JPH011952A - Piosensor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Industrial application field.

The present invention relates to a biosensor that can quickly and easily quantify specific components in various samples.

BACKGROUND OF THE INVENTION In recent years, biosensors have come into use that combine enzyme reactions and electrode reactions to measure the concentration of specific components in a sample.

A conventional biosensor will be explained below.

Figure 6 is a cross-sectional view of a conventional biosensor.
3, insulating substrates J4 and 16 are a measurement electrode and a counter electrode, respectively, embedded in the insulating substrate. 16 is a structure installed on the electrode section, 17 and 18 are a liquid retaining layer and a filtration layer fixed on the structure 16, and the filtration layer is a porous polycarbonate membrane with a pore diameter of 1 μm and a film thickness of 1 μm. . 19 is a holding frame, and 20 is a reaction layer fixed within the holding frame 19, in which an enzyme and a conjugated electron acceptor are supported on porous cellulose as a carrier.

The operation of the biosensor configured as described above will be described below. When the sample solution is dropped from the top, first, an enzymatic redox reaction proceeds between a specific component in the sample solution, an enzyme, and an electron acceptor in the reaction layer in the reaction layer 20, and the electron acceptor is reduced. .

The amount of reduced electron acceptor produced at this time is proportional to the amount of the specific component in the sample solution. Next, the sample solution after the reaction is removed by a filter layer 18 to remove macromolecules that would interfere with the measurement.
After passing through the liquid retaining layer 1γ, it descends to ', f, and the highest point 14°15.

On the electrode, the nil-reduced electron acceptor is oxidized by an electrographic reaction, and the concentration of a specific component in the sample liquid is detected from the oxidation current value.

Problems to be Solved by the Invention However, in the conventional configuration described above, the membrane thickness of the filtration layer is very thin in order to speed up the passage of the reaction liquid and reduce the amount of reaction liquid retained within the filtration layer. , the reaction solution is 7
The filtration layer is pushed down toward the electrode, and the thickness of the reaction liquid film on the electrode is not constant.Also, when the reaction liquid falls, it falls unevenly to the electrode, causing air bubbles and wet residue on the electrode. This caused problems in that measurement accuracy and reproducibility were poor.

In addition, when the 11'4 structure was used to widen the distance between the filtration layer and the electrode part as a means of mitigating the effect when the filtration layer was pushed down, the reaction liquid did not descend sufficiently, and variations in the measured values were also observed. Ta.

The present invention solves the problems of the prior art as described above, and it is possible to stabilize the thickness of the liquid film even if the filtration layer is pushed down, and to supply a sufficient amount of liquid to the electrode part. The purpose of the present invention is to provide a biosensor that can perform measurements with good accuracy and reproducibility by descending uniformly.

Means for Solving the Problems In order to achieve this object, the biosensor of the present invention uses an adhesive structure that forms a space above an electrode part provided with an electrode system consisting of at least a measurement electrode and a counter electrode. 0.1 to 0
．． It has a configuration that provides a height difference of 3 rran.

Function: With this configuration, even if the filtration layer is pushed down, the effect on the electrode section is alleviated, the thickness of the liquid film is stabilized, and a sufficient amount of liquid can be supplied to the electrode section. = 1, because the reaction liquid always falls from the adhesive structure side that is lower in height, the reaction liquid fills the electrode part from one direction, and no bubbles remain on the electrode or residual wetness occurs. Therefore, stable measurements can be made.

EXAMPLE Hereinafter, as an example of the present invention, a glucose sensor, which is an example of a biosensor, will be described with reference to the drawings.

FIG. 2 shows a plan view of an electrode section of a glucose sensor according to an embodiment of the present invention. In Figure 2, 1
is an insulating substrate made of polyethylene terephthalate. Reference numerals 1o and 11 denote a measurement electrode lead and a counter electrode lead, which are formed by screen printing conductive carbon paste on the insulating substrate 1 and heating and drying it. 3 is an insulating layer, in which an insulating resin paste is applied to an insulating substrate 1. Measuring pole lead 10. The opposite paper was printed and dried on the card 11 in the same manner as described in mI.

2 and 12 are a measurement electrode and a counter electrode formed by the insulating layer 3 to have a constant area.

FIG. 1 shows a cross-sectional view of the glucose sensor of this example. In the figure, 1 is an insulating substrate, 2 is a measurement electrode, and 3 is an insulating layer. 4 and 6 are adhesive structures using double-sided adhesive tape. Here, the heights of 4 and 5 are each 0-1
59 and 0.3. A difference in height was established between the two, and the distance between the two locations was set at 3.0. Hereinafter, 4 will be referred to as adhesive h'77 structure (low), and 6 will be referred to as adhesive structure (high).

6 is a liquid retaining layer, using a band-shaped rayon paper with a width of 2.0,
One end thereof was fixed onto the adhesive structure (low) 4. In addition,
The liquid retaining layer 6 is for quickly securing a sufficient amount of liquid. A filtration layer 7 is fixed on the adhesive structure 4.5 and is made of a porous polycarbonate membrane with a thickness of 101 tl11. 9 is a reaction layer held in a holding frame 8, in which cellulose as a carrier is impregnated with a highly concentrated solution of glucose oxidase, an enzyme, in a phosphate buffer (pHts, e), and after drying, it is further impregnated with a highly concentrated solution of glucose oxidase, an enzyme, in a phosphate buffer (pHts, e). It is manufactured by impregnating it with a highly concentrated bath solution of potassium ferricyanide in phosphate buffer (pH 5, 6), immersing it in an organic solvent such as ethanol, and vacuum drying.

The operation of the glucose sensor of this embodiment configured as described above will be described below. First, when blood is dropped onto the top of the sensor shown in FIG. 1, it permeates into the reaction layer 9, and an enzymatic redox reaction proceeds between glucose in the blood and glucose oxidase and potassium ferricyanide in the reaction layer 9. Potassium ferrocyanide is produced. The amount of potassium ferrocyanide produced at this time is proportional to the amount of glucose in the blood. The blood that has completed this reaction is filtered through the filter layer 7.
When passing through, macromolecules such as blood cell components are filtered out. Next, since the filtration layer has a gradient, the reaction liquid always permeates into the liquid retaining layer 6 from the side that is in contact with the adhesive structure (low) 4, and then flows onto the electrode section from the adhesive structure (low) 4 side. descend from Potassium ferrocyanide is oxidized by an electrode reaction on the electrode, and the glucose concentration in the blood is measured from the oxidation current value.

FIG. 3 shows the oxidation current value of the same blood measured by each glucose sensor. In the figure, M shows an adhesive structure according to an embodiment of the present invention with a height difference of 0.15+1111, and B and C show adhesive structures of a conventional example with no height difference.

In addition, the height of the adhesive structure B is 0.16rIIllI
, C was set to 0.3III+1. As can be seen from the figure, there is little variation in current values among the glucose sensors for humans, indicating stable measured values. However, it can be seen that B has large variations among the glucose sensors and is not stably measured. This is thought to be because the filtration layer is pushed down onto the electrode, the thickness of the liquid film on the electrode is not stable, and the liquid falls unevenly from the liquid retaining layer onto the electrode, resulting in wet residue and air bubbles. It will be done. Further, in C, the variation in current value is even larger. This is considered to be due to poor fall of the reaction solution.

FIG. 4 shows the relationship between the oxidation current value of the same blood and the height difference of the adhesive structure, measured with a glucose sensor having a height difference in the adhesive structure.

In addition, the height of the lower side of the adhesive structure was all set to 0.16 mm. Further, measurements were performed 10 times at each height difference, and the average value and the width of dispersion are shown in the figure. From this figure, the difference in height of the adhesive structure of the present invention is from 0.1 to 0.
It can be seen that in the range of 3+m, the average value is constant and the variation is small, making it possible to obtain stable measured values. This is considered to be because a liquid film with a constant thickness is formed by providing a height difference in the adhesive structure, and the liquid resistance is stabilized. Further, the large variation in measured values when the height difference is 0.06 m indicates that this level of height difference is insufficient to alleviate the variation in liquid resistance caused by pushing down the filtration layer. On the other hand, when the difference in height is set larger than 0.3 mm, there is a large variation in the measured values, and when the measurement diameter is resolved and the electrode section is examined, it is found that the liquid is insufficiently falling onto the electrode section and the amount of liquid is insufficient. I found out that it was.

Since the liquid retaining layer 6 is used to quickly supply a sufficient amount of filtrate onto the electrode, the filtration membrane should be made of a material having a higher filtration rate than that of this embodiment, such as a polycarbonate membrane with a pore size that is suitable for blood cell filtration. If it is spread to 6 to 10 μm without any problem, a sufficient amount of liquid can be secured on the electrode within the measurement time, and the measurement can be performed stably even if the liquid retaining layer is removed.

As described above, according to this embodiment, the adhesive structure has 0.
By providing a height difference of 1 to 0.3 tm, even if the filtration layer is pushed down during measurement, there is little effect on the thickness of the liquid film on the electrode, and the thickness of the liquid film on the electrode is stabilized and sufficient. Because we were able to secure a sufficient amount of liquid and the filtration layer has a gradient, the reaction liquid fell onto the electrode from the lower side of the structure, causing bubbles and wet residue on the electrode. By not doing so, accurate and reproducible measurements can be made.

In this example, the electrode system is a two-pole system consisting of a measurement electrode and a counter electrode, but the electrode system may be a three-pole system in which a reference electrode is added. In this case, the potential is stable and measurement can be performed with higher accuracy.

As the electron acceptor, in addition to the potassium ferricyanide used above, P-benzoquinone, methylene blue, etc. can also be used.

Moreover, stable measurement is possible even when the height range of the lower side of the adhesive structure is from 0.05 to 0.2 mm, in addition to the above-mentioned range of 0 and 15 m.

Furthermore, when hydrophilic cellulose is held as a spreading layer on the reaction layer in the holding frame, when blood is dropped, the liquid quickly diffuses through the spreading layer and spreads uniformly, so that the -layer can be measured with high accuracy.

Effects of the Invention The present invention makes the thickness of the liquid film constant and stabilizes the measurement by providing a height difference in the range of 0.1 to 0.3 m in the adhesive structure that constitutes the space above the electrode part. In addition, by creating a gradient in the superlayer, when providing the reaction solution to the electrode part, the adhesiveness of the reaction solution is always maintained at 1.
'f/ falls from the lower side of the structure, and the reaction liquid is uniformly supplied to the electrode, so there is no air bubbles or wet residue on the electrode, making it an accurate and reproducible biosensor. This has the effect of being able to provide the following.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of a glucose sensor according to an embodiment of the present invention, FIG. 2 is a plan view of the electrode portion of the glucose sensor, FIG. 3 is a correlation characteristic diagram between each glucose sensor and response current, and FIG. 4 6 is a correlation characteristic diagram between the response current of the glucose sensor and the height difference of the adhesive structure, and FIG. 6 is a sectional view of a conventional biosensor. 1...Insulating substrate, 2...Measurement electrode, 4
... Adhesive structure (low), 6 ... Adhesive structure (high), 7 ... P vortex layer, 8 ...
- Holding frame, 9...reaction layer, 12... counter electrode. Name of agent: Patent attorney Toshio Nakao and 1 other person 2nd
Figure 3 Glucose Sens (NO) 1-5 throat ↓ 3 board binding --- bale 11 fixed whetstone / S -- 1 inch woodcutter / 6 --- sticky 1 going] mouth book / 7 --- lost warrior skin / 8 ---', 5” l1 /9-m=holding frame 20-11 counter-layer Fig. 5/415

Claims (2)

[Claims] (1) A filtration layer, an oxidoreductase, and an electron acceptor conjugated with the enzyme are placed on an electrode part provided with an electrode system consisting of at least a measurement electrode and a counter electrode, through a space formed using an adhesive structure. 1. A biosensor characterized in that the adhesive structure has a configuration in which a reaction layer containing a reaction layer is installed, and a height difference is provided in the adhesive structure. (2) The biosensor according to claim 1, wherein the height difference of the adhesive structure is in the range of 0.1 to 0.3 mm.