JP2000121551A - Concentration measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a concentration measuring device utilizing surface plasmon resonance (SPR), which can obtain reproducibility of a measurement result regardless of whether a sensor chip is attached or detached. SOLUTION: A glass plate 1b having a gold thin film 1a on one surface is provided on the gold thin film 1a side, and a measurement sample is placed on the gold thin film 1a.
A sensor chip 1 having storage portions 1d to 1f for receiving light from the other surface side of the glass plate 1b, a light emitting diode 2 for emitting light from the light emitting diode 2, and a glass plate 1b Lens 3 which is incident so that various angles of incidence can be obtained with respect to the interface between the substrate and the gold thin film 1a, a CCD camera 4 which detects the intensity of light reflected at the interface at each of various angles of reflection, and a CCD camera 4 Calculation units 5 to 7 for calculating the concentration of the measurement sample from the measurement results of the measurement sample obtained by the above, and a prism 8 for slidably mounting the sensor chip 1 on one surface via a matching oil. Features.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a concentration measuring device, and more particularly, to a concentration measuring device utilizing surface plasmon resonance (SPR).

[0002]

2. Description of the Related Art A compression wave generated by oscillation of free electrons on a metal surface is called a surface plasmon wave. The phenomenon that the surface plasmon wave is excited by a light wave such as evanescent light is called surface plasmon resonance (SPR).

[0003] Various sensors (SPR sensors) utilizing this surface plasmon resonance have been conventionally proposed. Among them, a particularly well-known one is disclosed in
An SPR sensor using a system called a Kretschmann configuration described in -82455.

The SPR sensor includes a prism, a metal film formed on one surface of the prism and brought into contact with a sample, a light source for generating light, and a light from the light source that passes through the prism. And an optical system for causing various angles of incidence to be obtained with respect to the interface, and light detecting means capable of detecting the intensity of light totally reflected at the interface at various angles of incidence.

[0005] Further, since exchanging the prism for each measurement increases the cost, as a means for forming a metal film on one surface of the prism, a metal film is formed on one surface of a glass plate having the same refractive index as the prism. A sensor chip that is formed and that can be pressed against a surface of the glass plate opposite to the surface on which the metal film is formed and a prism is used. Note that a matching oil or the like is used for pressure bonding between the prism and the sensor chip.

In the SPR sensor, when a surface plasmon wave generated on a metal surface is excited by evanescent light generated at an interface between a metal film and a measurement sample and resonated with light irradiated on the metal film, the SPR sensor resonates with the metal film. Since the light energy of the light component having a specific incident angle shifts to the surface plasmon wave in the light irradiated on the surface, when the light reflected from the metal film is detected, the light component having the specific incident angle shifted to the surface plasmon wave is detected. This is based on the fact that the light energy is weakly detected.

[0007] The SPR sensor is used particularly for detecting an interaction between biopolymers. That is, after immobilizing one of the biopolymers as a ligand (molecular recognition element) on the surface of the metal film, the other biopolymer is caused to act on the surface of the immobilized biopolymer, and the biomolecule to be acted on By measuring the change in the concentration of the polymer, the interaction between the biopolymers is detected.

[0008]

However, in the SPR sensor, even when the same measurement sample is measured using the same sensor chip, the measured values obtained before and after the attachment and detachment of the sensor chip vary, and the There is a problem that reproducibility of is not obtained. This is because when the sensor chip is replaced from the prism, the crimped state of the sensor chip and prism before replacement is slightly different from the crimped state of the sensor chip and prism after replacement. Because it has a large effect on

SUMMARY OF THE INVENTION An object of the present invention is to provide a concentration measuring apparatus utilizing SPR, which can obtain reproducibility of a measurement result regardless of whether a sensor chip is attached or detached. Is to do.

[0010]

SUMMARY OF THE INVENTION The present invention is a concentration measuring device, and has the following structure as means for solving the above-mentioned technical problem. That is, the concentration measuring device of the present invention has a metal film on one surface, includes a light transmitting medium having a desired refractive index, and has a housing portion for housing a measurement sample in contact with the metal film surface. A measuring element provided on the metal film side, a light source for irradiating light incident on a surface of the measuring element that is not in contact with the metal film, and transmitting the light emitted from the light source to the light Incident angle setting means for causing various angles of incidence to be obtained with respect to the interface between the medium and the metal film, and detecting means capable of detecting the intensity of light reflected at the interface for each of various reflection angles, An arithmetic processing unit that calculates the concentration of the unknown concentration sample from the measurement values of the unknown concentration and known concentration measurement samples respectively housed in different storage units, measured without detaching the measurement element by the detection unit. It is characterized by having.

Here, in the concentration measuring apparatus according to the present invention, when the measuring element has a moving means which can move relatively to the light source, the incident angle setting means and the detecting means, Preferred for performing measurements.

Further, the concentration measuring apparatus of the present invention is suitable for measuring the concentration of a polymer, and further suitable for measuring the concentration of a biopolymer. The measurement sample measured by the concentration measuring device of the present invention is a liquid, and when the measurement target is a liquid, it is good to make the solution having an appropriate concentration by a solvent that mixes the measurement target, and the measurement target is a solid. In some cases, it is preferable to use a solution having an appropriate concentration depending on the solvent in which the object to be measured is dissolved.

The metal film used for the measuring element is exemplified by a metal film formed on one surface of the light transmitting medium by vacuum evaporation. The metal film to be formed may be a single layer or a multilayer, and the metal forming the film is not particularly limited, but gold widely used for the SPR sensor can be used.

The light transmitting medium is a light transmitting medium having a refractive index suitable for measuring the measurement sample, and the refractive index required for the light transmitting medium differs depending on the object to be measured. For this reason, when performing the measurement,
It is preferable to appropriately select a light transmitting medium having a refractive index suitable for measuring a measurement target. As a light transmission medium,
Glass and plastics such as an acrylic plate and a polycarbonate plate can be exemplified, and glass is more preferable.

[0015] The accommodation section accommodates the measurement sample in contact with the metal film, and is preferably made of a material that is chemically inert to the measurement sample and the solvent. A plurality of through holes are provided in a silicon rubber having an appropriate thickness in the housing portion, and the through holes when the silicon rubber is fixed to the metal film side of the light transmitting medium can be exemplified.

The light source may be a light source capable of irradiating light that generates evanescent light at the interface between the metal film and the measurement sample housed in the housing when irradiated on the metal film. The light emitting diodes used can be used.

The incident angle setting means sets the incident angle of the light so that the light emitted from the light source has various incident angles with respect to the interface between the metal film of the measuring element and the light transmitting medium. For example, between the light source and the interface, located on the optical path of the light emitted from the light source,
A convex lens provided to irradiate one point of the interface while condensing light emitted from a light source can be exemplified.

Further, an appropriate slit is provided in the light source, the light leaking from the slit is condensed, and the metal film is located on the optical path of the light emitted from the light source and faces all of the plurality of housing portions. Can be exemplified as a convex lens set so as to irradiate the portion with a line at the interface.

As another example of the incident angle setting means, a light source having excellent directivity such as laser light is used as a light source, and this light source irradiates one point of the interface and changes the distance from the irradiated part. So that only the irradiation angle can be changed without
Means for moving relatively to the measuring element may be used.

The detection means may be means for detecting the intensity of light having various reflection angles reflected at the interface. A light component having an incident angle at which light reflected at the interface reflects at the interface without being absorbed by SPR;
Since the light component having a specific incident angle absorbed at the interface by the SPR is detected by light and dark, a CCD camera is exemplified as the detection means.

The arithmetic processing unit approximates the measured values of one or more, preferably two or more measurement samples whose concentrations are known, to an appropriate function, and uses the obtained approximate expression to determine the measurement sample whose concentration is unknown. Means for calculating the concentration of the object to be measured, for example, a numerical conversion unit for digitizing the measurement result detected by the detecting means, and a function operation for approximating the numerically measured value to an appropriate function And a density calculator that calculates the density of the unknown density measurement target using the obtained function.

The measurement values used in the calculation are obtained by measuring measurement samples of unknown concentration and of known concentration stored in different storage units without detaching the measuring element. By performing measurement without detaching the measuring element, it is possible to suppress variation in measured values due to detachment of the measuring element.

[0023] The moving means transmits, through the incident angle setting means, light radiated from the side of the measuring element not in contact with the metal film to the metal film facing a certain accommodating portion, to the metal of the measuring element. A means for moving the measuring element so that light is irradiated from the side of the surface not in contact with the film to the metal film facing the other housing portion.

The moving means has the same refractive index as that of the light transmitting medium, and is arranged so as to maintain a relative position with respect to the light source, the incident angle setting means, and the detecting means.
For example, a prism is provided on one surface opposite to the incident angle setting unit / detection unit via a matching oil so that the measuring element is movably mounted in the surface direction.

Further, the measuring element is fixed, and while maintaining the relative positions of the light source, the incident angle setting means, and the detecting means,
The light source / incident angle setting means / detection means may be movable in the plane direction of the measuring element, and these exemplified means may be used alone or in combination with a plurality of means. Is also good.

The concentration measuring device of the present invention is used for detecting, for example, specific binding between an antigen and an antibody in a biopolymer and for detecting a specific substance in an unknown sample by utilizing such a specific binding force. used. In order to use the concentration measuring device of the present invention for these exemplified detections, one of the biopolymers as a ligand is immobilized on the metal film, and the other biopolymer is caused to act on the biopolymer, thereby causing the biomolecule to act. It is good to measure the change in the concentration of the polymer.

[0027]

Embodiments of the present invention will be described with reference to the drawings and tables. In the present embodiment, by measuring the standard sample solution having a known concentration and an unknown sample model whose concentration is known but assumed to be an unknown concentration using a plurality of measurement elements, the present invention An embodiment for evaluating the reproducibility of the measurement result of the concentration measuring device will be described.

FIG. 1 shows a concentration measuring apparatus according to this embodiment.
Is configured as shown in FIG. This concentration measuring device comprises a sensor chip 1 and a light emitting diode 2 as a light source.
A convex lens 3 as a means for setting an incident angle of light emitted from the light emitting diode 2, and a CCD camera 4 as a detecting means
And

The sensor chip 1 has a gold thin film 1 on one surface.
a glass plate 1b having a thin film 1a
It has three storage sections 1d to 1f that are stored so as to be in contact with the storage section. The light emitting diode 2 is a light source which is provided on the other surface side of the glass plate 1b, and is provided so as to irradiate a part of the gold thin film 1a facing the housing from the surface side of the glass plate 1b. The convex lens 3 is located between the sensor chip 1 and the light emitting diode 2 and condenses the light emitted from the light emitting diode 2, and a part of the gold thin film 1a is located at one point of the interface between the gold thin film 1a and the glass plate 1b. It is installed to irradiate light. The CCD camera 4 is a glass plate 1
b, at a predetermined angle with respect to the optical axis of the optical system of the light emitting diode 2 and the convex lens 3 at the other surface side and at a position where the intensity of light reflected at the interface is detected at various reflection angles. Have been.

The CCD camera 4 is connected to a numerical value converter 5 for converting an image taken by the CCD camera 4 into a numerical value. The numerical value converter 5 is a function operation unit for calculating the converted numerical value into an appropriate function. 6 and a function operation unit 6
Is a concentration calculator 7 for calculating the concentration of the unknown sample model by substituting the value of the unknown concentration sample into the obtained function.
Is connected to

The sensor chip 1 is mounted on one surface of a prism 8 having the same refractive index as the glass plate 1b via a matching oil, and the sensor chip 1 is slidable in the direction of the mounting surface with the prism 8. is there. The light emitting diode 2, the convex lens 3, the CCD camera 4, and the prism 8 are fixed, and are arranged so that their positional relationship does not change.

Next, the sensor chip 1 used in this embodiment will be described with reference to FIG. The sensor chip 1 includes a glass plate 1b having a gold thin film 1a formed on one surface by vapor deposition, and a holder 1c which is a silicon rubber sheet provided with three through holes. The size of the glass plate 1b is 12 mm in length and 20 mm in width. The holder 1c has a size of 12 mm in length, 20 mm in width, and 5 mm in thickness, and is provided with three through holes of 5 mm in diameter centered on the center line in the horizontal direction. The sensor chip 1 is formed by fixing the holder 1c to the gold thin film 1a side of the glass plate 1b. When the glass plate 1b and the holder 1c are fixed, the three through holes provided in the holder 1c are accommodated. Parts 1d to 1f are formed.

Next, a method of measuring the concentration of a measurement sample using the concentration measuring apparatus of the present embodiment will be described by taking the measurement of the ethanol concentration in an aqueous ethanol solution as an example.
First, the preparation of the standard sample solution will be described. A 10% and 30% aqueous ethanol solution is prepared as a standard sample solution. A 10% ethanol aqueous solution was prepared by accurately weighing 10 g of ethanol in a 100 ml volumetric flask and then adjusting the volume with distilled water.

A 30% ethanol aqueous solution as another standard sample solution was prepared in the same manner as the 10% ethanol aqueous solution. Next, a 20% aqueous ethanol solution was prepared as a measurement sample having an unknown concentration in the same manner as the standard sample solution, and used as an unknown sample model. The standard sample solution is preferably prepared by adjusting two or more standard sample solutions having different concentrations. The standard sample solution has a concentration close to that of the unknown sample model, and the standard sample solution has an unknown sample model concentration. Is more preferably a value sandwiching the concentration of

After adjusting the standard sample solution and the unknown sample model, the 10% ethanol solution is stored in the storage unit 1d, the 30% ethanol aqueous solution is stored in the storage unit 1f, and the 20% ethanol aqueous solution as the unknown sample model is stored in the storage unit 1e. It is housed and light is emitted from the light emitting diode 2.

First, the sensor chip 1 on the prism 8 is slid so that the light emitted from the light emitting diode 2 illuminates one point of the interface between the thin gold film 1a and the glass plate 1b facing the housing portion 1d. The concentration of a 10% aqueous ethanol solution as a sample solution is measured.

Since the light emitted from the light emitting diode 2 has weak directivity, it is condensed by the convex lens 3 to irradiate one point of the interface. The convex lens 3 condenses the light and sets the incident angle of the light emitted from the light emitting diode 2 so that the converged light is applied to the interface at various incident angles.

The light condensed by the convex lens 3 irradiates the gold thin film 1a at the interface, and generates a surface plasmon wave on the surface of the gold thin film 1a facing the housing 1d side. At the same time, evanescent light is generated from the surface of the gold thin film 1a irradiated with light toward the housing 1d from the surface facing the housing 1d,
When surface plasmon waves are excited by the evanescent light, surface plasmon resonance (SPR) occurs.

Of the irradiated light, the light energy of the light component having a certain incident angle is absorbed by the SPR, but the light components having the other incident angles are reflected by the gold thin film 1a. When the intensity of the reflected light is detected for each angle of the reflected light, it can be detected by the brightness as shown in FIG. The dark part in FIG. 3 represents a light component having a specific incident angle absorbed by the SPR.

The CCD camera 4 has a compound eye structure in which a plurality of cameras are gathered, and forms one image as an aggregate of the images captured by the plurality of cameras. Further, the CCD camera 4 can determine the density (brightness and darkness) of each image captured by a plurality of cameras in 256 steps. Therefore, by assigning a pixel number to the camera that captures each image in the vertical direction or the horizontal direction of the image that is the aggregate, the light component (dark portion) absorbed by the SPR can be converted into a numerical value (pixel number).

Using the structure of the CCD camera 4, the numerical value conversion unit 5 converts the dark part, which is a light component having a specific incident angle, absorbed by the SPR into a numerical value. The S in the 10% aqueous ethanol solution thus housed in the housing part 1d
The PR absorption light component is measured.

After the measurement of the 10% ethanol aqueous solution is completed, the sensor chip 1 is slid so that light is irradiated to the portion of the gold thin film 1a facing the housing 1f, and the other standard sample solution of 30% ethanol The aqueous solution is measured in the same manner, and then the sensor chip 1 is slid so that light is irradiated to the portion of the gold thin film 1a facing the accommodation section 1e, and the unknown sample model 20% ethanol aqueous solution is similarly measured. After the measurement of the standard sample solution and the unknown sample model by one sensor chip is completed, the measurement is repeated by changing the sensor chip to obtain the measured values of the standard sample solution and the unknown sample model using five different sensor chips.

After measuring the standard sample solution and the unknown sample model, the measurement result of the standard sample solution is converted into a function, which is an appropriate approximate expression, by the function calculator 6. The function operation unit 6 sets n standard sample solutions as a ₁ , a ₂ ,..., _An, and calculates a measured value (pixel number) obtained by measuring the n standard sample solutions as Ra ₁ ( 1), Ra ₂ (1),..., Ra _n (1), and the measured values (Ra ₁ (1),
Ra ₂ (1),..., Ra _n (1)) approximate to an appropriate function,
An approximate expression F (1) is obtained. The numbers in parentheses indicate the identity of the sensor chip used for the measurement. When the numbers in parentheses are the same, the measured values (Ra ₁ (1), Ra ₂ (1),
, Ra _n (1)) and the approximate expression (F (1)) indicate that the same sensor chip was used, and that the sensor chip was not attached or detached and was measured under the same measurement conditions. .

Here, the measured value of the standard sample solution measured under m different conditions of the sensor chip by attaching / detaching the m sensor chips or the sensor chip m times is as follows: And each of these measurements (1 to
m) is approximated to an appropriate function to obtain approximate expressions F (1), F (2),..., F (m). FIG. 4 and Table 1 show approximate expressions of the standard sample solutions (10% aqueous ethanol solution and 30% aqueous ethanol solution) obtained by the function operation unit 6 in this manner.

[0045]

[Table 1]

The concentration calculator 7 calculates the concentration of the unknown sample model by substituting and calculating the measured value (pixel number) of the unknown sample model into the approximate expression of the standard sample solution.

That is, 1 unknown sample solution measured in the same manner as the standard sample solution under m different conditions is: , And each of the obtained measured values of the unknown sample solution is represented by the approximate expression (F (1), F (2),.
The concentration of the unknown sample solution is calculated by substituting and calculating (m)).

In the present embodiment, the unknown sample solution is used as an unknown sample model (20% ethanol aqueous solution). Since the concentration of this unknown sample model is known, the unknown sample model calculated from each of the five approximate equations is used. By performing statistical processing on the concentration, it is possible to evaluate the dispersion of the measurement in the concentration measuring device of the present embodiment. The relative standard deviation of the concentration of the unknown sample model calculated by the concentration calculator 7 was found to be 8%.

Here, as a comparative experiment with the present embodiment,
A description will be given of a case where an average value of five measured values of the standard sample solution (10% aqueous ethanol solution and 30% aqueous ethanol solution) obtained in the present embodiment is taken and an appropriate function is approximated by the average value. FIG. 5 shows the standard sample solution and the unknown sample model (20%) measured in the first embodiment.
2 shows a plot of aqueous ethanol solution).

The five measured values in the standard sample solution and the unknown sample model have a variation of about 30 in the measured values,
It can be seen that the degree of variation is also different. The straight line in FIG.
The average value of the five measured values of the 10% aqueous ethanol solution as the standard sample solution is calculated, and the other standard sample solution, 3
Take the average of 5 measurements of 0% ethanol aqueous solution,
The straight line equation is y = 3.421x + 95.867. As a result of statistically processing the concentrations of the unknown sample model obtained by substituting and calculating the measured values of the five unknown sample models into this linear equation and calculating the relative standard deviation, the relative standard deviation was 14
%Met.

From the above results, it can be seen that the measurement results of the unknown sample model using the concentration measuring apparatus of the present invention have less variation than the measurement results of the unknown sample model obtained in the comparative experiment. Was completed.

[0052]

According to the concentration measuring apparatus of the present invention, a plurality of housings are provided in a measuring element, and a standard sample solution having a known concentration and an unknown concentration are stored in the plurality of housings without attaching / detaching the measuring element. By accommodating and measuring the unknown sample solution, the concentration of the unknown sample solution can be relatively determined for each measurement, and a reproducible measurement can be made from the measurement values that vary due to the crimped state of the measuring element. It is a concentration measuring device that can obtain results. With this concentration measuring device, it has become possible to make a more accurate comparison between the measured values before and after the attachment / detachment of the measuring element or between the measured values measured with different measuring elements, which has been difficult to compare conventionally.

Further, in the concentration measuring apparatus of the present invention, one biopolymer as a ligand is immobilized on the metal film of the measuring element, and the other biopolymer is caused to act on this biopolymer.
By measuring the change in the concentration of the biological macromolecule to be acted on, for example, detection of specific binding found between an antigen and an antibody, and detection of a specific substance in an unknown sample using such specific binding force Can be used.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing a concentration measuring device according to an embodiment of the present invention.

FIG. 2 is a perspective view of a sensor chip according to one embodiment of the present invention.

FIG. 3 is a diagram illustrating an example of a detection result detected by a concentration measuring device according to an embodiment of the present invention.

FIG. 4 is a diagram showing measured values and functions obtained according to an embodiment of the present invention.

FIG. 5 is a diagram showing a function obtained according to another embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Sensor chip 1a Gold thin film 1b Glass plate 1c Holder 1d-1f Housing part 2 Light emitting diode 3 Convex lens 4 CCD camera 5 Numerical conversion part 6 Function calculation part 7 Concentration calculation part 8 Prism

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yutaka Sasaki 1000, Kamoshita-cho, Aoba-ku, Yokohama-shi, Kanagawa Prefecture Inside the Yokohama Research Laboratory, Ryobi Chemical Co., Ltd. (72) Inventor Kazuhiko Watanabe 1000, Kamoshita-cho, Aoba-ku, Yokohama-shi, Kanagawa (72) Inventor: Yasuo Koike 1000, Kamoshida-cho, Aoba-ku, Yokohama-shi, Kanagawa Prefecture F-term (reference) 2G059 AA01 AA05 BB04 CC17 DD03 DD13 DD20 EE01 EE02 FF03 GG01 GG02 JJ11 JJ12 JJ21 KK04 MM01

Claims (4)

[Claims] 1. A metal film is provided on one surface, a light transmitting medium having a desired refractive index is provided, and a housing for housing a measurement sample in contact with the metal film surface is provided on the metal film side. A measuring element, a light source that irradiates light incident on a surface of the measuring element that is not in contact with the metal film, and a light transmitting medium and the metal film that irradiate light irradiated from the light source. Incident angle setting means for causing various angles of incidence to be obtained with respect to the interface with the detection means; detection means capable of detecting the intensity of light reflected at the interface at various reflection angles; and An arithmetic processing unit for calculating the concentration of the unknown concentration sample from the measured values of the unknown concentration and known concentration measurement samples respectively housed in different storage units, which are measured without detaching the measuring element. Concentration measuring device. 2. The concentration measuring device according to claim 1, wherein the measuring element has a moving unit that can move relatively to the light source, the incident angle setting unit, and the detecting unit. Concentration measuring device. 3. The concentration measuring device according to claim 1, wherein the concentration of the polymer is measured. 4. The concentration measuring device according to claim 3, wherein the polymer is a biopolymer.