JP2003185572A - Surface plasmon resonance sensor device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a surface plasmon resonance sensor device by which a reaction in a sample can be detected with high accuracy so as to be grasped with satisfactory reproducibility by discriminating the kind of a reaction species adsorbed to the sample. <P>SOLUTION: The surface plasmon resonance sensor device is provided with an optical system in which the sample for biochemical reaction measurement is placed on one face, in which a reflecting surface is constituted of the other face, and in which a thin film used to generate surface plasmon resonance is formed; a light source by which light at a plurality of wavelengths used to generate the surface plasmon resonance on the reflecting surface of the thin film is irradiated toward the reflecting surface; a light splitting means by which reflected light on the reflecting surface is split into respective wavelengths; and a plurality-of-wavelengths-light detection means which detects an intensity of light at each wavelength split by the light splitting means. The sensor device is constituted in such a way that the mind of the reaction specifies contained in the sample can be discriminated based on a detection result of the light detection means. <P>COPYRIGHT: (C)2003,JPO

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 that utilizes the surface plasmon resonance phenomenon, and more particularly to a surface plasmon resonance sensor device that can identify the type of a sample in which a biochemical reaction has occurred.

[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. 4, 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 light 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 totally reflected at the interface between the glass substrate 1 and the metal thin film 2 sharply decreases. To do. FIG. 5 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 measurement that handles minute amounts that cannot be measured by general measurement methods. For example, as in the two characteristics shown in FIG. 5, D in the measurement unit 4 (see FIG. 4) is
Incident angle θ at which the intensity of the reflected light 5 decreases with or without NA coupling
Therefore, it is possible to detect a minute change in mass due to the binding of DNA.

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. Devices have been developed to do so. For example, as described in Japanese Patent Laid-Open No. 2000-65730, an apparatus has been developed that efficiently detects the surface plasmon resonance angle of many kinds of sample components. This will be briefly described with reference to FIG. 6. In this conventional device, a prism 8 is attached to a sensor chip 7 provided with a glass substrate 6 on which a metal thin film (not shown) for adsorbing a sample solution is provided. The light from the light irradiation device 9 is incident at a required angle, the light receiving device 10 receives the reflected light from the boundary surface of the metal thin film on the glass substrate 6, and the components of the sample solution are detected by the resonance angle at which the intensity is minimized. To detect. The glass substrate 6 is attached to the lower surface of the cell plate 11 in which a large number of cells 11a having open lower surfaces are arranged at a required interval so as to close the openings of the cells 11a to form the sensor chip 7. The glass substrate 6 in each cell 11a selected in a state where the prism 8 is formed in a size corresponding to the plurality of cells 11a and the glass substrate 6 corresponding to the plurality of cells 11a is selectively adhered to the prism 8 The respective metal thin film boundary surfaces are irradiated with respective lights in a required angular width. The resonance angle of the sample solution dispensed in each cell 11a selected based on the intensity of each reflected light from the metal thin film boundary surface of the glass substrate 6 corresponding to each cell 11a can be simultaneously detected.

[0007]

Since the conventional device is constructed as described above, the following problems exist. That is, in the conventional surface plasmon resonance sensor device, it was possible to determine whether or not a biochemical reaction occurred in the sample by detecting the mass change of the sample, but it was possible to determine the type of the reactive species adsorbed in the sample. I couldn't. For example, it was not possible to discriminate whether the adsorption reaction occurred in DNA or the adsorption reaction occurred in proteins other than DNA.

The present invention has been made in order to solve the above problems, and in particular, the reaction in a sample can be detected with high accuracy and reproducibility by determining the type of reactive species adsorbed in the sample. An object is to provide a surface plasmon resonance sensor device that can be grasped well.

[0009]

In the surface plasmon resonance sensor device of the present invention, a sample for biochemical reaction measurement is placed on one surface and a reflecting surface is formed on the other surface to generate surface plasmon resonance. An optical system in which a thin film is formed, a light source that irradiates light of a plurality of wavelengths for causing plasmon resonance on the reflective surface of the thin film toward the reflective surface, and light reflected by the reflective surface. Light splitting means for splitting for each wavelength, and a plurality of wavelength light detecting means for detecting the intensity of light of each wavelength split by the light splitting means, respectively, the sample based on the detection result of the light detecting means The configuration is such that it is possible to determine the type of reactive species contained, and a plurality of the samples are mounted on one surface of the thin film, and the photodetector is configured to determine whether surface plasmon resonance occurs due to the plurality of samples. At the same time A structure that is configured to detect individually.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Preferred embodiments of a surface plasmon resonance sensor device according to the present invention will be described in detail below with reference to the drawings. The same or equivalent parts as those of the conventional device are designated by the same reference numerals, and the description thereof will be omitted.

Embodiment 1. As shown in FIG. 1, in the surface plasmon resonance sensor device according to the first embodiment of the present invention, light 15a emitted from a mercury lamp 15 which is a light source capable of simultaneously emitting two types of light having wavelengths of 260 nm and 280 nm is: It is converged via the first lens 16 and guided to a prism 17 which is a columnar optical system having a semicircular cross section. A metal thin film 2 is formed on the plane of the prism 17, and a sample for biochemical reaction detection is applied onto the metal thin film 2. The light 15a incident on the prism 17 is irradiated to the back side of the metal thin film 2 at the position where the sample is applied, and the reflected light 15b is incident on the beam splitter 19 which is the light splitting means through the second lens 18 and is bidirectional. Will be divided.

The light 19 reflected by the beam splitter 19
a is the first filter 20 that transmits light having a wavelength of 280 nm.
And is guided to the first photodetector 21. On the other hand, the light 19b that has passed through the beam splitter 19 passes through a second filter 22 that transmits light of 260 nm and passes through a second photodetector 23.
Be guided to. Each light 19a (wavelength 280nm)
And 19b (wavelength 260 nm), the reflectance (reflected light intensity) is obtained in the first photodetector 21 and the second photodetector 23, which are photodetection means.

The mercury lamp 15 has wavelengths of 260 nm and 280
It is possible to irradiate two kinds of light of nm at the same time.
This is because DNA generally has an absorption peak at a wavelength of 260 nm, while protein has an absorption peak at a wavelength of 280 nm. By irradiating the mercury lamp 15 as a light source with light having an absorption wavelength peculiar to the sample, the mass change In addition to this, it is possible to determine the type of reactive species.

For example, when detecting a bond between DNAs, substances other than DNA, such as proteins, are affected, but the light 15a from the mercury lamp 15 containing two wavelengths of 260 nm and 280 nm at each wavelength is detected. The light is made incident on the prism 17 so as to have a converging angle equal to or larger than the plasmon resonance angle. The incident angle at this time is near the resonance angle.

As shown in FIG. 2, the plasmon resonance spectrum (FIG.
Characteristic a: wavelength 280 nm) and a resonance spectrum measured in advance only for DNA (characteristic c in FIG. 2: wavelength 260
nm) to create a calibration curve and measure the plasmon resonance spectrum (characteristic b in FIG. 2) in the case of an actual sample to understand the influence of substances such as proteins other than DNA at one survey point. Can and DNA
It becomes possible to detect the adsorption reaction between the proteins and the adsorption reaction between the proteins other than DNA with high accuracy and to grasp with good reproducibility.

In the above explanation, DNA has a wavelength of 26
Has an absorption peak at 0 nm, while protein has a wavelength of 28
I explained the case of having an absorption peak at 0 nm, but D
NA or a protein other than NA may have an absorption peak outside the above-mentioned wavelength region. In such a case, it is sufficient to change the irradiation light wavelength of the mercury lamp 15 which is the light source, and to change the transmission wavelength bands of the first filter 20 and the second filter 22 as well. Further, by using an optical system capable of separately detecting this, it is possible to discriminate the type of reactive species regardless of the sample having an absorption peak at any wavelength.

Further, if a light source capable of irradiating light of three or more kinds of wavelengths is used and an optical system capable of discriminating the types of a large number of reactive species is used by a method of providing a plurality of stages of beam splitters, for example, a greater number of optical systems can be used. It is possible to provide a surface plasmon resonance sensor device capable of discriminating the type of the reactive species. Furthermore, a plurality of samples are placed on the metal thin film 2,
If a two-dimensional array sensor is used as the first photodetector 21 and the second photodetector 23, a surface plasmon resonance sensor device capable of simultaneously determining the types of reactive species for a plurality of samples placed at a plurality of points is provided. Can be provided.

Embodiment 2. As shown in FIG. 3, the surface plasmon resonance sensor device according to the second embodiment of the present invention uses a short pulse laser 25 having a light intensity capable of generating a two-photon absorption process as a light source. For example, when detecting DNA, a wavelength of 520 nm, which is twice the absorption peak wavelength of 260 nm, is required. Similarly, in order to detect a protein, 560 nm, which is the absorption peak wavelength of 280 nm, is required. Become. Therefore, 520 nm
If the short pulse laser 25 capable of irradiating laser light having two wavelengths of 560 nm and 560 nm is used as the light source, the DN is used as in the surface plasmon resonance sensor device according to the first embodiment.
It is possible to detect the adsorption reaction between A and the adsorption reaction between proteins other than DNA with high accuracy and to grasp with good reproducibility.

[0019]

In the surface plasmon resonance sensor device of the present invention, a sample for biochemical reaction measurement is applied on one surface, and a thin film is formed on the other surface to form a reflection surface and generate surface plasmon resonance. An optical system, a light source for irradiating the reflecting surface with light of a plurality of wavelengths for causing plasmon resonance on the reflecting surface of the thin film, and dividing the reflected light on the reflecting surface for each wavelength. Light splitting means and a plurality of wavelength light detecting means for respectively detecting the intensity of light of each wavelength split by the light splitting means are provided, and the reactive species contained in the sample based on the detection result of the light detecting means. Since the type can be discriminated, the adsorption reaction between DNA and the adsorption reaction between proteins other than DNA can be detected with high accuracy and grasped with good reproducibility. Further, a plurality of the samples are placed on one surface of the thin film, and the photodetector is configured to be able to simultaneously and individually detect the presence or absence of occurrence of surface plasmon resonance by the plurality of samples. Therefore, it is possible to provide a surface plasmon resonance sensor device capable of simultaneously determining the types of reactive species for a plurality of samples placed at a plurality of points.

[Brief description of drawings]

FIG. 1 is a configuration diagram schematically showing a surface plasmon resonance sensor device according to a first embodiment of the present invention.

FIG. 2 is a characteristic diagram showing a plasmon resonance spectrum of each sample.

FIG. 3 is a configuration diagram schematically showing a surface plasmon resonance sensor device according to a second embodiment of the present invention.

FIG. 4 is a diagram conceptually showing a detection principle for detecting a mass change using surface plasmon resonance.

FIG. 5 is a characteristic diagram showing the relationship between the incident angle θ and the intensity of reflected light in detection of mass change using surface plasmon resonance.

FIG. 6 is a configuration diagram showing an apparatus for efficiently detecting surface plasmon resonance angles of various kinds of sample components described in JP-A-2000-65730.

[Explanation of symbols]

2 metal thin film, 15 mercury lamp, 15a light, 15b
Reflected light, 16 First lens, 17 Prism, 18
Second lens, 19 Beam splitter, 19a light (wavelength 280 nm), 19b light (wavelength 260 nm), 20
1st filter, 21 1st photodetector, 22 2nd filter, 23 2nd photodetector, 25 Short pulse laser.

   ─────────────────────────────────────────────────── ─── 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 Hiroyuki Nakayama             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center (72) Takuma Sakai             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center (72) Inventor Hiromitsu Inuzuka             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center F term (reference) 2G059 AA01 BB04 BB12 CC16 EE02                       EE11 EE12 GG01 GG08 GG10                       HH02 HH03 HH06 JJ02 JJ11                       JJ12 JJ22 KK03 KK04 MM12                 4B029 AA07 BB15 BB20 CC02 FA10

Claims (2)

[Claims] 1. An optical system in which a sample for biochemical reaction measurement is placed on one surface, and a thin film is formed on the other surface to form a surface plasmon resonance and a reflection of the thin film. A light source for irradiating the reflecting surface with light of a plurality of wavelengths for causing plasmon resonance on the surface, a light splitting means for splitting the reflected light on the reflecting surface for each wavelength, and the light splitting means. A surface characterized by comprising a plurality of wavelength light detecting means for respectively detecting the intensity of light of each wavelength divided by, and capable of discriminating the type of reactive species contained in the sample based on the detection result of the light detecting means. Plasmon resonance sensor device. 2. A plurality of the samples are placed on one surface of the thin film, and the photodetector is configured to detect the presence or absence of surface plasmon resonance caused by the plurality of samples simultaneously and individually. The surface plasmon resonance sensor device according to claim 1, which is configured.