JPH11118802A - Surface plasmon measuring method and device thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect and measure a specimen of low concentration and low molecule by using the light of wavelength equal to the absorption wavelength of the specimen or a pigment bonded to the specimen, as the excitation light of the surface plazmon. SOLUTION: For example, Coomassie brilliant blue as a pigment to be bonded to a specimen, is added to a sample solution containing human albumin as the specimen, and is stirred for a specific time, and then bovine albumin, for example, is added to adsorb the residual pigment. On the sample solution treated as mentioned in the above, a surface plasmon resonance curve is obtained by a specific method by using a sensor chip on which a human albumin antibody is fixed, with the light of near 580 nm which is the absorption wavelength of Coomassie brilliant blue, for example, the light of 575 nm as the excitation light. The human albumin in the sample solution is detected on the basis of the change of the intensity of reflection light, a half value width and a resonance angle in a first resonance curve 3 by a buffer liquid and a second resonance curve 4 by the sample solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and an apparatus for measuring surface plasmon.

[0002]

2. Description of the Related Art A conventional measuring method using surface plasmons will be described with reference to FIG. Gold or silver thickness about 50nm
A binding species (for example, an antibody) that specifically binds to an analyte is immobilized on a sensor chip surface (detection surface) having a thin film of, and the surface of the sensor chip (detection surface) is irradiated with surface plasmon excitation light having a wavelength of, for example, 660 nm. First, the sensor chip surface is filled with a buffer solution containing no analyte (antigen), and the change in the refractive index near the sensor chip surface at this time is measured to obtain a resonance curve 1 as a reference. Next, the sensor chip surface is filled with a buffer solution in which the antigen is present, and the antigen is reacted (bound) with the antibody immobilized on the sensor chip surface, and then the antigen and other substances that have not reacted with the antibody are washed away. The surface of the sensor chip is washed with a buffer solution containing no antigen, the surface of the sensor chip is filled with the buffer solution, and a change in the refractive index near the surface of the sensor chip at that time is measured to obtain a resonance curve 2. Thus, a resonance curve 1 of the buffer solution before the antigen-antibody reaction and a resonance curve 2 after the antigen-antibody reaction are obtained, and the incident angles θ1 and θ2 of the excitation light at which the reflection intensity of the reflected light with respect to the excitation light becomes a minimum value are obtained. The amount of antigen bound to the antibody on the sensor chip can be determined from the difference (θ2−θ1) of the resonance angle with
This is converted to the concentration of the antigen present in the buffer solution.

However, a change in the refractive index near the surface of the sensor chip is measured as in the above-described conventional measurement method using surface plasmon, and the resonance curve 1 before the antigen is bound to the antibody immobilized on the sensor chip surface is measured. Later resonance curve 2
In the method of measuring the concentration of the antigen from the difference θ2−θ1 in the resonance angle from the above, it was difficult to detect a substance having a concentration of about several hundred ng / ml or less or a low molecular substance having a molecular weight of 1,000 or less.

As a method of extending the measurement limit of the measurement method using surface plasmon, for example, Japanese Patent Publication No. 7-1114
No. 35, a conventional radiolabeled immunoassay (RI
The application of the indirect method used in A) and enzyme immunoassay (EIA) is described. In this method, the antibody immobilized on the sensor chip surface is combined with the analyte, and then a secondary antibody for the analyte is further bound to enhance the signal intensity by a sandwich method. The analyte molecule is immobilized on the sensor chip surface. A competition method is described in which a specific antibody for a sample is mixed in a sample containing the sample, and the binding of the antibody between the sample on the sensor chip surface and the sample in the sample is competed. The sandwich method makes it possible to measure low-concentration analytes, and the competition method makes it possible to measure not only low-concentration analytes but also low-molecular analytes.

[0005]

In the indirect method as described above, it is necessary to prepare an antibody solution in addition to the analyte. This complicates the measurement sequence, and generally increases the measurement cost because the antibody solution is expensive. Further, when storing the antibody, it is necessary to control the temperature and prevent evaporation of water.

The present invention solves the above-mentioned problems of the prior art, and has a configuration equivalent to that of a conventional device using surface plasmon, and is applied to light having the same wavelength as the light absorbed by the subject or the subject. By irradiating the surface of the sensor chip with light having a wavelength equivalent to the light absorbed by the bound dye and utilizing the fact that the analyte or the dye bound to the analyte absorbs light, the low It is an object of the present invention to provide a measurement method and an apparatus that enable detection and measurement of a concentration analyte and a low-molecular analyte.

[0007]

Means for Solving the Problems According to the first aspect of the present invention, there is provided a measurement method using a surface plasmon in which a binding species that specifically binds to an analyte is immobilized on a detection surface. In the method, the surface of the sensor chip is irradiated with light having a wavelength equivalent to the wavelength of light absorbed by the subject or light having a wavelength equivalent to the wavelength of light absorbed by the dye bound to the subject. A first resonance curve measurement step of measuring a curve, and light of a wavelength equivalent to light of a wavelength absorbed by the subject or light of a wavelength equivalent to light of a wavelength absorbed by a dye bound to the subject is applied to the sensor chip surface. A second resonance curve measurement step of measuring a surface plasmon resonance curve when the sample or the dye bound to the sample absorbs the light applied to the sensor chip surface by irradiating the surface with a light, An object detection step of detecting an object by comparing the first resonance curve obtained in the vibration curve measurement step with the second resonance curve obtained in the second resonance curve measurement step. It is characterized by the following.

In the present invention, the light irradiated to the surface of the sensor chip is made to have a wavelength equivalent to the light having a wavelength absorbed by the subject or a wavelength equivalent to the light having a wavelength absorbed by a dye bound to the subject, By measuring the surface plasmon resonance curve when the light irradiating the sensor chip surface is obtained by absorbing the subject or the dye bound to the subject,
In the conventional surface plasmon measurement method, since the second resonance curve is moderated, the resonance curve width is increased, and the reflected light intensity of the resonance curve is reduced, and the difference from the reference first resonance curve can be increased. It enabled detection of low-concentration analytes or low-molecular analytes that could not be measured (the lower measurement limit was expanded).

According to a second aspect of the present invention, in the surface plasmon measurement method according to the first aspect, a step of binding a dye to the specimen and a step of removing a free dye not bound to the specimen. And characterized in that:

In the present invention, the step of binding the dye to the analyte is provided, so that the dye is bound even in the analyte having a low absorbance of the analyte itself, so that the second resonance curve is moderated. It is possible to increase the resonance curve width and reduce the intensity of the reflected light of the resonance curve, thereby increasing the difference from the reference first resonance curve. Also,
By providing a step of removing the free dye that is not bound to the analyte, when adding the dye to the analyte of unknown concentration, add a certain amount of the dye Since it is possible to remove a coloring matter, it is not necessary to require a high precision in the step of binding the coloring matter to the analyte, and the method is simple.

According to a third aspect of the present invention, in the step of removing the free dye which is not bound to the analyte, the free dye is bound to a substance which does not specifically bind to a bound species on the detection surface. It is characterized by being adsorbed and removed.

In the present invention, the substance that removes the free dye that is not bound to the analyte is a substance that does not specifically bind to the binding species on the detection surface, thereby adsorbing the free dye. Since the separated substance can completely remove the free dye without binding to the detection surface, the detection of the analyte is not affected by the free dye.

According to a fourth aspect of the present invention, there is provided a measuring method using a surface plasmon in which a binding species that specifically binds to an analyte is immobilized on a detection surface, the wavelength being equivalent to the wavelength of light absorbed by the analyte. A third resonance curve measuring step of measuring the reference surface plasmon resonance curve by irradiating the sensor chip surface with light of the same type, and irradiating the sensor chip surface with light having a wavelength equivalent to the wavelength of light absorbed by the subject. Resonance curve measuring step of measuring the surface plasmon resonance curve when the object is obtained by absorbing the light radiated to the sensor chip surface by the subject, and light having a wavelength equivalent to the wavelength of the light absorbed by the subject Among them, the subject absorbs the light radiated on the sensor chip surface by irradiating the sensor chip surface with light having a different wavelength from the light radiated in the third resonance curve measuring step and the fourth resonance curve measuring step. A fifth resonance curve measuring step of measuring the surface plasmon resonance curve when obtained, and a third resonance curve and a fourth resonance curve measuring step obtained in the third resonance curve measuring step. An object detection step of detecting an object by comparing the obtained fourth resonance curve with the fifth resonance curve obtained in the fifth resonance curve measurement step. .

In the present invention, in the third resonance curve measuring step, the fourth resonance curve measuring step, and the fifth resonance curve measuring step, the wavelength of the light to be irradiated on the sensor chip surface is changed so that the object to be inspected is changed. It is possible to detect a subject even if the subject has a similar absorbance and cannot be specified only by a resonance curve by irradiation of light of one wavelength.

According to a fifth aspect of the present invention, there is provided a measuring method utilizing a surface plasmon by immobilizing a catalyst which selectively acts on an analyte on a detection surface, wherein the analyte changes a molecular structure by the catalyst. A sixth resonance curve measurement step of irradiating the sensor chip surface with light having the same wavelength as the light of the wavelength to be absorbed and measuring the surface plasmon resonance curve before the subject changes the molecular structure by the catalyst, After changing the molecular structure by the catalyst, the subject irradiates the sensor chip surface with light of the same wavelength as the light of the wavelength to be absorbed after the subject changes the molecular structure by the catalyst, and irradiates the sensor chip surface with the light. A seventh resonance curve measurement step of measuring the surface plasmon resonance curve when the object is absorbed and obtained, and a sixth resonance curve obtained in the sixth resonance curve measurement step Characterized by comprising a subject detection step of detecting the analyte by comparing the seventh resonance curve obtained in seventh resonance curve measurement step.

In the present invention, a catalyst that selectively acts on an analyte is immobilized on a detection surface, and surface plasmon resonance curves before and after the analyte changes its molecular structure by the catalyst are measured. By performing the detection of low-concentration analytes and low molecules that could not be detected by the method of comparing the surface plasmon resonance curve between the case where the analyte does not exist and the case where the analyte exists as in the prior art An object can be detected.

According to a sixth aspect of the present invention, there is provided a measuring apparatus using a surface plasmon in which a binding species that specifically binds to an analyte is immobilized on a detection surface, the wavelength being equivalent to the wavelength of light absorbed by the analyte. First resonance curve measuring means for measuring a reference surface plasmon resonance curve by irradiating the surface of the sensor chip with light of the same wavelength as that of the light absorbed by the dye or the dye bound to the subject, and the subject Irradiates the sensor chip surface with light of the same wavelength as the light of the wavelength that is absorbed by the subject or light of the same wavelength as the light that is absorbed by the dye bound to the subject. A second resonance curve measuring means for measuring a surface plasmon resonance curve when the dye bound to the analyte is obtained by absorption, a first resonance curve obtained by the first resonance curve measuring means, and a second resonance curve. Characterized by comprising a subject detecting means for detecting an object by comparing the second resonance curve obtained by resonance curve measurement means.

In the present invention, the light irradiated on the surface of the sensor chip is set to a light having a wavelength equivalent to the light having a wavelength absorbed by the subject or a wavelength equivalent to a light having a wavelength absorbed by a dye bound to the subject, By measuring the surface plasmon resonance curve when the light irradiating the sensor chip surface is obtained by absorbing the subject or the dye bound to the subject,
In the conventional surface plasmon measuring device, since the second resonance curve can be moderated, the resonance curve width can be increased, and the reflected light intensity of the resonance curve can be reduced, and the difference from the first resonance curve serving as a reference can be increased. This enabled detection of low-concentration analytes or low-molecular analytes that could not be measured.

According to a seventh aspect of the present invention, there is provided a measuring apparatus using surface plasmon according to the sixth aspect, wherein a means for binding a dye to an analyte and a free dye not bound to the analyte are used. Removing means.

In the present invention, by providing a means for binding a dye to the analyte, the second resonance curve is moderated by binding the dye even to the analyte having a low absorbance of the analyte itself. It is possible to increase the resonance curve width and reduce the intensity of the reflected light of the resonance curve, thereby increasing the difference from the reference first resonance curve. Also,
By providing a means for removing the free dye that is not bound to the analyte, when adding the dye to the analyte whose concentration is not known, Since it is possible to remove a dye, the method for binding the dye to the analyte does not require high precision and is simple.

According to an eighth aspect of the present invention, in the measuring apparatus using the surface plasmon according to the seventh aspect, the means for removing the free dye which is not bound to the analyte is:
It is characterized in that a dye in a free state is adsorbed and removed by a substance that does not specifically bind to a binding species on a detection surface.

In the present invention, the substance that removes the free dye that is not bound to the analyte is a substance that does not specifically bind to the binding species on the detection surface, thereby adsorbing the free dye. Since the separated substance can completely remove the free dye without binding to the detection surface, the detection of the analyte is not affected by the free dye.

According to a ninth aspect of the present invention, there is provided a measuring apparatus using surface plasmons in which a binding species that specifically binds to an analyte is immobilized on a detection surface, the wavelength being equivalent to the wavelength of light absorbed by the analyte. A third resonance curve measuring means for irradiating the surface of the sensor chip with light of the same to measure a reference surface plasmon resonance curve, and irradiating the surface of the sensor chip with light having a wavelength equivalent to the light having a wavelength absorbed by the subject. Resonance curve measuring means for measuring the surface plasmon resonance curve when the object is obtained by absorbing the light radiated on the sensor chip surface by the subject, and light having the same wavelength as the light absorbed by the subject Among them, the subject absorbs the light radiated on the sensor chip surface by irradiating the sensor chip surface with light having a different wavelength from the light radiated in the third resonance curve measuring step and the fourth resonance curve measuring step. Fifth resonance curve measuring means for measuring the surface plasmon resonance curve when obtained, and a third resonance curve obtained by the third resonance curve measuring means and a fourth resonance curve measuring means. An object detecting means for detecting an object by comparing the obtained fourth resonance curve with the fifth resonance curve obtained by the fifth resonance curve measuring means. .

In the present invention, the third resonance curve measuring means, the fourth resonance curve measuring means, and the fifth resonance curve measuring means change the wavelength of light irradiated on the surface of the sensor chip to change the object. It is possible to detect a subject even if the subject has a similar absorbance and cannot be specified only by a resonance curve by irradiation of light of one wavelength.

According to a tenth aspect of the present invention, there is provided a measuring apparatus utilizing a surface plasmon by immobilizing a catalyst which selectively acts on an analyte on a detection surface, wherein the analyte changes the molecular structure by the catalyst. Sixth resonance curve measuring means for irradiating the surface of the sensor chip with light having the same wavelength as the light of the wavelength to be absorbed and measuring the surface plasmon resonance curve before the subject changes the molecular structure by the catalyst, After changing the molecular structure by the catalyst, the subject irradiates the sensor chip surface with light of the same wavelength as the light of the wavelength to be absorbed after the subject changes the molecular structure by the catalyst, and irradiates the sensor chip surface with the light. Seventh resonance curve measuring means for measuring a surface plasmon resonance curve when the object is absorbed and obtained, and a sixth resonance curve obtained by the sixth resonance curve measuring means Characterized by comprising a subject detection step of detecting the analyte by comparing the seventh resonance curve obtained in the seventh resonance curve measurement means.

In the present invention, a catalyst that selectively acts on an analyte is immobilized on a detection surface, and surface plasmon resonance curves before and after the analyte changes its molecular structure by the catalyst are measured. By performing the detection of low-concentration analytes and low molecules that could not be detected by the method of comparing the surface plasmon resonance curve between the case where the analyte does not exist and the case where the analyte exists as in the prior art An object can be detected.

According to the eleventh aspect of the present invention, in the surface plasmon measuring device according to any one of the sixth to tenth aspects, the object detecting means for detecting the object detects a change in reflected light intensity of a resonance curve. It is characterized by measuring and detecting the subject

In the present invention, the object detecting means measures the change in the reflected light intensity of the resonance curve to detect the object, thereby detecting the change in the incident angle of the excitation light. Then, it was possible to detect an analyte that could hardly be detected.

According to a twelfth aspect of the present invention, in the surface plasmon measuring device according to any one of the sixth to tenth aspects, the subject detecting means for detecting the subject measures a change in a half width of a resonance curve. Then, the detection of the subject is performed.

In the present invention, the object detecting means measures the change in the half-width of the resonance curve and detects the object. This enabled the detection of a subject that could hardly be detected.

[0031]

Embodiments of the present invention will be described below in detail with reference to the drawings. In FIG. 2, 3 is obtained by irradiating the surface of the sensor chip with light having a wavelength equivalent to the wavelength of light absorbed by the dye bound to the subject while the surface of the sensor chip is filled with the buffer solution, and measuring the resonance curve. This is a reference resonance curve (first resonance curve). 4 stains a low-concentration analyte or a low-molecular analyte with a dye (binds the dye to the analyte), adsorbs (binds) it to an antibody on the sensor chip surface, and absorbs the dye bound to the analyte. The second resonance curve is obtained by irradiating the sensor chip surface with light having the same wavelength as the light having the desired wavelength and absorbing the light irradiated on the sensor chip surface with the dye bound to the subject. is there.

When comparing FIG. 1 with FIG. 2, the difference between the first resonance curve 3 and the second resonance curve 4 in FIG. Is larger. The conventional SPR sensor shown in FIG. 1 utilizes the fact that the refractive index in the vicinity of the sensor chip changes due to the adsorption of the subject to the antibody on the sensor chip surface, and the resonance angle θ1 of the resonance curve 1 and the resonance angle of the resonance curve 2 The difference (θ2−θ1) from the angle θ2 is measured to measure (detect) the concentration of the subject. In the case of a low-concentration subject or a low-molecule subject, the resonance curves 1 and 2 overlap. Therefore, it is difficult to measure the concentration of the analyte because there is almost no difference. In the present invention shown in FIG. 2, the surface of the sensor chip is irradiated with light having the same wavelength as the light having the wavelength absorbed by the subject or light having the same wavelength as the light having the wavelength absorbed by the dye bound to the subject. By utilizing the fact that the subject or the dye bound to the subject absorbs light, the obtained resonance curve is gradual, the resonance curve width increases, and the reflected light intensity of the resonance curve decreases, and the reference Since the difference between the first resonance curve 3 and the second resonance curve 4 becomes large, the detection ability of a low-concentration analyte or a low-molecular analyte is improved as compared with the conventional surface plasmon measurement method (lower measurement limit). Spread).

According to the present invention, the excitation light incident angle θ4 of the second resonance curve 4 and the excitation light incident angle θ of the first resonance curve 3
When the variation (difference) from 3 is small, the sensitivity can be further improved by capturing the variation (difference) in the reflected light intensity R or the variation (difference) in the half-width W of the resonance curve.

The amount of change (difference) in the reflected light intensity R of the resonance curve
In the case of capturing, for example, the change amount (difference) between the reflected light intensity R4 of the second resonance curve 4 and the reflected light intensity R3 of the first resonance curve 3 at the position of the excitation light incident angle of 75 degrees (FIG. 2).
(R4-R3) can be captured. Further, utilizing the fact that the resonance curve of the second resonance curve 4 is gentler than that of the first resonance curve 3, the reflected light intensity R6 of the second resonance curve 4 at the position of the excitation light incident angle of 80 degrees is used. (Difference) (R) between the first resonance curve 3 and the reflected light intensity R5 of the first resonance curve 3.
By capturing 6-R5), the sensitivity can be further improved compared to capturing the amount of change (R4-R3) in the reflected light intensity at the position of the excitation light incident angle of 75 degrees.

When the change amount (difference) of the half-width W of the resonance curve is captured, for example, the half-width W4 of the second resonance curve 4 and the half-width of the first resonance curve 3 at the position of the reflected light intensity 0.6 are obtained. The amount of change (difference) from W3 (W4-W3) can be grasped.

Next, a description will be given of a means for removing the adverse effect on the measurement by the dye which has been stained with the dye added to the sample solution in order to stain the sample and which is still excessive and released. Usually, the dye is not specifically selected as a target to be stained. For example, the dye rarely selectively stains the protein human albumin and other animal albumin. Therefore, it is conceivable that the surplus and released dye that stains the specimen stains other substances in the sample solution and substances immobilized on the sensor chip surface, such as antibodies. Since this acts as noise on the measurement value of the sensor, a means for removing the free dye is required.

First, a dye is added to a sample solution containing an analyte, for example, a protein as a solute. At this time, an amount sufficient to stain the subject is added. If the analyte is a protein and the antibody is immobilized on the sensor chip surface, a dye is added to the sample solution to stain the target protein, and then another type of protein is added to the sample solution, and the excess dye is removed from this protein. Removed by adsorption to Antibodies are usually expected to strictly discriminate between different types of proteins, so that only the stained target protein is adsorbed on the sensor chip surface, and another type of protein is not adsorbed. In this way, only the stained target protein (analyte) can be adsorbed on the sensor chip surface.

The surplus dye can be separated using a separation membrane, for example, a dialysis membrane, by utilizing the difference in molecular weight from the analyte.

[0039]

Example 1 An example in which human albumin is used as a subject and Coomassie brilliant blue is used as a dye will be described. Coomassie brilliant blue is a dye that can easily stain human albumin, which is a protein. Further, since the dye absorbs light near 580 nm, the wavelength of light that excites surface plasmon naturally has a wavelength of 580 nm.
The light source is a white light source such as a halogen lamp, which is separated by a spectroscope, or the crystal is GaP.
A monochromatic light source such as an InGaAlP-based LED or the like can be used. Particularly, an InGaAlP-based LED having a good monochromaticity and easy to handle is most suitable as a light source for this purpose.

Surface plasmons are usually excited on a gold or silver thin film having a thickness of about 50 nm. Since gold absorbs light of 580 nm, the resonance curve is broader (slower curve) and the light energy is absorbed as compared with a case where surface plasmons are excited on a gold thin film with light of another wavelength, for example, light of 660 nm. Will also be less. In this case, the shape of the resonance curve is made normal (excitation light 660) by setting the film thickness of gold to around 40 nm, which is a film thickness smaller than the normal 50 nm.
(when surface plasmon is excited with a gold thin film thickness of 50 nm). In addition, since silver has no absorption in the visible light region, the same phenomenon as in the case of gold does not occur, and the resonance curve does not suddenly become broad regardless of the wavelength selected in the visible light region.

As a light source, InGaAl having an emission wavelength of 575 nm is used.
Using a P-based LED, this light is irradiated to the surface of the sensor chip using a general optical system for exciting surface plasmons to excite surface plasmons. A dye with a known number of molecules (Coomassie brilliant blue) is added to the sample solution containing the test sample (human albumin) and the solution is allowed to stand for a certain period of time (about 5 minutes).
After stirring, a protein other than human albumin having a molecular number equal to or greater than the previously added dye and having a known molecular number, such as bovine albumin, is added to the sample solution. And for a certain period of time
For minutes). Thus, the human albumin antibody is immobilized on the sensor chip surface.

First, the sensor chip surface is filled with a buffer solution and the resonance curve is measured to obtain a first resonance curve 3 (FIG. 2) as a reference. Next, the surface of the sensor chip is filled with the previously prepared sample solution. After a certain period of time (about 1 to 3 minutes), the surface of the sensor chip is washed with a buffer solution, and a resonance curve is measured to obtain a second resonance curve 4. Due to the effect of light absorption of the dye, the minimum value of the reflected light intensity of the second resonance curve 4 becomes smaller (lower) than the resonance curve of only the buffer solution (resonance curve 3), the resonance curve becomes broad and the half value width becomes large. Is increasing. Furthermore, the resonance angle (incident angle at which the reflected light intensity becomes a minimum value) increases due to the adsorption of the subject on the sensor chip surface. Needless to say, the detection method is not limited to Coomassie brilliant blue and albumin, respectively.

[0043]

Embodiment 2 An example in which hemoglobin is measured as a subject will be described. Hemoglobin includes oxyhemoglobin and deoxyhemoglobin, and it is known that the absorption spectrum in the visible light region has a wide absorption centering on 560 nm as a whole although the details differ in both. 580n
Since the relative absorbance of the light of m and m is 5 or more, the measurement method of the present invention can be applied without dyeing with a dye.
A hemoglobin antibody is immobilized on the sensor chip surface using an LED having an emission wavelength of 580 nm as a light source, and surface plasmons are excited using an ordinary optical system of a surface plasmon sensor. Fill the buffer solution on the sensor chip and measure the resonance curve. Next, a sample solution containing hemoglobin is left on the sensor chip for a certain period of time (about 3 minutes), and then washed with a buffer solution to measure a resonance curve. The amount of hemoglobin adsorbed on the sensor chip is converted from the difference in excitation light reflection intensity (reflectance) at an incident angle having a resonance curve before and after flowing the sample liquid.

Oxyhemoglobin has a higher light absorption at 580 nm than at 560 nm, and deoxyhemoglobin has a smaller light absorption at 580 nm than at 560 nm. Therefore, a resonance curve obtained by exciting surface plasmon at 560 nm and a resonance curve obtained by exciting surface plasmon at 580 nm are compared. It is possible to determine whether the hemoglobin adsorbed on the chip surface is oxyhemoglobin or deoxyhemoglobin. It goes without saying that this detection method is not limited to hemoglobin.

[0045]

Example 3 An example in which bilirubin oxidase, which is an enzyme, is immobilized on a sensor chip surface (detection surface) as a catalyst that selectively acts on an analyte, and bilirubin is measured as an analyte will be described. Bilirubin is an orange-yellow substance whose light absorption peaks at 446 nm. Bilirubin is oxidized by bilirubin oxidase to produce bilurubin. Bilbergine is a green substance whose light absorption peak is 640n
m. As the plasmon excitation light source that irradiates the sensor chip surface, a light source having a wavelength of about 640 nm is used.

First, the surface of the sensor chip on which bilirubin oxidase is immobilized is filled with a buffer solution, and the resonance curve (reference resonance curve) is measured. Next, the buffer solution is replaced with a sample solution containing bilirubin, and the resonance curve is measured at regular intervals (about 1 second). Bilirubin in the sample solution is oxidized by bilirubin oxidase, which is an enzyme, on the surface of the sensor chip and is converted into bilurubin. At that time, the vicinity of the sensor chip surface is covered with virbergine which absorbs 640 nm excitation light. Due to the presence of Bilbergine, the resonance curve is broader and wider at half-width than without Bilbergine (in the case of bilirubin). If this change is monitored at regular intervals (about one second), the state of the generation of bilurubin can be detected, and from this, the presence of bilirubin can be detected. It goes without saying that this measurement method is not limited to the detection of bilirubin.

[0047]

According to the present invention, the light having the same wavelength as the light absorbed by the subject or the wavelength absorbed by the dye bound to the subject has the same configuration as that of the conventional device using surface plasmon. By irradiating the surface of the sensor chip with light having the same wavelength as the light, and utilizing the fact that the analyte or the dye bound to the analyte absorbs light, detection of low-concentration analytes and low-molecular analytes, It enables measurement. In addition, by providing a step of binding the dye to the analyte, the second resonance curve is moderated and the resonance curve width is increased by binding the dye even in the analyte having low absorbance of the analyte itself. At the same time, the reflected light intensity of the resonance curve can be reduced, and the difference from the first resonance curve serving as a reference can be increased. In addition, by providing a step of removing the free dye that is not bound to the analyte, when adding the dye to the analyte of unknown concentration, add a certain amount of the dye, Since the excess dye can be removed, the process of binding the dye to the analyte does not require high precision and is simple.

In addition, since the substance that removes the free dye that is not bound to the analyte is a substance that does not specifically bind to the binding species on the detection surface, the substance that adsorbs the free dye can be Since the free dye can be completely removed without binding to the detection surface, detection of the analyte is not affected by the free dye. In addition, by changing the wavelength of the light irradiating the sensor chip surface, even if the subject itself is similar in absorbance of the subject itself and can not be specified only by the resonance curve by light irradiation of one type of wavelength, The detection of the subject was made possible.

Further, a catalyst that selectively acts on the analyte is fixed on the detection surface, and the surface plasmon resonance curves before and after the analyte changes the molecular structure by the catalyst are measured, and the detection of the analyte is performed. By doing so, the method of comparing the surface plasmon resonance curve of the case where the analyte does not exist and the case where the analyte does not exist as in the prior art can not detect the low concentration analyte or low molecular analyte which could not be detected Detection can be performed. In addition, since the object detection means measures the change in the reflected light intensity of the resonance curve to detect the object, the object detection means which conventionally measures the change in the incident angle of the excitation light can hardly detect the change. The detection of the subject was made possible. Further, the subject detection means measures the change in the half width of the resonance curve to detect the subject,
Conventionally, it has become possible to detect an object which could hardly be detected by the object detecting means which measures the change in the incident angle of the excitation light.

[Brief description of the drawings]

FIG. 1 is a diagram showing a surface plasmon resonance curve obtained by a conventional measurement method using surface plasmons.

FIG. 2 is a diagram showing a surface plasmon resonance curve obtained by a measurement method using surface plasmons of the present invention.

[Explanation of symbols]

1 ... resonance curve as a reference in the prior art; 2 ... resonance curve after reacting (binding) the antigen of the prior art with the antibody immobilized on the sensor chip surface; 3 ... the first resonance curve; Second resonance curve

Claims (12)

[Claims] In a measurement method using a surface plasmon in which a binding species that specifically binds to an analyte is immobilized on a detection surface, light that has a wavelength equivalent to the wavelength of light absorbed by the analyte or binds to the analyte is used. A first resonance curve measuring step of measuring the reference surface plasmon resonance curve by irradiating the surface of the sensor chip with light having a wavelength equivalent to the light of the wavelength absorbed by the dye, and equivalent to light having a wavelength absorbed by the subject. Irradiates the sensor chip surface with light of the same wavelength or light of a wavelength that is absorbed by the dye bound to the subject, and the light radiated to the sensor chip surface is absorbed by the subject or the dye bound to the subject A second resonance curve measuring step of measuring the surface plasmon resonance curve when obtained, and a first resonance curve obtained in the first resonance curve measuring step and a second resonance curve obtained in the second resonance curve measuring step. An object detection step of detecting an object by comparing the obtained second resonance curve with the obtained second resonance curve. 2. The surface plasmon measurement method according to claim 1, further comprising: a step of binding a dye to an analyte; and a step of removing a free dye not bound to the analyte. Surface plasmon measurement method. 3. The step of removing the free dye that is not bound to the analyte is characterized in that the free dye is adsorbed and removed by a substance that does not specifically bind to the bound species on the detection surface. The method for measuring surface plasmon according to claim 2, wherein 4. In a measurement method using surface plasmons in which a binding species that specifically binds to an analyte is immobilized on a detection surface, light having a wavelength equivalent to light absorbed by the analyte is irradiated onto the surface of the sensor chip. A third resonance curve measuring step of measuring a surface plasmon resonance curve to be a reference,
The fourth step is to irradiate the surface of the sensor chip with light having the same wavelength as the wavelength of light absorbed by the subject, and to measure the surface plasmon resonance curve obtained when the subject absorbs the light applied to the sensor chip surface. Of the resonance curve measurement step, and light having a wavelength different from the light irradiated in the third resonance curve measurement step and the fourth resonance curve measurement step, of the light having the same wavelength as the light having the wavelength absorbed by the subject. A fifth resonance curve measuring step of measuring a surface plasmon resonance curve when the subject absorbs light irradiated on the sensor chip surface by irradiating the sensor chip surface, and the third resonance curve measuring step By comparing the third resonance curve obtained in step 4, the fourth resonance curve obtained in the fourth resonance curve measurement step and the fifth resonance curve obtained in the fifth resonance curve measurement step Subject Surface plasmon measurement method characterized by comprising a subject detection step of detecting. 5. A measurement method using a surface plasmon in which a catalyst that selectively acts on an analyte is immobilized on a detection surface, the light having a wavelength that the analyte absorbs after the molecular structure is changed by the catalyst. A sixth resonance curve measuring step of irradiating light of the same wavelength to the sensor chip surface to measure the surface plasmon resonance curve before the subject changes the molecular structure by the catalyst, and the subject has a molecular structure by the catalyst. After changing the molecular structure by the catalyst, the subject is absorbed by irradiating the sensor chip surface with light having the same wavelength as the light having the wavelength to be absorbed after changing the molecular structure. A seventh resonance curve measurement step of measuring the surface plasmon resonance curve when the first and second resonance curves obtained in the sixth resonance curve measurement step and the seventh resonance curve measurement step An object detection step of detecting an object by comparing the obtained resonance curve with a seventh resonance curve. 6. A measuring apparatus using surface plasmons in which a binding species that specifically binds to an analyte is immobilized on a detection surface, and the binding species is bonded to light having a wavelength equivalent to the light absorbed by the analyte or to the analyte. First resonance curve measuring means for measuring a reference surface plasmon resonance curve by irradiating light having a wavelength equivalent to the wavelength of light absorbed by the dye to the sensor chip surface, and equivalent to light having a wavelength absorbed by the subject. Irradiates the sensor chip surface with light of the same wavelength or light of a wavelength that is absorbed by the dye bound to the subject, and the light radiated to the sensor chip surface is absorbed by the subject or the dye bound to the subject A second resonance curve measuring means for measuring the surface plasmon resonance curve obtained by the first and second resonance curve measuring means obtained by the first resonance curve measuring means. And a subject detecting means for detecting the subject by comparing the obtained second resonance curve with the obtained second resonance curve. 7. The measuring apparatus using surface plasmon according to claim 6, further comprising: means for binding a dye to an analyte; and means for removing a free dye not bound to the analyte. A surface plasmon measuring device characterized by the following. 8. The measuring device using surface plasmon according to claim 7, wherein the means for removing the free dye that is not bound to the analyte is a substance that does not specifically bind to the bound species on the detection surface. A surface plasmon measuring device characterized in that a free dye is adsorbed on a surface plasmon and removed. 9. A measuring device using surface plasmons in which a binding species that specifically binds to an analyte is immobilized on a detection surface, and the surface of the sensor chip is irradiated with light having a wavelength equivalent to the wavelength of light absorbed by the analyte. Third resonance curve measuring means for measuring the surface plasmon resonance curve as a reference,
The fourth step is to irradiate the surface of the sensor chip with light having the same wavelength as the wavelength of light absorbed by the subject, and to measure the surface plasmon resonance curve obtained when the subject absorbs the light applied to the sensor chip surface. Of the resonance curve measuring means, and light of a wavelength different from the light irradiated in the third resonance curve measurement step and the fourth resonance curve measurement step, of the light having the same wavelength as the light having the wavelength absorbed by the subject. Fifth resonance curve measuring means for measuring a surface plasmon resonance curve when the subject absorbs light irradiated on the sensor chip surface and irradiated on the sensor chip surface, and the third resonance curve measuring means By comparing the third resonance curve obtained by the above with the fourth resonance curve obtained by the fourth resonance curve measuring means and the fifth resonance curve obtained by the fifth resonance curve measuring means, Subject Surface plasmon measurement apparatus characterized by comprising a subject detecting means for detecting. 10. A measuring device using a surface plasmon in which a catalyst that selectively acts on an analyte is fixed on a detection surface, wherein the analyte absorbs light having a wavelength which is absorbed after the molecular structure is changed by the catalyst. Sixth resonance curve measuring means for irradiating the surface of the sensor chip with light of the same wavelength to measure the surface plasmon resonance curve before the subject changes the molecular structure by the catalyst, and the subject has the molecular structure by the catalyst. After changing the molecular structure by the catalyst, the subject is absorbed by irradiating the sensor chip surface with light having the same wavelength as the light having the wavelength to be absorbed after changing the molecular structure. The seventh resonance curve measuring means for measuring the surface plasmon resonance curve at the time of the first, the sixth resonance curve and the seventh resonance curve measuring means obtained by the sixth resonance curve measuring means An object detection step of detecting an object by comparing the seventh resonance curve obtained in the step (c). 11. The surface plasmon measuring device according to claim 6, wherein the object detecting means for detecting the object measures a change in reflected light intensity of a resonance curve to detect the object. A surface plasmon measuring device characterized by performing. 12. The surface plasmon measuring device according to claim 6, wherein the object detecting means for detecting the object detects a subject by measuring a change in a half width of a resonance curve. A surface plasmon measuring device, characterized in that: