JPS63144245A - Biosensor - Google Patents

Abstract

PURPOSE:To obtain a stable biosensor of uniform quality by installing and uniting a porous body which holds oxidoreductase and electron acceptors on an electrode plate selected by measuring the electric characteristics. CONSTITUTION:Conductive carbon paste is printed in parallel belts on an insulating substrate 1, and heated and dried to form an electrode system consisting of a counter electrode 2, a measurement electrode 3, and a reference electrode 4. Then, the electrode system is covered partially and an insulating layer 5 is formed by printing insulating paste so that parts 2'-4' of the respective electrodes which perform electrochemical operation are left; and the exposed parts 2'-4' are polished and a heat treatment is carried out. The electrode system uses only an electrode system which is uniform in response by taking a measurement as well as preliminary measurement using inspection liquid. Then, a holding frame 6 made of synthetic resin is adhered to the insulating layer 5 and the porous body 7 which carries enzyme and electron acceptors is held in a hole so that the electrode systems 2'-4' are covered, further, a cover 8 which is made of resin and has an opening part with a diameter smaller than the external diameter of the porous body 7 is adhered to unit the whole body.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The biosensor of the present invention can quickly and easily quantify a specific component in a biological sample with high precision, and can be widely applied to the medical field, food engineering, etc.

BACKGROUND OF THE INVENTION In recent years, various biosensors that utilize the specific catalytic action of enzymes have been developed, and attempts have been made to apply them particularly to the field of clinical testing. Nowadays, as the number of transverse strain items and the number of specimens increases, a biosensor that can measure quickly and accurately is desired.

Taking glucose sensors as an example, in today's world where diabetes is rapidly increasing, measuring and managing blood glucose levels requires a lot of time to centrifuge blood and convert it into plasma, as was done in the past. , there is a need for a sensor that can measure whole blood. A simple type, similar to the transversely strained paper used for pressure measurement, is a stick-shaped support containing an enzyme that reacts only with sugar (glucose) and a dye that changes during the enzyme reaction or by the products of the enzyme reaction. Some have a carrier containing them installed. This method involves adding ζ blood to this carrier and measuring the change in pigment visually or optically after a certain period of time.
Accuracy is low due to large interference from colored substances in the blood.

Therefore, a multilayer analytical carrier as shown in FIG. 5 has been proposed (Japanese Utility Model Publication No. 178496/1983).

This consists of a transparent support 9, a reagent layer io, a developing layer 11,
It has a structure in which a waterproof layer 12 and a filtration layer 13 are laminated in this order. When a blood sample is dropped from the top, it first passes through the filtration layer 1.
3, solid components such as red blood cells and platelets in the blood are removed and uniformly permeate into the spreading layer 11 through the small holes 14 in the waterproof layer 12, and a reaction proceeds in the reagent layer 1o. After the reaction is completed, light is irradiated through the transparent support 9 in the direction of the arrow, and the substrate concentration is measured by spectroscopic analysis.

Although it has a more complex structure than the conventional simple stick-shaped carrier, it has improved accuracy due to blood cell removal. However, since it takes time for blood to permeate and react, a waterproof layer 12 is required to prevent the sample from drying out, and it is necessary to incubate at a high temperature to speed up the reaction, making the apparatus and carrier complicated. be.

On the other hand, a biosensor as shown in Fig. 6 has been proposed as a method for quantifying specific components in biological samples such as blood with high precision without performing operations such as diluting or stirring the sample solution ( For example, Japanese Unexamined Patent Publication No. 59-166852). This biosensor has leads 18 on an insulating substrate 16.
．． A measuring electrode 16 and a counter electrode 17 made of platinum or the like having 19 are buried, and the exposed portions of these electrode systems are covered with a porous body 2o carrying an oxidoreductase and an electron acceptor. When a sample solution is dropped onto a porous material, the oxidoreductase and electron acceptor in the porous material are dissolved in the sample solution, an enzymatic reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. . After the enzymatic reaction is completed, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention In the conventional configuration, the porous body can be easily measured by changing the throat for each measurement, but the electrode system requires operations such as cleaning. Particularly after measuring blood or the like, proteins adhering to the electrode surface cannot be completely removed by washing with water alone, leading to deterioration of response and affecting measurement accuracy.

Means to Solve the Problems In order to solve the above problems, the present invention provides an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, and integrates it with a porous body carrying an enzyme and an electron acceptor. It became. Further, regarding the electrode system, only those that have been tested in advance with a test solution containing at least the electron acceptor in the same manner as in the measurement and have a consistent response are used.

Since the working electrode system and the porous body containing the enzyme and electron acceptor are integrated, the electrodes are also replaced after each measurement, making the measurement operation extremely simple and requiring only dropping the sample. Although the response depends on the area of the measurement electrode, a calibration solution containing electron acceptors was used to select electrodes with consistent responses, so a highly accurate response was obtained.

Example (Example 1) A glucose sensor will be described as an example of a biosensor. FIG. 1 shows an embodiment of a glucose sensor, and is an exploded view of the constituent parts. A conductive carbon paste containing a resin binder is printed in parallel strips on an insulating substrate 1 made of polyethylene terephthalate by screen printing, and then heated and dried to form a counter electrode 2, a measurement electrode 3. An electrode system consisting of a reference electrode 4 is formed. Next, 2', 3', 4' partially cover the electrode system and become the electrochemically active part of each electrode.
An insulating paste mainly composed of polyester is printed in the same manner as described above so that (1-) is left, and the insulating layer 5 is formed by heat treatment. Next, the exposed portions 2', 3', and 4' were polished and then heat treated in air at 100'C for 4 hours.

As a test solution, dissolve potassium ferricyanide and potassium ferrocyanide in a phosphate buffer solution with a pH of 5.6 to 0.1.
Prepare two equimolar solutions of M and place 2' on the electrode.

Added 30 μ on top of 3.4′. When a pulse voltage of 700 mV is applied with respect to the reference electrode 4', potassium ferrocyanide is oxidized on the measuring electrode 3', and an oxidation current flows. This oxidation current is affected by the area of the measurement electrode 3' when the concentration of potassium ferrocyanide is constant, so it is difficult to check the area of each measurement electrode when multiple electrode systems are created by screen printing. can. Since the measurement electrode is very small at 1-, it is difficult to judge the difference in area even by visual inspection or using a microscope. Moreover, the surface of the electrode is not smooth;
Since the surface condition changes depending on the printing conditions (temperature, humidity, mixing state with the resin binder), it is necessary to inspect it before measurement. By using the above method, the surface condition can be easily inspected, and after washing and drying, only electrodes with a uniform response can be used for measurement, improving measurement accuracy.

A holding frame 6 made of synthetic resin such as polyester with holes drilled on the selected electrodes is adhered to the insulating layer 6, and the electrode systems 2', 3
A porous body 7 carrying an enzyme and an electron acceptor is held in the hole so as to cover ', 4'. Furthermore, a resin cover 8 having an opening having a diameter smaller than the outer diameter of the porous body 7 is adhered to integrate the whole body.

A cross-sectional view along the measurement electrode 3 of this integrated biosensor is shown in FIG. The porous body used above has a nylon nonwoven fabric as a base material, and contains 200 mg of glucose oxidase as an oxidoreductase and aoomg of potassium ferricyanide as an electron acceptor at a concentration of 0.25 W.
H5,6 phosphate buffer containing t% surfactant (polyetherene glycol alkyl phenyl ether) 1
After impregnating the base material with the solution dissolved in rnl, the concentration was 0.25.
It was produced by immersing it in ethanol containing wt% surfactant to crystallize it, and then drying it under reduced pressure.

A glucose standard solution was dropped as a sample solution into the porous body of the glucose sensor configured as described above, and 2 minutes after dropping, a pulse voltage of 700 mV was applied with respect to the reference electrode to polarize the measurement electrode toward the anode. .

In this case, the added glucose reacts with potassium ferricyanide by the action of glucose oxidase supported on the porous body 7 to produce potassium ferrocyanide. Therefore, by applying the pulse voltage in the direction of the anode, an oxidation current proportional to the concentration of potassium ferrocyanide produced is obtained, and this current value corresponds to the concentration of glucose, which is the substrate.

FIG. 3 shows an example of the response characteristics of the sensor with the above configuration.
It shows the relationship between the current value 10 seconds after voltage application and the glucose concentration, and showed extremely good linearity.

(Example 2) One portion of glucose oxidase was added to the test solution used in Example 1.
In addition, the response current of each electrode was examined and the electrodes were selected in the same manner as in Example 1. A glucose sensor was constructed on this electrode in the same manner as in Example 1. Approximately 90 m97 for 10 glucose sensors with the above configuration
A serum sample containing dl of glucose was dropped into each sample, and after 2 minutes, a pulse voltage of 700 mV was applied and measurements were carried out in the same manner as in Example 1. As shown in Figure 4, good reproducibility was obtained. On the other hand, when a serum sample is dropped onto a glucose sensor composed of selectively selected electrodes that do not contain glucose oxidase and measured in the same manner as described above, the response is lower than that of humans, as shown in B in Figure 4. The current fluctuation was large. This difference is thought to be due to adsorption of adsorbed substances such as proteins in the serum sample to the electrode. Therefore, if the response of the electrode is checked in advance with a test solution containing glucose oxidase, glucose oxidase can be adsorbed to the electrode surface and proteins in the serum sample can be prevented from being adsorbed.

10 albumin instead of glucose oxidase
mji7cc In addition, the selected electrodes also showed good reproducibility.

The protein to be included in the test solution is not limited to glucose oxidase and albumin shown in the above examples.

In Example 911, a three-electrode force type electrode system was described, but measurement can also be performed using a two-electrode force type consisting of a counter electrode and a measurement electrode.

Potassium ferricyanide used in the above example is suitable as an electron acceptor supported on a porous material or contained in a test solution because it reacts stably, but P-benzoquinone can be used to speed up the reaction because it has a fast reaction rate. suitable for father, 2
・6-cyclophenol indophenol, methylene blue, phenazine methosulfate, β-naphthoquinone potassium 4-sulfonate, etc. can also be used.

Note that the sensor in the above embodiment can be used in systems involving redox enzymes, such as glucose sensors, alcohol sensors, and cholesterol sensors. Although glucose oxidase was used as the oxidoreductase, other enzymes such as alcohol oxidase and xanthine oxidase were used.

Cholesterol oxidase and the like can also be used.

Effects of the Invention A transversely strained liquid containing electron acceptors necessary for measuring a sample liquid is deposited on an electrode plate and its electrical characteristics are measured, and the oxidoreductase and the electron acceptor are deposited on the selected electrode plate. By installing and integrating a support plate that holds a biosensor, it is possible to obtain a biosensor that is stable and of uniform quality.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of a glucose sensor according to an embodiment of the present invention, FIG. 2 is a vertical cross-sectional view of FIG. 1, and FIGS. 3 and 4 are response characteristic diagrams of the same glucose sensor. Figures 5 and 6
The figure is a cross-sectional view of a conventional biosensor. 1...Substrate, 2...Counter electrode, 3...
... Measuring electrode, 4 ... Reference electrode, 5 ... Insulating layer, 6 ... Holding frame, 7 ... Porous body,
8...Resin cover. Name of agent: Patent attorney Toshio Nakao and one other person/N
-('%J rtsu Dimensions Figure 3 Z lucose thick, mourning (Hard Vd1) Figure 4 Full Gose Gunbu (澗υ Figure 5 Figure 6

Claims (3)

[Claims] (1) At least a measurement electrode and a counter electrode are formed on an insulating substrate to form an electrode plate, a test liquid containing an electron acceptor necessary for measuring a test liquid is adhered to the electrode plate, and a gap between the measurement electrode and the counter electrode is applied. The electrode plates are selected by measuring their electrical properties, and a carrier holding the oxidoreductase and the electron acceptor is placed and integrated in contact with the selected electrode plate. sensor. (2) The biosensor according to claim 1, wherein the test liquid is an aqueous solution containing oxidized and reduced electron acceptors. (3) The biosensor according to claim 1, wherein the test liquid is an electron acceptor plus protein.