JPS59128443A - Bio-sensor - Google Patents

Abstract

PURPOSE:To achieve the miniaturization of an apparatus and the enhancement of measuring sensitivity, by a method wherein a structure capable of holding a liquid is provided between a living body catalyst electrode and an opposed electrode and a small amount of a buffer solution is held between both electrodes in measuring while a specimen is added to said buffer solution. CONSTITUTION:For example, each of both electrodes 12, 13 is formed of a reticulated body or a plate body having a large number of holes and mutually confronted at a short interval so as to be capable of holding a small amount of a liquid therebetween by utilizing surface tension. In measuring, both electrodes are at first immersed in a buffer solution and drawn up therefrom and a small amount of the buffer solution is held between both electrodes by surface tension. In the next step, potential difference is preliminarily generated between both electrodes by using a potentiostat and a predetermined amount of a specimen is dripped to the living body catalyst electrode 12. When the current flowing between both electrodes 12, 13 is measured, the concn. of the specimen is known. As mentioned above, because the buffer solution is used in a small amount, measuring sensitivity is good and constitution is simple and can be miniaturized.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an iosensor used for measuring the concentration of a substance.

The industrial use of biological reactions carried out by microorganisms, enzymes, organelles, etc. has traditionally been popular in the pharmaceutical and food fields, but in recent years, along with its older brothers in genetic engineering, it has been used in textiles, chemicals, etc. It is now being used in fields other than those mentioned above.

The biological reactions described above have many advantages, such as only specific substances reacting at room temperature and pressure (substrate specificity), only specific reactions proceeding (reaction specificity), and fewer by-products. have.

However, reactions using enzymes have the following problems. In other words, enzymes are water bath compatible, and conventionally the enzyme reaction was carried out with the enzyme dissolved in water. Therefore, after the reaction is complete, only the enzyme can be separated and recovered from the reaction bath solution and reused. is a technically extremely difficult problem.

Furthermore, enzymes have a short lifespan.

Since enzymes are expensive, this is also very disadvantageous from a cost standpoint.

Therefore, in order to solve this problem, techniques for immobilizing enzymes (such as making enzymes water-insoluble in some way) have been developed.
Immobilized enzyme technology was developed.

This technology makes it possible to recover, reuse, and use enzymes in continuous reactions. It has also become possible to extend the lifespan of enzymes. In addition to enzymes, techniques for immobilizing microorganisms have also been developed.

Biosensors such as enzyme sensors are created using technology that immobilizes biocatalysts such as enzymes and microorganisms, and the concentration of the analyte can be determined regardless of the presence of the analyte and other impurities. It has excellent features such as being able to perform measurements, etc.

A biosensor generally includes a biocatalyst electrode such as an enzyme electrode in which a biocatalyst is fixed to a plate-shaped or rod-shaped conductor, and a counter electrode made of a plate-shaped or rod-shaped conductor. However, when measuring the concentration of a substance using such a conventional iosensor, various problems have arisen, and therefore improvements to the iodine sensor have been required.This will be explained below.

The reaction format of a biosensor is generally divided into two types: the tsute type and the flow type.
It is carried out as shown in the figure. As shown in the figure, a biosensor 3 is immersed in a predetermined amount of buffer solution 2 placed in a container 1. Next, a potential difference is created between the biocatalyst electrode and the counter electrode using a potentiostat or the like (not shown). After that, put a predetermined amount of sample into buffer solution 2, and while stirring using stirrer (magnetic stirrer) 5, etc.
Measure the current value (output current) flowing between both electrodes with an ammeter (omitted in the figure). This current value determines the concentration of the sample. However, with this method, it is necessary to put enough buffer solution in a container to immerse the biosensor and to change the buffer solution every time a sample is measured, so a large amount of buffer solution is required. There was a problem. Furthermore, during measurement, it is necessary to stir the sample and buffer solution so that they are evenly mixed.

Furthermore, when there is only a small amount of sample, there is a problem in that the sample is diluted with a large amount of buffer solution, which inevitably reduces measurement sensitivity.

Next, the case of a flow set will be explained. Figure 2 shows
Hail the measuring device used in the flow set. As can be seen, the device has a container 6 in which a buffer solution 2 is placed. A pipe 7 extends from the container 6, and a pump 8. Syringe9. Flow cells (liquid sample cells) 10 are connected by vibrators 7 in this order. A biosensor 11 is arranged in the flow cell 10. Biosensor 11
A means for creating a potential difference between the two electrodes, such as a potentiostat, and an ammeter are connected, but these are omitted from the diagram. This device is used as follows. The buffer solution 2 contained in the container 6 is made to flow through the vibrator 7 using a pump so as to maintain a constant flow rate.

Next, the sample is injected into the vibe 7 using a syringe.

A potential difference is generated between the two electrodes of the biosensor 11,
Measure the current flowing between the two poles. This method requires a large amount of buffer solution to flow through the vibrator, so like the batch method, a large amount of buffer solution is required, and when there is only a small amount of sample, the measurement sensitivity is reduced due to dilution with the buffer solution. There was a problem with it falling. Furthermore, this method required a vibrator and a pump, which led to the problem that the entire measuring device became too complicated.

This invention was made in view of the above-mentioned circumstances, and it is an object of the present invention to provide a biosensor that can perform measurements without causing the above-mentioned problems. That is,
The present invention is a biosensor equipped with a biocatalyst electrode and a counter electrode thereof, and the biocatalyst electrode and the counter electrode have a structure capable of retaining a liquid between them, and a small amount of buffer solution is retained between both electrodes during measurement. The gist of the biosensor is that the biosensor is characterized in that the sample is added to the biosensor. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the accompanying drawings.

FIG. 3 is an enlarged perspective view of the biosensor according to the present invention. As shown in the figure, this biosensor is equipped with a rectangular biocatalyst electrode 12 and a rectangular counter electrode 13, and both electrodes 12.13 have conductive wires 14 for connecting them to a potentiostat, an ammeter, etc. .15 is connected.

The biocatalyst electrode 12 and the counter electrode 13 face each other at a short distance by passing the conducting wires 14, 15 through and fixing them in a rod 16 made of an electrically insulating material or the like. Both electrodes 12.13 are, for example, a net-like body as shown in FIG. 4 or a plate-like body with many holes as shown in FIG. It is now possible to hold a small amount of liquid by using tension. Platinum and the like can be cited as the quartz metal used in the net-like body or the plate-like body, but the type is not particularly limited. The type of biocatalyst immobilized on the conductor is not particularly limited as long as it is related to an electrochemically detectable substrate or product. Examples of enzymes include invertase and glucose. Two or more types of biocatalysts may be immobilized at the same time. For example, a small fermentation system consisting of two or more enzymes, such as a combination of invertase and glucose, may be immobilized, or enzymes and microorganisms may be immobilized at the same time. Biological f! ill
! There are no particular limitations on the method for immobilizing the medium, and commonly used methods such as crosslinking methods can be used.

This biosensor is used in the following manner. Soak the crystals in a buffer solution and pull them up. 1
A small amount of buffer is retained at the +I41υ pole due to surface tension. Next, a potential difference is created between the two electrodes using a potentiometer or the like, and a predetermined amount of the sample is dropped onto the biocatalyst electrode. The concentration of the sample can be determined by measuring the current flowing between the two electrodes.

In this way, if the biosensor shown in FIG. 3 is used for measurement, the buffer solution only needs to be in an amount that is held between both electrodes, and there is no need to stir the buffer solution. It is also easy to operate. In addition, unlike a flow set, a pump or pipeline is not required, so the entire measuring device becomes smaller. Moreover, since the amount of buffer held between the two electrodes is small, even when there is only a small amount of sample, there is no problem such as a decrease in measurement sensitivity due to dilution.

The structure of the biosensor according to the present invention is not limited to that shown in FIG. In short, it is sufficient that the biocatalyst electrode and its counter electrode have a structure capable of retaining a liquid between them.

FIG. 6 shows an example of another biosensor. As shown in the figure, this biosensor comprises a biocatalytic electrode 17 in the form of a coil and a counter electrode 18 in the same shape. A straight conducting wire portion 19 is connected from one end of both electrodes 17 and 18.
．． 20 extends and is passed through and fixed in the rod 21, so that both electrodes 1 are slightly shifted from each other in the axial direction.
7.18 is fixed. This biosensor can also hold a small amount of liquid between the biocatalyst electrode 17 and the counter electrode by utilizing the surface tension of the liquid. The method of using this biosensor is the same as that of the biosensor shown in FIG.

The biosensor according to the present invention is constructed as described above, and has a structure in which the biocatalyst electrode and the counter electrode can retain liquid between them, so that only a small amount of buffer is required during measurement, and the sample There is no need to stir the buffer after adding. Furthermore, even when there is only a small amount of sample, the sensitivity is high because the output current is large. Moreover, the entire measuring device can be downsized, and the sensor can be easily cleaned.

Next, examples and comparative examples will be described.

[Example 1] The biosensor of Example 1, which has a structure as shown in FIG. 3 and both electrodes have a net structure as shown in FIG. I made it.

Two 0.5 mm mesh nets (platinum mesh) were made by assembling platinum wires in a mesh pattern with 5 wires in length and 5M in width.

Using one network as a carrier, 8
XIO-21 ng of glucose oxidase was immobilized. That is, after surface oxidation of the network, silane coupling treatment, and then cross-linking of glucose oxidase and the network with gluculaldehyde, fixation is performed in three steps to create a biocatalytic electrode (enzyme electrode). Ta. The remaining mesh was placed on the opposite electrode, and conductive wires were fixed to both electrodes. Next, the conductive wires of both electrodes were placed in glass tubes, and both glass tubes were fixed to each other so that the distance between the electrodes was 1 m, thereby obtaining a biosensor.

Using this biosensor, we measured the output current of glucose solutions of various concentrations as described above. first,
Both electrodes were immersed in 67mMU acid buffer (pH 7,5),
I pulled it up. Approximately 20 μl of buffer was held between both electrodes due to surface tension. A voltage was applied so that the biocatalyst electrode side was +7V, and 5 μl of glucose solution was dropped into the buffer solution held between both electrodes, and the flowing current was measured. The measurement results are shown in Figure 7.

[Example 2] The biosensor of Example 2 had a structure as shown in FIG. 3, and both electrodes had a porous plate structure as shown in FIG. That is, two platinum plates measuring 5 mm in length and 5 mm in width with nine holes of 1 mm diameter were used, and glucose oxidase was immobilized on one platinum plate in the same manner as described in Example 1. As a biocatalytic electrode,
Another platinum plate was used as a counterpoint. The distance between both electrodes was set to 111 nn.

[Example 3] A biosensor as shown in FIG. 6 was used as the biosensor of Example 3. That is, the length 5 made of platinum wire
Two coiled bodies of 1.0 m were used, glucose oxidase was immobilized on one coiled body in the same manner as described in Example 1 to serve as a biocatalyst electrode, and the other coiled body was used as a counter electrode. did.

[Comparative example]

Glucose oxidase was immobilized on a platinum plate measuring 5 mm x 5 m x 50 μm in the same manner as described in Example 1 to serve as a biocatalyst electrode, and a biosensor of a comparative example was prepared using the platinum plate as a counter electrode.

Using the biosensors of Examples 2 and 3, the output currents of glucose solutions of various concentrations were measured in the same manner as described in Example 1. Furthermore, using the biosensor of the comparative example, output currents of glucose solutions of various concentrations were measured using two methods: batch type and flow type. However, the voltage should be applied so that the bioelectrode side is +0.7V, and in the batch type, the buffer capacity should be 10TnI! , for the flow set, the buffer flow rate is 30 rnl! /min. The measurement results are shown in Figure 7. However, the output current value is a peak current value only for the flow set, and a steady current value for the others.

From FIG. 7, it can be seen that the measurements using the biosensors of Examples 1 to 3 have higher sensitivity than the measurements using the biosensors of the comparative example.

[Example 4] An iosensor having a structure as shown in FIG. 3 and an electrode having a network structure as shown in FIG. 4 was manufactured as follows. It was made into a new sensor.

That is, a biosensor was produced in the same manner as described in Example 1, except that a complex enzyme system consisting of inher p-setof-glucose oxidase was immobilized.

Using the obtained biosensor, the output current of sucrose bath solutions of various concentrations was measured in the same manner as described in Example 1. However, the amount of the sucrose bath solution added was 5 μl. The measurement results are shown in FIG.

From FIG. 8, it can be seen that the sensitivity is high when the biosensor of Example 4 is used for measurement.

[Brief explanation of the drawing]

Figure 1 is an explanatory diagram of a conventional biosensor (Tutch type measurement); Figure 2 is an explanatory diagram of a flow set measurement performed using a conventional biosensor; FIGS. 4 and 5 are enlarged perspective views of electrodes, respectively. FIG. 6 is an enlarged perspective view of other examples of the biosensor according to the present invention, and FIGS. Figure 8 is a graph showing the relationship between sample concentration and output current. 12.17...Biocatalyst electrode 13.18...Counter electrode agent Patent attorney Takehiko Matsumoto Figure 3 13 Figure 4 5 Figure 6 Drip glucose concentration (M) ・ Figure 7 Figure 8 Procedural amendment (voluntary) 1. Display of incident 13 Seven mouths 5841 Niu ￥ Zeng 1 Moan tribute '! 004385 No. 2
, Name of the invention Biosensor 3, Relationship with the case of the person making the amendment Patent applicant Location 1048 Oaza Kadoma, Kadoma City, Osaka Name (!583) Matsushita Electric Works Co., Ltd. Representative Iku-yaku Kobayashi Iku 4, Agent 1) On page 7, lines 10 and 11 of the specification, "glucose" is corrected to "glucose oxidase." (2) On page 7, line 13 of the specification, the text ``1 glucose'' is corrected to ``glucose oxidase.'' (3) On page 8, lines 1 and 2 of the specification, the text ``potentiometer'' is changed to ``glucose oxidase.'' Potentiostat,” he corrected. (4) On page 12, line 17 of the specification, the phrase "buffer capacity" is corrected to "buffer volume." (5) 130m on page 12, line 18 of the specification! ! /m
in" should be corrected to read "3mj2/min J.

Claims (2)

[Claims] (1) An iosensor equipped with a biocatalyst electrode and its counter electrode, which has a structure capable of holding a liquid between the biocatalyst electrode and the counter electrode, and a small amount of buffer solution is placed between the two electrodes during measurement. A novel iosensor is characterized in that a sample is added to the iosensor. (2) The biosensor according to claim 1, wherein both electrodes are one selected from a net-like body, a coil-like body, and a plate-like body with many holes.