JPH0466873A - Method of processing and measuring specimen and apparatus for measuring the same - Google Patents

Abstract

PURPOSE:To promote agglutination reaction efficiency of a carrier by a method wherein a mixture of the carrier and a specimen is irradiated with light having an intensity gradient and the carrier is concentrated near a position of the light irradiation while the position of light irradiation to the mixture is chamber relatively. CONSTITUTION:Laser light leaving an optical path 1 is branched with a beam splitter 4 and one luminous flux reaches an objective lens 13. The laser light passing through the lens 13 form a beam waste 11 having an intensity gradient at a part to be inspected of a reaction cell 31 sealed up with an reaction object to be inspected, namely, a mixture of latex LX particles sensitized by a monoclonal antibody and the specimen. Moreover, an oscillation mechanism 41 and a vibration mechanism 42 vary a relational position between the waste 11 and the reaction object to be inspected relatively in a two-dimensional manner. With such an arrangement, the LX particles gather near the center of the waste 11 by a light trapping phenomenon to promote agglutination of the particles with a higher contacting probability therebetween. In addition, relatively varying of the light irradiation position to the object to be inspected causes the light trapping phenomenon in a wide range thereby further promoting the agglutination reaction of the LX particles.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for measuring 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 so-called incubation is performed at a constant temperature for a sufficient period of time for the agglutination reaction to occur (usually about 20 to 30 minutes). By measuring the aggregation state of the carrier using an optical method, the target antigen in serum can be qualitatively or quantitatively measured. This is generally called the particle immunoassay method and is widely known.
693, JP 54-108594, JP 54-108 [i95, JP 54-1094]
It is described in detail in JP-A No. 94, JP-A-55-159157, JP-A-52-81567, etc.

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

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 object of the present invention is to further improve the above method and to promote the aggregation reaction more effectively in a simple manner.

[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 irradiating the mixture with light having an intensity gradient and concentrating the carrier in the vicinity of the light irradiation position by the light pressure, thereby increasing the aggregation reaction efficiency of the carrier; The method is characterized by comprising a step of relatively changing the light irradiation position on the carrier, and a step of measuring the target substance by detecting the aggregation state of the carrier in the mixture after the aggregation reaction.

[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, the liquid has a very large light intensity and an intensity gradient, such as a laser beam with a crow's distribution. It is known that when a beam waist is formed using a light beam having , as shown in FIG. 1(b), a centripetal force due to the optical pressure of the laser beam acts, 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 to increase particle density, thereby promoting aggregation reactions and increasing measurement speed and measurement sensitivity. It is something to do.

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 the first embodiment of the present invention. In the figure, 1 is a light source for an optical trap that generates irradiation light with an intensity gradient, and in this embodiment, the wavelength is 10.
64 nm% Nd3+ laser (YA
G laser) is used.

Note that the light source that can be used is not limited to the Nd'' laser, but it is preferable to use a light source in the infrared region because if a laser beam in the visible wavelength range or below is used, the particles may be destroyed. A density filter, 3 a beam expander, and 4 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 sent to a photodetector 7. Light intensity is detected.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. .

Further, the other beam split by the beam splitter 4 reaches the dichroic mirror 5 and the objective lens 13. The objective lens 13 is an immersion type objective lens with a high numerical aperture (NA), and in this example, NA=1.25.
It is. The laser beam that has passed through the objective lens 13 passes through the immersion oil 12, and then passes through the small beam waist 11 with a diameter of about 0.8 μm, which has an intensity gradient in the test area within the reaction cell 31.
form. 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.

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 approximately 5 μm in size, and this and a sample (blood components, urine, saliva, etc.) are prepared. It is made by mixing body fluids such as More details are described in the above publication, so a detailed explanation will be omitted here.

Furthermore, in order to cause the aggregation reaction to occur over a wide range, the dichroic mirror 5 is provided with a swing mechanism 41, and the reaction cell 31 is provided with a vibration mechanism 42. The swing mechanism 41 one-dimensionally scans the beam waist 11 in the reaction cell 31 by swinging the dichroic mirror 5, and the vibration mechanism 42 one-dimensionally vibrates the reaction cell 31 in a direction substantially perpendicular to the scanning. let In this way, the relative positional relationship between the beam waist 11 and the reaction sample sealed in the reaction cell 31 can be changed two-dimensionally.

Note that either the vibration mechanism 42 or the rocking mechanism 41 may be made to vibrate two-dimensionally, so that the reaction cell 31 is vibrated two-dimensionally on a plane orthogonal to the irradiation optical axis. In addition, by repeating turning on and off the Nd'+ laser beam at regular intervals in synchronization with the two-dimensional scanning of the beam waist 11, light irradiation for optical trapping is performed at substantially a plurality of fixed positions. It's okay.

When the beam waist 11 having an intensity gradient is formed in the test area in the reaction cell 31, due to the optical trap phenomenon,
A large number of latex particles gather near the center of the beam waist, increasing the density of latex particles and increasing the local concentration. 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. In this embodiment, since the beam waist 11 is moved two-dimensionally within the reaction sample enclosed in the reaction cell 31, a wider range of latex particles existing within the beam movement range is irradiated with light, and the trajectory of the beam waist is A larger number of latex particles can be collected by optical traps.

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 very small, and as a result, it was not enough to complete the latex aggregation reaction. 20 minutes~
Incubation for about 30 minutes was required. In contrast, in this embodiment, as described above, the phenomenon of optical trapping is used to move latex particles two-dimensionally or to concentrate them near the waist of a beam that is irradiated to multiple positions, thereby promoting the latex aggregation reaction. Therefore, the time required for incubation can be shortened. In addition, even if the overall concentration of latex particles is low, the optical trap increases the local concentration and causes an agglutination 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 blocks the laser light or weakens its intensity, and accordingly the light traps. 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. Furthermore, it is effective to vibrate and stir the liquid in the reaction cell 31 using the vibration excitation means 42 or a separately provided stirring means so that the dispersion can be carried out more efficiently in a shorter time. .

Next, the function of the measuring means for measuring the degree of latex agglutination reaction in a specimen after completion of incubation will be explained.

An image of latex particles illuminated over a wide area with visible light having a wavelength of 400 nm to 800 nm by an illumination optical system (not shown in FIG. 1) is focused by an objective lens 13 and reaches the dichroic mirror 5 again. Since the dichroic mirror 5 has the property of reflecting laser light with a wavelength of 11064n and transmitting visible light, the visible image of the latex particles is transmitted through the band pass filter 14 and the eyepiece lens 15, and then sent to the light receiving element array. An image is formed on a dimensional image sensor 16 (hereinafter referred to as rCCDJ).

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. The image processing device 18 performs image processing for analysis based on the contents of the frame memory 17, and outputs the results to a CRT 20 or a printer (not shown). In addition, the image of VTR19 is also CRT2
It is possible to output to 0. In addition, -dan VT
All images were taken on R19, and the output signal that was later played back is A.
It is also possible to perform /D conversion and store 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 image is ψ(α.

β) is ψ(α, β) = i! ':: f (x+a, y
+β) f (x, y) dx dy.

When many particles of a single diameter exist randomly on an image, the correlation function becomes maximum when the distance wiF♂+6 = o, and 1,
exp(-a
+8/ζ).

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. On the other hand, if the latex particles are bonded to each other by 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 FIG. 5, where the solid line A shows the correlation function when no latex aggregation reaction is occurring, and the dashed line B shows the correlation function when the latex agglutination reaction is occurring. As is clear from the figure, either the correlation function or the correlation immediate motion ζ that is 1/e can be found, or the constant distance 1m (α,
β) By determining the value of the correlation function when moving, the degree of latex agglutination reaction can be investigated.

Specifically, an agglomerate of latex particles was formed using an optical trap, the intensity of the laser beam was weakened, and a single image was captured and captured at the point when the latex particles that had been concentrated in one place were sufficiently dispersed. Binarize the image based on a certain threshold. Then, the autocorrelation is obtained by shifting the positions of two identical binarized images and calculating the product of the images.

The second image processing function is a function of calculating the product 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 ψ(1) with time t as a parameter is ψ(t) = SL f(x, y, T+t) 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 agglomeration can be measured. However, in this case, since the correlation time also depends on the degree of positional fluctuation due to Brownian motion, etc., 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.

This is an application of the third image IA filling function and 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, when the latex is sufficiently dispersed,
- Capture two images, binarize the captured images using a predetermined darkness value, and extract contour lines from the binarized images by image processing. Since the contour line is approximated to a circle, or the radius of the circle is known as the radius of the latex, the approximation can be easily made. 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, or in this case, it 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.

Note that in the above, a method of approximating a contour line larger than a latex particle with a plurality of circles has been shown, or there is also a method of simply introducing it as a latex agglomerate without approximating it. In this case, the image processing algorithm becomes very simple, which has the advantage of increasing processing speed. Although the binarization method is shown as a method for roughly 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.

[Effects of the Invention] As described above, according to the present invention, when increasing the agglutination reaction efficiency by utilizing the optical trapping phenomenon, by relatively changing the light irradiation position with respect to the specimen, the optical trapping phenomenon is caused over a wider range, and thereby The aggregation reaction can be further promoted.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram of the optical trap phenomenon, Fig. 2 is a configuration diagram of an embodiment of the present invention, and Fig. 3 is a graph diagram showing an autocorrelation function. ... Nd" laser 3 ... Beam expander 11 ... Beam waist 12 ... Night oil 13 ... Objective lens 14 ... Bandpass filter 16 ... CCD 17... Frame memory 18... Image processing device 19... VTR 20... CRT 31... Measurement cell 41... Rocking mechanism, 42... Vibration Mechanism, Figure 2, (η) et al.2 (at,) Distance M, (time)

Claims (4)

[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 aggregation reaction efficiency of the carrier by concentrating the carrier near the light irradiation position by the light pressure, and a step of relatively changing the light irradiation position of the mixture. Specimen processing method. (2) 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 carrier by concentrating the carrier near the light irradiation position using the light pressure; a step of relatively changing the light irradiation position of the mixture; and a step of relatively changing the light irradiation position of the mixture; A method for measuring a specimen, comprising the step of measuring a target substance by detecting an agglomerated state of a carrier therein. (3) 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, the mixture in the measurement cell having an intensity gradient. Light irradiation means for irradiating light to concentrate the carrier near the irradiation position and increasing the aggregation reaction efficiency; means for relatively changing the light irradiation position on the mixture in the measurement cell; A specimen measuring device comprising: a detection means for detecting an aggregation state of a carrier in a mixture; and an analysis means for measuring a target substance based on a detection value of the detection means. (4) The method for measuring an analyte according to claim 2 or the apparatus for measuring an analyte according to claim 3, wherein the aggregation state is detected by image-detecting the reaction mixture using a light-receiving element array.