JP2924707B2 - Optical waveguide type fluorescent immunosensor and method of manufacturing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sensor for measuring the concentration of a substance contained in a trace amount in a sample solution, and more particularly to an optical waveguide fluorescent immunosensor.

[0002]

2. Description of the Related Art An immunoassay method is known as a method for measuring trace components utilizing a specific reaction between an antigen and an antibody.
An immunosensor using an optical waveguide by applying this method has been proposed. As the optical waveguide immunosensor, two types, a label type and a non-label type, are known. As an unlabeled type, for example, an optical waveguide diffraction grating biochemical sensor as disclosed in JP-A-4-152249 is known. FIG. 5 shows the structure of this sensor.

A diffraction grating (grating) 22 is formed on a thin-film waveguide 21. When a laser light 23 is irradiated on the diffraction grating 22, it becomes a guided light 24 and propagates in the waveguide 21.
When the sample solution 25 is introduced onto the diffraction grating 22, proteins and ions are adsorbed on the diffraction grating 22, thereby changing the refractive index and the intensity of the guided light 24. Therefore, it is possible to measure the protein and ion concentrations in the sample from the intensity of the guided light 24. This diffraction grating 22
When an antibody-immobilized film is formed thereon, an immunosensor can be realized by forming an immune complex with the antigen in the sample 25, whereby the concentration of the antigen can be measured. However, such an unlabeled optical waveguide immunosensor works well when the sample contains a small amount of substances other than the substance to be measured, but, for example, in a sample composed of multiple components such as blood, Since components other than the substance to be measured also adsorb similarly (non-specific adsorption phenomenon), it is considered that they do not show an accurate response.

A labeling type using a fluorescent dye is described in "1984, Clinical Chemistry, Vol. 30, pages 1533 to 1538 (Suther).
land R. M. et. al. “Optical d
selection of antibody-anti
gen reactions at a glass-
liquid interface "Clin. Che
m. 30 (1984) p. 1533-1538) ". FIG. 6 shows a method for measuring the antigen to be measured contained in the sample solution by this method.

First, as shown in FIG. 6A, a first antibody 32 is immobilized on the surface of an optical waveguide substrate 31 (actually, a slide glass is used). Next, as shown in FIG. 6B, when the substrate 31 is brought into contact with a sample solution 33 containing the antigen 34 to be measured, the first antibody 32 and the antigen 34 to be measured are bound.
Further, as shown in FIG. 6C, when the second antibody 35 (fluorescently labeled) is added, an immune complex composed of the first antibody, the antigen to be measured, and the second antibody is formed on the surface of the substrate 31. . Then, as shown in FIG. 6D, when light is introduced into the substrate 31 via the prism 36, an evanescent wave is generated on the surface of the optical waveguide, and the evanescent wave excites a fluorescent dye to generate fluorescence. Therefore, the concentration of the antigen 26 to be measured can be determined by measuring the fluorescence intensity.

[0006] The evanescent wave is an electromagnetic wave that propagates only on the surface generated at the interface between the waveguide and the outside when light is totally reflected at the interface between the waveguide and the outside. The distance affected by the evanescent wave is about the wavelength (1 μm or less) from the surface of the waveguide, and when this light is used as excitation light, only the fluorescent dye near the surface of the optical waveguide can be excited. Therefore, when the first antibody is immobilized on the surface of the waveguide, only the fluorescent dye of the second antibody bound to the antigen-first antibody complex to be measured is excited. Method) is possible. Furthermore, in the case of the labeling type, since only the fluorescent dye can be detected, the method is not affected by nonspecifically adsorbed proteins and the like, and is a method suitable for a sample containing multiple components such as blood. .

A practical proposal based on this method is disclosed in, for example, Japanese Patent Application Laid-Open No.
2. Description of the Related Art An optical measuring device as disclosed in Japanese Patent No. 2263 is known. FIG. 7 shows an example of the structure of this device. A vessel composed of the optical waveguide 41, the wedge-shaped prism 42, and the solution container 43 to be measured is manufactured by integral molding of plastic (for example, polymethyl methacrylate), and is used as a fluorescent immunosensor. There is also an attempt to make evanescent waves efficiently by reducing the thickness of the optical waveguide layer.

[0008] For example, "1992, Analytical Chemistry, Vol. 64, No. 1, Choquette S.
J. et. al. “Planar Waveguide
Immunosensor with Fluore
sent Liposome Amplificat
ion "Analytical Chemistry,
64 (1) (1992)], a sodium ion in a glass substrate is exchanged for a silver ion to increase the refractive index and form a 6 μm-thick waveguide layer. Applied to immunosensors.

Also, "Sensors and & Co., 1990"
Actuators, Volume B1, 589-591 (Sloper AN et al. "Planar"
Indium Phosphate Monomod
e Waveguide Eventual Fi
eld Immunosensor "Sensors
nd Actuators, B1 (1990) p. 58
No. 9-591), a waveguide layer of 1 μm or less is formed by spin-coating indium phosphate on a glass substrate and used as a fluorescent immunosensor. However, since all of these sensors use a prism for optical coupling, there is a problem that downsizing of the sensor is difficult and the cost is high.

On the other hand, a grating other than a prism is conventionally known as an optical waveguide coupling device. This is described, for example, in "Applied Puzzix Letters, 1971, Vol. 18, No. 12 (" Format
ion of OpticalWaveguides
in Photoresist Films "Appl
ied Physics Letters, 18 (1
2) (1971) ", and many other examples are known. FIG. 8 shows an optical waveguide in this document.

A glass substrate 51 having a refractive index n ₃ = 1.512
An optical waveguide layer 52 having a refractive index n ₂ = 1.618 and a thickness t ₂ = 620 nm is formed thereon, and a thickness t _g = 60 nm and a grating pitch Λ = 660 nm on a part of the surface of the optical waveguide layer 52.
Is formed, and the refractive index n ₁ =
It is in contact with the 1.00 air layer. With this configuration, the light 54 incident on the grating coupler 53 at an angle θ _i
Propagates through the optical waveguide layer 52 as light having an angle θ _d .

Here, the coupling condition of the grating coupler to the optical waveguide layer is generally expressed by the following condition: n ₃ sin θ _i = n ₂ sin θ _d -qλ / Λ (1) Where q = 0, ± 1, ± 2
.. Is the order of diffraction, and λ is the wavelength of light.

On the other hand, the waveguide condition of the optical waveguide layer (TE mode)
Is represented by the following equation. 2mπ = 2K ₂ T- ₂ Tan ^-1 (K ₁ / K ₂ ) -2 Tan ^-1 (K ₃ / K ₂ ) (2) K ₁ = (| β ₂ -ni ² ko ² |) ^1/2 ( 3) β = k ₀ n ₂ sin θ _d ko = 2π / λ (4) where m = 0, 1, 2,...

Therefore, θ _d for guiding light is obtained from the equations (2) to (4), and then θ _i is calculated based on the equation (1).
It is possible to design a grating coupler such that the light incident at is θ _d .

[0015]

In an optical waveguide fluorescent immunosensor, if a grating is used for optical coupling instead of a prism, the sensor can be miniaturized and manufactured at low cost. However, in the conventional optical waveguide type fluorescence immunosensor, since plastic such as glass, indium phosphate, or polymethyl methacrylate is used as a material of the optical waveguide layer, a grating structure of about sub-micron by a fine processing technology. Was difficult to form. For this reason, it is difficult to realize an optical waveguide type fluorescence immunosensor provided with an optical coupling device using a grating, and as a result, there is a problem in that it is difficult to reduce the size of the sensor and the cost is high.

[0016]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a waveguide-type fluorescent immunosensor and a method of manufacturing the same, which enable optical coupling by a grating, thereby reducing the size and realizing a low cost. .

[0017]

An optical waveguide type fluorescence immunosensor of the present invention comprises a substrate and an optical waveguide layer formed on the substrate and having a grating formed at one end of the surface by etching. And an antibody-immobilized film formed on the optical waveguide layer. Here, the optical waveguide layer is composed of a silicon oxide film or a polyimide film.

Further, the method for producing an immunosensor of the present invention comprises:
Forming a grating by forming an optical waveguide layer on a substrate and selectively etching one end of the surface of the optical waveguide layer using a mask. In this case, it is preferable to form a silicon oxide film by a sol-gel method as the optical waveguide layer.

[0019]

By adopting a silicon oxide film or a polyimide film as the optical waveguide layer, a grating as an optical coupling device can be finely and easily formed by an etching method. Cost reduction can be realized.

[0020]

Next, an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a sectional view of an optical waveguide fluorescent immunosensor 5 according to one embodiment of the present invention. An optical waveguide layer 2 is formed of a silicon oxide film on a barium borosilicate glass substrate 1, and a grating 3 is formed at one end thereof.
An antibody immobilization film 4 is formed on the surface of the optical waveguide layer.

FIG. 2 is a diagram showing a method of manufacturing the optical waveguide fluorescent immunosensor shown in FIG. 1 in the order of steps. First, FIG.
1A, a silicon oxide film 2A is formed on a barium borosilicate glass substrate 1 by a sol-gel method. That is, the silicon oxide coating agent is spin-coated (2500 rpm, 30 seconds) on the substrate 1, dried, and baked at 300 ° C. for 20 minutes to form the silicon oxide film 2A.

Next, as shown in FIG. 2B, a photoresist PR is spin-coated on the silicon oxide film 2A, and exposed and developed using a mask to form a grating pattern. Subsequently, as shown in FIG. 2C, the silicon oxide film 2A is etched using a potassium hydroxide solution using the photoresist PR as a mask.
Thereafter, when the photoresist PR is removed by a stripping agent, as shown in FIG. 2D, an optical waveguide layer 2 made of a silicon oxide film having a thickness of 1 μm and having a grating 3 is completed.

Here, when forming the silicon oxide film 2A, various techniques such as a CVD method and a silicon oxidation method used in the semiconductor manufacturing technology field can be adopted. The silicon oxide film 2A can be etched by dry etching such as reactive ion etching in addition to wet etching such as potassium hydroxide.

In the substrate having the formed optical waveguide layer 2 made of a silicon oxide film, the refractive index n ₃ of the barium borosilicate glass substrate 1 is 1.53, and the refractive index n of the optical waveguide layer 2 is n.
_{Since 2} = 1.75, the grating pitch す る と = when calculated using the above equations (1) to (4).
At 500 nm, it can be seen that the light propagates through the optical waveguide layer 2 at an incident angle θ _i = 49 degrees and θ _d = 81.8 degrees.

Then, as shown in FIG. 1, an antibody immobilization film 4 is formed on the surface of the optical waveguide layer 2 made of the silicon oxide film. The composition and manufacturing method of the antibody-immobilized film 4 differ depending on the type of antibody. For example, when an antibody-immobilized film made of a protein is formed, silane coupling is performed on the surface of the optical waveguide layer 2. The agent is spin coated and selectively etched to leave only the required areas. Then, the protein is cross-linked to the amino group exposed on the surface using a cross-linking agent, whereby the antibody-immobilized membrane 4 can be formed.

FIG. 3 is a block diagram of an optical waveguide type fluorescence immunosensor measuring apparatus using the immunosensor 5 shown in FIG. Argon ion laser light (488 nm) from laser device 6
Is incident on the immunosensor 5 at a fixed angle. Further, a photomultiplier tube 7 for detecting fluorescence generated in the immunosensor 5 is provided. Although the fluorescence is measured from the lower surface of the immunosensor 5 in this example, the same applies to the measurement of the fluorescence from the upper surface of the sensor.

FIG. 4 is an explanatory diagram of a method for measuring the concentration of an antigen in a sample solution using the fluorescence immunosensor measuring device of FIG.
As shown in FIG. 4A, 10 μl of the sample solution 8 is dropped on the antibody-immobilized film 4 on the optical waveguide layer 2 of the immunosensor 5. Then, as shown in FIG. 4B, the antigen 9 contained in the sample solution 8 binds to the first antibody 4a of the antibody immobilization film 4 by an immunoreaction. Next, as shown in FIG. 4C, a solution 11 containing the second antibody 10 labeled with a fluorescent dye is dropped. The fluorescent dye is selected depending on the laser light to be used, and FITC is most suitable for an argon ion laser. The second antibody 10 binds to the antigen 9 at a site different from the first antibody 4a.
As a result, as shown in FIG.
An immune complex consisting of the antibody-antigen-second antibody is formed.

When the laser beam 12 incident from the grating 3 propagates in the optical waveguide layer 2, an evanescent wave is generated on the surface of the optical waveguide layer. The fluorescence generated when the fluorescent dye is excited by the evanescent wave is detected by a photomultiplier tube. Since there is a correlation between the fluorescence intensity and the concentration of the antigen contained in the sample solution, the concentration of the antigen can be determined.

Incidentally, in the immunosensor of the present invention, the optical waveguide layer may be formed of polyimide. In this case, the optical waveguide layer can be formed in the same manner as the case of the silicon oxide film by applying polyimide on the substrate and performing selective etching. However, the refractive index n _{2 of the} polyimide is 1.64.
Therefore, when calculated using the above equations (1) to (4), when the grating pitch Λ = 500 nm, light enters the waveguide at an incident angle θ _i = 80.77 degrees and θ _d = 80.7.
It will propagate at 7 degrees.

[0030]

As described above, the optical waveguide fluorescent immunosensor according to the present invention has a grating as an optical coupling device at one end of the surface of the optical waveguide layer, so that a prism or the like is not required. In addition, there is an effect that the size and cost of the sensor can be reduced. In particular, by using a silicon oxide film or a polyimide film as the optical waveguide layer, the grating can be formed finely and easily, and the miniaturization and cost reduction of the sensor can be promoted.

Further, in the manufacturing method of the present invention, since the grating is formed by the etching method after the formation of the optical waveguide layer, it is possible to easily perform the fine processing of the grating, and to reduce the size and the size of the sensor. Enables cost reduction. Particularly, by forming a silicon oxide film as an optical waveguide layer by a sol-gel method, there is an effect that the optical waveguide layer itself can be easily formed.

[Brief description of the drawings]

FIG. 1 is a sectional view of an immunosensor of the present invention.

FIG. 2 is a sectional view illustrating a method of manufacturing the immunosensor of FIG. 1 in the order of steps.

FIG. 3 is a conceptual configuration diagram of an immunosensor measurement device using the immunosensor of FIG. 1;

FIG. 4 is a schematic diagram for explaining a measurement method using the immunosensor measurement device of FIG. 3;

FIG. 5 is a cross-sectional view showing an example of a conventional optical waveguide diffraction grating biosensor.

FIG. 6 is a schematic diagram for explaining a measuring method using a conventional label type optical waveguide immunosensor.

FIG. 7 is a cross-sectional view illustrating an example of a conventional immunosensor measurement device using a prism.

FIG. 8 is a cross-sectional view showing an example of an apparatus using a grating for coupling of an optical waveguide.

[Explanation of symbols]

 Reference Signs List 1 glass substrate 2 optical waveguide layer 3 grating 4 antibody immobilization film 5 immunosensor 6 laser device 7 photomultiplier tube 8 sample solution 9 antigen 10 second antibody 11 solution 12 laser light

Claims (5)

(57) [Claims] And 1. A substrate is formed on the substrate, the table
An optical waveguide layer in which a grating is formed in one end portion by etching of the surface, is composed of an antibody immobilized membrane formed on the optical waveguide layer, it is introduced into the optical waveguide through the grating of the surface of said substrate An optical waveguide fluorescent immunosensor for measuring the intensity of fluorescence generated from a labeled fluorescent dye that forms an immune complex with an antibody immobilized on the optical waveguide surface by being excited by an evanescent wave generated by generated light. Wherein said optical waveguide layer is composed of a silicon oxide film
The optical waveguide fluorescent immunosensor according to claim 1 , wherein: Wherein of construction the optical waveguide layer is a polyimide film
The optical waveguide fluorescent immunosensor according to claim 1 , wherein 4. A step of forming an optical waveguide layer on a substrate;
A mask is formed on the optical waveguide layer, said optical waveguide layer
Forming a grating by selectively etching one end of the surface; and selectively forming an antibody-immobilized film corresponding to the antibody to be measured on the surface of the optical waveguide layer. Of manufacturing a fluorescent immunosensor of the type. 5. The method according to claim 4 , wherein the step of forming the optical waveguide layer is a step of forming a silicon oxide film by a sol-gel method.