JPS63168552A - Biosensor - Google Patents

Abstract

PURPOSE:To make the construction simple and compact with a simplified handling, by forming a reaction film chip in which a specified biologic reaction membrane is previously held at a circular holder member detachable to a recess of a planar measuring electrode body. CONSTITUTION:An enzyme immobilized membrane C1 with an interference blocking film C2 laminated thereon is held integral on the bottom of a circular holder E of a reaction membrane chip Z. When carrying out a measurement, the chip Z held in a buffer solution is taken out with a pincette or the like to be fitted into a recess A of a planar measuring electrode body X and a sample (solution containing glucose) is dripped with a micropipette P. A reaction is caused in a biologic reaction membrane C to form an electrode activating substance, which is diffused into a buffer solution layer S to generate a response current in a response electrode surface Y. Thus, the current is taken out as output signal corresponding to the concentration of glucose in the sample.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is a type of membrane-attached and removable measuring electrode, and is used to measure the concentration of biological components in urine or blood, such as glucose, albumin, uric acid, lactic acid, and urea. In enzyme electrodes and enzyme-immune electrodes used in measurements, enzyme-immobilized membranes and enzyme labels are placed on the response electrode surface of the measurement electrode body, which is composed of a bipolar hydrogen peroxide electrode, etc. Regarding so-called biosensors, which are configured to perform predetermined measurements by removably attaching a predetermined bioreaction membrane such as an immune reaction treatment membrane, they are extremely easy to handle compared to conventional ones, and This was done with the aim of providing a biosensor that has a completely new configuration that is very simple and compact, and that can use an innovatively simple measurement method.

[Conventional technology]

A conventional biosensor of this kind is shown in Fig. 6 (a) showing its decomposed state a (state without bioreaction membrane attached) and Fig. 6 (a) showing the measurement state with bioreaction membrane attached. As is clear from (b), in order to perform the prescribed measurement,
Response electrode surface y at the tip of Rondo-type measurement electrode body X
For example, a predetermined bioreactive membrane C (for example, an enzyme-immobilized membrane cut to an appropriate size or an antibody-immobilized carrier membrane is allowed to act on an antigen as a sample to be measured, or vice versa). When attaching an immunoreaction-treated membrane (such as an immunoreaction-treated membrane that is treated and labeled with an enzyme using the Sanderuch method or a competitive method), first apply the bioreaction membrane C itself to the response electrode surface y, and then , a fixing ring member d made of synthetic resin is attached to the outer peripheral surface of the tip of the measuring electrode body X.
By fitting the biological reaction membrane C so that the periphery of the biological reaction membrane C is sandwiched between r7u between the outer peripheral surface of the real tip of the measurement electrode body and the inner peripheral surface of the fixing ring member d, the biological reaction membrane C
is fixed in close contact with the response electrode surface y,
On the other hand, when removing the biological reaction membrane C from the response electrode surface y of the measurement electrode main body First, the fixing ring member d is removed from the outer peripheral surface of the tip of the measuring electrode main body X, and then the biological reaction membrane C itself is peeled off from the responsive electrode surface y. It was configured to perform attachment and detachment operations for biological reaction 11Qc.

In addition, during measurement, as shown in FIG. 6 (b) above, the tip of the measurement electrode main body In some cases, it is a mixed solution of sample and buffer solution,
When the bioreaction membrane C is an enzyme-labeled immunoreaction treated membrane, a batch measurement method may be used in which the membrane is immersed in a reaction tank f such as a beaker containing a mixed solution of a substrate and a buffer solution, or a method (not shown) may be used. However, a continuous measurement method was adopted in which the tip of the measurement electrode main body X equipped with the biological reaction membrane C was set in a flow cell configured to continuously flow the same sample as described above.

[Problem that the invention seeks to solve]

However, in the conventional biosensor described above, since the biological reaction membrane C itself must be handled independently, various treatments are required for it (for example, fixation treatment of enzymes, antibodies, or antigens, blocking treatment, immune reaction treatment, etc.). process.

It is very difficult to handle when carrying out buffer separation treatment, etc.), and the biological reaction membrane C after that treatment cannot be stacked on top of each other, so considerable care is required for storage and a large space is required. Furthermore, the attachment and detachment of the biological reaction membrane C to the response electrode surface y of the measurement electrode main body X must be performed by complicated manual operations as described above.
Therefore, not only is there an inconvenience that the bioreaction membrane C and the response electrode surface y are easily contaminated, but especially in the case of an enzyme immunoelectrode, etc., where the bioreaction membrane C needs to be replaced for each sample, it is difficult to attach and remove the bioreaction membrane C. The operation is extremely troublesome, and furthermore, as mentioned above, each time a measurement is made, the fixing ring member d is attached to the measuring electrode main body Since the bioreactive membrane C is fixed to the response electrode surface y by fitting onto the outer peripheral surface of the tip of the bioreactive membrane C, it is almost impossible to always set the tension of the bioreactive membrane C constant. Therefore, there have been various problems, such as the state of attachment of the biological reaction membrane C and the reproducibility of the measurement results, particularly when re-measuring the same biological reaction n*c.

Moreover, since the measuring electrode main body X has a rond-like configuration, the measuring electrode main body X itself has to be relatively large and complicated, and also, as shown in FIG. 6 (b) above, Whether a batch measurement method or a continuous measurement method is used, a relatively large-scale sampling system is required, making the entire measurement system large and complex, and requiring a large amount of samples. There was also the problem of.

An object of the present invention is to develop and provide an epoch-making biosensor having a configuration that can solve these conventional problems at once.

[Means for solving problems]

In order to achieve the above object, the biosensor according to the present invention includes a flat measuring electrode body formed of four parts on the top side with a response electrode surface provided at the bottom, and a recessed part of the flat measuring electrode body. It consists of a reaction membrane chip, which is formed by integrally holding a predetermined biological reaction membrane in an annular holder member having a shape and size that allows it to be removably inserted into the holder, and stored in a buffer solution when not measuring. In the measurement state in which the reaction membrane chip is fitted and placed in the recess of the flat measurement electrode body, a constant thickness is formed between the response electrode surface and the lower surface of the bioreaction membrane at the bottom of the recess. It is characterized in that it is configured to form a buffer solution layer.

[Effect]

The effects achieved by this characteristic configuration are as follows.

That is, in the biosensor according to the present invention, a reaction membrane chip is constructed by integrally holding a predetermined bioreaction membrane in advance in an annular holder member (thereby, when handling the bioreaction membrane, It can be handled as a whole reaction membrane chip, and there is no need to handle the bioreaction membrane itself separately as in the past. Therefore, when performing various treatments on the bioreaction membrane,
By holding the annular holder member, which is a component surrounding the reaction membrane chip, by hand or with tweezers, it is possible to perform the reaction very easily without directly touching the biological reaction membrane itself. The chips can be stacked together with the annular holder member that is a surrounding component serving as a spacer, so they can be stored easily and in a small space. A shape and size that allows the annular holder member, which is a surrounding component, to be removably fitted into a recess formed on the top side of the flat measuring electrode body and having a response electrode surface at its bottom. Therefore, when attaching and detaching the bioreaction membrane to the response electrode surface of the measurement electrode main body, the complicated and troublesome operation of handling the bioreaction membrane itself and the fixing ring separately as in the conventional method is eliminated. By having an annular holder member around the reaction membrane chip, the entire reaction membrane chip can be attached and removed from the response electrode surface without directly touching the biological reaction membrane itself (by fitting and placing it). This can be done with a very simple one-touch operation compared to, for example, a screw-in type.Therefore, there is no risk of contaminating the biological reaction membrane or the response electrode surface, and it is extremely easy to do. Moreover, the attachment and detachment operations can be carried out reliably, and therefore, it is very convenient especially in the case of an enzyme immunoelectrode, etc., in which the biological reaction membrane needs to be replaced for each sample. Furthermore, even when re-measuring the same bioreaction membrane, it is only necessary to place and reinstall the entire reaction membrane chip on the response electrode surface of the measurement electrode body. This eliminates the inconvenience of changing the tension of the bioreaction membrane, which would occur when the bioreaction membrane is re-fixed to the response electrode surface using a fixing ring member. It has become possible to easily ensure good reproducibility and to significantly improve the reliability of measurements.

Moreover, by configuring the measurement electrode body in a flat plate shape,
The measurement electrode body itself can be made much simpler and more compact than the conventional rond-shaped one, and when making measurements, as will become clearer from the description of the examples below, Drop the sample (sample solution if the bioreaction membrane is an enzyme-immobilized membrane, substrate solution if the bioreaction membrane is an enzyme-labeled immunoreaction membrane) onto the bioreaction membrane in the reaction membrane chip using a micropipette, etc. Since it can be carried out using an extremely simple method, there is no need to set up a large-scale sampling system like in the past, and the entire measurement system can be configured much simpler and more compact, and the amount of sample required for measurement is also extremely small. Now you can get away with it.

〔Example〕

Specific embodiments of the present invention are shown below in the drawings (Figures 1 to 5).
The explanation will be based on Figure).

FIG. 1 shows a biosensor constructed by applying the present invention, and this biosensor includes a flat measuring electrode main body X as shown in FIG. It is a member that can be attached to and detached from the reaction membrane chip Z as shown in FIG. 1 (b).

That is, as shown in FIG. 1(a), the flat measuring electrode main body , a recess A (circular in plan view in this example) is formed, and a platinum electrode (anode) 2 at the center and an annular silver electrode (cathode) 3 around the center are formed at the bottom of the recess A, respectively. A flat, buried response electrode surface Y is provided to form a so-called bipolar Bolaro type hydrogen peroxide electrode.

In addition, in the figure, 4 is a temperature compensation electrode embedded in the silver electrode 3 (as shown in the figure, this is molded with epoxy resin 4 or the like having high thermal conductivity).
Also, 2B, 3B, 4B are lead wires 2A, 3A, 4A
The platinum electrode 2, the silver electrode 3. Temperature compensation electrode 4
These are terminals for taking out signals connected to the terminals, respectively, and are arranged in the connector section 5. Further, although the connector section 5 provided with these signal extraction terminals 2B, 3B, and 4B is provided on the lower surface of the flat member 1 in this example, it may also be provided on the side surface of the flat member 1. Good, yet again.
The platinum electrode (anode) 2 and the silver electrode (cathode) 3 are not limited to the arrangement described above, but may be arranged, for example, so as to interlock with each other in a comb-teeth shape and face each other.

On the other hand, as shown in FIG.
Fitting and unfitting (fitting, mounting and removing)
An annular holder member E (circular in this example) made of synthetic resin (e.g., vinyl chloride, ABS, polycarbonate, polypropylene, etc.) and having a shape and size that can be attached and detached by A predetermined bioreactive membrane C (in this example, a hydrophilic or hydrophilic-treated polymer film selected from cellulose acetate, polycarbonate, polypropylene, silicone, Teflon, etc.) or a porous film thereof is applied in advance to the stepped portion 6. Then, an interference blocking layer 111c2 (for blocking interference other than the measurement target) is laminated on the lower surface of enzyme-immobilized llCl, which has glucose oxidase, which is an example of an enzyme, bonded, thermally fused, or ultrasonic fused. The biological reaction membrane C is integrally held by the annular holder member by directly fixing it by means such as bonding. A tapered surface 7 for guiding when the reaction membrane chimp Z is fitted into and placed in the upper surface side recess A of the measurement electrode main body X is formed on the outer lower end circumference of the annular holder member E.

By the way, when the reaction membrane chip Z is not being measured, it is stored in a tank T containing a buffer solution B, as shown by the imaginary line in FIG. At the time of measurement, it is taken out from the buffer solution B of the tank T and fitted into the recess A of the flat measuring electrode main body X as shown in FIG. 1(c).

Therefore, in the measurement state, between the response electrode surface Y at the bottom of the recess A of the flat measurement electrode main body X and the lower surface of the biological reaction membrane C in the reaction membrane chip Z, the biological A buffer solution layer S having a constant thickness determined by the installation height of the reaction membrane C and the height of the inner step 6 is formed.

Now, the biosensor configured as described above has a biological reaction Hc in the reaction membrane chip Z, which is a component of the biosensor.
Since the enzyme-immobilized membrane C1 is used as the enzyme-immobilized membrane C1, it functions as an enzyme electrode in this case, as shown in Fig. 1 (c).
As shown in FIG. 2, an instrument body Q consisting of a preamplifier 8.9, an arithmetic circuit 10, etc. is connected to the connector portion 5 of the flat measuring electrode body The membrane chip Z is fitted and mounted manually using a pinscent or the like, and then a small amount of sample (in this case, a sample solution containing glucose) is poured from above the reaction membrane chip Z using, for example, a micropipette P.
By just performing a very simple measurement operation of dropping , a reaction occurs on or inside the biological reaction membrane C (enzyme immobilization membrane C1) in the reaction membrane Chimp Z, and an electrode active substance (hydrogen peroxide) is generated. The electrode active substance diffuses into the buffer solution ls, and a response current is generated at the response electrode surface Y (platinum electrode 2 and silver electrode 3), which is taken out as an output signal corresponding to the glucose concentration in the sample. It is possible.

By the way, FIG. 2 is a graph showing raw data obtained when the thickness t of the buffer solution layer S is changed to various values and measurements are made on the same sample. It can be seen that if the thickness t of the buffer solution layer S is too small, overshoot tends to occur, and if it is too large, the response becomes extremely slow. Therefore,
From this experimental result, it can be said that it is desirable to set the thickness t of the buffer solution layer S to 1100 II to 200 μm.

One way to easily set a relatively small appropriate thickness of this buffer solution layer S is as follows.
As shown in the modified example of FIG. 3, while the installation height of the biological reaction membrane C in the reaction membrane chip Z (the height of the inner circumferential side step 6) is set to a relatively large predetermined thickness, flat measurement The central part A' of the concave part A of the electrode body X is raised by a predetermined height,
The height of the inner peripheral step portion 6 in the reaction membrane chip 2,
It is conceivable to set the thickness of the buffer solution layer S to a desired value based on the difference in height between the height of the central portion A° of the recessed portion A and the height of the central portion A° of the recessed portion A. In addition, the annular holder member E in the reaction membrane chip Z
In order to hold the bioreactive membrane C integrally with the membrane, instead of directly fixing it by means of adhesive, heat fusion, or ultrasonic fusion as described above, in the modified example shown in FIG. As shown by the dotted line, a fixing ring member may be fitted into the annular holder member E to sandwich and fix the biological reaction membrane C between them.The other configurations are the same as those described above. Therefore, members having the same functions will be designated by the same reference numerals, and their explanation will be omitted.

Figure 4 is a graph showing the results of verification measurement experiments for standard glucose at various concentrations using the biosensor configured as described above. Accuracy and linearity are obtained.

Further, FIG. 5 shows that standard albumin (antigen) of various concentrations as a sample to be measured is applied to a carrier film on which an anti-albumin antibody is immobilized as a biological reaction membrane C in the reaction membrane chip Z. After the reaction has occurred, an immunoreaction treated membrane consisting of a sandwich-bound glucose oxidase-labeled anti-albumin antibody is used, and a substrate solution is dropped from above the reaction membrane tube Z (in this case, This is a graph showing the results of a verification measurement experiment (this biosensor is used as an enzyme immunoelectrode). A certain S-shaped calibration curve was obtained according to theory, and sufficient measurement accuracy was ensured in this case as well.

〔Effect of the invention〕

As is clear from the detailed description above, according to the biosensor according to the present invention, the inside of the recess in the flat measuring electrode main body, which is formed on the upper surface side of the recess with the response electrode surface at the bottom, is Since the reaction membrane chip is constructed by integrally holding a predetermined bioreaction membrane in advance in an annular holder member having a shape and size that allows it to be removably fitted and placed on the There is no need to handle the bioreaction membrane itself; various treatments for the bioreaction membrane, storage of the reaction membrane chip, attachment/detachment of the bioreaction membrane to the response electrode surface of the measurement electrode body, etc., are performed on the bioreaction membrane itself. It can be carried out very easily without direct contact, and therefore without the risk of contaminating the biological reaction membrane or the response electrode surface.Moreover, since the measuring electrode body is constructed in a flat plate shape, the measuring electrode body itself can be replaced with a conventional Rondo shape. It can be made much simpler and more compact than conventional devices, and it can be measured using an extremely simple method of dropping the sample onto the biological reaction +1Q on the reaction membrane chip using a micropipette. There is no need to set up a large-scale sampling system as in It has come to pass.

[Brief explanation of the drawing]

Figures 1 to 5 show specific embodiments of the biosensor according to the present invention, where Figure 1 (a) is a longitudinal cross-sectional side view of the measurement electrode body, and Figure 1 (b) is a longitudinal cross-sectional view of the reaction membrane chip. Side view,
Figure 1 (c) is an explanatory diagram of the measurement state, and the second
The figure is a graph of measurement results examining the influence of the thickness of the buffer solution layer, Figure 3 is a longitudinal cross-sectional side view of a modified example, and Figure 4 is a graph of measurement results when used as an enzyme electrode.
FIG. 5 is a graph of measurement results when used as an enzyme immunoelectrode. In addition, the 6th session is for explaining the technical background of the present invention and the problems of the prior art, and is shown in Figure 6 (a).
6 shows an exploded side view of a biosensor having a conventional configuration, and FIG. A: Concave portion, X: Flat measuring electrode body, Y:
...Responsive electrophotographic surface, C...Biological reaction membrane, E...Annular holder member, Z...
...Reaction membrane chip, B...Buffer solution, S...Buffer solution layer. Applicant: Horiba Manufacturing Co., Ltd. Agent: Hideo Fujimoto A: Concavity, X: Flat measuring electrode body, Y:
...Response electrode surface, C...Bioreaction membrane, E...Annular holder member, Z...
...Reaction membrane chip, B...Buffer solution, S...Buffer solution layer. Fig. 1 A: Concavity, X: Flat side constant voltage model body, Y:
...Response electrode surface, C...Bioreaction membrane, E...Annular holder member, Z...
...Reaction membrane chip, B...Buffer solution, S...Buffer ts liquid layer e Fig. 1 (c) Fig. 2 1 Season' 3 Figure 6 (a) (b) Ants and koji throat ↓

Claims (1)

[Scope of Claims] [1] A flat measuring electrode body having a recess formed on the top side with a response electrode surface provided at the bottom, and a flat measuring electrode body that is removably fitted into the recess of the flat measuring electrode body. A reaction membrane chip is formed by integrally holding a predetermined biological reaction membrane in advance in an annular holder member having a shape and size that allows the reaction membrane to be placed, and the reaction membrane chip is stored in a buffer solution when not measuring. In the measurement state in which the membrane chip is fitted and placed in the recess of the flat measurement electrode main body, a buffer solution layer of a constant thickness is formed between the response electrode surface and the lower surface of the biological reaction membrane at the bottom of the recess. A biosensor characterized in that it is configured to [2] The thickness of the buffer solution layer is 100 μm to 200 μm.
Claim No. 1 that is configured to be μm
Biosensor as described in Section.