JPH11237337A - Specified constituent and its concentration measurement method for multiple-constituent substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a specific constituent and its concentration measurement method for multiple-constituent substance for making the specific substance and its concentration detectable by surface plasmon resonance method. SOLUTION: A surface plasmon resonance measuring device 1 measures the amount of adsorption of a multiple-constituent substance in a sensor chip 3 based on the resonance angle change of reflection light from the sensor chip 3 by applying a non-absorbent light to a specific constituent and other constituents for a multiple-constituent substance consisting of the mixed form of the specific constituent with a characteristic for absorbing light with a specific wavelength being adsorbed to the sensor chip 3 where a metal thin film is deposited on a glass plate 5 and other constituents with the non-absorption characteristics of light. Light with an absorption characteristic to the specific constituent is applied to the sensor chip 3, the specific constituent in the multiple-constituent substance is specified based on a resonance angle from the sensor chip 3, and at the same time the concentration of the specific constituent is measured according to the ratio of transmittance before and after the absorption of multiple-constituent substance for the sensor chip 3.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a specific component in a multicomponent substance and a method for measuring the concentration of the specific component.

[0002]

SUMMARY OF THE INVENTION For example, liquid crystals, semiconductors,
In various technical fields such as polymer thin films and medical tests, it is necessary to detect a specific component and its concentration in a multi-component substance. Conventionally, as a method for detecting a specific component in a multi-component substance, an ellipsometry, an optical waveguide method, a surface plasmon resonance method, and the like are generally known.

[0003] Among them, the ellipsometry is a method of irradiating a substance on a substrate with polarized light and analyzing the polarization state of the reflected light to detect a specific component. Due to the scattering of light, a specific component of the multi-component substance in contact with the liquid could not be detected.

The optical waveguide method is a method for detecting a specific component based on the attenuation of the intensity of light emitted due to a change in reflection conditions when a multi-component substance is adsorbed outside the optical waveguide. However, when a halogen, xenon, or the like was used as a light source for white light introduced into the optical waveguide, it could not be used as a spot light source. As a result, light could not be condensed so as to satisfy the condition of total reflection at all wavelengths, and could not be introduced into the optical waveguide.

Further, in the surface plasmon resonance method, a glass substrate on which a metal thin film of gold, silver, or the like is deposited is irradiated with light to detect a resonance angle at which the reflectance is minimized. However, a specific component cannot be detected because light of a single wavelength cannot distinguish each component by reflecting the contribution of all components. Also, the concentration of the specific substance could not be detected.

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned conventional drawbacks, and an object thereof is to provide a specific substance by a surface plasmon resonance method and a multi-component substance capable of detecting its concentration. And a method for measuring the concentration of the specific component.

[0007]

Accordingly, the present invention provides a specific component having a characteristic of absorbing light of a predetermined wavelength adsorbed on a sensor chip having a metal thin film deposited on a glass plate, and a non-absorbing component of the light. For a multi-component substance composed of a mixed form of other components having characteristics, a specific component and other components are irradiated with non-absorbing light, and a change in the resonance angle of the reflected light from the sensor chip is used in the sensor chip. Measure the amount of adsorption of the multi-component substance, and then irradiate the sensor chip with light having light absorption characteristics for the specific component, and specify the specific component in the multi-component substance based on the resonance angle from the sensor chip In addition, the concentration of the specific component is measured by the transmittance ratio before and after the adsorption of the multi-component substance on the sensor chip.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention will be described below with reference to the drawings.

FIG. 1 is an explanatory view showing an example of a detection apparatus embodying the method of the present invention.

FIG. 2 is an enlarged view of a portion shown in FIG. 1A.

According to the present invention, a sensor chip 3 of a surface plasmon resonance measuring device (hereinafter referred to as an SPR measuring device) 1 is provided.
Above, when there is a multi-component material of the specific component with absorbance and the specific component without the absorbance, the total amount of the multi-component material is measured with light having a wavelength that does not have absorbance for the specific component, and On the other hand, the principle is that a specific component in a multi-component substance is detected by light having a wavelength having an absorbance and the concentration is measured by a specific component ratio to the whole.

The sensor chip 3 of the SPR measuring device 1 using the above measurement has a film thickness of 5 ± 0.
5 nm gold thin film 7a (shown in FIG. 2) and film thickness 36 ± 3n
m of silver thin film 7b (shown in FIG. 2) is deposited in two layers.
Further, a prism 9 is adhered to the non-deposited metal thin film surface of the glass substrate 5 by a matching oil (not shown).

The metal thin film deposited on the glass substrate 5 has a two-layer structure of a gold thin film and a silver thin film for the following reasons.
That is, the detection range of the reflection angle by the surface plasmon resonance measurement apparatus 1 using light of different wavelengths is limited to about 10 ° width. The reflection angle of the light having the wavelength is about 10 as described above.
It does not appear in the range of °. In order to avoid this, a gold thin film for preventing oxidation was provided on the silver thin film deposited on the glass substrate 5.
A layer structure was adopted. Thereby, as shown in Table 2, the red laser light (wavelength 636 nm) and the green laser light (wavelength 538)
nm) could be expressed simultaneously within a range of about 10 °.

A light irradiation device 11 is arranged on the left side of the prism 9 in the vertical direction of the lead. The light irradiation device 11 irradiates a white laser light including a red laser light having a wavelength of 636 nm, a green laser light having a wavelength of 538 nm, and a blue laser light having a wavelength of 441 nm. Interference filter 1 to select
1b, and a lens system 11e that converts the light transmitted through the optical fiber 11c and the collinometer 11d into parallel rays, transmits the prism 9, and collects light at the boundary between the glass substrate 5 and the silver thin film 7b. The laser beam condensed by the lens system 11e has an incident angle width with respect to the prism 9 set within a range of about 60 to 80 degrees. In this measurement method, red laser light having a wavelength of 636 nm and green laser light having a wavelength of 538 nm were selected and used by the interference filter 11b.

The white laser light emitted from the white laser light source 11a is attenuated to a required intensity by an ND filter (not shown) as required.

On the other hand, a light detecting device 13 is disposed on the right side of the prism 9 in the drawing. The light detection device 13
Is reflected by the gold thin film 7a and the silver thin film 7b,
Lens system 13 for converting a laser beam emitted from a laser beam into a parallel beam
a, and a light receiving member 13b such as a CCD for detecting the light intensity of the parallel light beam.

Hereinafter, an example of measuring a specific component in a multi-component substance and its concentration using the SPR measuring device 1 will be described.

The multi-component substance is a solution in which a specific substance to which a fluorescent label having an absorbance with respect to green laser light is added and an unlabeled component are mixed, specifically, a labeled specific component and non-labeled other components. An IgG protein having a molecular weight of about 150,000 was used, and a fluorescent substance having a molecular weight of 448 was added as an labeling substance to the average of 2.6 molecules per IgG molecule. Then, while the total protein concentration in the mixed solution was fixed at 1 mg / ml, the concentration of the labeled specific component in the mixed solution was reduced to 0 mg / ml.
/ Ml, 0.1mg / ml, 0.25mg / ml, 1m
g / ml.

As shown in Table 3, the labeled specific component of the multi-component substance has absorbance with respect to the green laser light having a wavelength of 536 nm, and the unlabeled other components have absorbance with respect to this green laser light. Absent.

The change in reflectance before and after the adsorption of the fluorescent labeled molecule when using the green laser light was measured. First, pure water (n = 1.333) is brought into contact with the sensor chip 3 to obtain equilibrium.
Green laser light was selected by the interference filter 11b, and the incident angle dependence of the reflectance was measured. Next, 1 mg / ml of the fluorescent-labeled molecule solution was replaced with pure water, and the molecules were adsorbed to the sensor chip 3 to obtain equilibrium. In order to detect only the influence of the molecules adsorbed on the sensor chip 3, the solution was replaced with pure water again to obtain an equilibrium, and the incident angle dependence of the reflectance was measured in the same manner. Table 4 shows the results.

As described above, the green laser light is a marker characteristic,
Therefore, since it is absorbed by the label specific component, the amount of adsorption of the multicomponent substance composed of the label component and the non-label component on the sensor chip 3 can be measured by measuring the resonance angle.

Next, the concentration of the fluorescent labeling molecule was set to 0 mg / m
1, 0.1 mg / ml, 0.25 mg / ml, 0.5 m
g / ml and 1.0 mg / ml, and using a red laser beam and a green laser beam, the resonance angle and the change in reflectance at the resonance angle were measured, respectively. Table 5 shows the change in the resonance angle with respect to the concentration of the specific component of the label before and after the adsorption when using the red laser beam. Since the concentration of the multi-component substance adsorbed on the sensor chip 3 is adjusted to a constant value of 1 mg / ml in any case, the resonance angle should originally be constant. In the case of 0.5 mg / ml and 1 mg / ml,
0mg / ml, 0.1mg / ml, 0.25mg
/ Ml, the resonance angle was changed, but this was because the amount of adsorption to the sensor chip 3 increased due to the hydrophobicity of the fluorescent group contained in the surface of the sensor chip 3 and the labeling specific component. by.

Next, Table 6 shows the normalized transmittance ratio with respect to the concentration of the label specific component before and after the adsorption when using the green laser light. The normalized transmittance ratio is obtained by calculating the transmittance from the reflectance before and after adsorption at the resonance angle when using green laser light, dividing the transmittance after adsorption by the transmittance before adsorption, and then using the red laser light. The transmittance was determined from the reflectance before and after the adsorption at the resonance angle at the time, and the transmittance after the adsorption was divided by the value obtained by dividing the transmittance before the adsorption. The measurement using the red laser light and the green laser light was selected by the interference filter 11b and performed almost simultaneously. The normalized transmittance ratio indicates a transmittance ratio before and after the adsorption that does not depend on the amount of adsorption of the multi-component substance adsorbed on the sensor chip 3.

From the above Table 6, since the normalized transmittance decreases in proportion to the concentration of the label component in the multi-component substance, the concentration of the label specific component adsorbed on the sensor chip 3 can be measured. Was completed.

In the present embodiment, the change in resonance angle is determined by using light having a wavelength that absorbs a specific component to be detected in the surface plasmon resonance method and light that does not absorb the specific component and other components. By measuring the specific component, a specific component can be detected from the multi-component substance and the concentration thereof can be measured.

In the above description, green laser light adsorbed to a specific component and red laser light that does not absorb light are used as light of different wavelengths. However, the present invention is not limited to these, and the present invention is not limited thereto. The measurement may be performed using an ultraviolet laser beam having a different wavelength according to the light absorption characteristics.

[0027]

According to the present invention, it is possible to detect a specific substance and its concentration by the surface plasmon resonance method.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of a detection device embodying the method of the present invention.

FIG. 2 is an enlarged view of a portion of FIG. 1A.

[Explanation of symbols]

1 SPR measurement device, 3 sensor chip, 5 glass plate, 7a gold thin film, 7b silver thin film

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

Claims (6)

[Claims] 1. A mixed form of a specific component having a characteristic of absorbing light of a predetermined wavelength adsorbed on a sensor chip having a metal thin film deposited on a glass plate and another component having a non-absorbing characteristic of the light. Irradiating the non-absorbing light to the specific component and other components to the multi-component material, and measuring the adsorption amount of the multi-component material on the sensor chip based on the change in the resonance angle of the reflected light from the sensor chip, By irradiating the sensor chip with light having light absorption characteristics for the specific component, the specific component in the multi-component material is specified based on the resonance angle from the sensor chip, and before and after the adsorption of the multi-component material on the sensor chip. And a method for measuring the concentration of a specific component in a multi-component substance, wherein the concentration of the specific component is measured by a transmittance ratio in the method. 2. A method according to claim 1, wherein the metal thin film deposited on the sensor chip is a specific component in a multi-component substance comprising a gold thin film and a silver thin film, and a method for measuring the concentration thereof. 3. The sensor chip according to claim 1, wherein the sensor chip is provided with a prism on the surface of the glass plate on which the metal thin film is not deposited, and converts the parallel light with a required incident angle width into a multi-component material adsorbed by the metal thin film. A method for measuring a specific component in a multi-component material and its concentration, wherein the light can be collected at an interface and the reflected light from a sensor chip can be spread at a required angle. 4. The method for measuring the concentration of a specific component according to claim 1, wherein the specific component is made absorbable by adding a light-absorbing substance that absorbs light of a specific wavelength. 5. The method according to claim 4, wherein the light-absorbing substance is a specific component consisting of a fluorescent substance and its concentration measurement method. 6. The method according to claim 4, wherein the light-absorbing substance is a specific component comprising a metal substance and its concentration measurement method.