JP2540689B2 - Biological capacitance sensor - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a biological capacitance sensor for measuring high molecular weight analytes such as antigens, antibodies and enzyme inhibitors. In particular, the present invention has been specially devised so that it is possible to measure a physiologically active substance by detecting a change in dielectric property caused by a biological specific binding between an analyte and a biochemical binding system. The present invention relates to a biological capacitance sensor having a structure. The biochemical binding system is selected so that it has a specific affinity for the specific analyte to be measured.

[0002]

2. Description of the Related Art In order to measure high molecular weight analytes such as antigens, antibodies and oxygen inhibitors, using a binding substance having a specific affinity for the analytes. Conventionally, various techniques have been tried.
Immunoassays are used to identify analytes such as haptens, antigens and antibodies in liquid media. These immunoassays are based on the biological specific binding between reaction pairs such as antigens and antibodies. In many cases, a very small amount of substance is measured, and a radioisotope is used as a labeling reagent. However, it has various problems such as safety, and new immunoassays that do not use radioactive substances are being actively developed. For example, a labeled immunoassay method using an enzyme, a fluorescent agent, or the like as a labeling agent has been put to practical use.

However, it is difficult to say that any of the methods is simple and quick. Therefore, an immunosensor was devised for the purpose of performing a quick and easy immunoassay. These sensors generally assay for changes in physical and electrical or optical properties when one of the binding pairs binds to the partner pair with biological specificity. Various types of immunosensors have been prepared using only immunosensors for assaying changes in electrical properties.

Naoyama Yamamoto et al. Utilize the phenomenon that the electrode potential changes by immobilizing an antibody or an antigen on the surface of an electrode and binding the antigen or the antibody to this to form an antigen-antibody complex. Antigen or antibody detected (C
lin. Chem. 26, 1569 (1980)).

[0005] In Taniguchi, et al., A polytyramine-modified electrode on which an antibody (antigen) is immobilized is dipped in a phosphate buffer solution in which the antigen (antibody) and an enzyme-labeled antigen (enzyme-labeled antibody) are dissolved, and after a certain time, an enzyme substrate is added. In addition, the electrode current based on the enzyme reaction was measured to detect the antigen (antibody) (JP-A-2-179).
461 publication).

Yoshihiro Kumagai et al. Detected an antigen by immobilizing an antibody on the gate electrode of a field effect transistor, reacting the antigen with a second charged antibody, and measuring the potential change of the gate electrode (JP-A-2-129542). Issue). Although the above-mentioned immunosensor uses direct current measurement, an immunosensor using alternating current measurement has also been created.

[0007] Iver-Gia-Ever et al. Produce an insoluble reaction by adding an enzyme-labeled antigen or enzyme-labeled antibody and a suitable enzyme substrate on an electrode having a binding site specific to the antigen or antibody in the sample solution. Gave birth. The presence of the antigen or the antibody was quantitatively detected by measuring the impedance change caused thereby (JP-A-2-24548).

Arnold El-Newman has created a capacitance sensor that utilizes an open capacitor that produces one high electric field in the capacitance measurement region that contacts the biological coupling layer. The electrodes of this open-type capacitor are coated with an electrically insulating layer, and the bio-binding layer is fixed on the electrically insulating layer. The biospecific binding reaction pulls in or releases biological molecules from the binding layer and replaces the molecules in the fluid medium with different dielectric constants, resulting in a change in the capacitance of the sensor. No. 501446).

[0009] Pierre Batairard et al., The electrode which has immobilized the antibody on the SiO ₂ oxide film on the surface of the silicon substrate,
The antigen was detected by utilizing the phenomenon that the capacitance of the electrode changes when the antigen reacts with the antigen to form an antigen-antibody complex (Anal. Chem., 60, 2374 (198).
8)).

It is an object of the present invention to provide a biological capacitance sensor having a simple structure for measuring high molecular weight analytes such as antigens, antibodies and enzyme inhibitors.

[0011]

The present invention provides an antigen, an antibody,
The present invention relates to a biological capacitance sensor using a titanium substrate for measuring high molecular weight analytes such as enzyme inhibitors. In the present invention, a TiO ₂ oxide film is formed on the surface of a titanium substrate, and a detection electrode in which an organic compound capable of biologically specific binding to an analyte is fixed on the oxide film is used. When the detection electrode is immersed in a liquid containing the analyte, the analyte will bond with the immobilized organic compound. When measuring the capacitance of the detection electrode with a device that uses a potentiostat, lock-in amplifier, etc., the capacitance of the detection electrode changes before and after binding the analyte, so measure the analyte concentration in the liquid. be able to.

The present invention uses the following compounds as analytes and organic compounds capable of biologically specific binding thereto. When the analyte is an antigen, an antibody against it as an organic compound, when the analyte is an antibody, the antigen against it as an organic compound, when the analyte is an enzyme inhibitor, an enzyme against it as an organic compound, When the analyte is a polysaccharide or glycoprotein, the lectin corresponding to it is used as the organic compound.

The detection electrode of the present invention can be produced with the following simple structure. The surface of the titanium substrate is heated in an electric furnace,
A TiO ₂ oxide film is formed on the surface. The TiO ₂ oxide film is fixed to the hole part of the perforated plastic container with an adhesive so that the TiO ₂ oxide film comes into contact with the liquid. Then, an organic compound capable of biologically specific binding to the analyte is fixed on the TiO ₂ oxide film. A conductive metal film is formed on the back surface of the titanium substrate by a method such as vapor deposition,
Electrically connected to an electrical circuit that responds to changes in capacitance.
In this way, a detection electrode having a simple structure obtained by simply adhering the titanium substrate to the plastic container can be obtained.

[0014]

[Function] The biological capacitance sensor is designed to cause a large change in capacitance when an analyte binds to an organic compound capable of biologically specific binding to the analyte fixed to the oxide film of the detection electrode. There must be. Therefore, it is necessary to observe how the electric capacities of different films influence each other. The capacitance of the oxide film (electrical insulating film) and the biological film of the organic compound and the analyte can be regarded as two capacitors in series. Series capacitance C is the capacitance C ₁ of the electrically insulating film is expressed by the following equation by using the capacitance C ₂ of the biological membrane.

C = 1 / (1 / C ₁ + 1 / C ₂ ) What is understood from this equation is that in measuring the total capacitance of two films, a small capacitance has a greater effect than a large capacitance. That's what it means. In capacitance sensors, the desired capacitance change occurs within the biobinding membrane. Therefore, in order to obtain the maximum change in the overall capacitance, it is necessary to maximize the capacitance of the oxide film. Two parameters that change the capacitance of an oxide film are the thickness of the oxide film and its dielectric constant. In order to increase the capacitance of the oxide film, the thickness of the oxide film should be reduced or a material having a high dielectric constant should be selected.

Therefore, in the present invention, there is a limit in obtaining a large electric capacity by reducing the thickness of the oxide film.
An oxide film made of a material that is chemically stable and has a large dielectric constant is used to create a biological capacitance sensor that can obtain a large capacitance change. By using an oxide film made of a material having a large dielectric constant, it is possible to produce a capacitance sensor having a simple structure.

[0017]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a sectional view showing the structure of a detection electrode 18. The detection electrode 18 is created as follows. The surface of the titanium substrate 2 is heated in an electric furnace to form a TiO ₂ oxide film 4. A gold film 6 was added to the back surface of the titanium substrate 2 by a vacuum deposition method. The surface of the titanium substrate 2 thus prepared is fixed by adhering the surface of the hole of the plastic container 8 having holes with the adhesive 10.

Then, an organic compound capable of biospecific binding with an analyte is fixed to the TiO ₂ oxide film 4 to add an organic compound film 12, and a copper wire 14 is electrically connected to a gold film 6 with a silver paste 16. Connected to.

An organic compound capable of biologically specific binding to a high molecular weight analyte such as an antigen, an antibody or an enzyme inhibitor is fixed on the surface of the TiO ₂ oxide film 4 to form a detection electrode 18. The reason why the analyte concentration in the liquid of the aqueous solvent can be estimated by measuring the electric capacity is based on the following principle.

When the electric capacitance of the TiO ₂ oxide film 4 of the detection electrode 18 is C ₀ , the electric capacitance of the organic compound film 12 is C _m , and the total of the two electric capacitances is C _t , 1 / C _t = 1 / C ₀ + 1 / C _{m When the} analyte reacts with the organic compound film 12, the electric capacitance of the analyte film formed on the organic compound film 12 is C
_{When n} , the capacitance changes to _{Ct '} .

[0021] 1 / C _t measured _{'= 1 / C t + 1} / C n capacitance C _t and C _t', the amount of change from C _t to C _{t ',} the analyte amount in response to the organic compound film Will be estimated.

The capacitance of the detection electrode 18 is measured by the device shown in FIG. 2 as follows. The detection electrode 18 and the counter electrode 20 composed of a platinum electrode are dipped in a sample solution 22, and a reference electrode 24 composed of a silver-silver chloride electrode.
Is immersed in a saturated aqueous solution of potassium chloride 26, and both solutions are mixed with salt bridge 2
Connected with 8. The salt bridge 28 was used to prevent the protein in the sample solution from adhering to the silver-silver chloride electrode and deteriorating the silver-silver chloride electrode.

The three electrodes were connected to a potentiostat 30, and the potentiostat 30 kept the bias potential of the detection electrode 18 with respect to the reference electrode 24 at a set potential. The set potential was applied to the potentiostat 30 from an external function generator 32. Then, the frequency was changed from the AC oscillator 34 to the potentiostat 30 to apply a minute AC voltage. Next, the potential and current of the detection electrode 18 were drawn from the potentiostat 30 and sequentially connected to the lock-in amplifier 36, and only the potential of the detection electrode 18 and the AC component of the current were measured. Using the AC voltage and the AC current of the detection electrode 18 thus measured, the electric capacity of the detection electrode was obtained by a calculation formula.

The calculation formula required to obtain the electric capacitance of the detection electrode is as follows. If the impedance of the equivalent circuit in which the resistance of R ohm and the capacitor of C farad are connected in series is Z and the admittance is Y, there is a relation of Z = 1 / jωC + R Y = 1 / Z = G + jB, so G = ω ² C ² R / (1 + ω ² C ² R ² ) B = ω C / (1 + ω ² C ² R ² ) where ω (= 2πf) is the angular frequency. When the alternating current frequency f is used as a variable and the G-B relationship is plotted on the complex plane, the same form as in FIG. 3 is obtained. When an AC voltage e is applied to this equivalent circuit, an AC current i flows. Also Y = i /
Since it is e, G and B can be obtained by measuring e and i, so C can be obtained from the above two equations. When ω = ∞, G = 1 / R, and R is obtained. Detection electrode 18
When a G-B complex number plane is obtained in the same manner as above in a measurement system using the above, FIG. 3 is obtained.
The electric capacitance of the detection electrode can be obtained by using one of the equations.

The organic compound capable of biologically specific binding with the analyte is fixed on the oxide film 4 of the detection electrode 18 as follows. In the field of industrial application of enzymes and microorganisms, many techniques for immobilizing them on polymer carriers or inorganic carriers have already been developed. Therefore, an appropriate technique can be selected from these techniques to fix an organic compound such as an antigen, an antibody or an enzyme on the oxide film 4 of the detection electrode 18.

Experimental Example 1 HBs antigen (antigen of hepatitis B virus coat) and HBs antibody capable of biologically specific binding are used to detect antigen-antibody reaction by using a detection electrode 18 having a TiO ₂ oxide film 4 fixed thereto. The HBsAg was detected by the change in the capacitance that was generated by.

The detection electrode of FIG. 1 was prepared under the following conditions.
A square titanium substrate 2 having a thickness of 0.25 mm and a side of 1 cm was heated to 750 ° C. in an electric furnace to form a TiO ₂ oxide film 4. The plastic container 8 had a cylindrical shape with a diameter of 19 mm and a height of 17 mm, and was used with a hole having a diameter of 5 mm.

The HBs antibody was immobilized on the TiO ₂ oxide film 4 as follows. First on the TiO ₂ oxide film 4 10% 3-aminopropyltriethoxysilane solution (pH
7) was added dropwise with a microsyringe, dried in air, and washed with water.

Next, an aqueous glutaraldehyde solution was added dropwise onto the TiO ₂ oxide film 4 with a microsyringe. Then, after this, an HBs antibody aqueous solution is formed on the TiO ₂ oxide film 4.
After adding dropwise with a microsyringe and air-drying, wash solution (PBS-
Washed with Tween). The detection electrode 18 thus prepared was immersed in a phosphate buffer solution in which the HBs antigen was dissolved, and the electric capacity was measured by the measuring device shown in FIG. The occurrence of the antigen-antibody reaction caused a change in capacitance, which is a direct indicator of HBs antigen concentration.

Experimental Example 2 Using the detection electrode 18 in which trypsin (proteolytic enzyme) was immobilized on the TiO ₂ oxide film 4, soybean trypsin inhibitor (enzyme inhibitor: protein) reacted with trypsin to change the electric capacity. Soybean trypsin inhibitor was detected. Trypsin was immobilized on the same detection electrode as in Experimental Example 1 (in the case of HBs antibody) by the same method. The detection electrode thus prepared was immersed in a phosphate buffer solution in which soybean trypsin inhibitor was dissolved, and the electric capacity was measured by the measuring device shown in FIG. The reaction of soybean trypsin inhibitor with trypsin caused a change in capacitance, which is a direct indicator of soybean trypsin inhibitor concentration.

The present invention has been described above with reference to the illustrated embodiments and specific experimental examples. However, the present invention is not limited to the above-described embodiments and experimental examples, and various types are possible within the scope of the technical idea of the present invention. Can be changed.

[0032]

As, according to the present invention oxide layer made of an electrically insulating film capacitance sensor so far, SiO ₂ oxide film or a dielectric constant having a relative dielectric constant of 3 to 5 a SiO ₂ to Si ₃ N ₄ 6-8 Overlaid two-layer membranes were used. On the other hand, in the first invention, a TiO ₂ oxide film that is insoluble in water and most of the acid and has a large dielectric constant is used as the oxide film, and the relative dielectric constant is It has a value of 86. By using such an oxide film having a very high relative dielectric constant, a large capacitance change can be obtained, and a biological capacitance sensor having a simple structure can be provided.

According to the second invention, T having a large dielectric constant is used.
It becomes possible to measure an antigen or an antibody by using the detection electrode according to the first invention using the iO ₂ oxide film.

According to the third invention, T having a large relative dielectric constant is used.
An enzyme inhibitor can be measured using a detection electrode using an iO ₂ oxide film.

According to the fourth aspect of the invention, it is possible to measure high molecular weight analytes such as antigens, antibodies and enzyme inhibitors by using a detection electrode having a simple structure and compact size.

[Brief description of drawings]

FIG. 1 is a vertical sectional view showing a structure of a detection electrode.

FIG. 2 is a circuit diagram showing connection of a measuring device.

FIG. 3 is a graph showing a G-B relationship when obtaining the electric capacitance of a detection electrode.

[Explanation of symbols]

2 Titanium substrate 4 TiO ₂ Oxide film 6 Gold film 8 Plastic container 10 Adhesive 12 Organic compound film 14 Copper wire 16 Silver paste 18 Detection electrode 20 Platinum electrode 22 Sample solution 24 Silver-silver chloride electrode 26 Saturated potassium chloride aqueous solution 28 Salt bridge 30 Potentiostat 32 Function Generator 34 AC Oscillator 36 Lock-in Amplifier

Claims (4)

(57) [Claims] 1. A biological capacitance sensor for detecting the presence of high molecular weight analytes such as antigens, antibodies and enzyme inhibitors, which comprises a detection electrode, a counter electrode, a reference electrode, and these three electrodes. An electric circuit electrically connected to the detection electrode and responsive to a change in the capacitance of the detection electrode, wherein the detection electrode is a titanium substrate and T on the surface of the titanium substrate.
A biological capacitance sensor comprising an iO ₂ oxide film and an organic compound fixed to the surface of the TiO ₂ oxide film and capable of biologically specific binding to an analyte. 2. The analyte is an antigen, the organic compound is an antibody capable of biologically specific binding to the antigen, or the analyte is an antibody,
The biological capacitance sensor according to claim 1, wherein the organic compound is an antigen capable of biologically specific binding to the antibody. 3. The biology according to claim 1, wherein the analyte is an enzyme inhibitor and the organic compound is an enzyme capable of biologically specific binding to the enzyme inhibitor. Capacitance sensor. 4. The detection electrode comprises a container whose case has a hole, and the titanium substrate is bonded and fixed to the container so that the TiO ₂ oxide film on the surface of the titanium substrate faces the hole of the container. The TiO ₂ oxide film is brought into contact with the liquid through the hole, and
₍₂₎ An organic compound capable of biologically specific binding to the analyte is fixed on the oxide film, and a conductive metal film is formed on the back surface of the titanium substrate, and the metal film responds to the change in capacitance. The biological capacitance sensor according to claim 1, wherein the biological capacitance sensor is adapted to be electrically connected to an electric circuit.