JPH10267841A - Surface plasmon resonance-sensing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a resonance angle economically and stably by forming a multilayer film of a metallic thin film and a dielectric thin film on a prism and forming a polymer film of polysaccharide or the like as a fixation carrier for biologic molecules on the multilayer film. SOLUTION: The surface plasmon resonance-sensing device has a first metallic thin film layer 16 formed on a prism or substrate, a dielectric layer 17 on the first metallic thin film layer and a second metallic thin film layer 18 on the dielectric layer. The prism is formed of, e.g. quarts or glass which is transparent to ultraviolet rays, visible rays and near infrared rays and has a larger index of refraction than a liquid sample. The metallic thin film layers 16, 18 are formed of, e.g. gold or silver in a uniform thickness, and preferably have flat surfaces and good adhesion to a layer therebelow. The dielectric thin film layer 17 is formed of a material transparent to ultraviolet rays, visible rays and near infrared rays, for example, SiO2 , SiO or the like. The polysaccharide is glucose or the like using, for instance, dextran sulfate as an antibody-fixing carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a surface plasmon resonance sensing device for measuring the concentration of biomolecules in a sample solution from a change in the refractive index of a medium near the surface of a metal thin film formed on a prism. And a measuring method using the same.

[0002]

2. Description of the Related Art When a light beam is incident on a prism having a metal thin film formed on one side at a total reflection angle from the other side to the surface on which the metal thin film is formed, evanescent light and surface plasma waves are generated on the surface of the metal thin film. The phenomenon that the intensity of the reflected light decreases due to the resonance of the light is already known. Using this phenomenon, a surface plasmon resonance sensing device for measuring the refractive index of the medium near the surface of the metal thin film, and the mutual use of biomolecules such as an antigen-antibody reaction generated on the surface of the metal thin film using the device. Devices and measuring methods for determining the effect are already known.

[0003] This device is used in various fields taking advantage of its features, but is particularly actively used in the field of measurement of antigen-antibody reactions. Hereinafter, a surface plasmon resonance type antigen sensing device, an antigen detection device using the device, and an antigen detection method using the device will be described in detail. Some surface plasmon resonance sensing devices do not use a prism but use a diffraction grating or an optical fiber. However, the subject of this application is limited to those using a prism.

FIG. 1 shows a conventional surface plasmon resonance sensing device. A conventional surface plasmon resonance sensing device generally has a polygonal or semicircular prism 1 having a metal thin film 2 formed on one side surface. In particular, when an antigen is detected by an antigen-antibody reaction using this device, the device has a structure in which an antibody is immobilized on a metal thin film. 50nm thickness as metal thin film
A gold or silver thin film of about 60 to 60 nm is often used, but in order to improve the adhesion between glass and these metal thin films, usually a metal thin film layer of chromium or titanium of about 1 to 5 nm is interposed. Form. In order to simplify the handling of the device, a metal thin film 2 is formed on a glass plate 3 of the same material as the prism 1 instead of actually forming a metal thin film directly on the prism as shown in FIG. In many cases, the glass plate is closely attached to the prism via a matching oil 4 having the same refractive index as glass when used, but in this application, the glass plate is attached to the prism via the matching oil for convenience. Things are simply described as prisms.

The prism type surface plasmon resonance sensing device 5 as described above includes a monochromatic light source 6 such as a semiconductor laser (LD) or a light emitting diode (LED), a charge coupled device (CCD) or a phototransistor as shown in FIG. Is used as an integrated device in combination with the photodetector 7 and the polarizing plate 33. In the surface plasmon resonance sensing device, monochromatic light source, and photodetector, light from the monochromatic light source enters from the glass surface of the prism, is totally reflected by the metal thin film forming surface, is emitted again from the glass surface, and is detected through the polarizing plate. It is arranged to be detected by a vessel. However, the polarizing plate is arranged so that only P-polarized light (described later) is transmitted.
P-polarized light means that when this light beam is totally reflected on the side of a prism on which a multilayer film composed of a metal layer and a dielectric layer is formed, the plane of vibration of the electric field of this light beam is perpendicular to the multilayer film. Means things. On the other hand, S-polarized light means that the vibration plane of the magnetic field of this light beam is perpendicular to the multilayer film. Of these, only P-polarized light can cause surface plasmon resonance when incident on a surface plasmon resonance sensing device, resulting in a decrease in reflected light intensity.

As shown in FIG. 4, the output 8 of the photodetector of the device in which such an optical system is disposed has an absorption 9 of the reflected light 9 despite the total reflection due to the surface plasmon resonance phenomenon. Observed. At this time, the angle (resonance angle) θ at which the reflected light intensity becomes minimum and the refractive index n of the medium in contact with the metal thin film
Is known to be expressed by the following equation: Kp · sin (θ) = ω / c · ｛ε · n ² / (ε +
n ² )｝ ^1/2 where Kp is the wave number of the incident light, ω is the angular frequency of the incident light, C is the speed of light, and ε is the dielectric constant of the metal thin film.
From this equation, the refractive index of the medium in contact with the metal thin film and the amount of change thereof can be known from the resonance angle θ. However, the medium layer that affects the surface plasmon resonance phenomenon is limited to a range of at most several hundred nm from the surface of the metal thin film. Therefore, by measuring the resonance angle, the refractive index of the medium within a range of at most several hundred nm from the surface of the metal thin film and its change can be known.

[0007] The medium in which the refractive index and the amount of change thereof can be measured can be any medium having a refractive index lower than that of a prism. As shown in FIG. 5, an antigen-antibody reaction detection device incorporating the surface plasmon resonance sensing device 5 has a flow cell 11 formed on a metal thin film and is often used as a flow system. In this case, the antibody 12 (or antigen) is fixed on the metal thin film, and when the solution 14 containing the corresponding antigen 13 (or antibody) enters the flow cell, an antigen-antibody reaction occurs on the surface of the metal thin film, and the vicinity of the surface of the metal thin film Changes the resonance angle θ. By measuring this θ, the change in the refractive index near the surface of the metal thin film can be determined, and from this, the concentration of the antigen or antibody in the solution can be determined.

[0008] Antibodies are generally prepared by physical adsorption,
It is immobilized on a metal thin film on a prism using a chemical bonding method or a polymer carrier method. Here, the physical adsorption means that the antibody is adsorbed on the metal thin film due to an electrostatic interaction between the antibody and the metal thin film, or a physical interaction such as van der Waalska. This is not a practical immobilization method because antibodies and the like fall off from the thin film when exposed to the solution for a long time. On the other hand, the chemical bonding method refers to immobilizing an antibody to a metal thin film with a chemical bonding force. In this method, gold is used as the metal thin film layer, and the antibody is often immobilized on the metal thin film via a gold mercaptide bond.
It is rare to immobilize by other means such as a silane coupling agent. In the polymer carrier method, an antibody is bound to a polymer carrier immobilized on a metal substrate. Also in this case, the polymer carrier itself is often immobilized on the gold thin film via a gold mercaptide bond.

As described above, a gold thin film is suitable for immobilizing an antibody, and is not easily deteriorated as in the case of a silver thin film, and thus can be said to be the most practical thin film material. However, when a gold thin film is formed on a prism, the angle at which the intensity of reflected light decreases tends to be about five times larger than in the case of a silver thin film. Since the measurement accuracy of the resonance angle is inversely proportional to the dispersion width, there is a problem that the measurement accuracy of the resonance angle is lower in the case of a gold thin film than in the case of a silver thin film.

In the flow system, when measuring the concentration of an antigen or an antibody in a sample solution, a solution containing no antigen or antibody is usually kept flowing, and the sample solution is injected during the measurement. And the change in the resonance angle is measured. That is, the amount of change in the resonance angle before and after the injection of the sample solution is measured, which is based on the premise that the resonance angle is stable over time except when the sample is injected. However, in practice, it is not easy to keep the resonance angle stable due to temperature fluctuation, pressure fluctuation, and composition fluctuation of the solution. For example, when the temperature of water rises by 1 ° C., the refractive index changes, so that the resonance angle decreases by 0.015 degrees. On the other hand, in the conventional method,
The only solution is to keep the temperature of the device or flow cell constant. This has led to a huge device.

If the composition of the liquid constantly flowing in the flow system and the composition of the sample solution are slightly different,
Since the refractive indices of the two are different, there is a problem that the value of the resonance angle changes due to a different reason from the antigen-antibody reaction, for example, temperature fluctuation or pressure fluctuation of the solution. There is no solution.

[0012]

SUMMARY OF THE INVENTION In view of the prior art surface plasmon resonance sensing device, apparatus and method as described above, the problem to be solved by the present invention is to stably hold an antibody or an antigen and to achieve high precision. Providing a surface plasmon resonance sensing device, apparatus and method capable of measuring a resonance angle, and incorporating a module such as a temperature control system to make the resonance angle stable over time without making the apparatus huge and complicated. To provide a surface plasmon resonance sensing device, apparatus, and method capable of measuring the surface plasmon resonance.

[0013]

The above object is achieved, as shown in FIG. 6, by inserting a dielectric into a metal thin film formed on a prism and further using a biomolecule-immobilizing carrier. It was found to be solved.

That is, according to a first aspect, the present invention provides a polysaccharide, which is a carrier for immobilizing biomolecules, on which a multilayer film composed of a metal thin film and a dielectric thin film is formed on a prism. By forming a polymer film such as
Provided is a surface plasmon resonance sensing device that can be used for a method for optically measuring the concentration of a specific biomolecule in a liquid sample.

In the present invention, the liquid sample is a liquid in which a specific biomolecule to be measured coexists, and the liquid itself may be a solution or an emulsion. Specific examples include body fluids such as blood, serum, plasma, urine, and cerebrospinal fluid, and foods such as fruit juice.

In the present invention, the term "biomolecule" refers to a molecule or a molecular aggregate derived from a living body, and a molecule or a molecular aggregate that complementarily binds to these molecules or a molecular aggregate. Specific examples include proteins such as enzymes, antibodies and lectins, nucleic acids such as DNA and RNA, sugars such as starch and dextran, inorganic substances such as calcium and magnesium, and organic substances such as urea and lactic acid.

In the present invention, the surface plasmon resonance sensing device is, as shown in FIG. 7 or 8, a first metal thin film layer 16 is formed on a prism 15 or a substrate 20, and a dielectric layer 17 is formed thereon. Is formed, and the second metal thin film layer 18 is further formed thereon. Further, as shown in FIG. 6, it also means that a polymer film 25 which is a carrier for immobilizing biomolecules is further formed on a metal thin film layer.

The prism used in the surface plasmon resonance sensing device of the present invention is transparent to ultraviolet, visible, and near-infrared light such as quartz, glass, and polymethyl methacrylate. An optical component made of a material having a large refractive index. Although the shape is not particularly limited, those having a shape such as a semicircular column, a triangular column, or a trapezoidal column are used in many cases. In many cases, a metal thin film is formed on a substrate made of the same material as the prism, and the substrate is brought into close contact with the prism via a matching oil having the same refractive index as the prism or the substrate when used. In this application document, for convenience, a substrate mounted on a prism via a matching oil is also simply referred to as a prism.

The metal thin film used in the surface plasmon resonance sensing device of the present invention means a metal thin film made of a material such as gold, silver, copper, aluminum, chromium, and titanium. The thickness of the thin film is such that it can transmit light in the ultraviolet, visible, and near-infrared regions, and is usually not more than 100 nanometers. It is desirable that the metal thin film has a uniform thickness, a flat surface, and good adhesion to a lower layer. Such a metal thin film is often formed by a vacuum deposition method, a sputtering method, or the like, but the present invention is not limited to a specific film forming method. However, it is particularly preferable that the outermost layer of the surface plasmon resonance sensing device is a gold thin film, in which case a biomolecule such as an antigen or an antibody or a polymer carrier for immobilizing the biomolecule is bonded via a gold mercaptide bond. It becomes easy to fix.

The dielectric thin film used in the surface plasmon resonance sensing device of the present invention is SiO ₂ , Si
It refers to a thin film of an inorganic substance such as O, MgF ₂ , or CaF ₂ made of a material transparent to ultraviolet, visible, and near-infrared rays. The thickness of the thin film is about half to several times the wavelength of light in the ultraviolet, visible, and near-infrared regions, and is usually from one hundred nanometers to several thousand nanometers. It is desirable that the dielectric thin film has a uniform thickness, a flat surface, and good adhesion to a lower layer. Such a dielectric thin film can be formed by a vacuum deposition method, a sputtering method, a chemical vapor deposition method, a sol-gel method, or the like. The present invention is not limited to a specific film forming method.

The above-mentioned dielectric thin film layer is used as an optical waveguide when the thicknesses of the first metal thin film layer, the dielectric thin film layer, and the second metal thin film layer are optimally selected with respect to the wavelength of incident light. In order to function, only light rays of a specific incident angle propagate in a direction parallel to the thin film. Therefore, the dispersion of the resonance angle due to the surface plasmon resonance phenomenon is narrower than that of the surface plasmon resonance sensing device having no dielectric layer. FIG. 9 illustrates this state. In this figure, chromium (1 nm) and silver (30 nm) are placed on a prism (material BK7).
nm), chromium (1 nm), SiO ₂ (580 nm),
The graph shows the reflected light intensity of P-polarized light when monochromatic light having a wavelength of 660 nm is incident on a device in which a thin film of chromium (1 nm) and gold (25 nm) are formed in this order. In this figure, for comparison, a conventional device, that is, a device in which chromium (1 nm) and gold (50 nm) are formed on a prism, and a device in which chromium (1 nm) and silver (50 nm) are formed on a prism.
(nm) is also shown. As can be seen from FIG. 9, when P-polarized light is incident, the device according to the present invention shows sharp absorption comparable to that of the device formed with a silver thin film, even though the outermost layer is gold. Thus, the device of the present invention has a narrower dispersion of the resonance angle than the conventional device, so that the estimation of the resonance angle is more accurate, and as a result, the change in the refractive index of the medium near the device surface is also accurately detected. Become. Therefore, when the surface plasmon resonance sensing device according to the present invention is used for detecting an interaction between biomolecules, a lower concentration of biomolecules in a solution can be detected.

Conventionally, only P-polarized light has been used for measurement, and only a change in the refractive index in a medium having a thickness of at most several hundred nanometers near a metal thin film on a device can be measured. Therefore, when the refractive index of the medium changes due to fluctuations in temperature, angle, etc. of the medium, whether this is the change in the refractive index of the entire medium including the bulk or the change in the refractive index near the metal thin film It was difficult to distinguish between them. Therefore, when a solution containing a biomolecule to be measured is applied to the device, the concentration of the biomolecule estimated from the change in the resonance angle is not always reliable. On the other hand, in the case of the surface plasmon resonance sensing device of the present invention, when P-polarized light is incident, not only the reflected light intensity decreases due to the surface plasmon resonance phenomenon as in the conventional device, but also when S-polarized light is incident. Also, the reflected light intensity decreases. FIG. 10 illustrates this state. In this figure,
Chrome (1 nm), silver (30 nm), chromium (1 nm), SiO ₂ (580 n) are placed on the prism (material BK7).
m), chromium (1 nm), and gold (25 nm) thin films formed in this order show the intensity of reflected S-polarized light when monochromatic light having a wavelength of 660 nm is incident on the device. In this figure, for comparison, a conventional device, that is, a device having chromium (1 nm) and gold (50 nm) formed on a prism, and a device having chromium (1 nm) and silver (50 nm) formed on a prism. The reflected S-polarized light intensity of the device is also shown. The device of the present invention can measure the change in the refractive index in the bulk of the medium at a distance of 1000 nm or more from the metal thin film on the outermost surface of the device from the angle of absorption of the reflected S-polarized light. Therefore, when a solution containing a biomolecule to be measured is applied to the device, the S-polarized light is incident upon the S-polarized light from the refractive index change Δnp near the device obtained by entering the P-polarized light and measuring the resonance angle. Is obtained by subtracting the refractive index change Δns in the medium bulk obtained by measuring
np-Δns is a refractive index change caused only by the vicinity of the device, and the concentration of biomolecules in the solution calculated from this value is a reliable one that is not affected by changes in the temperature, pressure, etc. of the solution. Become.

In the present invention, the polysaccharide used as a carrier for immobilizing an antibody (or antigen) in a surface plasmon resonance sensing device is a polysaccharide in the ordinary sense in which a large number of monosaccharides such as glucose are linearly or branched and bonded. It means sugars or those obtained by chemically modifying them, such as sulfated dextran and soluble starch. FIG.
When the antibody 22 (or antigen) is directly or physically immobilized on the metal layer 21 on the outermost surface of the device by the method described above, the antibody (or antigen) is separated from the surface of the metal thin film by at most about 10 nm. Not. However, the layer in which the surface plasmon resonance sensing device can detect a change in the refractive index is generally in a range where the evanescent light 24 reaches, that is, in a range of several hundred nanometers from the surface of the metal thin film, and the antibody (or antigen) is immobilized. Most of the area is not used except for some layers. However, when the antibody 22 (or antigen) is immobilized on the polysaccharide 25 having a large molecular weight and a long length as shown in FIG. 12, the antibody (or antigen) is distributed over a range of several hundred nanometers from the surface of the metal thin film. In addition, since the refractive index changes in all the regions due to the complementary binding reaction of the antigen and the antibody, the measurement sensitivity of the antigen 23 (or the antibody) is increased. In particular, when a polysaccharide having a refractive index close to that of water is used, a change in the temperature, pH, ionic strength, etc. of the solution causes a very small change in the three-dimensional structure of the carrier, and does not cause a change in the refractive index. Measurement is possible.

According to a second aspect, the present invention is an apparatus for measuring the concentration of a specific biomolecule in a liquid sample by an optical method, comprising the surface plasmon resonance sensing device, flow cell, and injection device of the present invention. Provided is a measuring device having a valve, a liquid feed pump, a light source, a photodetector, a beam splitter, and a polarizing plate.

FIG. 1 schematically shows the configuration of such a measuring apparatus.
3 is shown. The measuring device of the present invention is usually used in the following usage form. First, the flow cell 27 is mounted on the side surface of the surface plasmon resonance sensing device 26 where the multilayer film composed of the metal thin film and the dielectric is formed. At this time,
It is assumed that a polysaccharide to which an antibody (or antigen) is bound is immobilized on the device before use. The solution is injected from the liquid sending pump 28 into the flow cell via the injection valve 29, and the solution discharged from the flow cell is discharged into the waste liquid reservoir 3.
Discharged to zero. Light is incident on the surface plasmon resonance sensing device from a light source 31, the incident light is totally reflected at an interface with the flow cell, and the reflected light is detected by a photodetector 34 through a beam splitter 32 and a polarizing plate 33.
However, one polarizing plate is arranged to pass only P-polarized light, and the other polarizing plate is arranged to pass only S-polarized light. Accordingly, one photodetector detects P-polarized reflected light, and the other photodetector detects S-polarized reflected light. It is assumed that the angles at which the reflected light intensity becomes minimum from the outputs of both detectors are simultaneously monitored in real time for each of the P-polarized light and the S-polarized light. An algorithm for calculating the minimum angle of the reflected light intensity from the intensity of the reflected light detected by the detector can use an existing method, is well known to those skilled in the art, and does not require further explanation. A sample containing an antigen (or antibody) is injected from an injection valve, flows into a flow of a solution sent from a pump, enters a flow cell, and an antigen-antibody reaction occurs on the surface of a metal thin film on the outermost surface of the device. At this time, when the minimum angles of the reflected light intensity of the P-polarized light before and after the injection of the sample solution are θp and θ′p, and the minimum angles of the reflected light intensity of the S-polarized light are θs and θ ′s, respectively, conventionally, (θ′p Although the concentration of the antigen (or antibody) in the sample solution was calculated from (−θp), when using the present measurement device, (θ′p−θp) −α (θ′s−θs)
The antigen (or antibody) concentration is calculated from the above. However,
α is a coefficient. Variations derived from the surface of the metal thin film such as antigen-antibody reactions appear only in (θ′p−θp) (θ′s−θ
Although the variation does not appear in (s), but does not originate in the metal thin film surface, it appears in both (θ′p−θp) and (θ′s−θs), so that the influence can be removed.

[0026]

Example 1 A surface plasmon resonance sensing device of the present invention was immersed in a 10 μM aminoethanethiol solution and washed, and then a sulfated dextran solution was mixed with a 2% sodium periodate solution at a weight ratio of 5: 2. It was immersed in the solution stirred overnight and then washed. Next, the device was added to a human albumin solution (fraction V, 0.5 mg / m2).
After immersion in 1) for washing, the substrate was immersed in a 10 mM sodium borohydride solution (pH 7.5 to 8, 4 ° C.) for washing.
This device is mounted on a surface plasmon resonance measurement device,
The operation of injecting 50 μL of anti-human albumin (goat) from an injection valve while injecting a 20 mM phosphate buffer (pH 7) and then injecting a glycine-hydrochloric acid buffer (pH 2.5) is repeated. Data obtained.

[0027]

Example 2 A surface plasmon resonance sensing device (sulfated dextran-immobilized membrane carrier method) prepared in the same manner as in Example 1 was immersed and washed in a 10 μM aminoethanethiol solution, and then washed with a 1% glutaraldehyde solution. After immersion and washing, the albumin solution (0.5 mg / m
1) Using a device (chemical bonding method) immersed and washed in an albumin solution (0.5 mg / ml) and a device washed (physical adsorption method) in an albumin solution (0.5 mg / ml), 50 μL of anti-human albumin (goat) was injected from an injection valve. The operation of injecting a glycine-hydrochloric acid buffer (pH 2.5) after the injection is repeated, and the amount of change in the resonance angle with respect to anti-human albumin at this time is plotted against the number of sample solution injections in FIG. .

[0028]

Example 3 Using a surface plasmon resonance sensing device manufactured by the same method as in Example 1 and a measuring apparatus having the device, a 20 mM phosphate buffer (pH
FIG. 16 shows the result of repeatedly performing the operation of injecting 50 μL of anti-human albumin from the injection valve and then injecting a glycine-hydrochloric acid buffer (pH 2.5) while changing the concentration of anti-human albumin. Show.

[0029]

By using the surface plasmon resonance sensing device, measuring apparatus and measuring method of the present invention, biomolecules in a sample solution are less sensitive to fluctuations in temperature, pressure, composition and the like of a solution and highly sensitive. Can be measured. This makes it possible to detect specific components contained in body fluids such as blood, serum, plasma, urine, and cerebrospinal fluid, and foods such as fruit juices more quickly, more accurately, and in real time than before. Become.

[Brief description of the drawings]

FIG. 1 is a schematic diagram schematically showing one form of a conventional surface plasmon resonance sensing device.

FIG. 2 is a schematic view schematically showing another embodiment of the conventional surface plasmon resonance sensing device.

FIG. 3 is a schematic diagram schematically showing a minimum apparatus configuration for observing a surface plasmon resonance phenomenon using a conventional surface plasmon resonance sensing device.

FIG. 4 is a diagram showing the angle dependence of reflected light intensity observed by a photodetector when a surface plasmon resonance phenomenon is observed using a conventional surface plasmon resonance sensing device.

FIG. 5 is a schematic diagram schematically showing an antigen-antibody reaction detection device incorporating a conventional surface plasmon resonance sensing device.

FIG. 6 is a schematic view schematically showing one embodiment of the surface plasmon resonance sensing device of the present invention.

FIG. 7 is a schematic diagram schematically showing one embodiment of the surface plasmon resonance sensing device of the present invention.

FIG. 8 is a schematic view schematically showing one embodiment of the surface plasmon resonance sensing device of the present invention.

FIG. 9 is a diagram showing the reflected light intensity of P-polarized light when water is used as a sample using a conventional surface plasmon resonance sensing device and the surface plasmon resonance sensing device of the present invention.

FIG. 10 is a diagram showing the intensity of reflected S-polarized light when water is used as a sample, using a conventional surface plasmon resonance sensing device and the surface plasmon resonance sensing device of the present invention.

FIG. 11 is a diagram showing a state near a metal thin film surface when an antibody (or antigen) is directly immobilized on the metal thin film surface of a surface plasmon resonance sensing device.

FIG. 12 is a diagram showing a state near a metal thin film surface when a polysaccharide as an antibody (or antigen) immobilization carrier is used for a surface plasmon resonance sensing device.

FIG. 13 is a schematic diagram schematically showing an apparatus for measuring the concentration of a specific biomolecule in a sample solution using the surface plasmon resonance sensing device of the present invention.

FIG. 14 is a diagram showing a time change of a resonance angle when anti-human albumin is injected into a surface plasmon resonance sensing device of the present invention in which human albumin is immobilized by using a sulfated dextran-immobilized membrane carrier method.

FIG. 15 is a diagram showing the repetitive response to anti-human albumin of the surface plasmon resonance sensing device of the present invention in which human albumin is immobilized using a physical adsorption method, a chemical bonding method, and a sulfated dextran-immobilized membrane carrier method. .

FIG. 16 is a graph showing the relationship between the concentration of anti-human albumin and the response of the surface plasmon resonance sensing device of the present invention in which human albumin is immobilized using a sulfated dextran-immobilized membrane carrier method.

[Explanation of symbols]

Reference Signs List 1 prism 2 metal thin film 3 glass plate 4 matching oil 5 conventional surface plasmon resonance sensing device 6 light source 7 photodetector 8 surface plasmon resonance curve 9 reflected light intensity absorption 10 critical angle 11 flow cell 12 antibody 13 antigen 14 solution 15 according to the present invention Surface Plasmon Resonance Sensing Device 16 Metal Thin Film 17 Dielectric 18 Metal Thin Film 19 Matching Oil 20 Glass Plate 21 Metal Thin Film 22 Antibody 23 Antigen 24 Energy Distribution of Evanescent Light 25 Polysaccharide 26 Surface Plasmon Resonance Sensing Device According to the Present Invention 27 Flow Cell 28 Liquid Transfer Pump 29 injection valve 30 waste liquid reservoir 31 light source 32 beam splitter 33 polarizing plate 34 photodetector

Claims (16)

[Claims] A first metal thin film formed on the prism, a dielectric thin film formed on the metal thin film, and a second metal thin film formed on the dielectric thin film;
A surface plasmon resonance sensing device capable of sharply detecting a change in refractive index in the vicinity of a thin film and a bulk of a medium in contact with the thin film. 2. The surface plasmon resonance sensing device according to claim 1, wherein a material having a refractive index of the dielectric thin film smaller than that of the prism with respect to the wavelength of light incident on the prism is used. 3. A prism made of transparent quartz glass, Vycor, Pyrex, aluminosilicate glass, aluminoborosilicate glass, borosilicate glass, BaO—Al ₂ O ₃ —B ₂
Using any of O ₃ —SiO ₂ glass, side-by-side glass, low alkali glass, flint glass, soda-lime glass, and the like, the dielectric thin film is LiF, NaF, Na ₃ AlF ₆ ,
3. The surface plasmon resonance sensing device according to claim 2, comprising a layer containing at least one component of SiO ₂ , SiO, MgF ₂ , CaF _{2 and the} like. 4. The surface plasmon resonance sensing device according to claim 1, wherein the metal thin film formed between the prism and the dielectric thin film is a silver single layer film or a metal multilayer film including a silver layer. 5. The surface plasmon resonance sensing device according to claim 4, wherein the multilayer film constituting the metal thin film formed between the prism and the dielectric thin film includes a talom layer or a titanium layer. 6. The surface plasmon resonance sensing device according to claim 1, wherein a metal single-layer film or a metal multilayer film including a gold layer is used as the metal thin film formed on the dielectric thin film. 7. The surface plasmon resonance sensing device according to claim 6, wherein the multilayer film forming the metal thin film formed on the dielectric thin film includes at least one layer of chromium, titanium, and aluminum. 8. A refractive index near a metal thin film of a medium in contact with an outermost metal thin film of the device using the surface plasmon resonance sensing device according to claim 1 as P-polarized light incident on a prism. Device to measure. 9. The apparatus according to claim 8, wherein the refractive index of the bulk of the medium in contact with the outermost metal thin film of the surface plasmon resonance sensing device is measured using S-polarized light as incident light on the prism. 10. A method for measuring a difference between a refractive index near a metal thin film of a medium in contact with an outermost metal thin film of a surface plasmon resonance sensing device and a bulk refractive index of the medium using the apparatus according to claim 9. . 11. The method according to claim 8 or 9, wherein a change in the refractive index of the medium in contact with the outermost metal thin film of the surface plasmon resonance sensing device in the vicinity of the metal thin film indicates that the biomolecules immobilized on the metal thin film are different from each other. , A method for measuring the interaction with another biomolecule in a medium. 12. A polymer having a refractive index close to that of water, such as a polysaccharide having a molecular weight of tens of thousands or less, is fixed on the metal surface obtained by the method according to any one of claims 2 to 8, and the carrier is immobilized on the polymer. A surface plasmon resonance sensing device and a method for preparing the same, wherein an antibody (or an antigen) is immobilized thereon so that a remarkable refractive index change accompanying the antigen-antibody reaction occurring here can be detected sharply. 13. The immobilization carrier according to claim 1, wherein a polysaccharide having a molecular weight of 50,000 daltons or less is used so as to effectively use the evanescent wave that decays exponentially.
A surface plasmon resonance sensing device having a thin film surface obtained by the method 2 and a method for preparing the same. 14. The method according to claim 1, wherein the polysaccharide having a molecular weight of tens of thousands or more is used.
A method for preparing a support, characterized by being radially arranged on the surface of a thin film obtained by the method 2 or 13, and a surface plasmon resonance sensing device obtained by the method. 15. The surface plasmon resonance sensing device according to any one of claims 12 to 14, wherein the polysaccharide is any of soluble starch and sulfated dextran, and a method for preparing the same. 16. The method according to claim 9, wherein the difference between the refractive index near the metal thin film of the medium in contact with the outermost metal thin film of the surface plasmon resonance sensing device and the refractive index of the bulk of the medium is determined from the change in the refractive index. Biomolecules immobilized on a thin film,
A method for examining the interaction with a biomolecule that performs complementary binding in a medium.