JPH02249480A - Bio-sensor - Google Patents

Abstract

PURPOSE:To accurately, continuously and readily measure the concentration of a substrate in a test solution by passing the test solution through a tube having a biological catalyst adhered to the inner wall thereof and transmitting light through the inner portion of the tube to detect the light transmittance of the test solution. CONSTITUTION:A test solution-charging device 3 and a test solution-discharging device 4 are disposed at one end of a tube 1 having a biological catalyst adhered to the inner wall thereof and at the other end thereof, respectively. A means 5 for radiating a light beam directly or through an optical fiber and a means 7 for receiving the light beam passed through the tube directly or through an optical fiber are disposed at one end of the tube 1 and at the other end thereof, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a biosensor that measures the concentration of a substrate (analyte) in a test liquid. INDUSTRIAL APPLICATION This invention is utilized for process control, environmental measurement, medical diagnosis, measurement, etc. of pharmaceuticals, food industry, chemical industry, etc.

[Conventional technology]

Conventional a storage measurement devices include electrode methods (devices) used for oxygen snow scraping and hydrogen peroxide electrodes, electrical methods (devices) using thermistor methods, and optical methods (devices) using absorbance methods and fluorescence methods that use colored or luminescent substances. A method (apparatus) is known.

[Department notification that the invention attempts to solve]

Devices based on the electrode method described above are susceptible to electromagnetic noise because they measure minute currents and potentials. The device based on the thermistor method uses a precise and delicate bridge circuit, so it has drawbacks in portability and noise resistance. Furthermore, the apparatus based on the optical method requires a special coloring agent and has a complicated apparatus configuration, making it difficult to put it to practical use.

The present invention has been made in view of the above-mentioned viewpoints, and it has been discovered that the propagation characteristics of light change due to the generation of a 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 is complete.

The present invention is suitable for continuous measurement and is suitable for receiving electrical noise.
The present invention aims to provide a biosensor that is simple and inexpensive, can be applied to many reaction systems, and can be remotely controlled as necessary.

[Means to solve the problem]

The biosensor of the present invention includes a tubular body having at least a catalyst on the inner wall that promotes the reaction of a substrate contained in a test liquid;
A test liquid introducing means and a deriving means are attached to one end and the other end of the tubular body, respectively, and a light emitting element and a light receiving element are arranged at the one end and the other end of the tubular body, respectively. It is characterized by

Here, "having a catalyst on at least the inner wall" means that a catalyst layer may be formed on the inner peripheral surface of the tubular body, or a catalyst material may be supported (fixed) on the entire interior of the tubular body itself or on its surface. It is used in the sense that it may be permitted to This catalyst layer is
Usually, a porous support layer is formed and a predetermined catalyst substance is supported on this layer.

Incidentally, various auxiliary agents, dispersants, etc. can be used as necessary. This catalyst layer may be formed on a part of the inner wall surface. The thickness, porosity, method of forming the coating layer, etc. are not limited.

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

[Effect]

When a test liquid containing a substrate is introduced into the tubular body, the substrate undergoes a catalytic reaction by an enzyme or the like on the inner wall surface, and new substances are generated as the substrate is decomposed. Along with this reaction, each reaction and product substance generates a concentration distribution that is symmetrical to the central axis of the tubular body, as shown, for example, in FIGS. 2(a) to (C). These substances have different concentration diffusion rates due to differences in their properties such as molecular weight, molecular shape, and solvent affinity. Therefore, a refractive index distribution that is symmetrical about the central axis is produced as a whole. The type of this refractive index distribution is shown in Figures 4 and 2.
As shown in figure (d), when the maximum is on the center side (a)
There is a case (b) where it becomes the minimum. As shown in Figure 3,
In the former case, the light incident on the sample liquid is bent inward, reducing reflection, absorption, and scattering on the wall surface, and the number of reflections also decreases, reducing the amount transmitted and absorbed by the inner wall. , the amount of light received increases. In the latter case, it is bent outward and the relationship is opposite to that described above. For comparison, refraction,
A conventional example without a rate distribution is shown in dotted line (c).

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

〔Effect of the invention〕

As shown in the above action, this biosensor shows good proportionality, especially linearity, over a wide concentration range of substrates due to the combination of a simple optical system part and a tubular body. Can perform highly accurate measurements. Also,
Unlike optical methods, it allows continuous measurement, is not affected by pH, and is less susceptible to electromagnetic noise than electrical methods, allowing stable measurements. Furthermore, since the catalytic reaction can be freely selected and a large number of reaction systems can be used, the range of applications is wide.

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

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 This biosensor measures sucrose concentration, and as shown in FIG. 1, it consists of a tubular body 1, a catalyst layer 2, an introduction section 3 as a means for introducing a test liquid, and an output section as an extraction means. 4
, a He-Ne laser 5 , an optical meter 61 , and a constant temperature layer 8 .

This tubular body 1 has an inner diameter of 2 ff1 mφ and an outer diameter of 3 ff1
It is an alumina tube (purity 99°9%) with mφ and length 100m+a with both ends open. A catalyst tank 2 in which an enzyme (invertase) is immobilized on an alumina porous membrane is formed on the inner wall of the tubular body 1. On both end sides of this tubular body 1, a flange 31.41 disposed on the inside, a glass window 33.43 disposed at the end, a cylindrical portion 32.42 disposed therebetween, and a cylindrical portion 32.42 disposed therebetween. An inlet 34 attached to the side of the
Alternatively, the medium introduction section 3 or the medium outlet section 4 consisting of the outlet port 44 is attached so as to be removable. The test liquid A enters the inlet 34 and exits from the outlet 44. Note that the introduction and extraction of the medium may be reversed to the above. A sealing material was disposed at the contact portion between the tubular body 1 and the inlet portion 3 or the outlet portion 4 to ensure sealing performance.

Then, a predetermined laser device 6 is installed on the glass window 33 on the inlet side.
were placed opposite to each other, and a plastic optical fiber (1 mmφ) 7 was inserted through the other glass window 43 to be placed opposite to the other end surface of the tubular body, which was further connected to an optical meter 61. still,
The optical fiber 7 may be arranged opposite to the glass window 43 without passing through it. The He-Ne laser 6 serves as a light emitting source, and this laser light is transmitted into the tubular body l through the glass window 33, and is transmitted to the optical fiber 7 disposed at the other end.
The amount of light received is detected by an optical meter 61 via the optical meter 61.

This device was manufactured as follows. That is, first, the above-mentioned alumina tube was prepared, and an alumina porous membrane was baked on the inner wall of the tube in the following manner. That is, alumina (purity 99
．． 9%, commercially available product with an average particle size of 0.6 μm), 80 g of deionized water, and 1 g of ammonium polycarboxylate (dispersant) were mixed with alumina raw stone (10 mmφ) with a purity of 99.9%.
Along with 300g, the internal volume is 500mj! The mixture was placed in a polyethylene container and mixed and dispersed at 12 ORPM for 48 hours. After passing the slurry thus obtained through a 200 mesh sieve,
After transferring to a graduated cylinder and immersing the alumina tube,
Pull it up and apply the inner surface. Next, this was naturally dried for 15 hours and then fired in an electric furnace at 1150° C. for 6 hours to form a porous alumina membrane. This alumina film has a thickness of 35μ
The m1 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.

Thereafter, this was immersed for 15 hours in a 10% T-aminopropyltriethoxysilane a7tone solution for silanization treatment. It was air-dried for 1 hour, and then mixed with 5% glutaraldehyde in 005M phosphate buffer (pH
7,0) for 4 hours. Next, add this to the above enzyme 1
60m of 0.05M phosphate buffer (pH 7.0) containing g
to immobilize the enzyme. If you wish to preserve it, immerse it in 0.05M phosphate buffer (pH 70) at 4°C.

The introduction section 3, outlet section 4, and constant temperature bath 8 were attached to this tubular body, and then the laser device W5, the optical fiber 7, and the optical meter 61 were arranged to produce the present biosensor.

In addition, sucrose was dissolved in a phosphate buffer solution at various concentrations to prepare predetermined test solutions. Then, the temperature of the constant temperature bath 8 is set to 35°C, and the test liquid is introduced using the pump 9 at a flow rate of 0.33 m1/min, and is continuously supplied to the inlet 34 to test the inside of the tubular body. While maintaining the liquid in a laminar flow state, a laser beam with a wavelength of 543 nm and an output of 1 mW was made to enter approximately the center of the tubular body 1, and the amount of light received was measured. Note that various known means other than the pump may be used as the test liquid supply means.

In this example, reactions according to the following formulas were carried out, and the respective reactions, concentration distributions of produced substances, etc. are conceptually shown in FIGS. 2(a) to 2(c).

Sucrose (a) Chiki → α-low glucose (b) + fructose (c) α-D
- Glucose and fructose have a high diffusion rate because their molecular weight is about half that of sucrose. Therefore, the gradients (b) and (c) of this concentration distribution are gentler than that of sucrose (a).
Therefore, the entire refractive index distribution (d) exhibits a maximum convex shape at the central axis.

From the above, the results of the relationship between sucrose concentration and the amount of light received can be summarized as
Shown in the figure. Next, as a comparative example, a test was conducted in the same manner as in the above example except that no catalyst was used, and the results are also shown in the same figure.

As shown in this figure, in the comparative example, even if the substrate concentration is increased, the gradient (change) in the relationship between the amount of received light and the concentration is extremely small, so sufficient sensitivity for that concentration cannot be obtained and it is not suitable for detection. do not have. On the other hand, in this embodiment, the refractive index on the center side of the tubular body is smaller than that on the inner wall side, and the light is bent inward as shown in FIG.
The amount of absorbed energy decreased and the amount of light received increased. A good linear relationship with a large slope was observed over a wide concentration range.

Therefore, by using this biosensor, the sucrose concentration can be measured satisfactorily and with high sensitivity over a wide concentration range, and furthermore, it is possible to perform continuous measurement at high speed without being affected by electrical noise.

Embodiment 2 In this embodiment, as shown in FIG.
An LED lamp 51 is used as a light emitting element for the outlet port 4a, and an optical fiber 7 is also used for the inlet port 3a, and the tips of both optical fibers 7.71 are directly connected to the inside of the tubular body via the rubber seal 13.14. It is located in Note that the introducing means or the deriving means may also be a medium introducing part or a medium deriving part as in the first embodiment.

In this case, the overall structure can be made simple and compact, which is convenient for continuous measurement.

It should be noted that the present invention is not limited to what is shown in the above-mentioned specific examples, but can be variously modified within the scope of the present invention depending on the purpose and use. That is, the above-mentioned tubular body may be one that allows the test liquid to pass through, and its size, length, overall shape, cross-sectional shape, material, etc. can be selected from various types depending on the purpose and use. . For example, its overall shape may be curved rather than straight, and its cross-sectional shape is usually a perfect circle, but it can also be square, hexagonal, elliptical, etc., and even honeycomb-shaped or lotus root-shaped. It may have a plurality of flow passage holes as shown in FIG. This material is not limited to ceramics, but may also be glass, metal, etc.

Further, the porous membrane can also be made of glass, resin, metal, or the like. When measuring glucose in blood, a material that prevents blood coagulation, such as hydroxyapatite, silicone resin, chitin, chitosan, etc., can be used for the porous membrane. Biocatalysts include, but are not limited to invertase, urease, sikolate decarboxylase,
Alcohol oxidase, maltase, glucose oxidase, etc. can be used.

The medium for the test liquid may be gas instead of liquid. Other known elements can also be used as the light emitting element and the light receiving element. These elements may be directly attached to the tubular body. Various lengths, thicknesses, materials, shapes, mounting positions, etc. of the optical fibers can be selected; for example, the material is not limited to resin, but may also be glass. Furthermore, the method of irradiating light by the light emitting element may be such that the entire end surface of the tubular body is irradiated almost uniformly, and in the case of a laser, irradiation is usually performed almost at the center as in Example 1, but the method is not limited to this. , near the pipe wall,
It is also possible to irradiate areas close to the center. A location close to the tube wall has the effect of improving sensitivity. Further, the diameter of the light beam is also selected depending on the purpose and the like.

[Brief explanation of drawings]

FIG. 1 is an explanatory cross-sectional view of a biosensor according to Example 1,
Figure 2 shows the concentration distribution of reactants and the overall refractive index distribution in Example 1, (a) is sucrose, (b) is α
-D-glucose, (c) is a conceptual explanatory diagram showing each concentration distribution of fructose, (d) is the entire refractive index distribution, 3rd
The figure is an explanatory diagram showing the trajectory of light passing through the tubular body, and Figure 4 is an explanatory diagram showing the distribution of refractive index occurring in the radial direction of the tubular body. FIG. 5 is an explanatory cross-sectional view of the biosensor according to Example 2, and FIG. 6 is a graph showing the relationship between the sucrose concentration and the amount of light received in Example 1. l: tubular body, 2: catalyst layer, 3; introduction part (means) 4; outlet part (means), 5; laser device, 51; LED lamp, 6
; Light receiving element, 61; Optical meter, 7; Optical fiber, 8; Constant temperature chamber. Patent applicant: NGK Spark Plug Co., Ltd. Agent: Kiyoji Kojima, Figure 3, Figure 5, Figure 6 Sucrose; 1 degree (mm-1:, 1) Procedural amendment (voluntary) l. 2゜3゜Indication of the case 1999 Patent Application No. 72770 Name of the invention Person who makes biosensor correction Relationship to the case Patent applicant 14-18 (454) Takatsuji-cho, Mizuho-ku, Nagoya City Representative of NGK Spark Plug Co., Ltd. Person Suzukitei - 4゜

Claims (1)

[Claims] (1) A tubular body having at least a biocatalyst on its inner wall that promotes the reaction of a substrate contained in the test liquid, and an introduction means attached to one end of the tubular body for introducing the test liquid into the inside of the tubular body. and a derivation means attached to the other end of the tubular body to derive the test liquid from the tubular body, and a derivation means disposed at the one end of the tubular body, either directly or via a light transmission optical fiber. A biosensor comprising: a light-emitting element; and a light-receiving element disposed on the other end side of the tubular body directly or via a light-receiving optical fiber.