JP2003287536A - Optical waveguide type protein chip and protein detecting apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical waveguide type protein chip capable of both shortening a reaction time between proteins in a specimen solution such as blood and an antibody and highly sensitively detecting protein molecules even through the use of the specimen solution of a very small quantity. <P>SOLUTION: The optical waveguide type protein chip is provided with a substrate, a first optical waveguide layer formed on the surface of the substrate, gratings each formed on the surfaces of both end parts of the first optical waveguide layer, a second optical waveguide layer made of a transparent conductive material having a refractive index higher than that of the first optical waveguide layer and formed on the first, optical waveguide layer located between the gratings, an antibody fixing film formed on the second optical waveguide layer, a first insulating member formed on the substrate containing the first optical waveguide layer and having a recessed well at a part at which the antibody immobilizing film is located, and an electrode membrane formed in such a way that its part is close to the well on the first insulating member for generating electric charge in-between the second optical waveguide layer. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an optical waveguide type protein chip and a protein detection device for detecting protein molecules in a sample solution.

[0002]

2. Description of the Related Art In the enzyme-linked immunosorbent assay (ELISA), an antibody is immobilized on the inner wall of a container called a microplate made of transparent plastic, a sample solution is injected, and a target protein molecule is reacted with the antibody to form a protein molecule. Is being detected. This reaction is spent at 4 ° C. for about 4 hours.

To measure protein molecules with high sensitivity,
A target protein molecule that has reacted with the antibody on the inner wall of the container is reacted with a secondary antibody labeled with an enzyme. Further, a dye that produces color or fluorescence with an enzyme is added to measure the absorbance or fluorescence intensity.

[0004]

However, the conventional protein microplate has a problem that the reaction time between the immobilized antibody and the protein molecule is long.

The present invention can shorten the reaction time between a protein molecule and an antibody in a sample solution such as blood, and can detect a protein molecule with high sensitivity even when a small amount of sample solution is used. It is intended to provide a possible optical waveguide type protein chip.

The present invention provides a protein detection device capable of simultaneously reacting protein molecules in a plurality of sample solutions such as blood with an antibody in parallel, and shortening the detection time and increasing the sensitivity. Is what you are trying to do.

[0007]

An optical waveguide type protein chip according to the present invention is formed on a substrate, a first optical waveguide layer formed on the surface of the substrate, and both end surfaces of the first optical waveguide layer. And a second optical waveguide layer formed on the first optical waveguide layer located between the gratings, the second optical waveguide layer being made of a conductive material having a higher refractive index than the first optical waveguide layer and being transparent. An antibody-immobilized film formed on the optical waveguide layer, and a first insulating member formed on the substrate including the first optical waveguide layer and having a well recessed at a position where the antibody-immobilized film is located, A part of the electrode is formed on the first insulating member so as to be close to the well, and an electrode thin film for generating charges between the second insulating layer and the second optical waveguide layer is provided. .

A protein detection device according to the present invention includes a substrate, a plurality of first optical waveguide layers formed on the surface of the substrate, and gratings formed on both end surfaces of each of the first optical waveguide layers. A plurality of second optical waveguide layers each formed on each of the first optical waveguide layers positioned between the gratings and made of a conductive material having a refractive index higher than that of the first optical waveguide layers; A first plurality of antibody-immobilized films respectively formed on the optical waveguide layer, and a well formed on the substrate including the first optical waveguide layer and having a well recessed at a position where each of the antibody-immobilized films is located; An insulating member; and a plurality of electrode thin films, which are formed on the first insulating member so as to be partially close to the respective wells and generate charges between the second optical waveguide layer and the second optical waveguide layer. Optical waveguide type protector A laser chip for injecting laser light into one end of each first optical waveguide layer of the protein chip; a light receiving element for receiving light emitted from the other end of each first optical waveguide layer of the protein chip; and A polygon mirror arranged between the protein chip and the laser element is provided.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION An optical waveguide type protein chip and a protein detection device of the present invention will be described in detail below with reference to the drawings.

FIG. 1 is a plan view showing an optical waveguide type protein chip used in this embodiment, and FIG. 2 is II- of FIG.
A sectional view taken along line II, and FIG. 3 is a sectional view taken along line III-III in FIG.

A substrate 1 made of glass, for example, has a plurality of first optical waveguide layers 2 having a higher refractive index than the substrate 1, for example, four first optical waveguide layers 2 formed on the surface in parallel with each other. These first optical waveguides 2 are formed, for example, by immersing in an ion exchange solution such as a molten salt of potassium nitrate at 380 to 400 ° C. to ion exchange high refractive index elements such as potassium and sodium. The grating 3 includes the first optical waveguide layer 2
Has a refractive index equal to or higher than that of the first optical waveguide layer 2 and is formed on both end surfaces of the respective first optical waveguide layers 2. These gratings 3 are made of photoresist, titanium oxide, zinc oxide, lithium niobate, GaAs, for example.

As shown in FIG. 2, a plurality of, for example, four second optical waveguide layers 4 each having an inclined end in the length direction have a higher refractive index than each of the first optical waveguide layers 2, and It is formed on each of the first optical waveguide layers 2 located between the two gratings 3. These second optical waveguide layers 4 are made of a transparent conductive material such as ITO or tin oxide.

A plurality of, for example, four antibody-immobilized films 5 are formed on the flat surface of the second optical waveguide 4, respectively. These antibody-immobilized membranes 5 are, for example, dextrin,
Made from silanes with carboxyl groups.

The first insulating member 6 is formed on the substrate 1 including the first optical waveguide layer 2, the second optical waveguide layer 4 and the antibody immobilizing film 5, and the antibody immobilizing film 5 is formed. The wells 7 are formed by denting the places where are located.
The first insulating member 6 is made of, for example, a photoresist containing fluorine. Specifically, the solution of the photoresist is applied onto the substrate 1 including the first and second optical waveguide layers 2 and 4 and the antibody-immobilized film 5, dried, and then exposed and developed. By the treatment, the first insulating member 6 having the outer shape shown in FIG. 1 and having the wells 7 formed by recessing the positions of the antibody-immobilized films 5 is produced.

A plurality of, for example, four electrode thin films 8 are formed on the first insulating member 6 located between the first optical waveguide layers 2 in parallel with the first optical waveguide layers 2 and near the center. It has an extending portion 9 extending near the opening of the well 7. These electrode thin films 8 are made of a metal such as Au, Pt, or Ti.

For example, another electrode thin film (reference electrode) 10 made of a metal such as Au, Pt, or Ti and a standard electrode 11 made of an Ag / AgCl thin film are provided on the first insulating member 6 located around the substrate 1. Has been formed. That is, the electrode thin film 8, the reference electrode 10, and the standard electrode 1
1 constitutes an electrochemical triode structure.

For example, the rectangular plate-shaped second insulating member 12 is formed on the first insulating member 6 including the wells 7 in a direction orthogonal to the plurality of first optical waveguides 2. The second insulating member 12 applies the sample solution to the well 7
A supply hole 13 and a discharge hole 14 for supplying and discharging to and from each well 7 are opened for each well 7.

The good heat conductive coating 15 is formed on the rear surface of the substrate 1 except the light incident region and the light emitting region. Examples of the good thermal conductive film 15 include a metal film such as copper and aluminum, and a ceramic film such as aluminum nitride and boron nitride.

Next, a protein detection device equipped with the above-mentioned optical waveguide type protein chip 16 will be described with reference to FIG.

This protein detection device emits a laser beam arranged on one end face side (right end face side) of the optical waveguide type protein chip 16 where one end of the plurality of first optical waveguide layers 2 is exposed. A laser element (for example, a semiconductor laser having a wavelength of 650 nm) 21 is provided. A collimator lens 22, a polarizing plate 23 and a polygon mirror 24 are sequentially arranged on the laser light emitting side of the laser element 21. A galvanometer mirror may be used instead of the polygon mirror 24. A first cylinder lens 25 is arranged on the laser light emitting side of the polygon mirror 24 so as to be parallel to the right end surface of the protein chip 16. The second cylinder lens 26 is arranged on the other end surface side (left end surface side) of the protein chip 16 where the other ends of the plurality of first optical waveguide layers 2 are exposed. The second polarizing plate 2 is provided on the laser light emitting side of the second cylinder lens 26.
7 and the light receiving element 28 are sequentially arranged.

In the protein detection device, the optical waveguide type protein chip 16 is detachable, and an accommodating portion (not shown) is provided at the disposition position.

Next, the operation of the above-mentioned optical waveguide type protein chip and protein detection device will be described.

First, the needle of the auto sampler (not shown)
The second insulating member 12 of the optical waveguide type protein chip 16
The antibody is supplied into the four wells 7 through the needles of these autosamplers and immobilized on the antibody-immobilized membrane 5 at the bottom of each well 7. In this case, even if the antibody immobilized on each antibody-immobilized membrane 5 is the same,
May be different.

Next, these autosampler needles are removed, and another autosampler needle (not shown) is inserted into the supply hole 13 of the second insulating member 12 in the same manner as shown in FIG.
As shown in FIG. 5, blood 29, which is a sample solution, is supplied into each of the wells 7 through the needles of these autosamplers, so that protein molecules in blood are placed in each well 7 according to the ELISA method such as silane having a carboxyl group. The antibody immobilized on the antibody-immobilized film 5 such as a coat is reacted. At the same time, by applying a voltage of, for example, 0.2 to 0.3 V to the reference electrode 11, a current flows between a plurality of, for example, four electrode thin films 8 and the reference electrode 10, so that the extension of each electrode thin film 8 is increased. Electric charges are generated between the output portion 9 and the second photoconductive layer 4 made of a conductive material.
At this time, since the protein molecules in the blood have an electric charge according to the pH, the protein molecules are directed toward the second photoconductive layer 4, that is, the antibody immobilized on the second photoconductive layer 4 is immobilized. It is drawn toward the chemical film 5. For this reason,
The reaction between the antibody immobilized on each antibody-immobilized membrane 5 and the target protein molecule in the blood takes place in a short time. In this reaction, by attaching the good heat conductive coating 15 to the back surface of the substrate 1, it becomes possible to make the temperature of the blood in each well 7 uniform.

Optical waveguide type protein chip 16 after reaction
4 was incorporated into the protein detection apparatus shown in FIG. 4, and a secondary antibody with a dye marker was supplied into each of the wells 7 through a needle of an autosampler (not shown) to convert the protein molecules reacted with the antibody on the antibody-immobilized membrane 5. On the other hand, an intercalator with a dye marker is acted on to develop a color.

In this state, as shown in FIG. 4, a laser beam having a wavelength of 650 nm is emitted from the laser element 21 to the polygon mirror 24 which is rotationally driven through the collimator lens 22 and the polarizing plate 23. At this time, the laser light is collimated by the collimator lens 22, adjusted so that the light intensities in the TE and TM modes are the same by the polarizing plate 23, reflected by the polygon mirror 24 that is driven to rotate, and reflected by the four of the protein chips 13. It is distributed toward the first optical waveguide layer 2. The distributed laser light is incident on the back surface side of the substrate 1 where the first optical waveguide layer 2 of the protein chip 16 is located as shown in FIG. 5, and passes through the substrate 1 at the interface between the grating 3 and the first optical waveguide layer 2. It is refracted and propagated through the first optical waveguide layer 2. The laser light propagated through the first optical waveguide layer 2 has two modes (TM) at the interface with the second optical waveguide layer 4 having a higher refractive index than the first optical waveguide layer 2.
Mode, TE mode) and propagate through the first and second optical waveguide layers 2 and 4. At this time, the antibody and the protein molecule react with each other on the antibody-immobilized film 5, and a change (for example, a change in absorbance) accompanying the color development causes a change in the light that propagates through the second optical waveguide layer 4 immediately below the antibody-immobilized film 5. The intensity changes. Thus, the light propagating through the first and second optical waveguide layers 2 and 4 is recombined and interferes at the interface between the optical waveguide layers 2 and 4 at the opposite end of the second optical waveguide layer 4. The change in the intensity of the light propagating through the second optical waveguide layer 4 can be amplified. As a result, the reaction between the antibody in the antibody-immobilized film 5 and the protein molecule in the blood, and the minute change of the light propagating through the second optical waveguide layer 4 due to the coloring caused by the dye marking also cause the second cylinder lens 26 and the second cylinder lens 26. Polarizing plate 27
It becomes possible to detect with the light receiving element 28 through. In addition, such a detection operation is performed simultaneously and in parallel in the plurality (four) of the first optical waveguide layers 2 of the protein chip 16 to identify the protein molecule.

As described above, according to the optical waveguide type protein chip of the present invention, an electric charge is generated between the extending portion 9 of each electrode thin film 8 and the second photoconductive layer 4 made of a conductive material, and the antibody is formed on the bottom. The protein molecules in the blood in each well 7 in which the immobilization film 5 is located are directed toward the second photoconductive layer 4, that is, the second photoconductive layer 4.
Since it can be attracted toward the antibody-immobilized film 5 located on the photoconductive layer 4, the reaction time between the antibody immobilized on each antibody-immobilized film 5 and the target protein molecule in blood is significantly shortened. Can be converted.

Also, by attaching the good thermal conductive coating 15 to the back surface of the substrate 1, the temperature of the sample solution in each well 7 can be made uniform, and more accurate detection can be performed.

On the other hand, FIG. 1 having a plurality of chip units.
~ According to the protein detection device in which the optical waveguide type rotein chip 16 shown in Fig. 3 is incorporated as shown in Fig. 4, the protein molecules in the blood as the sample solution are reacted with the antibody in each well 7 of the protein chip 16. After the color development reaction, light is incident on the first optical waveguide layer 2 located below each well 7 and split into two modes (TM mode and TE mode) at the interface with the second optical waveguide layer 4 during propagation. By merging, and measuring the transmitted light intensity of the light emitted from the first optical waveguide layer 2, the protein molecule in the blood in each well 7 can be detected. Therefore, protein molecules in blood can be detected by supplying blood into each well 7 in such a small amount as to be located above the surface of the antibody-immobilized film 5. As a result, compared with the conventional case where light is transmitted in the depth direction of the coloring solution in the well,
It is possible to reduce the amount of blood and reagent used as the sample solution. In addition, the first insulating member 6 having the well 7 can be formed into a photoresist containing fluorine, for example, by a photolithography technique, and the well dimension can be reduced to the micron order. Since the amount of blood used can be significantly reduced, the amount of blood used can be reduced.

Further, by using the protein detection device incorporating the protein chip 16 as shown in FIG. 4, it is possible to identify protein molecules of a plurality of sample solutions simultaneously in parallel with high sensitivity and shorten the detection time. You can

In the above-described embodiment, the first optical waveguide layer 2 in which the antibody-immobilized film 5 and the second optical waveguide layer 4 are laminated under the well 7 constitutes one chip unit, and this chip unit is formed on the substrate 1 in plural. Although arranged, the protein chip may be configured by disposing only one chip unit on the substrate.

In the above-described embodiment, the second insulating member 12
Although the supply hole 13 and the discharge hole 14 are provided so as to communicate with each well 7 of the first insulating member 6, the second insulating member may be omitted.

[0033]

As described in detail above, according to the present invention, it is possible to shorten the reaction time of a protein and an antibody in a sample solution such as blood, and to use a small amount of sample solution. It is possible to provide an optical waveguide type protein chip capable of detecting protein molecules with high sensitivity.

Further, according to the present invention, protein molecules in a plurality of sample solutions such as blood can be simultaneously reacted with an antibody in parallel, and the detection time can be shortened and the sensitivity can be improved. A device can be provided.

[Brief description of drawings]

FIG. 1 is a plan view showing an optical waveguide type protein chip of the present invention.

FIG. 2 is a sectional view taken along line II-II in FIG.

FIG. 3 is a sectional view taken along the line III-III in FIG.

FIG. 4 is a plan view showing a protein detection device of the present invention.

FIG. 5 is a cross-sectional view for explaining the operation of the protein detection device of the present invention.

[Explanation of symbols]

1 ... substrate, 2 ... the first optical waveguide layer, 3 ... Grating, 4 ... a second optical waveguide layer, 5 ... Antibody-immobilized membrane, 6 ... the first insulating member, 7 ... well, 8 ... electrode thin film, 12 ... a second insulating member, 15. Good thermal conductive film 16 ... Optical waveguide type protein chip, 21 ... Laser element, 24 ... Polygon mirror, 28 ... Light receiving element, 19 ... Sample solution (blood).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Ichiro Higashino             33, Shinisogo-cho, Isogo-ku, Yokohama-shi, Kanagawa             Inside the Toshiba Production Technology Center F-term (reference) 2G054 AA06 AA07 CA23 CE01 EA01                       FA16                 2H047 KA02 LA01 NA01 PA01 PA24                       PA30 QA01 QA04

Claims (6)

[Claims] 1. A substrate, a first optical waveguide layer formed on the surface of the substrate, gratings formed on both end surfaces of the first optical waveguide layer, and the first optical waveguide layer located between the gratings. A second optical waveguide layer formed on the optical waveguide layer and made of a conductive material having a higher refractive index than that of the first optical waveguide layer; and an antibody immobilization film formed on the second optical waveguide layer, A first insulating member having a well formed on the substrate including a first optical waveguide layer and recessed at a position where the antibody-immobilized film is located; and a part of the well on the first insulating member is close to the well And an electrode thin film for generating electric charges between the second optical waveguide layer and the second optical waveguide layer. 2. The first optical waveguide layer, the grating, the second optical waveguide layer, the antibody-immobilized film, the well, and the electrode thin film form one chip unit, and the chip unit is provided on the substrate surface as the first chip unit. The optical waveguide type protein chip according to claim 1, wherein one optical waveguide layer is formed so as to be parallel to each other. 3. The first insulating member is made of a photoresist, and the photoresist is applied to the substrate, dried, and then exposed and developed to be processed into a predetermined outer shape, and the well is formed. The optical waveguide type protein chip according to claim 1, which is formed. 4. A second insulating member having two holes for supplying and discharging a sample solution to and from the well is further formed on the first insulating member including at least the well. The optical waveguide type protein chip according to claim 1 or 2. 5. The optical waveguide type protein chip according to claim 1, wherein the good heat conductive coating is further formed on a desired portion of the back surface of the substrate. 6. A substrate, a plurality of first optical waveguide layers formed on the surface of the substrate, a grating formed on each end surface of each of the first optical waveguide layers, and located between the gratings. The first optical waveguide layer is formed on each of the first optical waveguide layers.
A plurality of second optical waveguide layers made of a conductive material having a higher refractive index than the optical waveguide layers, a plurality of antibody-immobilized films respectively formed on the respective second optical waveguide layers, and the first optical waveguide layer A first insulating member having a well that is formed on the substrate including a recess and is located at a position where each antibody-immobilized film is located; and a part of the first insulating member on the first insulating member so as to be close to each well. An optical waveguide type protein chip formed and provided with a plurality of electrode thin films for generating electric charges between the second optical waveguide layer; and a laser beam incident on one end of each first optical waveguide layer of the protein chip. And a light receiving element for receiving light emitted from the other end of each first optical waveguide layer of the protein chip; and a polygon mirror arranged between the protein chip and the laser element. Protein detection apparatus according to claim and.