JPH10274654A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biosensor that can be manufactured and handled easily and can provide reliable measurement data when it is used for the quantitative analysis of an antibody or an antigen. SOLUTION: A sensor consists of a piezoelectric vibrator (a crystal vibrator), an electrode (a gold electrode) 2 that is formed on the surface of the piezoelectric vibrator and a, functional film that is formed oh the surface of the electrode 2 and is connected to an antigen or an antibody a-IgA. In this case, the electrode 2 is formed by at least one kind of metal being selected from platinum, gold, and silver and the antigen or the antibody a-IgA is connected to the functional film by a covalent bonding via an organic compound and is formed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a piezoelectric vibrator having an antigen or antibody immobilized on a surface thereof, and for example, to be effectively used in a method for quantifying a specific antibody or antigen present in an aqueous phase system. The present invention relates to a biosensor that can be used.

[0002]

2. Description of the Related Art Conventionally, quantitative analysis of antibodies or antigens in an aqueous phase system, for example, in saliva, has been performed, for example, by radioimmunoassay (RIA) using a radioactive element, enzyme immunoassay (EIA) using an enzymatic reaction, and fluorescence. It is performed by a method such as a microcapsule immunoassay (MCIA) using a microcapsule in which a substance is encapsulated (JP-A-3-206962). In addition, as a quantification method (measurement method) of an antibody or an antigen other than the above method, for example, a method of adsorbing and immobilizing an antibody or an antigen on the surface of a quartz oscillator (JP-A-63-11835), and a method of quantifying a specific antigen or A method of measuring an antigen-antibody reaction using a combination of an insoluble carrier carrying an antibody and a quartz oscillator not immobilizing the antibody or antigen (Japanese Patent Application Laid-Open No. 3-77061) is also known.

[0003]

However, the conventional methods for quantifying antibodies (antigens) (measuring methods) have various problems. That is, the RIA requires the installation of expensive radiation-dedicated equipment, and this radiation-dedicated equipment can be handled only by an operator who has a license for radiation. In addition, in the measurement by EIA, there is a problem that it takes a long time to stabilize the enzyme reaction and its optical measuring device. Furthermore, when using microcapsules,
There is a risk that the non-specific reaction other than the antigen-antibody reaction will destroy the microcapsules and impair the reliability of the measurement. On the other hand, in a conventional immunosensor using a quartz oscillator,
As the measurement is repeated, the sensitivity of the antibody immobilized by adsorption decreases, and the measurement is unstable. In addition, the measurement using a quartz oscillator that does not immobilize the antigen or antibody requires a skilled technique and complicated procedures such as preparation of an insoluble carrier particle solution carrying the antigen or antibody and preparation of a calibration curve, and an aqueous phase system. It is not suitable for quantitative analysis of antigens or antibodies in saliva, especially in saliva.

[0004] The present invention has been made to solve the above-mentioned problems of the prior art, and it is an object of the present invention to make it easy to manufacture and handle, and to reduce the reliability of measurement data when used for quantitative analysis of antibodies or antigens. Another object of the present invention is to provide a biosensor having high performance.

[0005]

That is, a biosensor according to the present invention comprises a piezoelectric vibrator, an electrode formed on the surface of the piezoelectric vibrator, and an antigen or an antibody formed on the surface of the electrode and bound to an antigen or an antibody. In a biosensor comprising a functional film, the electrode is made of at least one metal selected from platinum, gold and silver, and the antigen or antibody is attached to the functional film by a covalent bond via an organic compound. It is characterized by being combined. The biosensor of the present invention is oscillated in an aqueous phase system, and an antibody or an antigen present in the aqueous phase system and an antigen or an antibody immobilized on a functional membrane of a piezoelectric vibrator.
By measuring the change in the oscillation frequency of the piezoelectric vibrator due to the antibody reaction, the amount of the antibody or antigen present in the aqueous phase system can be determined.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the biosensor of the present invention,
A biosensor in which the antigen is immunoglobulin A (IgA) and the antibody is anti-immunoglobulin A (a-IgA) is preferred. Further, the biosensor of the present invention is preferably used for quantifying a protein antigen in saliva, wherein the aqueous phase system is saliva.

[0007] Examples of the piezoelectric vibrator that can be used in the biosensor of the present invention include barium titanate, potassium sodium tartrate, and quartz. The type, size and shape of these piezoelectric vibrators are appropriately selected according to the purpose of use and the environment. As the electrode, at least one kind of metal selected from platinum, gold, and silver is used. 2 as electrodes
It is possible to use more than one kind of the above metals, and the type, size and shape of the electrode are appropriately selected. The electrodes may be manufactured by a conventional manufacturing method, for example, a plating method, a vapor deposition method, a coating method, or the like.

[0008] The antigen and antibody used in the biosensor of the present invention may be appropriately selected according to the purpose of use. For example, antigens include immunoglobulins G, A, and M, and antibodies include anti-immunoglobulins G, A, and M.

[0009] The antigen or antibody is covalently attached to the functional membrane using a suitable organic compound. Covalent attachment of the antigen or antibody to the functional membrane and formation of the functional membrane may be performed sequentially or simultaneously. As the organic compound, various compounds can be used alone or in combination depending on the purpose of use.

When quantifying antibodies or antigens present in an aqueous phase system using the biosensor of the present invention, an oscillator is used to oscillate the piezoelectric vibrator at a predetermined frequency.
The change in the oscillation frequency when quantifying the antibody or antigen is measured using an oscillation frequency measuring device. These oscillators and oscillation frequency measuring devices may be appropriately selected.

[0011]

The present invention will be described in more detail with reference to the following examples and comparative examples. In the following examples and comparative examples, in order to show the effects of the present invention with specific examples, a quartz oscillator was used as a piezoelectric oscillator, a gold electrode was used as an electrode, and human saliva was used as a subject. The invention is not particularly limited to these. In the following Examples and Comparative Examples, immunoglobulin A (IgA) was used as an antigen in saliva.
Was used as a measurement target, and anti-immunoglobulin A (a-IgA) was used as a corresponding antibody immobilized on the surface of a quartz oscillator.

I. Example 1: Immobilization of a-IgA on the surface of a quartz oscillator Example 1: Covalent immobilization method The quartz oscillator biosensor of Example 1 shown in FIG. 1 was produced. That is, the gold electrode (φ4.5 mm, 16 mm ² ) 2 of the quartz oscillator 1 subjected to ultrasonic cleaning was applied to a cysteamine ethanol solution (HSCH ₂ CH ₂ NH ₂ 1 mM, 11.3 m).
g / 100 ml, pH 6.4) at room temperature for 1 hour to react. After washing with water, 15 μl of a-IgA sodium acetate solution (1-2 mg / ml) and water-soluble carbodiimide [C ₈ H ₁₈ ClN ₃ : 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide HCl] (5 mg
/ Ml) at 4 ° C for 24 hours. The gold electrodes 2 (two) are connected to an oscillator and an oscillation measuring instrument 3. In FIG. 1, it can be seen that immunoglobulin A (IgA) is selectively captured by anti-immunoglobulin A (a-IgA). Comparative Example 1: Adsorption and immobilization method a-Ig was placed on a gold electrode (φ4.5 mm, 16 mm ² ) of a quartz oscillator that was ultrasonically cleaned with ethanol and acetone.
A sodium acetate solution (1-2 mg / ml)
1 drop and store at 37 ° C. for 24 hours, then 10 mM H
After washing with Cl and adsorbing and fixing a-IgA, a quartz oscillator type biosensor of Comparative Example 1 was produced.

II. Preparation of IgA sample solution and a-IgA
As a sample solution, 0.5-20 μg of human-derived IgA was used as a sample solution.
A PBS solution was prepared at a concentration of / ml. Also, a-I
As a dissociation solution of gA and IgA, a 10 mM hydrochloric acid solution was used.

III. Antigen quantification The antigen was measured in a phosphate buffer using the biosensor of Example 1 and the biosensor of Comparative Example 1. afterwards,
The antibody and the antigen were dissociated with hydrochloric acid. After waiting for the oscillation frequency to stabilize, the antigen in the phosphate buffer was measured again. Such measurements were repeated many times to evaluate the stability and durability of the biosensor.

FIG. 2 shows the relationship between the number of measurements and the rate of change of the measured values. Based on the measured value of the frequency at the start of measurement (100%)
As a result of observing how much the measurement frequency fluctuates as the number of measurements increases, in the biosensor of Comparative Example 1, the measured value greatly fluctuates in several times, and stable measurement cannot be performed. I understood. In contrast, the biosensor of Example 1 was found to withstand 15 or more re-measurements. From the above, it was found that the biosensor produced by the covalent immobilization method (method of the example) had much better stability in measurement than the adsorption immobilization method (method of the comparative example).

Example 2 Using the quartz-crystal vibrating biosensor of Example 1 produced by the covalent immobilization method, the frequency change of an IgA phosphate buffer (concentration: 1 μg / ml) was performed using the quartz-crystal oscillator measuring system shown in FIG. Was measured. The frequency change value was 26 Hz. In the quartz oscillator measuring system, the quartz oscillator type biosensor 4 (a-IgA is immobilized) is replaced with a sample solution 5 (IgA
), And the sample solution 5 is stirred by the Stirling magnet 6 and the magnet stirrer 7. An oscillator and a computer 8 are connected to the quartz-crystal vibrating biosensor 4. The discharge of the sample solution 5 is performed by the bio-micro pump 9. After the measurement was completed, the sample solution 5 was discharged by the biomicro pump 9, and hydrochloric acid solution was added to dissociate a-IgA and IgA immobilized on the surface of the quartz vibrating biosensor 4. Next, the hydrochloric acid solution was discharged by the biomicropump 9, and this time, a phosphate buffer solution (S-IgA) obtained by diluting human saliva by a factor of 100 was added as the sample solution 5 and measured. The frequency change value is 29
Hz. The sample solution 5 is discharged again, and a hydrochloric acid solution is added to the sample solution a fixed to the surface of the quartz vibrating biosensor 4.
-IgA and S-IgA were dissociated. Then, a mixed solution of the IgA phosphate buffer solution (concentration: 1 μg / ml) and the phosphate buffer solution (S-IgA) obtained by diluting the saliva by a factor of 100 was added as a sample solution, and the frequency change was observed.
As a result, the frequency change value was 55 Hz. this is,
It is equal to the sum of the change width due to IgA (26 Hz) and the change width due to S-IgA (29 Hz). As described above, the antigen (S-IgA: about 11) in human saliva was actually measured using the quartz oscillation type biosensor prepared by the covalent immobilization method.
0 μg / ml) with good reproducibility.

[0017]

The biosensor of the present invention is easy to manufacture and handle, and has high reliability (accuracy and reproducibility) of measurement data when used for quantitative analysis of antibodies or antigens. Therefore, the antigen or antibody in the aqueous phase system, particularly in saliva, can be simply, quickly and accurately determined using the biosensor of the present invention.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram illustrating a schematic configuration of a quartz oscillator type biosensor according to a first embodiment of the present invention.

FIG. 2 is a diagram showing the relationship between the number of measurements and the rate of change in measured values of the biosensor of Example 1 and the biosensor of Comparative Example 1.

FIG. 3 is an explanatory diagram illustrating a schematic configuration of a measurement system using the quartz oscillator type biosensor according to the first embodiment of the present invention.

[Description of Signs] 1: Quartz crystal oscillator 2: Gold electrode 3: Oscillator and oscillation measuring instrument 4: Quartz crystal vibration type biosensor 5: Sample solution 6: Stirling magnet 7: Magnet stirrer 8: Oscillator and computer 9: Biomicropump

Claims (1)

[Claims] 1. A biosensor comprising a piezoelectric vibrator, an electrode formed on a surface of the piezoelectric vibrator, and a functional film formed on a surface of the electrode and having an antigen or an antibody bound thereto, wherein the electrode Is a biosensor comprising at least one metal selected from platinum, gold and silver, wherein the antigen or antibody is bound to the functional film by a covalent bond via an organic compound.