JP2003042946A - Surface plasmon resonance sensor, surface plasmon resonance sensor system using the surface plasmon resonance sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure and process a large number of samples at once, to measure with proper precision, and to reduce cost to enable disposable use due to its simple constitution. SOLUTION: This surface plasmon resonance sensor 20 is provided with a light guide panel 21 provided with a light reflecting face 21a, a light incident face 21b for making light beam get incident into the light reflecting face 21a, and a light-emitting face 21c for emitting reflected light of the light beam reflected by the light-reflecting face 21a. A metal thin film 23 for causing surface plasmon resonance, and a biochemical reaction detecting portion 24 provided on the metal thin film 23 to contact with a DNA sample 25, are provided in the light-reflecting face 21a. The surface plasmon resonance sensor 20 is also provided with a light-extracting part 22, arranged in the emitting face 21c of the light guide panel 21, and comprising a transparent thin film, such as resin having a refractive index which is higher than that of the light guide panel 21.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to detecting a minute mass change of a liquid sample which is a target of biochemical measurement.
The present invention relates to a surface plasmon resonance sensor device utilizing a surface plasmon resonance phenomenon, and more particularly, to a surface plasmon resonance sensor which is inexpensive, disposable, and capable of rapidly measuring and processing a large number of samples.

[0002]

2. Description of the Related Art Conventionally, a surface plasmon resonance sensor device using a surface plasmon resonance phenomenon has been known as a method for detecting the amount of physicochemical change of a substance with the progress of biochemical reaction. For example, as shown in FIG. 6, a metal thin film 2 of gold or silver is formed on the surface of a parallel glass substrate (or glass prism) 1 having a diffraction grating by using a film forming technique such as vacuum deposition. When the laser beam 3 is irradiated from the glass substrate 1 side toward the interface with the metal thin film 2 at an angle satisfying the total reflection condition, surface plasmon resonance is excited in the metal thin film 2 at a specific incident angle θ. That is.

When surface plasmon resonance is excited, optical energy is absorbed by plasmon waves on the metal thin film 2, so that the intensity of the reflected light 5 reflected at the interface between the parallel glass substrate 1 and the metal thin film 2 sharply decreases. To do. FIG. 7 shows the relationship between the incident angle θ and the intensity of the reflected light 5. Here, there is a relation that the incident angle θ at which the surface plasmon resonance occurs depends on the density of the medium such as the sample solution of the measurement unit 4 in contact with the metal thin film 2 (that is, the mass of the metal thin film surface).

Therefore, the reflected light 5 reflected from this optical system
By specifically determining the reflection angle at which the surface plasmon resonance phenomenon occurs, and the incident angle θ when the surface plasmon resonance phenomenon occurs, the density of the medium, that is, the metal It is possible to obtain the change in mass on the surface of the thin film.

As described above, a minute change in the mass of the surface of the metal thin film can be sensitively detected as a large change in the amount of light due to surface plasmon resonance absorption of the metal thin film. Therefore, according to the measurement method to which the surface plasmon resonance phenomenon is applied, it is possible to measure an extremely small amount, and a mass change of 1 picogram can usually be detected. Therefore, it is also applied to biochemical components that handle minute amounts that cannot be measured by general measurement methods. For example, as shown in FIG. 7, the presence or absence of DNA binding in the measurement unit 4 (see FIG. 6) appears as a difference in the incident angle θ at which the intensity of the reflected light 5 decreases, and thus a slight change in mass due to DNA binding. Can be detected.

Generally, the above-mentioned surface plasmon resonance sensor device individually measures a biochemical reaction carried out at one detection site, and since the measurement work efficiency is low, a large number of samples are measured. Multi-point detectors have been developed. For example, US Pat. No. 5,629,921.
There are biosensor devices as described in US Pat. This will be briefly described with reference to FIG.
Are arranged on the same plane on the metal thin film 14 deposited on the glass substrate 13, and the light beam from the light source 17 is incident on the prism 16 as parallel light through the collimating lens 18 and reflected by the inspection unit 11. By detecting the light with the photodetector 19, it is possible to measure the number of sample portions 15 at one time.

[0007]

However, since the prism 16 is used as the light guide plate for causing the surface plasmon resonance phenomenon and the sample portion 15 is divided into a large number, the apparatus is large-scale and very large. Since the cost is high, there is a case where disposable is desired depending on the sample to be inspected, but there is a problem that disposable is uneconomical.

Further, in order to maintain the measurement accuracy when the prism 16 is used as the light guide plate, two surfaces of the prism 16 on which the incident light from the light source 17 is incident and on which the reflected light is emitted are polished. However, there was a problem that it took time to process. Further, when divided into a large number, the cross-sectional width in the optical path of the prism 16 becomes wide, various reflected lights are mixed at the measurement point, and the measurement accuracy is reduced.

Therefore, the present invention has been made in order to solve the above-mentioned problems of the prior art, and it is possible to measure a large number of samples at a time, the measurement accuracy is good, and the surface is inexpensive and disposable. Its main purpose is to provide a plasmon resonance sensor.

Another object of the present invention is to provide a surface plasmon resonance sensor device for measuring a large number of samples at once using the above-mentioned surface plasmon resonance sensor.

[0011]

In order to achieve the above-mentioned object, a surface plasmon resonance sensor according to a first aspect of the present invention is a thin film that causes surface plasmon resonance, and a thin film provided on the thin film. A light-reflecting surface having a biochemical reaction detection site in contact with the light-reflecting surface, a light-incident surface for making a light beam incident on the light-reflecting surface, and an emission surface for emitting reflected light of the light beam reflected by the light-reflecting surface. A plate-shaped light guide plate,
And a light extraction part which is disposed on the emission surface of the light guide plate and is made of a thin film having a refractive index higher than that of the light guide plate.

It is preferable that a plurality of biochemical reaction detection sites are provided on the thin film of the light reflecting surface. Further, it is preferable that the light incident surface of the light guide plate is inclined at an acute angle with respect to the light reflecting surface. The light extraction part may be made of transparent resin.

According to another aspect of the present invention, a surface plasmon resonance sensor device according to a fifth aspect includes the surface plasmon resonance sensor according to any one of the first to fourth aspects,
It is characterized by comprising a light source unit for irradiating the surface plasmon resonance sensor with a light beam, and a light detection unit for receiving the reflected light from the surface plasmon resonance sensor.

The light detecting means is preferably an array sensor extending in the longitudinal direction of the light extraction portion of the surface plasmon resonance sensor. The light beam emitted from the light source unit is preferably parallel light having a width that covers at least the width of the biochemical reaction test site. The light source of the light source unit may be a variable wavelength light source.

Further, the surface plasmon resonance sensor is
It is possible to swing the light beam around an axis perpendicular to the optical path of the light beam, and swing the light detection unit integrally with the surface plasmon resonance sensor.

Further, the light source unit includes a light source and a reflection plate for reflecting the light beam emitted from the light source to the surface plasmon resonance sensor, and the reflection plate is used for reflecting the light beam from the light source. It is also preferable that the biochemical reaction inspection site is swung so as to be deflected along the longitudinal direction.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Next, preferred embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, the same reference numerals indicate the same or corresponding parts.

First, an embodiment of a surface plasmon resonance sensor according to the present invention will be described with reference to FIG. The surface plasmon resonance sensor 20 includes a plate-shaped light guide plate 21 formed of glass or resin, and a light emitting surface 21 of the light guide plate 21.
and a light extraction part 22 attached to c.

The light guide plate 21 has a substantially trapezoidal shape,
The longer one of the two parallel surfaces is the light reflecting surface 21a, the shorter one is the light emitting surface 21c, and the inclined surface is the light incident surface 21.
b. The light reflection surface 21a is a flat surface, and a metal thin film 23 such as gold or silver is formed thereon by a well-known method such as an ion plating method, a sputtering method, or a vapor deposition method. . As the thin film, not only a metal but also a semiconductor thin film made of, for example, silicon can be used. When a light beam having a specific incident angle is incident on the light reflection surface 21a having the metal thin film 23, the surface plasmon resonance phenomenon is excited.

On the lower side of the metal thin film 23, that is, on the open side, a biochemical reaction detection site 24 that comes into contact with a liquid DNA sample 25 to be inspected is provided in the longitudinal direction of the light reflecting surface 21a of the light guide plate 21. Therefore, a plurality, in this embodiment, 6
They are arranged in a row. Biochemical reaction detection site 2
4 is formed by depositing a substance (reagent) such as a polymer on the metal thin film 23. In addition, since it is formed on a flat surface, the interval between adjacent parts of the biochemical reaction detection parts 24 arranged in a line is 10 μm to 1 c.
They can be arranged very close to each other at an arbitrary interval of about m.

The light incident surface 21b of the light guide plate 21 is inclined at an acute angle with respect to the light reflecting surface 21a. This light reflecting surface 21
The angle α of the light incident surface 21b with respect to a is radiated from the light source 31 of the light source unit 30, and the light beam that has become parallel light with a width that covers at least all biochemical reaction test sites 23 via the lens 32, It is preferable that the angle is such that the light is totally reflected by the light reflecting surface 21a. This is because the utilization efficiency of the light beam from the light source 31 is improved. Since the light incident surface 21b is inclined, it is possible to easily adjust the angle at which the light beam from the light source 31 is totally reflected by the light reflecting surface 21a.

A light extraction portion 22 is provided on the light emitting surface 21c. The light extraction part 22 is composed of a thin film of a transparent material such as resin having a refractive index higher than that of the light guide plate 21. In addition, the light extraction part 22 has a light reflection surface 21a.
It is attached along the longitudinal direction of the light emitting surface 21c over the length of the reflected light reflected by the light emitting surface 21c.

Light guide plate 2 of surface plasmon resonance sensor 20
Reference numeral 1 is a general plate-shaped glass substrate or resin substrate having a thin cross section in the width direction. When the light guide plate 21 having such a thin cross section is used, since the light beam from the light source passes through a two-dimensional path from being incident to being emitted,
The spread of light can be narrowed, and unnecessary reflected light does not mix. Therefore, the measurement accuracy is improved as compared with the case where a prism is used as in the related art. The light guide plate 21
As for the plate thickness, the thinner it is, the more unnecessary reflected light is not mixed, which is preferable, but in practice, it is 0.3 to 1.
The plate thickness is preferably about 0 mm.

The light guide plate 21 of the surface plasmon resonance sensor 20 can be manufactured very simply by performing general precision cutting on a general glass substrate or resin substrate. The light incident surface 2 can be simply cut by such precision cutting.
It is possible to obtain the necessary and sufficient accuracy for the 1b and the light emitting surface 21c. However, only the light reflection surface 21a on which the metal thin film 23 is deposited needs to be polished. However, when a prism is used as in the prior art, it is necessary to polish two surfaces, a light incident surface and a light reflecting surface, and the light guide plate 21 of the present application has only one surface to be polished, so Is easy.

The surface plasmon resonance sensor according to the present invention is obtained by vapor-depositing the metal thin film 23 on the light guide plate 21 formed only by such simple processing, and by coating, for example, resin which functions as the light extraction portion 22. 20 can be formed. Therefore, the sensor main body can be made very inexpensively, and the surface plasmon resonance sensor for detecting a plurality of samples can be disposable.

Next, a surface plasmon resonance sensor device using the surface plasmon resonance sensor 20 having such a configuration will be described. Referring again to FIG. 1, the surface plasmon resonance sensor device includes a surface plasmon resonance sensor 20, a light source unit 30 that irradiates the surface plasmon resonance sensor 20 with a light beam, and a light extraction unit 2 of the surface plasmon resonance sensor 20.
2 and a light detecting means 40 for receiving the reflected light emitted from the light source 2.

The light source unit 30 irradiates the light reflecting surface 21a with a light beam for obtaining various incident angles.
1 and a lens 32 for converting the light beam from the light source 31 into parallel light having a width that covers at least all biochemical reaction test sites 23. On the other hand, the light detection means 40 arranged on the light extraction section 22 side is an array sensor extending in the longitudinal direction of the light extraction section 22. That is, the array sensor 40 as the light detecting means is arranged so as to extend in a direction in which the reflected light of the light beam reflected by the light reflecting surface 21a can be received at various reflection angles.

The light source 31 and the array sensor 40 are electrically connected to a measuring unit, an arithmetic unit, etc., which are not shown, and the DNA is detected by detecting the incident angle at which the surface plasmon resonance angle detected by the array sensor 40 is generated. It is possible to identify whether or not a specific component of DNA is present in the sample 25.

A measuring method using the surface plasmon resonance sensor device configured as described above will be described. First, the DNA sample 25 to be inspected is attached to the biochemical reaction inspection site 23. The method will be described with reference to FIG.
The surface plasmon resonance sensor 20 is infiltrated in the inspection container 26 containing a small amount of the A sample 25. At that time, the gap between the biochemical reaction inspection site 23 and the bottom of the inspection container 26 is 100 μm.
By setting a microchannel to about m, the test DNA is quickly attached to the biochemical reaction test site 23. The substances to be attached to the biochemical reaction detection site 27 in advance include proteins such as antibodies and enzymes, DN
Various substances such as A-derived substances or other substances that selectively adsorb or react with other substances can be considered.

Next, the surface plasmon resonance sensor 20 to which the DNA sample 25 is attached is detachably attached to a predetermined position of the optical system of the surface plasmon resonance sensor device. Next, a light beam is emitted from the light source 31, and the light beam that has passed through the lens 32 is reflected by the light reflecting surface 21a above the plurality of biochemical reaction detection sites 24 to which the DNA sample 25 is attached. Then, the reflected light that has been reflected and passed through the light extraction unit 22 is received by the array sensor 40. Then, the difference in the intensity of the received reflected light is detected to detect whether surface plasmon resonance has occurred. From the phase shift of the incident angle θ at which this surface plasmon resonance occurs, the minute mass change of the substance on the metal thin film 23 is detected. As a result, the biochemical reaction detection site 24
It is discriminated whether or not a specific component was present in the DNA sample 25 attached to the.

In this embodiment, when the light beam from the light source 31 is made incident at a specific angle (angle at which surface plasmon resonance occurs) peculiar to each specific component to be detected in the DNA sample, the angle is constant. The difference in the intensity of the reflected light from each inspected part in was examined. However, it is possible to detect various components by changing the incident angle of the incident light from the light source.

Next, another embodiment of the multipoint detecting device using the surface plasmon resonance sensor 20 will be described with reference to FIG. In FIG. 3, the surface plasmon resonance sensor 20 and the light detection means 40 have the same configurations as those of the previous embodiment, and therefore their explanations are omitted.

The light source section 30 'is composed of a variable wavelength light source 31 and a reflection plate 34 for reflecting the light beam from the variable wavelength light source 31 to the surface plasmon resonance sensor 20. Although it is described as a single light in the drawings,
The light beam emitted from the light source is parallel light having a width that entirely covers the plurality of biochemical reaction detection sites 24 attached, as in the previous embodiments. The variable wavelength light source 3
1 and the light detection means 40 are connected to the processing device 50.

The measuring method of the surface plasmon resonance sensor device thus constructed will be described.
A light beam having a predetermined wavelength is irradiated, and the intensity of reflected light at each biochemical reaction detection site is measured. Then, by sequentially changing the wavelength of the light beam emitted from the variable wavelength light source 31, the change in the intensity of the reflected light at each biochemical reaction detection site for each wavelength is measured, and the wavelength at which the surface plasmon resonance occurs is measured. Ask. This utilizes the attribute that the surface plasmon resonance depends on the frequency of light, and the same relationship as the graph shown in FIG. 7 can be derived. Thereby, whether or not a specific component is present in the DNA sample 25 attached to the biochemical reaction detection site 24 is discriminated from the deviation of the wavelength of incident light that causes surface plasmon resonance. Although the reflecting plate 34 is used in this embodiment, the light beam may be directly emitted from the variable wavelength light source 33 to the surface plasmon resonance sensor 20.

Next, still another embodiment of the multipoint detecting device using the surface plasmon resonance sensor 20 will be described with reference to FIG. In FIG. 4, the surface plasmon resonance sensor 20, the light detection means 40, and the processing device 50 have the same configurations as those of the previous embodiment, and therefore their explanations are omitted.

In this embodiment, the light source section 30 ″ is composed of a single light source 35 and a reflection plate 36 for reflecting the light beam from the light source 35 to the surface plasmon resonance sensor 20. The reflecting plate 36 is adapted to swing by a driving means (not shown). More specifically, the reflector is swung so as to deflect the light beam from the light source 35 along the longitudinal direction of the biochemical reaction test site. That is, for each biochemical reaction detection site,
The incident angle of incident light is configured to swing within ± 5 ° of the angle at which surface plasmon resonance is excited. In addition,
A piezoelectric element or the like is used as the driving means.

A measuring method of the surface plasmon resonance sensor device configured as described above will be described. Each time the light beam is emitted from the light source 35, the angle information of the reflection plate 36 at that time is input to the processing device 50 as a control signal for controlling the driving unit. At the same time, the intensity information of the reflected light is also input to the processing device 50 from the light detection means 40, and the signal of the width information of the light detection means 40 is also input to the processing device 50. Based on this width information, it is determined whether the intensity of the reflected light detected at the specific position is the one in which the inclination of the incident light is shifted by the driving means or the one in which the surface plasmon resonance is actually excited. to correct. In this way, it is possible to test a plurality of DNA samples at once.

Further, still another embodiment of the multipoint detecting device using the surface plasmon resonance sensor 20 will be described with reference to FIG. In FIG. 5, the surface plasmon resonance sensor 20, the light source 35, and the processing device 50 have the same configurations as those of the previous embodiment, and therefore the description thereof will be omitted. This embodiment is different from the previous embodiment in that FIG.
In the example shown in (1), the reflecting plate is swung in order to detect the surface plasmon resonance angle, but in this embodiment, the surface plasmon resonance sensor is swung.

In this embodiment, the light source section 30 '''is
It is composed of a single light source 35 and a reflection plate 38 that reflects the light beam from the light source 35 to the surface plasmon resonance sensor 20. On the other hand, the surface plasmon resonance sensor 20 is oscillated by ± 5 ° around an axis in the direction perpendicular to the optical path of the light beam from the light source 35 by a driving unit (not shown). Further, the light detecting means 40 'is also swung in synchronization with the surface plasmon resonance sensor 20 in synchronization with the surface plasmon resonance sensor 20 by a driving means (not shown). As described above, by swinging the surface plasmon resonance sensor 20, the light reflecting surface itself swings, and the incident angle of light at that portion swings within a predetermined range. The same effect as that of the form can be obtained.

In this embodiment, whether or not a specific component was present in the DNA sample 25 attached to the biochemical reaction detection site 24 was identified. Depending on the type of substance (reagent) attached, protein or nucleic acid hybridization, antibody assay, or DNA chip (or DNA array) can be detected.

[0041]

Since the surface plasmon resonance sensor according to the present invention includes the plate-shaped light guide plate and the light extraction portion formed of a thin film having a refractive index higher than that of the light guide plate, the cross section in the width direction is thin. Since unnecessary reflected light does not mix and the measurement accuracy improves, it can be processed by simple precision cutting, so the structure is simple and it can be inexpensive and disposable.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a surface plasmon resonance sensor using surface plasmon resonance according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing a method of attaching a DNA sample to the surface plasmon resonance sensor of FIG.

FIG. 3 is a schematic view of a surface plasmon resonance sensor device according to an embodiment of the present invention, which uses the surface plasmon resonance sensor of FIG.

FIG. 4 is a schematic diagram of a surface plasmon resonance sensor device according to another embodiment of the present invention, which uses the surface plasmon resonance sensor of FIG. 1.

5 is a schematic view of a surface plasmon resonance sensor device according to still another embodiment of the present invention, which uses the surface plasmon resonance sensor of FIG.

FIG. 6 is a schematic diagram of a detection principle using surface plasmon resonance.

7 is a characteristic diagram showing a relationship between a light incident angle and a reflected light intensity in the surface plasmon resonance in FIG.

FIG. 8 is a schematic diagram of a conventional biosensor device for measuring a large number of samples.

[Explanation of symbols]

1 ... Glass substrate, 2 ... Metal thin film, 3 ... Laser beam, 4 ...
Measuring unit, 5 ... Reflected light, 10 ... Biosensor device, 11 ...
Inspection part, 13 ... Glass substrate, 14 ... Metal thin film, 15 ... Sample part, 16 ... Prism, 17 ... Light source, 18 ... Collimating lens, 19 ... Photodetector, 20 ... Surface plasmon resonance sensor, 21 ... Light guide plate , 22 ... Light extraction part, 23 ... Metal thin film, 24 ... Biochemical reaction detection site, 25 ... DNA sample, 26 ... Container, 30, 30 ′, 30 ″, 30 ″ ′
... light source part, 31 ... light source, 32 ... lens, 33 ... variable wavelength light source, 34 ... reflector, 35 ... light source, 36 ... reflector, 4
0, 40 '... Photodetector (array sensor), 50 ... Processing device, α ... Angle.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Satoshi Tahara             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center (72) Inventor Takayuki Goto             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center (72) Inventor Hiroyuki Nakayama             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center F term (reference) 2G059 AA01 BB13 DD12 DD13 EE02                       FF03 GG04 JJ17 JJ21 KK04                       PP10

Claims (10)

[Claims] 1. A light reflecting surface having a thin film that causes surface plasmon resonance, and a biochemical reaction detection portion provided on the thin film and in contact with a sample, and a light incident surface for making a light beam incident on the light reflecting surface. And a plate-shaped light guide plate having an emission surface through which the reflected light of the light beam reflected by the light reflection surface is emitted, and a light guide plate disposed on the emission surface of the light guide plate and having a refractive index higher than that of the light guide plate. A surface plasmon resonance sensor, comprising: a light extraction section made of a thin film having a refractive index. 2. The surface plasmon resonance sensor according to claim 1, wherein a plurality of the biochemical reaction detection portions are provided on the thin film of the light reflecting surface. 3. The surface plasmon resonance sensor according to claim 1, wherein the light incident surface of the light guide plate is inclined at an acute angle with respect to the light reflecting surface. 4. The surface plasmon resonance sensor according to claim 1, wherein the light extraction portion is made of a transparent resin. 5. The surface plasmon resonance sensor according to claim 1, a light source unit for irradiating the surface plasmon resonance sensor with a light beam, and a reflected light from the surface plasmon resonance sensor. A surface plasmon resonance sensor device comprising: a light detecting unit that receives light. 6. The surface plasmon resonance sensor device according to claim 5, wherein the light detection means is an array sensor extending in the longitudinal direction of the light extraction portion of the surface plasmon resonance sensor. 7. The surface plasmon resonance sensor device according to claim 6, wherein the light beam emitted from the light source unit is parallel light having a width that covers at least the width of the biochemical reaction test site. 8. The surface plasmon resonance sensor device according to claim 7, wherein the light source unit includes a variable wavelength light source. 9. The surface plasmon resonance sensor oscillates about an axis perpendicular to the optical path of the light beam, and the light detecting means oscillates integrally with the surface plasmon resonance sensor. The surface plasmon resonance sensor device according to claim 7. 10. The light source unit includes a light source, and a reflection plate that reflects the light beam emitted from the light source to the surface plasmon resonance sensor, and the reflection plate includes the light beam from the light source. 8. The surface plasmon resonance sensor device according to claim 7, wherein the surface plasmon resonance sensor device is swung so as to be deflected along the longitudinal direction of the biochemical reaction test site.