JPH0714336B2 - Biosensor - Google Patents

Description

The present invention relates to a biosensor for measuring the concentration of a substrate (substance to be measured) in a test liquid. INDUSTRIAL APPLICABILITY The present invention is used for process control of pharmaceuticals, food industry, chemical industry, etc., environmental measurement or medical diagnosis, measurement, etc.

[Conventional technology]

As a conventional concentration measuring apparatus, an electrical method (apparatus) based on an electrode method and a thermistor method used for oxygen electrodes and hydrogen peroxide electrodes, and an optical method based on an absorbance method and a fluorescence method using a substance that emits color or emits light. (Device) is known.

[Problems to be Solved by the Invention]

The device using the electrode method is susceptible to electromagnetic noise because it measures minute currents and electric potentials. Since the device using the thermistor method uses a precise and delicate bridge circuit, it has a problem in portability and noise resistance. In addition, the device using the above optical method requires a special color former and has a complicated device structure, which is a problem in the practical method.

The present invention has been made in view of the above problems, and it has been found that the propagation characteristics of light are changed by the refractive index distribution in the radial direction of the test liquid inside the tubular body due to the catalytic reaction on the inner wall. It has been completed.

An object of the present invention is to provide a biosensor suitable for continuous measurement, less susceptible to electric noise, simple and inexpensive, applicable to many reaction systems, and capable of remotely controlling the process as needed. .

[Means for Solving the Problems]

The biosensor of the present invention comprises a tubular body having a catalyst for promoting the reaction of a substrate contained in the test solution only on the inner wall, and a test solution introducing means attached to one end side and the other end side of the tubular body, respectively. And a light emitting element and a light receiving element which are respectively disposed on one end side and the other end side of the tubular body.

Here, "having a catalyst only on the inner wall" may be formed by coating a catalyst layer on the inner peripheral surface of the tubular body, or may be supporting (including fixing) a catalyst substance on the surface portion. Used for meaning. This catalyst layer usually forms a porous support layer, and a predetermined catalyst substance is supported on this layer. If necessary, various auxiliaries, dispersants and the like can be used. This catalyst layer may be formed on a part of the inner wall surface. The film thickness of the coating layer, the porosity, the method of forming the same, and the like do not matter.

This catalyst is a biocatalyst that promotes the reaction of the substrate in the test liquid, various enzymes, yeasts, microorganisms, antibodies, etc. can be used, and it is appropriately selected depending on the type of the substance to be measured.

[Action]

When the test liquid containing the substrate is introduced into the tubular body, the substrate undergoes a catalytic reaction by an enzyme or the like on the inner wall surface thereof, and a new substance is generated along with the decomposition of the substrate. Along with this reaction, the respective reaction and product substances produce a concentration distribution symmetrical to the central axis of the tubular body, as shown in, for example, FIGS. These substances have different rates of concentration diffusion due to differences in properties such as molecular weight, molecule formation, and solvent affinity. Therefore, a refractive index distribution symmetrical with respect to the central axis is produced as a whole. As for the type of this refractive index distribution, as shown in FIG. 4 and FIG. 2 (d), there are cases of maximum (a) and minimum (b) on the center side. As shown in FIG. 3, in the former case, the light incident on the test liquid A is bent inward, and the reflection, absorption, and scattering on the wall surface are reduced, and the number of reflections is also reduced. The amount of light transmitted and absorbed decreases, and the amount of light received increases. In the latter case, it is bent outward, which is the reverse of the above. For comparison,
A conventional example having no refractive index distribution is shown by a dotted line (C).

From the above, in the case of the present invention, since the refractive index distribution in the radial direction of the tubular body, the amount of light transmission increases or decreases depending on the presence or absence of the catalyst or the substrate concentration, and both show a proportional relationship. Become. Incidentally, this tendency becomes larger as the concentration of the reactant becomes higher.

〔The invention's effect〕

As shown in the above action, in the present biosensor, the combination of the simple optical system portion and the tubular body shows a good proportional relationship up to a wide concentration range of the substrate, in particular, linearity, so that the biosensor has a high reliability in the wide concentration range. High measurement can be performed. Also,
Unlike optical methods, continuous measurement is possible and is not affected by pH,
Electromagnetic noise is less likely to occur as compared with the electrical method, so stable measurement can be performed. Furthermore, since the catalytic reaction can be freely selected and a large number of reaction systems can be used,
Its application range is wide.

Further, when an optical fiber is used, the process can be remotely controlled by extending the optical fiber, which is very useful.

〔Example〕

 Hereinafter, the present invention will be specifically described with reference to examples.

Example 1 The present biosensor measures the sucrose concentration, and as shown in FIG. 1, the tubular body 1, the catalyst layer 2, the introducing section 3 as the test solution introducing means, and the deriving section as the deriving means. Four
And a He-Ne laser 5, an optical meter 61 and a constant temperature layer 8.

The tubular body 1 is an alumina tube (purity 99.9%) having an inner diameter of 2 mmφ, an outer diameter of 3 mmφ and a length of 100 mm. On the inner wall of the tubular body 1, a catalyst layer 2 in which an enzyme (invertase) is immobilized on a porous alumina membrane is formed. On both end sides of the tubular body 1, the flange portions 31 and 41 arranged inside, the glass windows 33 and 43 arranged at the end portions, the cylindrical portions 32 and 42 arranged between them, and the cylindrical portions 32 and 42. The medium introducing section 3 or the medium ejecting section 4 including the introduction port 34 or the ejection port 44 attached to the side portion of is attached so as to be removable. The test liquid A enters the inlet 34 and exits the outlet 44. The introduction and the derivation of the medium may be reversed. A sealing material was arranged in the tubular body 1 and the catalyst portion of the introduction portion 3 or the extraction portion 4 to ensure the sealing property.

Then, a predetermined laser device 6 is placed in opposition to the glass window 33 on the inlet side, a plastic optical fiber (1 mmφ) 7 is inserted in the other glass window 43 to be placed in opposition to the other end surface of the tubular body, and this is further attached to the optical meter. Connected to 61. The optical fiber 7 may be arranged opposite to the glass window 43 without penetrating the glass window 43. The He-Ne laser 6 serves as a light emission source, and the laser light is transmitted to the inside of the tubular body 1 through the glass window 33, and the amount of light received is detected by the optical meter 61 through the optical fiber 7 arranged on the other end side. To do.

This device was manufactured as follows. That is, first, the alumina tube was prepared, and an alumina porous film was baked on the inner wall of the alumina tube as follows. That is, alumina (purity 99.9
%, A commercially available product having an average particle size of 0.6 μm) 100 g, deionized water 80 g and ammonium polycarboxylate (dispersant) 1 g and a purity of 99.9.
% Of alumina ball stone (10mmφ) 300g, internal volume 500m
Place in a polyethylene container (1) and mix and disperse at 120 RPM for 48 hours. The sludge thus obtained is passed through a 200-mesh screen, then transferred to a graduated cylinder, the alumina tube is dipped therein, and then the inner surface is pulled up and applied. Next, this was naturally dried for 15 hours and then baked in an electric furnace at 1150 ° C. for 6 hours to form a porous alumina film. This alumina film has a thickness of 35μ
m, the porosity was 45%, and the pore diameter was 0.3 μm (average).

Further, the tubular body is treated with concentrated hydrochloric acid for 1 hour. afterwards,
This was immersed in an acetone solution of 10% γ-aminopropyltriethoxysilane for 15 hours for silanization treatment.
Then, it was naturally dried for 1 hour, and then immersed in 5% glutaraldehyde 0.05M phosphate buffer (pH 7.0) for 4 hours. Next, this was immersed in 60 ml of 0.05 M phosphate buffer (pH 7.0) containing 1 g of the above enzyme to immobilize the enzyme. still,
If you want to store this, please add 0.05M phosphate buffer (p
H7.0).

The introduction part 3, the extraction part 4 and the constant temperature bath 8 were attached to the tubular body, and then the laser device 5, the optical fiber 7 and the optical meter 61 were arranged to manufacture the present biosensor.

Also, sucrose was dissolved in phosphate buffer at various concentrations to prepare a predetermined test solution. And the temperature of the constant temperature bath 8
The test liquid is set at 35 ° C., the test liquid is fed at a flow rate of 0.38 ml / min using the pump 9, and continuously supplied to the inlet 34 so that the test liquid inside the tubular body is kept in a laminar flow state. And wavelength 54
A laser beam having a wavelength of 3 nm and an output of 1 mW was incident on the tubular body 1 substantially at the center, and the amount of received light was measured. Note that various known means other than a pump can be used as the test liquid supply means.

In this example, the reactions of the following formulas are carried out, and each reaction, the concentration distribution of the produced substance, etc. are conceptually shown in FIGS. 2 (a) to (c).

Sucrose (a) + water → α-D-glucose (b) + fructose (c) α-D-glucose and fructose have a large diffusion rate because their molecular weight is about half that of sucrose. Therefore, since the gradients (b) and (c) of this concentration distribution are gentler than those of sucrose (a), the entire refractive index distribution (d) shows the maximum convex shape on the central axis.

From the above, the result of the relationship between the sucrose concentration and the amount of received light is
As shown in the figure. Next, as a comparative example, a test was conducted in the same manner as the above example except that the catalyst was not included, and the results are also shown in the same figure.

As shown in this figure, in the comparative example, the substrate concentration is increased and the gradient (change) in the relationship between the amount of received light and the concentration is extremely small. Therefore, sufficient sensitivity to that concentration cannot be obtained and it is not suitable for detection. . On the other hand, in the present embodiment, the refractive index of the center side of the tubular body is larger than that of the inner wall side, and the light is bent inward as shown in FIG. 3 (a), so that the amount of transmission and absorption at the tube wall is reduced. , The amount of received light has increased. Then, a good linear relationship with a large inclination was shown in a wide concentration range.

Therefore, by using the present biosensor, it is possible to measure the sucrose concentration satisfactorily and with high sensitivity in a wide concentration range, and it is also possible to perform continuous measurement at high speed without receiving electrical noise.

Example 2 In this example, as shown in FIG. 5, the inlet 3a and outlet 4a directly attached to the tubular body 1 were used as the test liquid introducing means and the discharging means, and the LED lamp 51 was used as the light emitting element. Moreover, the optical fiber 7 is also used on the side of the introduction port 3a, and the tips of both optical fibers 7 and 71 are directly disposed inside the tubular body via the rubber seals 13 and 14. The introducing unit or the deriving unit may be the medium introducing unit or the medium deriving unit as in the first embodiment.

In this case, the entire structure can be simple and small, which is convenient for continuous measurement.

The present invention is not limited to the specific examples described above, and various modifications may be made within the scope of the present invention depending on the purpose and application. That is, the tubular body only needs to pass the test liquid, and various sizes, lengths, overall shapes, cross-sectional shapes, materials, etc. can be selected depending on the purpose and application. . For example, the entire shape may be a curved tube instead of a straight tube, and the cross-sectional shape thereof is usually a perfect circle, but it may be a square, a hexagon, an ellipse, etc., and a honeycomb shape or a lotus root shape. You may have several flow-path holes like this. This material is not limited to ceramics, but may be glass, metal, or the like.

The porous film can also be made of glass, resin, metal or the like. When measuring glucose in blood or the like, a material that prevents blood coagulation, such as hydroxyapatite, silicon resin, chitin, chitosan, etc., can be used for the porous membrane. The biocatalyst is not limited to invertase, urease, oxalate decarboxylase,
Alcohol oxidase, maltase, glucose oxidase, etc. can be used.

The medium of the test liquid may be gas instead of liquid. As the light emitting element and the hole receiving element, other known ones can be used. These elements may be directly attached to the tubular body. The length, thickness, material, form, attachment position, etc. of the optical fiber can be variously selected, and the material is not limited to resin and may be glass. Further, the method of irradiating light by the light emitting element may irradiate the entire end surface of the tubular body substantially evenly, and in the case of a laser, it is normally irradiating the light beam substantially at the center as in Example 1, but not limited to this. It is also possible to irradiate a place near the tube wall, a place near the center, or the like. When it is near the tube wall, it has the effect of improving the sensitivity. Also, the diameter of the light beam is variously selected according to the purpose.

[Brief description of drawings]

FIG. 1 is an explanatory sectional view of a biosensor according to Example 1,
FIG. 2 shows the concentration distribution of the reactant and the entire refractive index distribution in Example 1, where (a) is sucrose and (b) is α-.
D-glucose, (c) each fructose concentration distribution,
(D) is a conceptual explanatory diagram showing the entire refractive index distribution, FIG. 3 is an explanatory diagram showing the trajectory of light passing through the tubular body, and FIG. 4 is that the refractive index distribution occurs in the radial direction of the tubular body. FIG. 5A is an explanatory view showing a state where the center side is large and (B) a large inner wall side, FIG. 5 is an explanatory sectional view of the biosensor according to Example 2, and FIG. It is a graph which shows the relationship between a sucrose concentration and the amount of light received. 1; tubular body, 2; catalyst layer, 3; introduction part (means), 4; derivation part (means), 5; laser device, 51; LED lamp, 6; light receiving element, 61;
Optical meter, 7; optical fiber, 8; constant temperature bath.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Otoshi Tsuneto 14-18 Takatsuji-cho, Mizuho-ku, Nagoya-shi, Aichi Nihon Special Ceramics Co., Ltd. (72) Inventor Junichi Tokumoto 14 Takatsuji-cho, Mizuho-ku, Aichi No. 18 Nihon Special Ceramics Co., Ltd. (72) Inventor Hideho Aoki 14-18 Takatsuji-cho, Mizuho-ku, Aichi Prefecture Nagoya, Japan Nihon Special Ceramics Co., Ltd.

Claims (1)

[Claims] 1. A tubular body having biological contact for promoting reaction of a substrate contained in a test solution only on an inner wall, and a test solution introduced into one end of the tubular body to introduce the test solution into the tubular body. Introducing means, lead-out means attached to the other end side of the tubular body and leading out the test liquid from the tubular body, and arranged at one end side of the tubular body directly or through an optical fiber for light transmission. And a light receiving element which is arranged on the other end side of the tubular body directly or through an optical fiber for receiving light.