JPH01119753A - Immunity sensor and preparation thereof - Google Patents

Abstract

PURPOSE:To enable the highly sensitive detection of a specified antigen alone without any hindrance by others, by selecting and fixing an antibody for the antigen to be detected. CONSTITUTION:An immune antibody is fixed by a covalent bonding on the surface of an electrode, particularly on the surface of a gate electrode of FET or the like. The electrode, e.g. the gate electrode of FET, is prepared by a publicly-known semiconductor manufacturing technique of ion implantation, evaporation, photoetching or the like with a silicon wafer or the like used, for instance. In order to fix the immune antibody on the surface of the electrode by the covalent bond, a method is employed wherein an epoxy group is fixed on the surface of the electrode by making a silane compound having the epoxy group and a group connected with a silicon atom and being active in reaction, such as hydrogen, alkyl or alkoxy, react with a hydroxyl group existing on the surface, the immune antibody is made to contact with the fixed epoxy group subsequently and thereby the epoxy group and an amino group in the immune antibody are made to react with each other, so as to produce the covalent bonding.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] Industrial Field of Use The present invention relates to a sensor for detecting the amount of a physiologically active substance, etc., using an antigen-antibody reaction.

2. Description of the Related Art A so-called biosensor is known that detects the presence of a trace amount of a chemical substance and outputs it as an electrical signal. Such a biosensor consists of a part where a substance reacts with an organism or an enzyme, and a part that converts the physical and chemical changes caused by this reaction into an electrical signal (transuser).
It is composed of. Transusers of such biosensors are known to use oxygen electrodes, ion electrodes, thermistors, optical sensors, etc.
Biological substances that react with chemical substances are usually immobilized on the sensitive parts of such transusers in various states.

With recent advances in semiconductor manufacturing technology, semiconductor chemical sensors have come to be manufactured, and a representative example thereof is an ion-sensitive field effect transistor (ISFET) that is sensitive to hydrogen ions. Such an 15FET is a metal oxide semiconductor field effect transistor (MOSFET).
An ion-sensitive membrane, such as a thin layer of silicon nitride, is provided at the gate electrode part of the ion-sensitive membrane, and when immersed in an aqueous solution, a potential is generated on the ion-sensitive membrane depending on the concentration of hydrogen ions, so the potential is different from that of the aqueous solution. By applying a constant voltage between a fixed reference electrode and, for example, the base of the 15FET, an electrical output corresponding to the pH can be obtained.

Furthermore, such 15FETs can be used with conventional oxygen electrodes or PL
A complex type that is used as a transducer in place of the + electrode, and is used in combination with various immobilized enzyme membranes to detect the concentration of specific ions, such as hydrogen ions, produced when chemical substances react with these enzymes using 15FET. A sensor has been proposed. In this type of sensor, a sensor with an immobilized enzyme membrane and a sensor without an immobilized enzyme membrane are fabricated using 15FETs having the same performance and are combined adjacently to produce a differential output. Some proposals have also been made to obtain the following.

Problems to be Solved In conventional biosensors, as mentioned above, the accumulated state of ions and heat generated as a result of the reaction of chemical substances with enzymes etc. is converted into electrical signals by the transducer section. Not only does it have an inherent drawback of slow response speed, but it also has the problem that the output is easily affected by measurement environmental conditions.

In view of the problems with the prior art biosensors, the present invention aims to provide a biosensor based on a new principle that essentially does not have these drawbacks.

Furthermore, the present invention aims to provide a biosensor that has better selectivity for a substance to be measured than conventional biosensors.

[Structure of the invention]

Means for Solving the Problems The above-mentioned objects of the present invention can be achieved by an immunosensor in which immune antibodies are immobilized by covalent bonds on the surface of an electrode, particularly on the surface of a gate electrode of a field effect transistor (FET), etc. Such an immunosensor forms a metal layer on the surface of an electrode on which hydroxyl groups can be generated, then oxidizes the surface of the metal layer to generate hydroxyl groups, and applies a silane compound containing an epoxy group to the surface. It can be produced by bringing the hydroxyl group and the silane compound into contact with each other to react with each other, and further bringing the epoxy group into contact with the immunized antibody solution under reaction conditions to covalently bond the epoxy group and the immunized antibody.

The electrode used in the present invention, such as the gate electrode of an FET, can be manufactured using, for example, a silicon wafer by conventional semiconductor manufacturing techniques such as ion implantation, vapor deposition, and photoetching. Of course, the substrate may be made of not only silicon metal, but also germanium, or a compound semiconductor material such as gallium arsenide. In the final stage of fabrication of such electrodes, such as F E 1', the electrode surface, particularly the gate electrode surface, is covered with a metal layer, but such a metalized electrode surface is oxidized to generate hydroxyl groups. For example, silicon or germanium metal is used. It is advantageous for electrodes whose surfaces are made of metallic silicon because hydroxyl groups are easily generated by contacting the surface with humid air.

To covalently immobilize immune antibodies on the electrode surface,
The epoxy group is fixed on the electrode surface by reacting the hydroxyl group existing on the surface with a silane compound having an epoxy group and a reactively active group such as hydrogen, alkyl group, or alkoxy group bonded to a silicon atom. Next, a method can be used in which an immunizing antibody is brought into contact with the epoxy group and an amino group in the immunizing antibody is reacted with each other to form a covalent bond.

The silane compound used to immobilize the immune antibody in this way has at least one epoxy group that remains unreacted when the hydroxyl group on the electrode surface reacts with the reactive group to form a Si-0 bond. Those having one or more are preferred, and for example, γ-glycidoxyprobyl trimethoxysilane and the like are preferably used. Such silane compounds are
It is brought into contact with the electrode surface having a hydroxyl group either as it is or diluted with an appropriate solvent, and allowed to react for a required period of time. At this time, the reaction time can be shortened by increasing the temperature.

In order to immobilize a desired immune antibody onto an electrode on which a reactive epoxy group is immobilized on the surface in this way, for example, the immune antibody or a peptide compound bound to the immune antibody is dissolved in a buffer solution having an appropriate pH value. etc. is brought into contact with the electrode surface and held for a required period of time to cause the amino groups of the immune antibody and the like to react with the epoxy groups on the electrode. At this time, pH
If pl+ is too low, the epoxy group will be hydrolyzed and the number of covalent bonds with amino groups will decrease, and if pl+ is too high, the Si-0 bond that fixes the epoxy group to the electrode surface will be severed, resulting in In order to reliably immobilize antibodies by covalent bonding, it is desirable to use a buffer solution of p(18 to 1o).In addition, the reaction time for immobilization can be shortened by appropriately increasing the temperature. .

The immune antibody used to obtain the immunosensor of the present invention can be selected from those having an antigen-antibody binding site that is complementary to the physiologically active structure of the substance to be detected by the sensor. If necessary, such an antibody can be used, for example, by injecting a substance that serves as an antigen into a mouse, and then separating and purifying the immunoglobulin produced by the proliferated lymphocytes. An immunosensor in which the thus obtained immune antibody is covalently immobilized on an electrode has the property of selectively binding only to a specific antigenic substance and not binding to other types of substances at all.

Operation When the immunosensor of the present invention is brought into contact with a solution containing an antigenic substance, only the specific antigenic substance is bound onto the electrodes of the immunosensor. At this time, if the antigen substance has a charge, a charge layer is formed on the electrode, and the electrode potential changes. On the other hand, sensors with reference electrodes that differ only in that they do not have the ability to bind to antigens are placed adjacent to each other in a mutually insulated state, and by differentially detecting the electrode potentials of these two sensors, it is possible to detect antigenic substances. can be detected. Furthermore, when the antigenic substance does not have a charge, it may be converted into a charged antigenic substance by pretreatment such as binding with a charged substance.

Furthermore, changes in electrode potential may be detected differentially as changes in unipolar potential; It is also possible to configure it as a pole and detect changes in electrode potential as the output of the FET. In this case, two FETs can be placed adjacent to each other in a mutually insulated state and the difference in their outputs can be detected. is preferred.

Example 1 (1) Preparation of antigen 100 mg of bovine serum albumin was diluted with 0.1 M at pH 7.5.
10ml of sodium phosphate buffer solution! The mixture was mixed with 0.5 me of an acetone solution containing 2 mg of dansyl chloride, and reacted at 4° C. for one day and night. The obtained reaction product was dialyzed against a 10 mM sodium phosphate buffer solution at pH 7.2 for 1 week to remove low-molecular weight substances and obtain a dansylated bovine serum albumin solution.

The dansylated bovine serum albumin thus obtained was 061
The solution was diluted to 1 mg/ml in M sodium hydrogen carbonate solution, and dialyzed for 1 day against 0.1 M sodium hydrogen carbonate solution to remove phosphate ions and the like to obtain a purified solution. 1 mg of N-succinimide ester of biotin prepared separately
/mj dimethyl sulfoxide solution was mixed at a ratio of 0.06 volume to 1 volume of the purified dansylated bovine serum albumin solution, and reacted at room temperature for 4 hours. The obtained reaction product was purified by dialysis for 3 days against a 10mMU triurium chloride buffer solution at pH 7.2 to remove solvents, other low molecular weight substances, and other types of salts.

(2) Preparation of antibodies After injecting the biotin-bound dansylated bovine serum albumin described above into mice as an antigen, 107 hybrid cells producing monoclonal antibodies against dansyl obtained by the cell fusion method were injected into the abdominal cavity of the mouse. After 3 weeks to 1 month, the accumulated ascites was collected and centrifuged to obtain a supernatant.

On the other hand, fill a column with a packing material in which Protein A, which specifically binds to IgG type antibodies, is immobilized on Sepharose, and swell it with 10mM phosphoric acid (pH 7.2)+Jum buffered saline. On the other hand, the immunoglobulin (antibody) contained therein was adsorbed and separated through the ascites supernatant, and then eluted with 0.1M acetic acid (pH 3,1) to remove and recover the antibody. The obtained antibody solution was
The pA of pl+ was adjusted to 7.2 by mixing the same amount of pl+6.5 with 0.5 M IJ sodium phosphate buffer solution. The antibody content is approximately 1 mg/mI! Met. The solution was stored at below 4°C and used in the following experiments.

(3) Creation of sensor 16mm cut from a high-purity silicon polycrystalline wafer
A +X16 mm silicon chip was prepared by exposing it to air under indoor conditions to generate hydroxyl groups on the surface.

After a lead wire was attached to the back surface of this silicon chip, a waterproof coating of silicone rubber was applied to cover the entire surface, leaving a window with a diameter of 10 mm on the surface.

This was washed sequentially with trichloroethylene, acetone, and ethanol, and after drying, 100 μl of T-glycidoxypropyl trimethoxysilane was added onto the surface.
The mixture was reacted for 15 minutes in a constant temperature chamber at 90° C. to obtain epoxidized silicon chips.

Next, the antibody solution obtained in (2) was mixed with IM sodium carbonate buffer solution of pH 9.5 to give an antibody concentration of 0.2 mg/
A solution of me was prepared, and each drop of 50 μm was dropped on the epoxidized silicon chip, reacted for 16 hours at room temperature in a clouded atmosphere with 100% humidity, and then added to bovine serum albumin in 10 mM sodium phosphate buffered saline. Unreacted epoxy groups were blocked by immersion in a 1% solution for 1 hour. Further, unreacted peptides and the like were removed by washing five times with 10 mM sodium phosphate buffered saline to obtain an antibody-immobilized silicon chip.

(4) Preparation of standard antigen N-succinimide ester of biotin and α-lysine were mixed in a molar ratio of 10=10 in a 0.1M sodium carbonate aqueous solution.
1, the pH was adjusted to 7.6 with hydrochloric acid, and the mixture was reacted at 50° C. for 3 hours. After completion of the reaction, the reaction product was lyophilized and separated by thin layer chromatography using a chloroform-methanol-water (2:1:1) solvent. The stationary phase portion bound to the product was extracted using dimethylformamide and lyophilized to obtain biotin-bound lysine.

Next, an acetone solution containing 10 mg of dansyl chloride 5
ml and 1 mp of a dimethylformamide solution containing biotin-bound lysine Img were added to 5 ml of a 0.1 M sodium carbonate-sodium hydrogen carbonate aqueous solution (pl+9°5 to 10.0).
l? was added to the solution, dissolved, and reacted at room temperature for 4 hours. After the reaction was completed, the reactants were separated by thin stone chromatography in the same manner as described above, and biotin-bound dansylated lysine was extracted from the stationary phase with methanol.

Subsequently, add avidin l10ff1 to 2mM at pH 7.4)
1 mg of the biotin-bound dansylated lysine dissolved in 0.5 mi of methanol-water (1:1) solvent was added to the solution dissolved in 7'ris buffer solution 2rr7, and the mixture was incubated at room temperature for 30 minutes. Made it react.

After the reaction is complete, the protein is adsorbed through a column filled with a gel for protein separation (Sephadex G-25, manufactured by Pharmacia), and then eluted using 10mMU sodium phosphate buffered saline to remove the corresponding avidin-biotin. The bound dansylated lysine fraction was separated and collected by fluorescence monitoring. This was subsequently used as a standard antigen in the following experiments.

(5) Creation of reference electrode (3) Using the same procedure as in creating the antibody-immobilized silicon chip for the odor C sensor, instead of immobilizing the antibody, bovine serum albumin was added in 10 mM sodium phosphate buffer at pl+7.2. A silicon chip fixed with bovine serum albumin was obtained by dissolving it in a solution at a concentration of 0.2 mg/m7!, and this was used as a reference electrode.

(6) Antibody-fixed silicon chip electrode prepared in sensor operation test (3) and (5)
Connect the bovine serum albumin-fixed silicon chip reference electrode prepared above to the terminals of a high input impedance differential amplifier circuit, use the silver/silver chloride electrode as a reference electrode, and connect these electrodes to the
It was immersed in a container containing 50 ml of a 2mM Tris-HCl buffer solution of H7,4.

After keeping the solution at 25°C and outputting a stable potential value, add 2m containing the standard antigen prepared in (4).
M)! A JS buffer solution was added little by little, and the relationship between the potential difference of the sensor electrode with respect to the reference electrode at °C1 and the standard antigen concentration in the solution was investigated, and the results shown in FIG. 1 were obtained.

This shows that the immunosensor of the present invention provides a potential output with good reproducibility in response to the antigen concentration.

Example 2 Using a P-type silicon wafer and applying a known NMO3 manufacturing technique, an FET with chip dimensions of 7.2 x 3.6 mm was manufactured. At this time, polysilicon stones were provided on the source, drain, and gate by the CVD method, and aluminum wiring was deposited on the source and drain electrodes, and then covered with a 1.2 μm thick silicon nitride insulating layer.
After that, the silicon nitride layer on the gate electrode was removed and the
A window of X 3 ml was created. It was confirmed that hydroxyl groups were formed on the surface of the polysilicon on the gate electrode due to exposure to indoor air.

On the gate electrode, 90% of r-glycidoxypropyl trimethoxysilane was applied using the method (3) of Example 1.
React at ℃ for 15 minutes to form an epoxidized layer,
Next, this surface was thoroughly washed with acetone, and then 0.5 μβ of 1M carbonic acid solution containing the antibody at 0.2 mg/mem was added dropwise to fix the antibody, and further unreacted The epoxy groups of were blocked by bovine serum albumin. Other procedures were the same as in Example 1 (3) to obtain an FET sensor.

In addition, the gate electrode surface of the FET on the control side was
After epoxidation using the method described in 5), bovine serum albumin was fixed.

The outputs of the pair of FETs obtained in this way were each amplified and inputted to a differential output circuit, and the output of the immunosensor was examined with respect to the antigen concentration. It was confirmed that an output corresponding to the charge of the antigen was obtained. I was caught.

(Effects of the Invention) The immunosensor of the present invention is made by covalently immobilizing an immune antibody on the electrode surface and has the ability to bind only to a specific antigen, so it generates an electrical output corresponding to the amount of charge that the antigen has. It shows. Therefore, by selecting and immobilizing an antibody against the antigen to be detected, it has become possible to detect only a specific antigen with high sensitivity without interference from the pond.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between antigen concentration and output potential difference in an example of the immunosensor of the present invention.

Claims (6)

[Claims] (1) An immunosensor in which immune antibodies are immobilized on the electrode surface by covalent bonds. (2) The immunosensor according to claim 1, wherein the covalent bond includes an Si-O bond as a part thereof. (3) The immunosensor according to claim 1 or 2, wherein the covalent bond includes a C-N-C bond as a part thereof. (4) The immunosensor according to any one of claims 1 to 3, wherein the electrode is a gate electrode of a field effect transistor. (5) An immunosensor in which mutually insulated electrodes are arranged adjacent to each other and an immune antibody is immobilized on one surface of the electrodes by covalent bonding. (6) Form a metal layer on the electrode surface on which hydroxyl groups can be generated, then oxidize the surface of the metal layer and generate hydroxyl groups, and contact the surface with a silane compound containing an epoxy group. A method for producing an immunosensor, which comprises reacting the hydroxyl group with the silane compound, and further contacting the epoxy group with an immune antibody solution under reaction conditions to covalently bond the epoxy group and the immune antibody.