JPH1123468A - Fluorescence analyzer on solid surface using optical waveguide - Google Patents

Abstract

(57) [Problem] To provide a fluorescence analyzer which is inexpensive, can be expected to have high detection sensitivity, and has a good detection limit. An optical waveguide made of a material transparent to fluorescent light from a fluorescent material to be analyzed and a material made of a material transparent to excitation light are expected to include an optical waveguide and an object to be analyzed inside. A container for containing the test solution to be tested, and a light emission source capable of emitting light having a wavelength capable of exciting the fluorescent substance by fluorescence, the surface being a surface facing the main side surface of the optical waveguide contained in the container. Fluorescence analyzer comprising a light radiation source and the like arranged to emit excitation light toward.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting a fluorescent signal from a fluorescent substance directly or indirectly bound on a solid phase surface or a fluorescent substance located near the solid phase surface. And a fluorescence analyzer using an optical waveguide as the solid phase. The present invention also relates to a fluorescence analyzer using an optical waveguide for performing an immunoassay for measuring the presence and concentration of an antigen and / or an antibody using a so-called sandwich method or competition method.

[0002]

2. Description of the Related Art Some fluorescent analyzers for measuring a fluorescent substance directly or indirectly bound on the surface of a solid phase using an optical waveguide as a solid phase have already been proposed. For example, there are known devices that detect a fluorescent substance directly or indirectly bound on the solid phase surface via an immune reaction or an adsorption reaction, or a fluorescent signal from a fluorescent substance located near the solid phase surface. Have been. More specifically, for example, the excitation light is incident from the side of the optical waveguide, and using so-called evanescent light, which leaks out of the optical waveguide during its total reflection, is directly or indirectly constrained on the optical waveguide surface. An apparatus as shown in FIG. 3 has been proposed as an apparatus for determining the amount of a specific component in a test solution by detecting a fluorescent signal from a fluorescent substance or a fluorescent substance located near an optical waveguide surface. ing.

In the device shown in FIG. 3, a prism (4
8) A reaction tank (47) having one surface as one section screen is provided, and the one surface of the prism is coated with a substance that promotes adsorption of a specific component, and a fluorescent substance is bonded inside the reaction tank. A test solution (49) containing a specific component is injected. By doing so, an amount of the fluorescent substance corresponding to the concentration of the specific component in the test solution is adsorbed on the coating surface of the prism. When the excitation light (43) is incident from a surface different from the above-mentioned coating surface of the prism so as to form an angle equal to or larger than the critical angle at the interface between the coating surface and the solution, the excitation light is once applied to the coating surface of the prism. Is totally reflected and exits from the other surface, but at the time of the total reflection, a part of the light seeps into the test solution as evanescent light. The seeping distance dp (the electrolytic strength is 1 /
e) is in accordance with the following equation, and is generally 100
Since it is around nm, it is possible to selectively excite a fluorescent substance bound to the coating surface of the prism.

[0004]

(Equation 1)

In the above equation, λ is the wavelength of light, n
1 is the refractive index of the prism, n2 is the refractive index of the solution, and θ is the incident angle of light incident on the interface between the prism and the solution. The fluorescence (44) induced in this way is detected from the direction facing the coating surface of the prism, and the physical quantity of the fluorescent substance bound to the coating surface and the physical quantity corresponding to the concentration of the specific component in the test solution are calculated. is there.

[0006] Further, specifically, for example, an apparatus as shown in FIG. 4 has been proposed. In this apparatus, a reaction tank having one surface of the slab-type optical waveguide (52) as one screen is provided, and a binding antibody or a binding antigen is previously fixed to the one surface of the slab-type optical waveguide, A labeled antibody or labeled antigen in which a test solution and a fluorescent substance are bound is injected into the inside of this reaction tank to carry out an immune reaction. By doing so, an amount of fluorescent substance corresponding to the concentration of the antigen or antibody in the test solution is bound to the coating surface of the slab-type optical waveguide. When the excitation light (43) is incident from one end face of the slab type optical waveguide, it propagates while repeating total reflection on the surface of the optical waveguide. At the time of the total reflection, evanescent light that leaks into the test solution is used for coating. It is possible to selectively excite the fluorescent substance bound to the surface.

In such an apparatus, since the total reflection occurs a plurality of times on the coating surface, the amount of fluorescence is larger than in the case of using one total reflection on the prism surface as shown in FIG. There is an advantage that highly sensitive measurement can be performed on the antigen or antibody in the test solution.

More specifically, for example, as shown in FIG. 5, a fiber type optical waveguide (53) is provided in a reaction tank (5).
An apparatus immersed in 1) has also been proposed. A labeled antibody or labeled antigen in which a test solution (49) and a fluorescent substance are bound inside a reaction vessel in which the binding antibody or binding antigen is preliminarily fixed on the surface of the fiber type optical waveguide and the fiber type optical waveguide is immersed. To perform an immune response. By doing so, an amount of the fluorescent substance corresponding to the concentration of the antigen or the antibody in the test solution is bound to the coating surface of the fiber type optical waveguide. When the excitation light (43) is incident from one end face of the fiber type optical waveguide, it propagates while repeating total reflection on the surface of the optical waveguide. At the time of the total reflection, evanescent light that permeates into the solution is used.
It is possible to selectively excite the fluorescent substance bound to the coating surface.

Also in such an apparatus, since the total reflection occurs a plurality of times on the coating surface, the amount of fluorescence is larger than that in the case of using one total reflection on the prism surface as shown in FIG. In addition, there is an advantage that highly sensitive measurement can be performed on an antigen or an antibody in a test solution.

In the apparatus shown in FIGS. 4 and 5, a labeling substance of a labeled antibody or labeled antigen is used as an enzyme, and an enzyme substrate (fluorescent substance precursor) which is converted into a fluorescent substance by the action of the enzyme is injected. For example, the fluorescent substance generated by the action of the enzyme constrained on the surface of the optical waveguide is temporarily present near the surface of the optical waveguide, so that the evanescent light seeping out of the optical waveguide is used to produce a fluorescent substance near the surface of the optical waveguide. The existing fluorescent substance can be selectively excited. Therefore, it becomes possible to quantify the enzyme or the antibody in the test solution that is bound by the optical waveguide.

It is known that the evanescent light that has permeated into the solution from the surface of the optical waveguide again enters the optical waveguide and propagates. This phenomenon is also caused by the fluorescence induced by the evanescent light. The same is true. That is, as shown in FIG. 6, the fluorescent light (105) generated in the range where the evanescent light seeps and penetrated into the inside of the optical waveguide (1) is more than the critical angle when reflected from the surface of the optical waveguide on its surface. Fluorescent light propagates through the optical waveguide while repeating total reflection. Therefore, not only slab type or fiber type,
When the excitation light is incident from the end face of the optical waveguide and the multiple reflection on the surface is used, the method of detecting the fluorescence from the direction facing the coating surface of the optical waveguide in the direction of detecting the fluorescence, the excitation light is emitted There are three methods, that is, a detection method on the end face side and a detection method on the end face side on which the excitation light is incident. Recently, as shown in FIGS. 4 and 5, a method of detecting the excitation light on the side of the end face on which the excitation light is incident has been often adopted. The intention is to improve the selectivity on the surface of the optical waveguide and to increase the excitation. In removing light background.

As is apparent from FIGS. 4 and 5,
Since the optical confinement in a slab optical waveguide is one-dimensional, while the optical confinement in a fiber optical waveguide is two-dimensional, the propagation of fluorescence in the optical waveguide is more efficient in the fiber optical waveguide. .

[0013]

A conventional solid phase detecting a fluorescent signal from a fluorescent substance directly or indirectly constrained on the surface of the optical waveguide or a fluorescent substance located near the optical waveguide surface. The fluorescence analyzer on the surface, which induces fluorescence using evanescent light that exudes when the excitation light incident from the side of the optical waveguide is totally reflected, as shown in FIG. There is such a problem.

First, most of the light propagating through the optical waveguide is confined inside the optical waveguide, the amount of light that leaks out from the optical waveguide into the solution as evanescent light is small, and a part of the excitation light from the light radiation source is emitted. Only can be used effectively. Since the fluorescence intensity in fluorescence analysis is proportional to the intensity of the excitation light, a large fluorescence signal cannot be expected if only a part of the excitation light is available, and high detection is required for the purpose of quantifying specific components in the test solution. I can't expect sensitivity.

Second, there is a problem that the optical waveguide itself emits fluorescence. Cheap glass, polystyrene,
In the case of an inexpensive plastic material such as polymethyl methacrylate, since a substance or an impurity that emits strong fluorescence is contained, autofluorescence from the optical waveguide itself propagates and becomes a background factor in fluorescence analysis. In the conventional method, the influence is large just because the distance traveled in the optical waveguide is long,
As a result, when used for the purpose of quantifying a specific component in a test solution, the detection limit is deteriorated. If a material such as synthetic quartz, which has extremely few impurities and whose fluorescence is almost negligible in the normal wavelength region, is used as the optical waveguide, the effect of the fluorescence of the optical waveguide itself can be neglected. This requires costly processing technology, which raises cost issues and is not suitable for disposable use.

Third, when pumping light is incident from the end face of the optical waveguide, there is a problem that the pumping light is scattered or reflected, so that the background rises and the S / N ratio of the signal deteriorates. In the method of detecting fluorescence at the end face side where excitation light is incident, which is often used in the conventional method, since the measurement direction of the fluorescence is substantially on the same axis as the direction of incidence of the excitation light, the incidence of the optical waveguide is It is difficult to sufficiently remove scattered light and reflected light from the end face and the output end face, and as a result, when quantifying a specific component in the test solution, the detection limit is deteriorated.

Fourth, there is a problem that the shape of the incident end face of the optical waveguide becomes complicated. In the conventional method, the incident end face is shaped into a prism shape or a lens shape in order to increase the incidence efficiency of the excitation light into the optical waveguide or reduce the reflection at the end face, but this increases the manufacturing cost as a result. is there.

[0018]

According to a first aspect of the present invention, there is provided a fluorescent substance which is directly or indirectly constrained on a solid phase surface. In a fluorescence analyzer on a solid phase surface for detecting a fluorescence signal from a fluorescent substance located in the vicinity of, a substance transparent to fluorescence from a fluorescent substance to be analyzed is provided as a material, and the solid phase surface is provided. An optical waveguide, a storage container containing a test solution expected to include the optical waveguide and the analysis object therein, made of a material transparent to excitation light for exciting the fluorescent substance to be analyzed; and A light radiation source capable of emitting light having a wavelength capable of fluorescence-exciting a substance, which is arranged so as to emit excitation light from a side facing a main side surface of an optical waveguide housed in a housing container toward the surface. Light source and the end face of the optical waveguide An optical signal emitted from the fluorescent substance is disposed so as to be detected through optical means for selecting a fluorescent wavelength radiated from the fluorescent substance, and is disposed so as to enter the optical waveguide and receive fluorescent light propagating through the optical waveguide. And a light receiving sensor.

The immunoassay apparatus according to claim 2 of the present invention has been made in order to solve the above-mentioned problem, and a binding antibody or a binding antigen is immobilized on the surface of a solid phase in advance, and the solid phase is immobilized. After indirectly binding the labeled antibody or labeled antigen on the solid phase surface by reacting with an antigenic substance in the test solution and a labeled antibody or labeled antigen to which a fluorescent substance or an enzyme has been previously bound, the bound labeled antibody or An immunoanalyzer for analyzing a physical quantity related to the presence or concentration of the antigenic substance in a test solution by detecting a fluorescent signal of a fluorescent substance bound to a labeled antigen or a fluorescent substance generated by an enzymatic action, comprising: An optical waveguide that provides a solid phase surface with a material transparent to fluorescence from the material,
A material transparent to excitation light that excites the fluorescent substance to be analyzed is a material, and a storage container containing the optical waveguide and the test solution therein, and light having a wavelength capable of exciting the fluorescent substance by fluorescence can be emitted. A light radiation source, wherein the light radiation source is arranged to emit the excitation light from the side facing the main side surface of the optical waveguide housed in the housing toward the surface, and the light emission source is emitted from an end face of the optical waveguide. The optical signal to be transmitted is arranged to be detected through optical means for selecting the fluorescence wavelength emitted from the fluorescent substance, and is arranged so as to be able to enter the optical waveguide and receive the fluorescent light propagating through the optical waveguide. It is characterized by comprising a light receiving sensor.

[0020]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the drawings.

FIG. 1 is a view showing one embodiment of the fluorescence analyzer of the present invention, and FIG. 2 is a view showing one embodiment of the immunoassay of the present invention.

Each device has an optical waveguide (1) made of a material transparent to the fluorescent light from the fluorescent substance to be analyzed, and an optical waveguide (1) transparent to the excitation light for exciting the fluorescent substance to be analyzed. A storage container (3) made of a substance and containing therein the optical waveguide and a test solution expected to contain the analysis object; and light (7) having a wavelength capable of exciting the fluorescent substance by fluorescence. A light radiation source capable of being disposed so as to be emitted toward the main waveguide from the side facing the main side surface of the light waveguide housed in the housing, and an end face of the light waveguide. An optical signal emitted from the fluorescent material is arranged to be detected through an optical means (5) for selecting a fluorescent wavelength emitted from the fluorescent substance, and fluorescence (8) penetrating into the optical waveguide and propagating through the optical waveguide is detected. Is the light receiving sensor (6) adapted to receive light? It is configured. The material of the optical waveguide is required to have a refractive index higher than that of the test solution expected to contain the analysis target, in addition to being transparent to the fluorescence from the fluorescent substance to be analyzed. Usually, the test solution is not particularly limited since it is often an aqueous solution represented by a biological sample such as blood or serum.

In these apparatuses, as an optical waveguide (1), a glass slide for optical microscope (size;
26 × 76 × 0.9 mm, refractive index = 1.52), a glass beaker having an inner diameter of φ28 × an outer diameter of φ30 × a height of 60 mm as a storage container (3), and a xenon flash lamp as a light radiation source (4). A photomultiplier tube is used as the light receiving sensor (6). In the apparatus shown in FIG. 1, a coating for binding a fluorescent substance to be analyzed is provided on the optical waveguide (1), and in the apparatus shown in FIG. is there. Here, for example, if one optical waveguide is divided into a plurality of fractions, and a coating is applied to each fraction to constrain different substances, or antibodies for different analytes are fixed, one Fluorescence analysis of a plurality of objects to be analyzed is possible only by using the optical waveguide of (1). That is, a plurality of light irradiation sources are arranged so as to be able to irradiate each of the above fractions, and a different analysis target is measured by switching the light irradiation sources. Of course, one light irradiation source may be movably arranged to irradiate each of the above fractions. Furthermore, it is also possible that a plurality of light guides can be provided in the housing container and one or a plurality of the above-mentioned fractions are provided on each light guide.

FIG. 7 is a diagram showing the above device in more detail. The optical waveguide (1) is made of ethylene tetrafluoride,
It is installed in a storage container (3) via an assembly part (21) having a black rubber coating treatment on its surface and having two upper portions on a taper portion. The storage container (3) is set on a table of the Z-axis stage, and the tapered portion of the assembly part (21) and the table (23) are operated by operating the Z-axis stage.
By moving the upper surface of b) so as to be in contact with the connection part (24), the positional relationship between the optical waveguide (1) and the light radiation source (4) and between the optical waveguide (1) and the light receiving sensor (6) can be determined.
Excitation light emitted from the light radiation source (4) is selected in wavelength by an optical filter (4b) installed on the light radiation source side, is adjusted in light flux by an optical lens (4c), and then has an optical waveguide (1). Illuminate a specific area above. The fluorescent signal emitted from the upper end surface of the optical waveguide (1) is selected only from the fluorescent material that propagates through the optical waveguide through the optical lens (5a, 5c), the dichroic mirror (5b), and the optical filter (5d). After that, it is detected by the light receiving sensor (6).

In the above example, a slide glass for an optical microscope was used as the optical waveguide (1) for providing the solid phase surface. In addition, a slab-shaped optical waveguide, a fiber-shaped optical waveguide, Any of the waveguides according to the above, transparent to the fluorescence from the fluorescent substance, and,
Any material having a refractive index larger than that of the test solution can be used without particular limitation. Here, if the optical waveguide is also transparent to the excitation light, it passes through the side (principal side surface) irradiated with the excitation light and goes to the opposite side, so that it is possible to excite the fluorescent substance also on the opposite surface. preferable.

The optical waveguide does not need to be entirely transparent to the excitation light, and at least a coating region for binding a fluorescent substance to be analyzed or an antigenic substance due to an immune reaction. It is sufficient that the region to be irradiated with the excitation light, which is the region where the antigen or antibody to be immobilized is immobilized, is transparent. Here, the coating region or the region where the antibody or the like is fixed is provided on the main side surface of the optical waveguide. In the present invention, one surface of the optical waveguide in which a coating region for binding a fluorescent substance or an antigenic substance or a region where an antibody or the like is immobilized is a main side surface, and enters the optical waveguide and propagates therein. The surface for guiding the fluorescence from the fluorescent substance to the light receiving sensor is the end surface.

With respect to the container (3), FIGS.
In the above example, a glass beaker or the like was used, but the container is made of a material transparent to the excitation light, and can accommodate the test solution inside, and fixes the above-mentioned coating region of the optical waveguide (1) or the antibody. The area that can be contacted with the test solution, and at least one end of the optical waveguide is not immersed in the test solution in order to enter the optical waveguide and guide the fluorescence from the fluorescent substance propagating therein to the light receiving sensor, There is no particular limitation as long as it has a shape. Note that the entire container does not need to be entirely transparent to the excitation light, and at least a part of the container is transparent so that the excitation light is applied to the coating region or the region where the antibody or the like is fixed in the optical waveguide. I just want it. The storage container (3) is, for example, as shown in FIG.
As shown in the above, instead of the glass beaker, a transparent cover glass (3) arranged so as to cover the above-mentioned coating area on the slide glass (1) or the area where the antibody or the like is fixed may be used. In this case, a space having an appropriate thickness can be arranged between the slide glass and the cover glass to provide a space for accommodating the test solution.

Further, in the above example, since the xenon flash lamp (4a) which emits light of a wide wavelength from the ultraviolet region to the visible region in all directions is used, the wavelength can be selected through the optical filter (4b) and the optical lens (4c). The luminous flux is being prepared, but this is not the case when using a light radiation source that emits light of a limited wavelength and luminous flux, such as a laser, for example, and an optical system suitable for the characteristics of the light radiation source to be used is adopted. can do. The light radiation source only needs to be able to irradiate at least the coating region or the region where the antibody or the like is fixed on the main side surface of the optical waveguide through the transparent portion of the container. In particular, it is preferable to arrange the light radiation source such that the excitation light can be irradiated at an angle such that the excitation light enters from the main side surface, passes through the opposite surface of the optical waveguide, and does not propagate through the optical waveguide. In addition, at least one light emission source of excitation light for fluorescently exciting the fluorescent substance may be arranged at least around the optical waveguide, but by increasing the number thereof, it is located at or near the surface of the optical waveguide. It is preferable because the excitation efficiency of the labeled antibody with respect to the fluorescent substance can be improved.

Further, in the above example, the fluorescence signal emitted from the upper end face of the optical waveguide is wavelength-selected through the optical filter and then detected by the light receiving sensor.
For c) and the dichroic mirror (5b), various conventionally known optical components can be used, and are not limited to the above examples. Means for determining the positional relationship between the optical waveguide and the light radiation source, and the positional relationship between the optical waveguide and the light receiving sensor, and other means such as using a connector are also conceivable.

Various immunoassays using the immunoassay apparatus according to claim 2 of the present invention shown in FIG. 2 (FIG. 7) will be described.

In the first method, first, an appropriate buffer solution, a solution containing a labeled antibody or a labeled antigen is sequentially or once injected into a sample solution such as serum or urine to be subjected to analysis, and the reaction solution is injected. Prepared and cause an immune reaction between a specific antigen or antibody (antigenic substance) in the sample solution and the labeled antibody or labeled antigen. Next, the reaction solution is brought into contact with an optical waveguide having a region in which the antibody or antigen for immobilizing the antigenic substance has been immobilized in advance, and the specific antigenic substance in the sample solution bound to the labeled antibody or labeled antigen is photoconductive. An immune reaction is generated between the restraining antibody or the restraining antigen fixed on the main side surface of the waveguide. As a result, an amount of the labeled antibody or labeled antigen corresponding to the concentration of the specific antigenic substance in the sample solution is restricted on the main side surface of the optical waveguide. Here, when the label is a fluorescent label, the reaction solution is used as a test solution, and when the label is an enzyme or the like, a precursor that is converted into a fluorescent substance by the action of the enzyme is put into the test solution. After being formed, the light guide irradiates the light guide with excitation light from the light emission source, and the fluorescent light from the induced fluorescent substance that penetrates the light guide and propagates while repeating total reflection is generated at the upper end face of the light guide. The light may be received from

When the label is an enzyme or the like, the reaction between the binding antibody or the like on the optical waveguide and the reaction solution can be carried out in the presence of the above-mentioned precursor. Further, if the test solution can be finally excited by fluorescence in the container, a part or all of the above-described reactions may be generated in a separately prepared container. Furthermore, in order to remove fluorescence that does not correspond to the concentration of the specific antigenic substance, it is preferable to remove a label other than the labeled antibody or the like indirectly constrained on the optical waveguide before removing the fluorescence. . For this purpose, for example, the contact between the reaction solution and the optical waveguide may be performed outside the container, the optical waveguide may be washed, and then placed in the container to perform fluorescence measurement.

The fluorescent light that penetrates the optical waveguide and propagates while repeating total reflection is generated in a range where the evanescent light exudes when the light propagates through the optical waveguide. Fluorescent substance bound to the labeled antibody or labeled antigen, or the fluorescent substance present in the vicinity of the enzyme bound to the labeled antibody or labeled antigen bound on the optical waveguide selectively contains fluorescence from Corresponds to the amount or rate at which the labeled antibody or labeled antigen is bound to the optical waveguide. These physical quantities are related to the concentration of the specific antigen or antibody in the sample solution as is clear from the above, and the calibration is performed by performing the same operation on a standard solution having a known specific antigen or antibody concentration. By collating with the line, the concentration of the specific antigen or antibody in the sample solution can be calculated.

In the second method, first, an immune reaction is caused between a specific antigenic substance in a sample solution and a binding antibody or a binding antigen immobilized on an optical waveguide, and then, a labeled antibody or a labeled antibody is generated. The solution of the antigen is injected to generate an immune reaction between the specific antigenic substance in the sample solution and the labeled antibody or labeled antigen. For other points, the same operation as in the first method may be performed.

In the third method, an immune reaction is simultaneously generated between a specific antigenic substance, a labeled antibody or a labeled antigen in a sample solution, and a binding antibody or a binding antigen immobilized on an optical waveguide. It is. For other points, the same operation as in the first method may be performed.

A fourth method is that, in each of the above methods, the specific antigen in the sample solution and the labeled antigen are allowed to compete with the binding antibody fixed on the optical waveguide, or the specific antigen in the sample solution is reacted with the specific antibody in the sample solution. The labeled antibody is allowed to compete with the binding antigen immobilized on the optical waveguide. For other points, the same operation as in the first method may be performed.

Example 1 Using the fluorescence analyzer shown in FIG. 7, fluorescence analysis of a fluorescent substance present on the surface of the optical waveguide was performed by the following method.

First, a slide glass (1) used as an optical waveguide, which also transmits excitation light, and a cover glass (3) used as a storage container are bonded with a 160 μm-thick ethylene tetrafluoride tape therebetween. 5 μM E
40 μl of u3 + chelate solution or 40 μl of blank solution without Eu3 + were injected. Thereafter, excitation light is irradiated from the light radiation source (4) to each solution in the container (3), and among the induced fluorescence from the Eu3 + chelate, the fluorescence enters the optical waveguide and propagates while repeating total reflection. Increasing fluorescence was received from the upper end surface of the optical waveguide.

The fluorescent light that enters the optical waveguide and propagates while repeating total reflection is generated in a range where the evanescent light exudes when the light is propagated through the optical waveguide. The signal from the Eu3 + chelate present in the optical waveguide selectively contains, but when light is propagated through the optical waveguide, the range where the evanescent light seeps out is as follows.
Calculated from the refractive index of the slide glass or solution used in this example, it is about 100 nm. Of course, 100n
One end of the fluorescence from Eu3 + existing at a distance of m or more from the surface also enters the optical waveguide, but most of the fluorescence exits the optical waveguide because the angle of incidence on the surface of the optical waveguide is smaller than the critical angle of total reflection. Then, since it attenuates as the reflection repeats, by detecting the fluorescence at the end face of the optical waveguide,
It is possible to selectively detect fluorescence from the Eu3 + chelate present in the range of the exudation depth of the evanescent light (about 100 nm in this embodiment).

For comparison, the same sample was used in the conventional method in which excitation light was incident from the end face of the optical waveguide and the fluorescent substance was excited by evanescent excitation light, using the conventional fluorescence analyzer shown in FIG. Was measured.

FIG. 9 shows the time-resolved data of the fluorescence signal measured by the apparatus of FIG. 7 and the time-resolved data of the fluorescence signal measured by the conventional apparatus of FIG.

Comparing the signal difference between the 5 μM Eu3 + chelate solution and the blank solution, the signal difference is clearly larger than that of the conventional apparatus, and becomes about 200 times when corrected by the excitation light intensity involved in the excitation within a range of 100 nm from the surface. That is, it is clear that a larger signal amount can be obtained than in the conventional device.

FIG. 10 shows a fluorescence signal obtained by measuring only the slide glass used as the optical waveguide in the apparatus shown in FIG. 7 or the apparatus shown in FIG. Since the fluorescence signal amount and its time change are almost the same as those of the blank solution in FIG. 9, the fluorescence signal observed when measuring the blank solution is controlled by the fluorescence from the slide glass itself used as the optical waveguide. You can see that it is done. Therefore, FIG.
By comparing the signal amount of the blank solution, it is possible to estimate the effect of autofluorescence from the optical waveguide serving as a disturbing signal. However, it is apparent that the disturbing signal is about 1/40, which is clearly smaller than the conventional device. Recognize.

[0044]

According to the present invention, the fluorescent analyzer of the present invention is used for performing a fluorescent analysis of a fluorescent substance directly or indirectly constrained on the surface of an optical waveguide or a fluorescent substance located near the optical waveguide surface. In this case, there are the following advantages as compared with the case where the conventional apparatus is used. First, since all of the excitation light can be used effectively, fluorescence can be induced with higher efficiency as compared with the conventional method using evanescent light that seeps into the solution from the optical waveguide, and as a result, a large fluorescence signal is obtained. It becomes possible. Therefore, in an apparatus such as an immunoanalyzer for quantifying a specific component in a test solution, the detection limit can be improved. In addition, by arranging a plurality of light emission sources around the optical waveguide, additional effects such as further increasing the fluorescence intensity and performing analysis of various antigenic substances continuously are produced.

Second, even when a glass material other than quartz and an inexpensive plastic material such as polystyrene or polymethyl methacrylate are used as the material of the optical waveguide, the excitation light travels a short distance in the optical waveguide, so that the optical waveguide is not used. The influence of interfering fluorescence from the waveguide itself is small, and the background included in the fluorescence signal can be reduced. As a result, the S / N ratio of the fluorescence signal increases, and the detection limit can be improved even when applied to quantitative determination of a specific component in a test solution such as immunoassay. For example, as shown in the embodiment, by using a slide glass for an optical microscope which is generally widely used, it is not necessary to use an expensive material such as quartz.

Third, since the excitation light is not incident from the end face of the optical waveguide, there is no scattering or reflection of the excitation light on the end face of the optical waveguide, and the background contained in the fluorescence signal can be reduced. It is possible. As a result, the S / N ratio of the fluorescence signal increases, and the detection limit can be improved even when applied to quantitative determination of a specific component in a test solution such as immunoassay.

Fourth, since the excitation light is not incident from the end face of the optical waveguide, it is not necessary to form the end face into a prism shape or a lens shape, so that the manufacturing cost can be suppressed.

Fifth, in order to detect the fluorescent light that enters the optical waveguide and propagates while repeating total reflection, the fluorescent light generated within the range where the evanescent light exudes when the excitation light enters the optical waveguide is selected. And the surface selectivity of the optical waveguide can be ensured.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing one embodiment of a fluorescence analyzer of the present invention.

FIG. 2 is a conceptual diagram showing one embodiment when the fluorescence analyzer of the present invention is applied to immunoassay.

FIG. 3 is a schematic diagram of a conventional fluorescence analyzer.

FIG. 4 is a schematic diagram of a conventional fluorescence analyzer.

FIG. 5 is a schematic diagram of a conventional fluorescence analyzer.

FIG. 6 is a conceptual diagram illustrating evanescent collection of fluorescence.

FIG. 7 is a detailed assembly view showing one embodiment of the fluorescence analyzer of the present invention.

FIG. 8 is a detailed assembly view showing a conventional fluorescence analyzer.

FIG. 9 is a view showing a result of an example.

FIG. 10 is a diagram showing the results of an example.

[Explanation of symbols]

 Reference Signs List 1; optical waveguide 2; region on which binding antibody or binding antigen is immobilized 3; storage container 4; light emitting source 4a; xenon flash lamp 4b; optical filter 4c; optical lens 5; 5b; dichroic mirror 5d; optical filter 6; light receiving sensor 7; excitation light 8; fluorescence 21; assembly part 22; Z-axis stage 23a, 23b; table 24 on Z-axis stage; Antigen of interest 103; Restricted labeled antibody 104; Unrestricted labeled antibody 105; Light signal from constrained labeled antibody 106; Light signal from unrestricted labeled antibody

Claims (2)

[Claims] 1. A fluorescence analyzer on a solid phase surface for detecting a fluorescence signal from a fluorescent substance directly or indirectly bound on a solid phase surface or a fluorescent substance located near the solid phase surface, A material that is transparent to the fluorescence from the fluorescent substance to be analyzed is made of a material, and the optical waveguide that provides the solid phase surface and a substance that is transparent to excitation light that excites the fluorescent substance to be analyzed is made of a material. A storage container for containing a test solution expected to contain the optical waveguide and the analysis object therein,
A light radiation source capable of emitting light having a wavelength capable of exciting the fluorescent substance by fluorescence, such that the excitation light is emitted from a side facing the main side surface of the optical waveguide housed in the housing container toward the surface. The light emission source is arranged, and an optical signal emitted from the end face of the optical waveguide is arranged to be detected through optical means for selecting a fluorescent wavelength emitted from the fluorescent substance. The above device comprising a light receiving sensor arranged so as to be able to receive fluorescent light propagating in the wave path. 2. A binding antibody or binding antigen is fixed on the surface of a solid phase in advance, and the solid phase is combined with an antigenic substance in a test solution and a labeled antibody or labeled antigen to which a fluorescent substance or an enzyme has been bound in advance. After reacting and indirectly binding the labeled antibody or labeled antigen on the solid phase surface, the fluorescent signal of the fluorescent substance bound to the bound labeled antibody or labeled antigen or the fluorescent substance generated by enzymatic action is detected. By doing so, in an immunoanalyzer for analyzing a physical quantity relating to the presence or concentration of the antigenic substance in a test solution, an optical waveguide that provides a material that is transparent to fluorescence from the fluorescent substance and provides the solid phase surface A storage container containing the optical waveguide and the test solution therein, which is made of a material transparent to excitation light for exciting the fluorescent substance to be analyzed, and a fluorescent excitation of the fluorescent substance. A light radiation source capable of emitting light of a possible wavelength, the light radiation source being arranged to emit excitation light from a side facing the main side surface of the optical waveguide housed in the housing container toward the surface. And an optical signal emitted from the end face of the optical waveguide is arranged to be detected through optical means for selecting a fluorescent wavelength radiated from the fluorescent substance, and the fluorescence that penetrates the optical waveguide and propagates through the optical waveguide is detected. The above device comprising a light receiving sensor arranged to receive light.