JP2597073B2 - Biological substance sensor, biological substance detection device, and method of quantifying biological substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a quantitative analysis of a biological substance (antigen) using a specific binding reaction (antigen-antibody reaction) between a test substance and the biological substance.

[0002]

2. Description of the Related Art There are several methods for immobilizing a biological substance in an apparatus in which a substance in which a biological substance is immobilized is provided on an end face of an optical fiber and an antigen-antibody reaction of the biological substance is used to perform quantitative analysis of an antigen. There are advantages and disadvantages as described below.

[0003] First, a physical adsorption method is used. This is a method of immobilizing biological substances by physically adsorbing them on a water-insoluble carrier. The immobilization conditions are relatively relaxed, and have the advantage that the destruction of active centers or changes in high-dimensional structures is small. It is. However, the physical adsorption method has disadvantages in that the amount of binding is low and the interaction with the biological material is weak, so that the biological material is easily detached from the carrier. Therefore, a method called a crosslinking method is considered as an immobilization method that does not have such a defect. this is,
In this method, a biological substance is immobilized by a chemical bonding method, but the immobilization is performed without using a carrier. That is, this is a method of immobilizing biological materials by crosslinking the biological materials with each other using a reagent having a plurality of functional groups. However, the cross-linking method also has serious drawbacks in that the substance to be immobilized is deactivated during the binding process and the binding activity after the binding is reduced.

[0004] As a method which does not have any disadvantages, there is a method called the lattice method of the inclusive method (for example, JP-A-61-272,661). This is a method in which a biological substance is wrapped in a lattice of a polymer compound gel and immobilized in a state where it cannot be detached. According to this method, the biological substance can be relatively easily immobilized, and the stability and binding ability of the immobilized substance are well maintained.

[0005]

However, in the comprehensive law,
Since the diffusion phenomenon is used to introduce the substance into the gel, there is a problem that the introduction of the substance is slow and it takes time to quantify the antigen.

Accordingly, the present invention provides an optical detection apparatus utilizing an inclusive method for immobilizing a biological substance, in which a substance is introduced into a gel of a high-molecular compound quickly and an antigen can be quantified in a short time. The purpose is to do.

[0007]

Means for Solving the Problems In order to solve the above problems,
The biological material sensor of the present invention includes a transparent electrode, a thin film formed of a gel-like polymer compound in which a biological material specifically formed on the surface of the transparent electrode and immobilized to a test substance is fixed, and a transparent electrode. An optical transmission member having an end face adhered to the opposite surface on which the thin film is formed.

Further, in order to solve the above-mentioned problems, a biological substance detecting apparatus according to the present invention is characterized in that a thin film made of a gel-like high molecular compound on which a biological substance that specifically binds to a test substance is immobilized has a light transmission property. A biological material sensor formed on a surface of a first electrode formed on an end surface of the member, a container for storing a solution containing a test substance, and a second electrode provided in the container and immersed in the solution And power supply means for applying a polarity reversible DC bias between the first and second electrodes.

In order to solve the above problems, a method for quantifying a biological substance according to the present invention comprises the steps of: forming an electrode having a thin film made of a gel-like polymer compound on which a biological substance is immobilized; A first step of immersing the sample in a mixed solution containing a known substance and a fluorescent labeling substance having the same polarity as the test substance, and applying a voltage having a polarity opposite to that of the test substance and the fluorescent labeling substance to the electrode. A second step of applying a voltage having a polarity opposite to that of the electrode to the mixed solution and allowing the mixture to stand for a predetermined time; a third step of applying a voltage having the same polarity as the test substance and the fluorescent labeling substance to the electrode; And irradiating the thin film formed on the surface of the electrode with excitation light to measure the amount of fluorescence generated by the fluorescent labeling substance.

[0010]

According to the above arrangement, on the surface of the transparent electrode,
A thin film made of a gel-like polymer compound on which a biological material is immobilized is formed, and an optical transmission member is provided on a surface opposite to the surface on which the thin film is formed. Therefore, if the thin film is immersed in a solution containing the test substance and a potential opposite to that of the test substance is applied to the electrode, the test substance can be electrophoresed in the thin film. Can be performed in a short time. Further, it is possible to irradiate the test substance with the excitation light through the light transmission member, and to measure the fluorescence through the light transmission member.

[0011]

Further, according to the above configuration, the biological material sensor is provided with the first electrode, the container is provided with the second electrode, and the potential difference between the two electrodes in the solution containing the test substance. Can be caused.

According to the above method, first, a voltage having a polarity opposite to that of the test substance and the fluorescent label substance is applied to the electrode.
The test substance and the fluorescent labeling substance can be electrophoretically incorporated into the gel-like polymer compound on which the biological substance is immobilized. Next, since a voltage having the same polarity as the test substance and the fluorescent labeling substance is applied to the electrode, the test substance and the fluorescent labeling substance that did not cause a binding reaction can be quickly discharged out of the gel polymer compound. it can. Furthermore, when the thin film is irradiated with excitation light, fluorescence is generated by the fluorescent labeling substance. If the amount of this fluorescence is measured, the concentration of the test substance is determined from the relationship between the quantitative ratio of the test substance and the fluorescent labeling substance. Can be measured.

[0014] Further, when the test substance is electrophoresed in this manner, a trace amount of the test substance can be concentrated in the gel-like polymer compound, so that the concentration is lower than the detection limit concentration of the conventional competitive method. It is possible to detect.

[0015]

Embodiments of the present invention will be described below with reference to the accompanying drawings. In the description of the drawings, the same elements will be denoted by the same reference symbols, without redundant description.

An optical detection device according to an embodiment of the present invention will be described with reference to FIGS. The optical detection device according to the present embodiment includes an electrolytic solution tank 1 for storing an electrolytic solution 4 containing a test substance, and an optical fiber cable 2 having a sensor section on one end surface. The opposite side of the optical fiber cable 2 from the sensor section is divided into two parts. A light emitter 5 for generating excitation light is provided on one of the divided end faces, and a short wavelength excitation light is provided on the other divided end face. Fluorescence intensity measuring device 7 via cut filter 6 for cutting light
Is provided. The sensor section includes a transparent electrode (ITO) 21 coated on the end face of the optical fiber 2 and a thin film 2 of a gel carrier coated on the transparent electrode 21.
And 2. The other electrode 3 forming a pair with the transparent electrode 21 is formed at the bottom of the electrolytic solution tank 1.

The gel carrier thin film 22 is formed of polyacrylamide incorporating anti-insulin antibody β.
That is, the thin film 22 of the gel-like carrier is formed by wrapping the anti-insulin antibody β in the lattice of the polyacrylamide gel so that it cannot be detached. The size of the lattice of the polyacrylamide gel varies depending on the concentration of the monomer and the crosslinking agent used in the polymerization reaction, but is about 10 to 40 Å on average. In addition, instead of polyacrylamide as the polymer used for the sensor part, using polyvinyl alcohol, a copolymer of methacrylic acid and 2-hydroxymethacrylic acid, a photocrosslinkable resin, a urethane polymer, pectin, starch, konjac powder, etc. Is also good.

Next, a method of quantifying an antigen using the biological substance detecting device according to the present embodiment will be described with reference to the flowchart of FIG.

First, as shown in FIG. 4, an electrolytic solution 4 containing an unknown concentration of insulin α1 (for example, contained in serum), which is a test substance, and an insulin having a known concentration. A predetermined ratio of an electrolyte solution 4 containing an insulin-FITC complex α2, which is a fluorescently labeled antigen to which fluorescein isothiocyanate (hereinafter referred to as FITC) bound to a fluorescent substance, is prepared to form a mixed solution;
This mixed solution is injected into the electrolytic solution tank 1 (step 30).
1).

Next, as shown in FIG. 5, the optical fiber 2 is immersed in the mixed solution, and the sensor section of the optical fiber 2 is completely immersed in the electrolytic solution 4 (Step 302).

A positive voltage is applied to the transparent electrode 21 of the sensor section, and a negative voltage is applied to the electrode 3 at the bottom of the electrolytic solution tank 1 (step 303). As a result, a potential difference occurs in the electrolytic solution tank 1, so that the positively charged insulin α1 and the insulin-FITC complex α2 (hereinafter, referred to as “insulin-FITC complex α2”)
Insulin or the like) is electrophoresed in the thin film 22 of the gel carrier in the sensor section.

As shown in FIG. 6, the gel carrier thin film 22
Insulin and the like incorporated therein cause an antigen-antibody reaction competitively with the anti-insulin antibody β immobilized in the lattice of the gel and bind to each at a fixed ratio. Here, the amount of the anti-insulin antibody β to be bound is limited, and the amount of insulin or the like is larger than the amount of the anti-insulin antibody β to be bound, so that a part of the insulin or the like binds to the anti-insulin antibody β. It remains in the thin film 22 without performing. The quantitative ratio in this state is determined by the quantitative ratio of insulin α1 and insulin-FITC complex α2 and the difference in binding ability.

Here, the voltage applied to the transparent electrode 21 and the electrode 3 is turned off (step 304). Next, as shown in FIG. 7, a negative voltage is applied to the transparent electrode 21 of the sensor section, and a positive voltage is applied to the electrode 3 at the bottom of the electrolytic solution tank 1 (step 305). As a result, a potential difference is generated in the electrolytic solution tank 1 in a direction opposite to that in the previous case, and the insulin and the like remaining in the thin film 22 without binding to the anti-insulin antibody β are removed by the thin film of the gel carrier in the sensor section. 22 is moved outside.

After the voltage applied to the transparent electrode 21 and the electrode 3 is cut off, as shown in FIG. Irradiate excitation light. This excitation light causes FIT
C is excited to emit long-wavelength fluorescence, and the fluorescence intensity measuring device 7
Quantifies the amount of FITC fluorescence (step 30).
6). At this time, a part of the excitation light may be reflected by the transparent electrode 21 or the thin film 22. Is not detected.

In general, the insulin-FITC complex α2
The amount of insulin α1 that reacts with anti-insulin antibody β is determined in proportion to the magnitude of the amount ratio between insulin and α1. That is, if the amount of insulin α1 is increased, insulin-binding to anti-insulin antibody β
The amount of FITC complex α2 is reduced, and as a result, the fluorescence intensity is also reduced. Therefore, the horizontal axis represents the amount of insulin α1,
Insulin-FIT bound to anti-insulin antibody β
If the amount of fluorescence generated by the C complex α2 is plotted on the vertical axis, the calibration curve becomes as shown in FIG. From such a relationship, by measuring the amount of fluorescence, the concentration of insulin α1 can be measured.

That is, in this embodiment, since the electrode 3 is provided in the electrolytic solution tank 1 and the transparent electrode 21 is provided in the sensor section, a potential difference is generated between the two electrodes in a solution containing insulin or the like. be able to. Therefore, since insulin or the like is electrophoresed and taken into the gel-like polyacrylamide on which the anti-insulin antibody β is immobilized, the antigen-antibody reaction between insulin or the like and the anti-insulin antibody β can be performed quickly. On the other hand, for the same reason, it becomes possible to rapidly discharge insulin and the like which have not caused an antigen-antibody reaction out of the gel-like polyacrylamide. This makes it possible to quickly introduce insulin into the gelled polyacrylamide in question and to perform high-level quantification. As a result, application in a wide range of fields such as clinical tests, food analysis, and water quality analysis becomes possible.

The quantification method used in the present example is generally called a competitive method. One of the drawbacks of such a competitive method is that it is not possible to perform an extremely small amount of quantification. This is because the ratio of the fluorescent substance to be detected is at most 5% in consideration of the actual data variation. The reason is that the sensitivity of the competition method depends on the affinity constant of the antigen-antibody reaction,
This is because the affinity constant is at most about 10 ⁹ M ⁻¹ .

Therefore, as for the detection sensitivity, the antigen concentration is p
M units were the limit. However, in this embodiment,
Although the amount of insulin and the like increases, a trace amount of insulin and the like can be concentrated in the gel, so that it is possible to detect the concentration at a concentration lower than the detection limit concentration of the conventional competitive method.

In this embodiment, the case where insulin, which is a kind of protein, is detected has been described. However, the apparatus according to the present invention is used to detect DNA having a specific sequence, enzymes having a specific structure, and the like. It is also possible.
In this case, an anti-DNA antibody, an anti-enzyme antibody, or the like is used as the biological substance.

[0030]

As described above, the present invention provides:
On the surface of the electrode, a thin film made of a gel-like polymer compound on which a biological substance is immobilized is formed. Therefore, if the thin film is immersed in a solution containing the test substance and a potential opposite to that of the test substance is applied to the electrode, the test substance can be electrophoresed in the thin film. Can be performed in a short time.

That is, since the biological material sensor is provided with the first electrode and the container is provided with the second electrode, a potential difference can be generated between the two electrodes with the solution containing the test substance. Therefore, if a voltage having the same polarity as the test substance is applied to the electrode, the test substance can be electrophoresed into the gel-like polymer compound on which the biological substance is immobilized. It becomes possible to quickly perform a binding reaction with a biological substance. On the other hand, for the same reason, if a voltage having the same polarity as the test substance is applied to the electrode, the test substance that has not caused a binding reaction can be quickly discharged out of the gel polymer compound.

In addition, when the test substance is electrophoresed as described above, a trace amount of the test substance can be concentrated in the gel-like polymer compound, so that the concentration is lower than the detection limit concentration of the conventional competitive method. It is possible to detect.

Furthermore, if the electrode is a transparent electrode and an optical transmission member is provided on the surface opposite to the surface on which the thin film is formed, the test substance can be irradiated with excitation light via the optical transmission member. Also, the amount of fluorescence can be measured via the light transmission member. By measuring the amount of fluorescence, the concentration of the test substance can be measured from the relationship between the quantitative ratio of the test substance and the fluorescent labeling substance.

As a result, it becomes possible to quickly introduce the substance into the gelled polymer compound in question, and it is possible to perform a high-level quantification.
As a result, application in a wide range of fields such as clinical tests, food analysis, and water quality analysis becomes possible.

[Brief description of the drawings]

FIG. 1 is a perspective view of an apparatus according to an embodiment.

FIG. 2 is a conceptual diagram of an apparatus according to the present embodiment.

FIG. 3 is a flowchart of a method for quantifying an antigen using the device according to the present embodiment.

FIG. 4 is an explanatory diagram of a use state of the device according to the present embodiment.

FIG. 5 is an explanatory diagram of a use state of the device according to the present embodiment.

FIG. 6 is an explanatory diagram of a use state of the device according to the present embodiment.

FIG. 7 is an explanatory diagram of a use state of the device according to the present embodiment.

FIG. 8 is an explanatory diagram of a use state of the device according to the present embodiment.

FIG. 9 is a calibration diagram showing the relationship between the fluorescence intensity and the light source concentration.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Electrolyte tank, 2 ... Optical fiber, 3 ... Electrode, 21 ... Transparent electrode, 22 ... Gel thin film, α1 ... Insulin, α2
... insulin-FITC complex, β ... anti-insulin antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location G01N 33/545 G01N 33/548 Z 33/548 27/26 301Z

Claims (3)

(57) [Claims] And 1. A transparent electrode is formed on a surface of the transparent electrode, a thin film biological substance that specifically binds to the test substance consisting of immobilized gel polymer compound, wherein the transparent electrode A biological material sensor, comprising: a light transmission member having an end face in close contact with the opposite surface on which the thin film is formed. 2. A thin film made of a gel-like polymer compound on which a biological substance that specifically binds to a test substance is immobilized is formed on a surface of a first electrode formed on an end face of an optical transmission member. A biological material sensor, a container for storing a solution containing the test substance, a second electrode provided in the container and immersed in the solution, and a polarity between the first and second electrodes. And a power supply means for applying a DC bias that can be switched. 3. An electrode, on which a thin film made of a gel-like polymer compound on which a biological substance is immobilized, is formed, on a test substance having an unknown concentration and a test substance whose concentration is known and the same as the test substance. First immersion in a mixed solution containing a polar fluorescent labeling substance
A step of applying a voltage having a polarity opposite to that of the test substance and the fluorescent labeling substance to the electrode, applying a voltage having a polarity opposite to that of the electrode to the mixture, and allowing the mixture to stand for a predetermined time; A third step of applying a voltage of the same polarity as the test substance and the fluorescent labeling substance to an electrode, removing the electrode from the mixed solution, irradiating a thin film formed on the surface of the electrode with excitation light, And a fourth step of measuring the amount of fluorescence generated by the fluorescent labeling substance.