increases. The local concentration increases. This increases the probability that the latex particles will come into contact with each other, and aggregation of the latex particles with each other via the target antigen in the serum will be further promoted. In this way, when the target antigen is present in the specimen, latex particles bind to each other to form a large number of aggregates each consisting of about 2 to 5 latex particles. Furthermore, if the target antigen is not present, no aggregate will naturally be formed.

Conventionally, accidental phenomena such as Brownian motion and stirring were used to cause this latex aggregation reaction, so the number of times the latex particles came into contact with each other was extremely small, and as a result, it was difficult to complete the latex aggregation reaction sufficiently. 20 minutes ~3
Incubation for approximately 0 minutes was required. In contrast, in this example, as described above, the optical trap phenomenon is used to concentrate latex particles near the center of the beam waist and promote the latex aggregation reaction, so the time required for incubation is reduced. Can be shortened. At the same time, even if the overall latex particle concentration is low, the optical trap increases the local concentration and causes an aggregation reaction, which has the effect of increasing measurement sensitivity.

After continuing to irradiate the light for a certain period of time to promote the latex agglomerate reaction, the control circuit cuts off the laser light or weakens its intensity, and the light trap is activated accordingly. As the force weakens, the latex particles that were concentrated near the beam waist gradually disperse throughout the liquid due to Brownian motion and other effects. At this time, the latex aggregate formed by the antigen-antibody reaction is dispersed while remaining agglomerated. Further, it would be even more effective to provide a stirring means or a vibration means to stir or vibrate the liquid in the reaction cell 31 so that the dispersion can be effected more effectively in a shorter time.

Next, the degree of latex agglutination reaction in the specimen after incubation is measured, and the function of the measuring means will be explained.

An image of the latex particles illuminated with visible light having a wavelength of 400 nm to 800 nm by an illumination optical system (not shown in FIG. 341) is focused by the objective lens 13 and reaches the dichroic mirror 5 again. As described above, the dichroic mirror 5 has the characteristic of reflecting laser light with a wavelength of 11064n and transmitting visible light, so that the dichroic mirror 5 passes through the latex lenses 15 and onto the two-dimensional image sensor 16 (hereinafter referred to as rccDJ) which is a light receiving element array. Form an image.

A plurality of image signals obtained by the CCD 16 are taken into a frame memory 17 and a VTR 19, and stored as digital and analog image information, respectively.An image processing device 18 processes data for analysis based on the contents of the frame memory 17. Image processing is performed, and the results are output to a CRT 20 or a printer (not shown). Also, VTR19 (7) images are also CR
It is possible to output at T20. Note that it is also possible to record the entire image on the VTR 19 and later reproduce the output signal, A/D converting it and storing it in the frame memory 17. By doing so, images at various points in time can be repeatedly analyzed with a minimum frame memory capacity.

In this example, a two-dimensional photodetector array was used to capture an image of the specimen. It is also possible to detect dimensional image information.

Now, the image processing device 18 mainly has the following three image processing functions. These functions are appropriately selected by the operator.

The first image processing function is a function of shifting the positions of two identical images and calculating the product of the images, that is, a function of calculating the autocorrelation of the images.

Generally, when the amount of deviation between two identical images is (α, β), the two-dimensional autocorrelation function of the images is ψ(α.

β) is ψ(α, β) bow = f(X・α,y+β) f(x,y)
Defined as dx dy.

When a large number of particles with a single diameter exist randomly on an image, the correlation function F&lIi a +I3 =O(Dtt
It is expressed in the form of exp (-(τ7T7/l)) which is maximum and decreases with the increase of a +137.

ζ is called a correlation distance and is a parameter representing the particle size. When latex particles are not aggregated, ζ is a predetermined value ζ1 corresponding to the latex particle diameter, and α2+β2=ζ
When 1', the correlation function becomes 1/e. However, if the latex particles are bonded to each other due to the latex aggregation reaction, it can be considered that the latex particle diameter has substantially increased, so the correlation distance ζ2 becomes a value larger than the predetermined value ζ.

This relationship is shown in Figure 5, where the solid line A shows the correlation function when no latex aggregation reaction occurs, and the - dotted line B shows the correlation function when the latex agglutination reaction occurs. As is clear from
, β) By determining the value of the correlation function when moving, the degree of latex agglutination reaction can be investigated.

Specifically, an optical trap is used to form agglomerates of latex particles, the intensity of the laser beam is weakened, and then a single image is captured and captured when the latex particles that were concentrated in one place have been sufficiently dispersed. The image is binarized based on a certain threshold. Then, the autocorrelation is obtained by calculating the product of two identical binarized images while shifting their positions.

The second image processing function is a function that calculates the width of different images that change over time, that is, a function that calculates the cross-correlation between images that change over time.

The cross-correlation function ψft) with time t as a parameter is ψ(t) = 'Ir-, f(x, y, T◆t)
It is defined as f(x, y, T) dx dy.

When there are many particles with a single diameter on an image, and their positions fluctuate stochastically due to Brownian motion, the correlation function is maximum at time 1 = 0, and decreases as t increases.
It is expressed in the form p(-t/τ). τ is called the correlation time and is also a parameter representing the particle size. Figure 5 shows the relationship.

Specifically, the laser beam is weakened to release the optical trap, and after a certain period of time has elapsed and the latex is sufficiently dispersed, two images are captured at a certain time interval, and a correlation function between both images is calculated. Accordingly, the degree of latex aggregation can be measured. However, in this case, since the correlation time τ also depends on the degree of positional fluctuation due to Brownian motion or the like, it is desirable to control the temperature of the reaction cell.

Furthermore, it is more preferable to change the time interval at which images are captured depending on the temperature.

The third image processing function is an application of the contour extraction technique. Similar to the second image processing function, the intensity of the laser beam is weakened to release the optical trap of the latex, and after a predetermined period of time has elapsed, when the latex is sufficiently dispersed, -
Two images are captured, the captured images are binarized using a predetermined threshold value, and the outlines are extracted. The contour line is approximated to a circle, and since the radius of the circle is known as the radius of the latex, the approximation can be easily performed. At this time, contour lines smaller than the latex particles are removed as image noise. When a latex aggregation reaction occurs, a contour line larger than the latex particles is generated, but in this case, the contour line is treated as a plurality of overlapping circles. In this way, by statistically processing the area, shape, etc. within the contour obtained as a result of the image processing, the number of latex particles in the image, the number of latex particles bonded to each other by latex aggregation reaction, and the ratio of both, etc. can be determined, and the degree of latex agglutination reaction can be measured.

In the above, a method of approximating an outline larger than a latex particle with a plurality of circles has been shown, but there is also a method of recognizing it as a latex agglomerate to a falcon without approximating it. In this case, the image processing algorithm becomes very simple, which has the advantage of increasing processing speed. Further, although the binarization method has been shown as a method for extracting the contour, another method may be adopted in which the contour is obtained by differentiating the image and performing edge enhancement processing.

In addition, in the first and third image processing functions of the above embodiments, in order to simplify the explanation, the image used is one image captured after a certain period of time has elapsed while the intensity of the laser beam is weakened. However, it is not necessary to limit the number of sheets to -, and by measuring continuously or intermittently, it is also possible to capture changes in the measurement results over time and further improve the reliability of the measurement.

Similarly, in the second image processing function, more accurate measurements can be made by continuously capturing a plurality of images in time series and calculating the cross-correlation between each image.

Further, when calculating autocorrelation and cross-correlation, it is not necessary to use a binary image, and a multivalued image captured by a CCD may be used to calculate the correlation function.

[Second Embodiment] Next, a second embodiment of the present invention will be explained using FIG.
The same reference numerals as in FIG. 2 represent the same or similar parts.

In the previous example, an image inside the measurement cell was captured using a CCD, which is a light receiving element array, to obtain image information, but in this example, a minute light beam spot is used to capture the image inside the measurement cell two-dimensionally. Image information is obtained by scanning and detecting the generated light in time series with a single light receiving element.

The mechanism for promoting the aggregation reaction of latex particles by the optical trapping phenomenon is the same as that of the embodiment shown in FIG. 2, in which a beam waist +1 is formed in the measurement cell 31 to promote the aggregation reaction of the carrier.

As the laser light source 21 which is a light source of scanning light, a semiconductor laser having a wavelength different from that of the laser light source 1 (YAG laser) for optical trapping, for example, a wavelength of 670 nm is used. The laser light emitted from the laser light source 21 is transmitted to the acousto-optic element 22.
(hereinafter referred to as r A OD J) in the main scanning direction, and after passing through the beam expander 23, the beam is scanned in the sub-scanning direction orthogonal to the previous main scanning direction by the vibrating mirror 24. In this way, a two-dimensional scanning optical system is formed by the combination of the AOD and the vibrating mirror.

This scanning light is directed toward the measurement cell 31 by the half mirror 25, and is formed into a minute imaging spot of about 0.5 μm in the measurement cell 31 by the objective lens 13, and the measurement part in the measurement cell 31 is formed into an imaging spot. Two-dimensional optical scanning is performed. FIG. 4 shows the situation at this time. Latex particles are concentrated on the beam waist 11 of the laser beam, and the imaging spot 30 is two-dimensionally scanned in the direction of the arrow.

At this time, the scattered light generated from the position where the imaging spot 30 is irradiated is filtered through the bandpass filter 14 and the condensing lens 15.
The light enters the photodetector 26, which is a single light-receiving element. Then, based on the synchronization signal that controls the two-dimensional scanning optical system, the output of the photodetector 26 is captured in time series and stored in the frame memory 17. In this way, two-dimensional image information substantially equivalent to that of the previous embodiment is obtained. The analysis method of this image information obtained in the frame memory 17 is the same as in the previous embodiment, and image processing is performed in the image processing device 18 using an analysis method appropriately selected by the operator.

[Effects of the Invention] According to the present invention, it is possible to effectively increase the agglutination reaction efficiency with a simple method, and since the aggregation state is measured by image analysis, very highly accurate measurements can be performed. .

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram of the optical trap phenomenon, Fig. 2 is a block diagram of the first embodiment of the present invention, Fig. 3 is a block diagram of the second embodiment of the present invention, and Fig. 4 is a block diagram of the second embodiment of the present invention. Figure 5 is a graph showing the autocorrelation function, and the main symbols in the figure are: 1... YAG laser 3... Beam expander 11... Beam waist 12... Immersion oil 13... Objective lens 14... Band pass filter 16... CCD 17... Frame memory 18... Image processing Device 19...VTR 20...CRT 21...Semiconductor laser 22...AOD 23...Beam expander 2'4...Vibration mirror 26...Photo detection Instrument 31...Measuring cell Figure 3 Distance (time)

Claims (8)

[Claims] (1) A step of creating a mixture by mixing a carrier sensitized with a substance that specifically reacts with the target substance with a sample containing the target substance, and irradiating the mixture with light having an intensity gradient. , a step of increasing the efficiency of the aggregation reaction of the carriers by concentrating the carriers near the light irradiation position using the light pressure; detecting the aggregation state of the carriers in the mixture after the aggregation reaction as image information; A method for measuring a specimen, comprising the step of measuring a target substance by analysis. (2) A measurement cell containing a mixture prepared by mixing a carrier sensitized with a substance that specifically reacts with the target substance with a sample containing the target substance, and an intensity gradient at a predetermined position in the measurement cell. A light irradiation means for irradiating light with a light beam to concentrate the carriers near the irradiation position and increase the efficiency of the aggregation reaction; a detection means for detecting the aggregation state of the carriers in the mixture after the aggregation reaction has been completed as image information; An analyte measuring device comprising: analysis means for measuring a target substance by analyzing image information obtained by the means. (3) The specimen measuring method according to claim (1) or the specimen measuring apparatus according to claim (2), wherein the image information is detected by detecting an image of the specimen using a light-receiving element array. (4) The specimen measuring method according to claim (1) or the specimen measuring apparatus according to claim (2), wherein the image information is detected by performing two-dimensional optical scanning of the specimen. (5) The specimen measuring device according to claim 2, wherein the detection means has means for storing detected image information digitally or analogously. (6) The specimen measuring method according to claim (1) or the specimen measuring device according to claim (2), wherein the analysis of the image information is performed using an image obtained by binarizing the detected image using a predetermined threshold value. (7) The sample measuring method according to claim (1) or claim (1), wherein the analysis of the image information calculates the degree of aggregation by calculating autocorrelation or cross-correlation of the detected image.
2) The specimen measuring device described above. (8) The specimen measuring method according to claim (1) or the specimen measuring apparatus according to claim (2), wherein the analysis of the image information calculates the degree of aggregation by extracting the contour of the detected image.