JP4129367B2 - Electrochemical biosensor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrochemical biosensor, and more particularly, to an electrochemical biosensor capable of measuring the amount of a measurement target substance present in a test solution indirectly via an electrochemically active substance.
[0002]
[Prior art]
Proteins such as albumin are usually reabsorbed by the kidney and excreted in the urine only in a small amount, but when renal failure or renal dysfunction occurs, the kidney cannot absorb albumin, so the amount of albumin excreted in the urine is reduced. To increase. For this reason, it is important to measure albumin in urine for the diagnosis of kidney disease. In particular, in diabetes, measurement of the amount of albumin excreted in urine is important because it may cause renal damage.
As the above-mentioned albumin measuring method, urine is dropped onto a membrane on which albumin antibody is immobilized, and a substance capable of detecting the interaction between albumin contained in urine and albumin antibody immobilized on the membrane as a change in light intensity. In general, an optical measurement method for detecting by further coupling is used.
However, the optical measurement method described above has a problem that the measurement system is complicated and expensive, and the development of a simpler and lower cost measurement method is required in the market.
As a simpler and less expensive measuring method, there is an electrochemical measuring method in which a chemical reaction of a substance is extracted as an electric signal. However, since albumin is not an electrically active substance, albumin itself is used as an electric signal. There is a problem that it cannot be measured using a chemical measurement method.
The above-mentioned problem is not limited to albumin but is a problem common to substances that are not electrochemically active.
[0003]
[Problems to be solved by the invention]
As a result of diligent research to solve the above-mentioned problems related to measurement of non-electrochemically active substances, the applicant generates an electrochemically active substance by reacting the antibody of the substance to be measured with a substrate. An enzyme-antibody-binding layer containing an enzyme-bound antibody to which an enzyme is bound, and a substance that binds to the enzyme-bound antibody, at least in an amount sufficient to cause a competitive reaction with the substance to be measured in the reagent solution, Providing an electrochemical reaction layer containing a substrate that generates an electrochemically active substance by reaction with the enzyme, and an electrode, introducing a test solution containing the substance to be measured from the enzyme antibody binding layer, The reagent introduced into the enzyme-antibody binding layer passes through the competitive reaction layer and is introduced into the electrochemical reaction layer so that the electrochemical reaction in the electrochemical reaction layer can be measured through the electrode. To make up The material is not more electrochemically active, indirectly it invented a method of electrochemical measurement using an electrically active material was patent the invention (Japanese Patent Application No. 2002-93526).
Applicants will continue to study the method described above, and if the value of the electrical signal that can be taken out at the electrode section can be increased even when the amount of the substance to be measured is extremely small, measurement of microalbumin in urine will be possible. As described above, the present inventors have come to the conclusion that it is extremely effective for measuring a very small amount of a substance and that a more precise measurement result can be obtained.
The present invention is an electrochemical biosensor that can extract a sufficiently large electrical signal with an electrode even when the amount of a substance to be measured is extremely small, and can accurately measure even a small quantity of substance. The purpose is to provide.
[0004]
[Means for Solving the Problems]
In order to achieve the above-described object, an electrochemical biosensor according to the present invention is an enzyme in which an enzyme that produces an electrochemically active reduced detection substance by reaction with a substrate is bound to an antibody to be measured. Reaction between the enzyme-antibody-binding layer containing the bound antibody, the amount of the substance that binds to the enzyme-bound antibody, and the immobilized competitive reaction layer, which is at least sufficient for a competitive reaction with the substance to be measured in the test solution, and the enzyme electrochemically active and electrochemical reaction layer containing a substrate to produce a reduced form detection substance laminated on an electrode formed on the substrate by, the substrate, electrochemical potential is higher than the target substance and material, the electrochemical reaction layer, the substrate was included more than the amount consumed by the enzymatic reaction with at least an enzyme-linked antibody, the electrochemical reaction layer located on the electrode, the enzyme reaction After reduction detecting substance produced Ri is oxidized at the electrode, in which again, is characterized by being configured to repeat the reaction cycle which reacts with the substrate is re-reduced to the reduced form detection substance.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of an electrochemical biosensor according to the present invention will be described below with reference to some examples shown in the accompanying drawings.
FIG. 1 shows a schematic perspective view of an electrochemical biosensor according to the present invention.
Reference numeral 1 in the drawing denotes a substrate, and an electrode 2 is screen-printed on the substrate 1.
Three layers are stacked on the electrode 2. In the following description, these three layers are referred to as an enzyme-linked antibody layer 3, a competitive reaction layer 4, and an electrochemical reaction layer 5 in the order in which the test solution passes.
The enzyme-linked antibody layer 3 is the uppermost layer that forms the first layer of the present invention. The enzyme-linked antibody layer 3 is formed by electrochemically reacting an antibody of a substance to be measured with a substrate by reaction with the substrate. Contains a substance bound with an enzyme that produces an active substance. In the present specification, a substance obtained by binding an enzyme to the antibody of the substance to be measured is referred to as an enzyme-linked antibody.
The amount of the enzyme-bound antibody contained in the enzyme-bound antibody layer 3 can be appropriately changed depending on the substance to be measured, but is larger than the amount that undergoes a competitive reaction with the substance to be measured during the development of the test solution in the competitive reaction layer. It is necessary to keep it. For example, 2.5 μg of β-galactosidase labeled albumin antibody can be included in the enzyme-linked antibody layer 3.
The competitive reaction layer 4 is a layer that forms the second layer according to the present invention, and is located downstream of the enzyme binding layer 3. A substance capable of binding to the enzyme-linked antibody contained in the enzyme-linked antibody layer 3 is fixed to the competitive reaction layer 4. This substance may be the same substance as the substance to be measured, but is not limited thereto, and may be any substance as long as it can bind to the enzyme-linked antibody contained in the enzyme-linked antibody layer 3.
The amount of the substance immobilized on the competitive reaction layer 4 can be appropriately determined according to the amount of the substance to be measured and the amount of the enzyme-bound antibody contained in the enzyme-bound antibody layer 3, but at least the enzyme-bound antibody The amount of the substance to be measured in the layer 3 is set to an amount sufficient for a competitive reaction with the surplus enzyme-linked antibody that has not been bound.
The electrochemical reaction layer 5 is a layer forming the third layer according to the present invention, is located on the most downstream side, and is in direct contact with the electrode 2. The electrochemical reaction layer 5 includes a substrate that reacts with an enzyme in the enzyme-linked antibody contained in the enzyme-linked antibody layer 3 to generate an electrochemically active substance.
As the above-described enzyme and substrate, those which generate an electrochemically active detection substance by reaction and whose substrate has an electrochemical potential higher than that of the detection substance are used. Specifically, β-galactosidase (β-Gal) is used as the enzyme, and paraaminofemilgalactoside PAPG having an electrochemical potential higher than that of the detection material paraaminophenol PAP is used as the substrate.
The amount of the substrate contained in the electrochemical reaction layer 5 needs to be larger than the amount of the substance to be measured, that is, the amount consumed by the reaction with the enzyme, like the first layer. .
[0006]
The operation of the electrochemical biosensor configured as described above will be described.
In the following description, as an example, the measurement target substance is albumin Alb, the antibody is albumin antibody Ab, the enzyme is β-galactosidase (β-Gal), and the substrate is paraaminophenylgalactopyranoside PAPG.
When urine is introduced from the enzyme-bound antibody layer 3 as a test solution, the enzyme-bound antibody Ab + Gal in which albumin antibody and β-galactosidase (β-Gal) are bound in the urine is dissolved in the enzyme-bound antibody layer.
The enzyme-linked antibody Ab + Gal dissolved in the urine is bound to albumin Alb in the urine, and an albumin-antibody complex in which β-galactosidase (β-Gal) is bound (hereinafter referred to as an enzyme-linked albumin-antibody conjugate). Into the competitive reaction layer 4.
The urine that has entered the competitive reaction layer 4 contains an enzyme-bound albumin-antibody conjugate and an enzyme-bound antibody Ab + Gal that could not be bound to albumin Alb. Since the albumin Alb is immobilized in advance in the competitive reaction layer 4 as a substance that binds to the enzyme-bound antibody Ab + Gal, the surplus enzyme-bound antibody Ab + Gal binds to the albumin Alb immobilized in the competitive reaction layer 4 to perform a competitive reaction. Stops at layer 4.
Therefore, the enzyme-bound antibody Al + Gal that does not bind to the measurement substance albumin by the competitive reaction in the competitive reaction layer 4 is removed from the urine, and the electrochemical reaction layer 5 contains the enzyme-bound albumin− generated from the albumin to be measured. Contains only antibody conjugates.
Since the electrochemical reaction layer 5 contains paraaminophenylgalactopyranoside PAPG which is a substrate of β-galactosidase (β-Gal), the enzyme-bound albumin-antibody conjugate of urine is contained. Paraaminophenol PAP is produced by an enzymatic reaction between β-galactosidase (β-Gal) and paraaminophenylgalactopyranoside PAPG.
Here, the chemical reaction in the electrochemical reaction layer 5 will be described in detail with reference to FIG. As shown in the drawing, the reduced paraaminophenol PAP produced by the enzymatic reaction of β-galactosidase (β-Gal) and paraaminophenylgalactopyranoside PAPG delivers electrons to the electrode 2 to give oxidized paraaminophenol PAP and Become.
On the other hand, since the electrochemical reaction layer 5 contains paraaminophenylgalactopyranoside PAPG having a higher electrochemical potential than paraaminophenol PAP, the oxidized paraaminophenol PAP is reduced by paraaminophenylgalactopyranoside PAPG. The reaction again becomes reduced paraaminophenol PAP. The reduced paraaminophenol PAP produced here is transferred to the electrode 2 to become oxidized paraaminophenol PAP, which is reduced again by paraaminophenylgalactopyranoside PAPG. Once generated, the number of electrons obtained at the electrode by repeating the reaction of reduced paraaminophenol PAP and paraaminophenyl galactopyranoside PAPG, that is, the value of the electrical signal extracted from the electrode is dramatically increased. Become.
As a result, even if the measurement object is a very small amount, it is possible to take out a large electrical signal from the electrode and output it to a measurement device (not shown).
The amount of reduced paraaminophenol PAP produced in this electrochemical reaction layer is proportional to the amount of albumin Alb contained in the urine. Even with a very small amount, it is possible to calculate a measurement value with high accuracy by the measuring device.
[0007]
Next, a second embodiment of the electrochemical biosensor according to the present invention will be described with reference to FIGS.
3 is a schematic top view of a second embodiment of the electrochemical biosensor, FIG. 4 is a partial cross-sectional schematic perspective view of the electrochemical biosensor shown in FIG. 3, and FIG. 5 is a cross-sectional view taken along line AA in FIG. FIG. 6 shows a cross-sectional view taken along the line BB of FIG.
In the figure, reference numeral 10 denotes a substrate made of an insulating plastic film. On this substrate 10, a working electrode 11 and a measuring electrode 12 formed by screen printing a conductive paste ink material are provided.
The two electrodes 11 and 12 described above are covered with an insulating resist film 13 except for electrode portions and terminal portions.
On the electrodes 11 and 12 configured as described above, an enzyme-linked antibody layer 14, a competitive reaction layer 15, and an electrochemical reaction layer 16 are provided as shown in FIGS. Hereinafter, the layout of each of the layers 14 to 16 will be described.
The electrochemical reaction layer 16 is provided on the electrodes 11 and 12 and is configured to be in direct contact with the electrode portion.
The competitive reaction layer 15 is composed of a lateral flow membrane, extends from the rear side of the substrate 10 (left side in FIG. 3) toward the front side of the substrate 10 (right side in FIG. 3), and has a fine gap 17 over the electrochemical reaction layer 16. It passes through, is bent so as to come into contact with the resist film 13 formed on the substrate 10 in front of the electrochemical reaction layer 16, and its tip is fixed to the resist film 13. The competitive reaction layer 15 is liquid-tightly coated on the exposed upper surface portion. Further, the joint portion between the competitive reaction layer 15 and the resist film 13 is coated so that the liquid does not leak to the outside (electrode terminal side), but the liquid leaks to the inside (electrode portion side).
The enzyme-linked antibody layer 14 is provided in the upstream portion of the competitive reaction layer 15 so as to be in direct contact with the competitive reaction layer 15.
In the figure, reference numeral 18 denotes a spacer.
Here, the flow of the reagent solution in the electrochemical biosensor configured as described above will be briefly described with reference to FIG. 7. The reagent solution introduced into the enzyme-linked antibody layer 14 is transferred to the competitive reaction layer 15. Inflow. Since the competitive reaction layer 15 is formed of a lateral flow membrane arranged so that the test solution flows in the front-rear direction of the sensor, the sample solution that has flowed into the competitive reaction layer 15 flows toward the tip of the competitive reaction layer 15.
The reagent solution that has reached the tip of the competitive reaction layer 15 leaks inward from the tip of the competitive reaction film 15 and forms an electrode lead portion by capillary action along the step portion generated on the substrate 10. And 12 are drawn into a fine gap 17 formed between the competitive reaction layer 15 and the electrochemical reaction layer 16, and uniformly electrochemically from the upper surface of the electrochemical reaction layer 16. The reaction layer 16 enters.
With the configuration as described above, the test solution can flow into the electrochemical reaction layer 16 evenly even though the lateral flow membrane is used, and a better measurement result can be obtained.
[0008]
FIG. 8 shows the results of measuring the amount of albumin actually contained in the test solution using the electrochemical biosensor configured as described above.
In this experiment,
The enzyme-linked antibody layer 14 contains 2.5 μg of enzyme-linked antibody Ab + Gal in which albumin antibody and β-galactosidase (β-Gal) are bound,
20 μg / cm ^{2 of} albumin Alb was fixed to the competitive reaction layer 15;
The electrochemical reaction layer 16 contains 22 μg of paraaminophenylgalactopyranoside PAPG,
A test solution containing 1 to 1000 mg / L of albumin in advance was introduced from the enzyme-linked antibody layer 14 and the amount of output current at the electrode was measured.
From the experimental results shown in FIG. 8, it can be seen that if the electrochemical biosensor configured as described above is used, a sufficient amount of output current can be obtained even for a test solution having a low albumin content.
[0009]
Next, the experimental result showing the amplification effect of the output current amount by using paraaminophenyl galactopyranoside PAPG is shown in FIG.
FIG. 9 is a graph showing the amount of output current according to the change in the amount of paraaminophenylgalactopyranoside PAPG, and FIG. 10 is a graph showing the amount of output current according to the change in paraaminophenol PAP.
In this experiment,
Only an electrochemical reaction layer is provided on the electrode, and the electrochemical reaction layer contains paraaminofemil galactoside PAPG in an amount of 0 to 100 mmol / L,
A test solution containing 0.25 to 1.0 mmol / L of paraaminophenol PAP was dropped, and the amount of output current at the electrode was measured.
As shown in FIGS. 9 and 10, the output value obtained from the electrode increases with the concentration of paraaminofemil galactoside PAPG, and it can be seen that there is an amplification effect by paraaminofemil galactoside PAPG based on this value. .
[0010]
In the above-described embodiment, the electrochemical biosensor is formed of three layers, an enzyme-linked antibody layer, a competitive reaction layer, and an electrochemical reaction layer. If necessary, the enzyme constituting the first layer On the upper part of the bound antibody layer, a substance that can be oxidized and removed in advance from a substance that is easily oxidized in the test solution may be laminated. This makes it possible to previously remove substances such as ascorbic acid, uric acid, and acetaminophen that are included in the test solution and are likely to be electrically oxidized, which may affect the measurement.
Examples of substances that can be oxidized and removed in advance from the electrochemically oxidizable substance include lead dioxide and tin dioxide.
[0011]
【The invention's effect】
As described above, the electrochemical biosensor according to the present invention includes an enzyme-linked antibody in which an enzyme that produces an electrochemically active reduced-type detection substance by a reaction with a substrate is bound to an antibody to be measured. An enzyme antibody-binding layer, and a substance that binds to the enzyme-bound antibody, at least in an amount sufficient to cause a competitive reaction with the substance to be measured in the test solution, is immobilized by the reaction between the enzyme and the enzyme. An electrochemical reaction layer containing a substrate that generates a reductive detection substance that is active in an active state, and is stacked on an electrode formed on the substrate, and the substrate has a higher electrochemical potential than the detection substance, the electrochemical reaction layer, the substrate was included more than the amount consumed by the enzymatic reaction with at least an enzyme-linked antibody, the electrochemical reaction layer located on the electrode, generating of the enzymatic reaction And after reduction detection substance is oxidized at the electrode, again, because then reacts with the substrate are configured to repeat the reaction cycle to be re-reduced to the reduced form detection material, amount of substance to be determined is very small Even so, the number of electrons obtained at the electrode, that is, the value of the electrode signal taken out from the electrode, increases dramatically when the detection substance repeats the oxidation-reduction reaction between the substrate and the electrode. Can also be measured accurately.
[Brief description of the drawings]
FIG. 1 is a schematic perspective view of an electrochemical biosensor according to the present invention.
FIG. 2 is a diagram showing a chemical reaction in an electrochemical reaction layer 5;
FIG. 3 is a schematic top view of a second embodiment of an electrochemical biosensor.
4 is a partial cross-sectional schematic perspective view of the electrochemical biosensor shown in FIG. 3. FIG.
5 is a cross-sectional view taken along line AA in FIG.
6 is a cross-sectional view taken along the line BB in FIG.
7 is an explanatory diagram showing the flow of the test solution with arrows in the partial cross-sectional perspective view of FIG. 4;
FIG. 8 is a graph showing experimental results obtained by actually measuring the amount of albumin in the test solution using the electrochemical biosensor shown in FIGS.
FIG. 9 is an experimental result showing an amplification effect of an output current amount by using paraaminophenyl galactopyranoside PAPG.
FIG. 10 is an experimental result showing an amplification effect of an output current amount by using paraaminophenyl galactopyranoside PAPG.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Substrate 2 Electrode 3 Enzyme binding layer 4 Excess enzyme removal layer 5 Electrochemical reaction layer 10 Substrate 11 Working electrode 12 Measurement electrode 13 Resist film 14 Enzyme binding layer 15 Excess enzyme removal layer 16 Electrochemical reaction layer 17 Gap 18 Spacer

Claims (4)

An enzyme-antibody-binding layer comprising an enzyme-linked antibody in which an enzyme that generates an electrochemically active reduced-type detection substance by reacting with an antibody of a substance to be measured is bound;
A substance that binds to the enzyme-linked antibody, at least in an amount sufficient for a competitive reaction with the substance to be measured in the test solution, and a fixed competitive reaction layer;
The electrochemical reaction layer containing an electrochemically substrate to produce an active reduced form detectable substance laminated on an electrode formed on the substrate by reaction with the enzyme,
The substrate is a substance having an electrochemical potential higher than that of the detection substance, and the electrochemical reaction layer contains the substrate at least in an amount consumed by an enzyme reaction with an enzyme-bound antibody ,
In the electrochemical reaction layer located on the electrode, after the reduced detection substance produced by the enzyme reaction is oxidized at the electrode, the reaction cycle is repeated again, which reacts with the substrate and is reduced again to the reduced detection substance. An electrochemical biosensor characterized by comprising The reduced detection substance produced by the reaction between the enzyme and the substrate is reduced paraaminophenol,
The electrochemical biosensor according to claim 1, wherein the substrate is paraaminophenylgalactopyranoside. The substance to be measured is albumin contained in urine,
The antibody is an albumin antibody;
The electrochemical biosensor according to claim 1 or 2, wherein the amount of albumin in urine can be indirectly measured through a reaction between an enzyme and a substrate. The substance to be measured is albumin contained in urine,
The antibody is an albumin antibody;
The electrochemical biosensor according to claim 2, wherein the electrochemical reaction layer contains 1 to 100 mmol / L of paraaminophenolgalactopyranoside as a substrate.

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