JPH049254B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a biochemically sensitive semiconductor sensor, a measuring device, and a method for measuring an analyte using the sensor.

BACKGROUND OF THE INVENTION Immunoassays are commonly used for the identification and quantification of haptens, antigens, and antibodies, all commonly referred to as analytes. The basic principle of all immunoassays lies in the specific binding between the components of a reaction pair (e.g., antigen/antibody, hapten/antibody, etc.), and in some cases one component is isolated by some external method. labeled for easy analysis by

Radioimmunoassay (RIA) is based on the use of radioisotopes as labels for one of the components of a specific binding pair. The radioisotope-labeled component is then measured using appropriate equipment by counting the radiation emitted by the isotope.

Other methods of labeling one member of a specific binding pair have also been developed. Enzyme and fluorescent labels have recently been used and are referred to as enzyme immunoassays (EIA) and fluorescent immunoassays (FIA), respectively. Also, using appropriate reagents and equipment, these labels can be utilized for the measurement of analytes in liquid media. Many variations on this basic operation are available, but many require reaction, separation, and label detection steps.

More recently, electrochemical sensors have improved the sensitivity obtained by these operations and
Efforts have been made to simplify the process. Basically, ion-selective electrodes are employed to detect reaction products of enzymes that are used as labels for one component of their specific binding pair.

Furthermore, Japanese Patent Application Laid-Open No. 10546/1983 describes an immunosensor using a field transistor in which an improved method for attaching an antigen or antibody to the surface of an insulating film is used.

The purpose of the present invention is to eliminate the preparation of a labeled component of a specific binding pair, the separation of this component from an assay system, and subsequent detection, thereby greatly simplifying the immunoassay method. . More specifically,
The present invention relates to a novel and useful improvement in the method of measuring analytes in liquid media by utilizing a biochemically sensitive semiconductor device as described below.

(Means for Solving the Problems) The present invention provides an apparatus used for measuring analytes in a liquid medium. More specifically,
The present invention relates to a sensor made of an electrically semiconducting substance, preferably a semiconducting organic polymer, and a device thereof, in which the binding of an analyte to a specific binding substance is caused by the electrical conductivity of the semiconducting substance. The specific binding substance of the analyte is preferably immobilized on the substance by a method that changes the target semiconductivity to a measurable extent. Furthermore, the present invention provides a method for detecting the presence of an analyte in a liquid medium using such a device.

The invention is specified as follows.

(a) an insulating structure; and (b) an element having an active portion of a polyacetylene semiconducting organic polymer disposed on the insulating structure for exposure to a medium and containing a specific binding substance for the analyte. and (c) at least two or more electrodes, including two electrodes in contact with said element.

(a) producing an element of polyacetylene semiconducting organic polymer; (b) treating the element with a specific binding substance; (c) exposing the element to a medium; and (d) increasing the electrical A method for identifying the type and amount of an analyte in a medium, characterized in that it comprises the step of measuring changes in properties.

(A) an active portion consisting of (a) an insulating structure and (b) a polyacetylene semiconducting organic polymer disposed on the insulating structure for exposure to a medium and containing a specific binding substance for the analyte; (B) a first variable resistance sensor for the measurement of an analyte in a medium comprising an element means having: (c) at least two or more electrodes, including two electrodes in contact with the element; (b) element means having an active portion of a polyacetylene semiconducting organic polymer disposed on the insulating structure for exposure to a medium and comprising a specific binding substance for the analyte; c) a second variable resistance sensor for measuring an analyte in a medium, comprising at least two or more electrodes, including two electrodes in contact with the element; (C) the first variable resistance sensor and the second variable resistance sensor; and a resistance comparison means for measuring the resistance between variable resistance sensors of the invention.

Since many specific binding substances for any particular analyte are biological in nature, this device is referred to as a biochemically sensitive semiconducting device or BSSD. These specific binding substances are generally of organic chemical type and exhibit measurable properties, one of which is a special charge. Furthermore, for a specific binding substance, its charge changes as a result of binding to a specific analyte; examples of a binding substance and its specific analyte include antibodies and their specific antigens. According to the invention,
The binding between a specific antibody (binding substance) and an antigen (analyte) is quantified by detecting and measuring the charge change of one or two components of the binding reactant. By placing one of the two components of a specific binding system in close proximity to a substance that is affected by an electric field, changes in the electric field as a result of the binding reaction can cause changes in the properties of that substance. become. If the properties of this substance can be measured, then this binding reaction can also be measured.

(Operation) The sensor, device and operation thereof of the present invention are best explained element by element as shown in FIGS. 1, 2 and 4, and the electrical operation of the sensor and device is explained in the It will be explained as shown in the figure.

The element 10 occupying the center of the sensor is
Consists of strips of semiconducting organic polymer. Any semiconducting organic polymer can be used for this. This relatively new class of compounds includes polyacetylenes,
Poly(p-phenylene), polypyrrole, poly(polyalkylpolydiyne) such as poly(hepta-1,6-diyne), poly(p-phenylene sulfide), polymetalophthalocyanines, preferably polyaluminophthalocyanine fluoride. and polyphthalocyanine silolanes. This list of semiconducting organic polymers is illustrative only and not limiting.

The organic polymer may be used in an undoped state or with commonly employed dopants such as arsenic pentafluoride, iodine, perchlorate, and the like.

Element 10 is exposed to a binding reagent. It is sufficient to immerse the element in an aqueous solution of binding reagent in a suitable buffer. As used herein, the term "binding reagent" includes antigen/coupling reactions of the type commonly understood in the biochemical arts.
All substances that carry out something like an antibody reaction are included.

Typical binding reagents include antibodies to IgG, enzymes such as β-galactosidase, concanavalin A, limulin, cofactor-type enzymes such as isocitrate dehydrogenase, alcohol dehydrogenase, NAD (nicotinamide adenine dinucleotide), enzymes. Substrates, e.g. isocitrate, Enzyme inhibitors, e.g. β-galactosidase inhibitors, Hormones, e.g. thyroxine, Enzyme substrate analogues, e.g. O-nitrophenyl-
β-D-galactopyrariside, an antigen such as human chorionic gonadotropin. These illustrations herein are not critical and are not intended to be limiting.

Although the illustration of the present invention only discloses a simple contacting of a polymeric element with a binding reagent, it is understood that under certain conditions activation treatment of the element substrate may be necessary for binding with certain binding reagents. will be obvious to a person skilled in the art. Such reactions are commonly performed and well known to those skilled in the art, and are also included within the technical scope of the present invention.

In a typical process for treating element 10 with a binding reagent, the newly synthesized polymer is placed in a suitable buffer containing a solution of the desired binding reagent, and the
Incubate at room temperature for ~24 hours, wash with saline, and store under nitrogen. Although storage under nitrogen is not required, it is known that many binding reagents are sensitive to oxygen and that their activity is best maintained by reducing exposure to this material as much as possible. .

One embodiment of the sensor of the invention is shown in FIG. In FIG. 1, element 10 includes a semiconducting material and a binding reagent bound thereto;
It is provided on an insulating material 14. Here, electrical contacts 22 and 24, which are electrodes, are connected to the element 10.
It is applied to When the device is activated and the medium 12, which is the analyte solution, is placed over it, additional electrical contacts 26, 28, which are electrodes, are applied to the medium 12. Here, the additional electrical contacts are used for measuring the conductivity of the medium or for amplifying the resistance change between the elements using the field transistor effect. This can be understood from FIG. That is, the positions of the contacts 22, 24 are associated with the element 10 and the insulating carrier 14 in its right-hand part. On its left hand side a similar arrangement is shown showing contacts 26. Contacts 28 would be applied similarly. It is particularly preferred for good bonding if this contact is made of gold plating or gold wire.

When using the sensor at its maximum capacity, the second
A slightly more complex device is used as shown in the figure. The last two digits of the drawing numbers correspond to the corresponding two digits in FIG.

Thus, FIG. 2 shows a substrate 114 of insulating material comprising an element 110 of a semiconducting organic polymer and a binding reagent. element 1
10 has a pair of metal contacts 122, each having leads 123 and 125 connected to each other.
124 are provided. The lower portion of the recess 111 is formed by a disc-shaped insulating element 162 having an L-shaped cross section. On the disk element 162, located opposite the recess 111, are electrodes 126, 128 with connected leads 127 and 129, respectively. These contacts 126, 128 occupy only a small part of the wall of the recess and are insulatively surrounded by a disk element 164, so that the medium containing the analyte 112 is transported to the recess 111 created in this way.
placed inside.

The sensor constitutes a device for measuring analytes in media according to the invention in combination with resistance comparison means for measuring changes in the electrical properties of the elements, for example the Wheatstone bridge circuit shown in FIG. In this circuit a positive potential is applied between contacts 32 and 36. One element 10 is connected between contacts 32, 34, and a similar element is connected between contacts 36, 38.
Connect between. Contact 34 connects to junction 48;
One end of the resistor 42 is connected to this. This resistor may be a variable resistor if desired. Similarly, one end of another resistor 44, also preferably a variable resistor, is connected at a contact 46, to which contact 38 is connected. Junctions 46 and 48 are differential amplifiers 5
2, which is connected to ground through a metering device 54.

In operation of the device of the invention, the two elements 10, connected respectively to 32/34 and 36/38, are balanced in the usual manner. The medium containing the analyte is then placed in the recess 111
and the resistance change of this element is measured. Resistance change has been found to be a factor of time and concentration, thus requiring calibration with standard solutions. To further increase the sensitivity of the measurement, additional electrical contacts 26 or 2
This can be done by applying a voltage to either of 8, or by applying the same voltage to both at the same time. Also, if desired, additional electrical contacts 26 and 28 are used to measure the conductivity of the medium in the usual manner.

The term "analyte" as used herein includes antigens, antibodies, haptens, enzymes, enzyme substrates, enzyme substrate analogs, agglutinins, lectins, enzyme cofactors, enzyme inhibitors, hormones, and the like.

Experimental Preparation A The IgG fraction of goat anti-rabbit IgG serum was
Garvey, Jay. Garvey, JS et al., “Methods in Immunology,” Vol.
33% ammonium sulfate precipitation and
It was isolated by a combination of DEAE cellulose chromatography. Furthermore, if necessary, purification was carried out by affinity chromatography technique, which is the method disclosed in the above-mentioned document.

Preparation AA Trans-type polymer of acetylene was prepared using a Ziegler-type catalyst using Ito et al.
Polymer, Science (Journal of Polymer
Science) Vol. 12, p. 11 (1974).

Preparation BB poly(P-phenylene) was prepared according to Shacklette et al.
It was prepared according to the method of Syn.Met. Vol. 1, p. 307 (1980).

The prepared CC polypyrrole was prepared by Kanazawa et al.'s Syn.Met.
It was prepared according to the method of Vol. 329 (1980).

Preparation DD poly(hepter-1,6-diyne) was prepared by Gibson et al., J.Chem.Soc. (Chem.Comm) 426
(1980).

Preparation EE Poly(p-phenylene sulfide) hexafluoroarsenate was prepared by Rabolt et al., Journal of Chemical Society (J.
Chem.Soc.) (Chem.Comm) 347 (1980).

Reference Example I A strip of newly synthesized polyacetylene approximately 4 x 10 mm was injected with 5 mg/ml of purified goat anti-rabbit IgG.
was placed in 0.05M carbonate-bicarbonate buffer, pH 9.5, containing 100% chloride and kept overnight at room temperature. The polyacetylene antibody strips were then washed with saline and stored under nitrogen.

Reference Example A strip of newly synthesized poly(p-phenylene) (approximately 4 x 20 mm) was added to PH containing 5 mg/ml of a monoclonal antibody against human chorionic gonadotropin.
9.5 in 0.05 M carbonate-bicarbonate buffer;
It was kept at room temperature overnight. poly(p-phenylene)
The strips were then washed with saline and stored under nitrogen.

Reference example Newly synthesized polypyrrole strips (approximately 4×
20mm) at pH 9.5 containing 5mg/ml of avidin.
Placed in 0.05M carbonate-bicarbonate buffer and kept at room temperature overnight. The polypyrrole/avidin strips were then washed with saline and stored under nitrogen.

Reference Example A strip of newly synthesized poly(hepta-1,6-diyne) (approximately 4 x 20 mm) was added to a 0.05 M carbonate-bicarbonate buffer at pH 9.5 containing 5 mg/ml of β-galactosidase. It was placed in a solution and kept at room temperature overnight.
The poly(hepta-1,6-diyne)/β-galactosidase strips were then washed with saline and stored under nitrogen.

Reference example V Newly synthesized poly(p-phenylene sulfide) hexafluoroarsenate (approximately 4×20
mm), 0.05M of PH9.5 containing linulin 5mg/ml
of carbonate-bicarbonate buffer and kept at room temperature overnight. Poly(p-phenylene sulfide)
Hexafluoroarsenate/linulin strips are
It was then washed with saline and stored under nitrogen.

Reference example A strip of newly synthesized polyaluminotalocyanine fluoride (approximately 4 x 20 mm) was mixed with NAD 5 mg/ml.
0.05M carbonate-bicarbonate buffer with pH 9.5 and kept at room temperature overnight. The polyaluminotalocyanine fluoride was then washed with saline and stored under nitrogen.

Example 1 A pair of elements prepared according to Reference Example I were connected to a pair of devices on the right-hand side of the device shown in FIG. 4 and continued into a third fiber circuit. The bridge circuit is balanced and rabbit IgG
Add 10 microliters (μ) of PBS buffer (IgG500μg/ml) to one element, and add the same amount of
PBS buffer was placed in the other element and care was taken to avoid contact of either sample with the metallic contact elements 22 and 24. Element 44 of the circuit was a variable transformer whose readings were as follows from T=0 to T=1 hours.

T=0 470 T=1 minute 498 T=2 minutes 488 T=3 minutes 563 T=9 minutes 731 T=20 minutes 687 T=1 hour 587 The voltage supplied was 5.1 volts.

The experiment was repeated using 20 μ of IgG with a supply voltage of 5.0 volts and the following readings were obtained.

T = 0 378 T = 1 minute 358 T = 5 minutes 403 T = 10 minutes 499 T = 20 minutes 499 T = 30 minutes 435 Example 2 As in Reference Example I, strips of polyacetylene antibody were used with goat anti-rabbit IgG prepared using the preparation method and placed in a Teflon holder. 3mm
A mesh cut out to the diameter was spread on a polyacetylene film and used to place an aqueous gelatin film on it. Care was taken not to connect the two conductive clamps with the gelatin film either physically or electrically. Measurement of special allanite is carried out in Example 1.
It was carried out using the same equipment as shown in .

Example 3 Polyacetylene antibody gelatin film strips were prepared as in Reference Example. However, additional electrical connections were made by placing pieces of platinum wire on the surface of the gelatin film. In this example, the device is similar in principle to a field effect transistor. This additional electrical connection will apply a voltage perpendicular to the flow of electrons through the polyacetylene antibody film. Binding of rabbit IgG to goat anti-rabbit IgG antibody using this device was confirmed in the same manner as shown in Example 1.

The operations in the above example were repeated using the apparatus of FIG. In that case, 2 to 10 volts were applied to contact point 126 of the device.

EXAMPLE 4 From the invention thus described, it will be obvious that the same may be varied in many ways. Such modifications are not to be considered as a departure from the spirit and scope of the invention, and all such modifications are intended to be included within the scope of the following claims.

(Effects of the Invention) The present invention relates to an apparatus and a measuring method used for measuring an analyte, and the binding between a specific binding substance (antibody) and an analyte (antigen) is caused by one or two of the binding reaction substances. It can be easily quantified by detecting and measuring changes in the charge of the two components.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of an element portion showing one embodiment of the sensor of the present invention, FIG. 2 is a cross-sectional view showing another embodiment, FIG. 3 is a circuit diagram for operating the device of the present invention, and FIG. The figure is a perspective view of the carrier portion of the sensor of the present invention. 10...Element, 12...Medium, 14...
Insulating material, 22, 24, 26, 28, 32, 3
4, 36, 38... Contact, 46, 48, 50...
Junction point, 52...Amplifier, 54...Meter device, 110...Element, 111...Recess, 1
22, 124... Contact, 123, 125, 12
7,129...Lead, 126,128...Electrode, 162,164...Disk element.

Claims (1)

[Claims] 1. (a) an insulating structure; and (b) a polyacetylene semiconducting organic polymer provided on the insulating structure for exposure to a medium and containing a specific binding substance for an analyte. (c) at least two or more electrodes, including two electrodes in contact with the element. 2. The device according to claim 1, wherein there are four electrodes, two electrodes are connected to the element, and the remaining two electrodes are in contact with the medium for measuring the conductivity of the medium. sensor. 3. The sensor according to claim 1, wherein there are two electrodes connected to the element. 4. Sensor according to claim 1, in which there are three electrodes, two electrodes are connected to the element and the remaining electrode contacts the medium in such a way as to produce a field transistor effect. 5. Claim 1, wherein the specific binding substance is selected from the group consisting of antibodies, antigens, enzymes, enzyme substrates, enzyme substrate analogs, agglutinins, lectins, enzyme cofactors, enzyme inhibitors, and hormones. or the sensor described in Section 4. 6 (a) producing an element of a polyacetylene semiconducting organic polymer; (b) treating the element with a specific binding substance; (c) exposing the element to a medium; and (d) exposing the treated element to a medium. A method for identifying the type and amount of an analyte in a medium, characterized in that it comprises the step of measuring changes in electrical properties. 7. The method according to claim 6, wherein two electrodes are brought into contact with the element and changes in electrical properties between the electrodes are measured. 8. A method according to claim 7, in which three electrodes are used, two electrodes are in contact with the element and the remaining electrode is in contact with the medium so as to produce a field transistor effect. 9. Claim 6, wherein the specific binding substance is selected from the group consisting of antibodies, antigens, enzymes, enzyme substrates, enzyme substrate analogs, agglutinins, lectins, enzyme cofactors, enzyme inhibitors, and hormones. The method described in any one of Items 7 to 8. 10 (A) (a) an insulating structure; (b) an active material comprising a polyacetylene semiconducting organic polymer disposed on the insulating structure for exposure to a medium and containing a specific binding substance for the analyte; (c) at least two or more electrodes, including two electrodes in contact with the element; (B) (a) an insulating structure; (b) element means disposed on the insulating structure for exposure to the medium and having an active portion consisting of a polyacetylene semiconducting organic polymer containing a specific binding substance for the analyte; (c) a variable resistance sensor for measuring an analyte in a medium, comprising at least two or more electrodes including two electrodes in contact with the element; (C) between the first variable resistance sensor and the second variable resistance sensor; and a resistance comparison means for measuring the resistance of an analyte in a medium. 11 There are eight electrodes, two electrodes are connected to the element of the first variable resistance sensor, two electrodes are connected to the element of the second variable resistance sensor, and two electrodes are connected to the element of the first variable resistance sensor. 11. The device of claim 10, wherein the sensor contacts the medium for measuring the conductivity of the medium and the remaining two electrodes contact the medium for measuring the medium conductivity of the second variable resistance sensor. 12. Claim 10, wherein there are four electrodes, two electrodes are connected to the element of the first variable resistance sensor, and the remaining two electrodes are connected to the element of the second variable resistance sensor. The device described in. 13 There are six electrodes, two electrodes are connected to the element of the first variable resistance sensor, two electrodes are connected to the element of the second variable resistance sensor, and one electrode is connected to the element of the second variable resistance sensor, and one electrode is connected to the element of the second variable resistance sensor. 11. The apparatus of claim 10, wherein the remaining one electrode contacts the medium of the second variable resistance sensor to produce a field transistor effect. 14. Claims 10 to 14, wherein the specific binding substance is selected from the group consisting of antibodies, antigens, enzymes, enzyme substrates, enzyme substrate analogs, agglutinins, lectins, enzyme cofactors, enzyme inhibitors, and hormones. 1
The apparatus according to any one of clauses 3 to 3.