JPH0697989B2 - Enzyme activity measuring sensor and measuring device and method using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an optical fiber sensor used for optical measurement of enzyme activity due to contact reaction with a sample, an enzyme activity measuring device incorporating the same, and a measuring method.

The measurement of enzyme activity plays a very important role in biochemistry and clinical analysis. Deviation from baseline activity means the occurrence of certain diseases. For example, the activity of the enzyme "acid phosphatase" is significantly increased when a bone mass or a prostatic mass occurs.

[Conventional technology]

Many methods are known for measuring the activity of enzymes. For example, "Enzymatic Methcds of Analysis," by GG Gilvault.
(Pergamon Press, Oxford, 1970),
And H. Bergmayer's "Grundlagen der enzymatischen Analyse" (Fairark Hemmie, Weinheim-New York, 19).
1977). Recently, there has been much interest in the confirmation of bacterial etiologies using protease substrates (COBURN, LYTLE, Huba (H
UBER) "Analytical Chemistry (Anal.Chem) 57" (1669, 198)
5 years))). For example, it is known to measure enzyme activity using synthetic dyes or fluorescent substrates. For this measurement an enzyme substrate is used which decomposes into a pigment product under the influence of the enzyme. The increase in color or fluorescence that changes with time is used as a measure of enzyme activity. Measurable hydrolases particularly suitable for this method include carboxylesterases, phosphatases, dealkylases, glycosidases and proteases.

A typical example is "Analytical Chemistry, 143, 146" by KOLLER and WOLFBEIS (1984).
According to the method for measuring phosphatase described in the above, a phosphate ester having the following structure A is decomposed into two components by the enzyme "acid phosphatase", and an ionic hydrolysis product B and a free phosphate ion are produced.

This henolate-ion B, unlike the starting substrate A, emits a strong yellow or blue-green fluorescence. The color change and fluorescence increase over time are used as a measure of the activity of "acid phosphatase".

This measurement method has high sensitivity and is
It allows for the photometric detection of 0.001 activity units of phosphatase and about 60 micro units per milliliter in fluorescence measurements.

Similarly, methods for measuring amylase such as carboxylesterase and sulfatase are known.

Proteases can be measured in a similar manner using synthetic peptides or amides, as described in the Cobain, Lytle, and Huber article, supra. All these methods have in common that the enzymatic reaction gives rise to various dyes or fluorescent products.

[Problems to be Solved by the Invention]

None of the above-mentioned known measuring methods are suitable for direct application to a living organism, that is, a living body, and enzyme activity measurement has hitherto been performed only in a test tube. However, for example, in-vivo measurement can be performed in real time, and thus it is desired for various reasons. On the contrary, the conventional measurement method requires sampling, and an error that occurs when sampling or preparing a sample cannot be avoided.

Furthermore, the above-mentioned measurement method using an enzyme substrate is suitable only when an optically transparent solution or sample is provided, and is not suitable for in vivo measurement of blood or the like.

Therefore, an object of the present invention is to solve the above-mentioned problems of the prior art, and particularly to provide an enzyme activity measuring sensor that enables in vivo measurement of enzyme activity, a measuring device and a measuring method using the same. To provide.

[Means for Solving the Problems]

In order to achieve the above object, according to the present invention, there is provided a sensor for measuring enzyme activity, which is an optical fiber sensor in which an enzyme substrate is arranged at the end of an optical fiber. The enzyme activity is measured by measuring the change in the optical spectral properties of the enzyme substrate or the reaction product from the enzyme substrate caused by the reaction. As a specific method of disposing the enzyme substrate on the end of the optical fiber, for example, a method of directly fixing the enzyme substrate to the fiber end surface, a method of peeling off the coating of the end of the optical fiber and fixing it to the outer peripheral surface of the core, a polymer, Ion conversion membrane, polyacrylamide, a method of fixing it on a carrier film such as cellulose and attaching it to the end of an optical fiber, or a method of fixing and containing an enzyme substrate on a water-soluble or swellable polymer carrier by an enzyme-permeable membrane. The method of enclosing and attaching to the end of the optical fiber is preferable. The enzyme activity measuring device is configured by including a photodetector for measuring the light output from the optical fiber sensor thus obtained and giving a measurement signal to the evaluation device, an optical system including a filter and a lens, and the like.

[Action / effect]

As described above, in-vivo measurement is possible by using the optical fiber sensor in which the enzyme substrate for the hydrolytically active enzyme is arranged at the end of the optical fiber. The enzyme substrate at the end of the optical fiber comes into contact with the sample in the living body, and the spectral characteristic of the enzyme substrate changes depending on the enzyme activity of the sample. This change is measured via an optical fiber. For the first time, this makes it possible to measure enzyme activity in blood or other non-transparent liquids without preparing a sample in advance.

In the embodiment of the measuring device and the measuring method according to the present invention, the change in the light absorption property of the enzyme substrate caused by the enzyme reaction is obtained through the optical fiber and used as the measured value of the enzyme activity. Alternatively, a spectral change of a fluorescent beam of an enzyme substrate caused by an enzymatic reaction, for example, a change in fluorescence intensity or a shift in fluorescence maximum value is obtained through an optical fiber and used as a measurement value of enzyme activity. In the former case, the intensity of the excitation light that is attenuated by the enzyme substrate or reaction product is measured, and the intensity depends on the absorption characteristics of the substrate which is affected by the enzyme activity due to the catalytic reaction. In the latter case, the change in the fluorescence intensity of the enzyme substrate due to the catalytic reaction of the enzyme is measured via an optical fiber using, for example, a filter or an energy dispersive detection system, and the measured value is evaluated.

As another embodiment of the measuring method according to the present invention, it is also preferable to measure the PH value in addition to the enzyme activity by a known method and use this measured value for correction of the previously measured enzyme activity.

As is well known, the rate of many enzymatic reactions is strongly dependent on the pH value of the solution. Furthermore, the degree of dissociation of the dye released during hydrolysis also depends on the PH value. Since the PH value of physiological samples varies within a certain range, it is necessary to correct the actually measured enzyme activity with respect to the PH value. This works best with empirical methods. The PH dependence of enzyme activity is known for many enzymes, but in the case of immobilized enzyme, it has to be measured newly. Individual of the sample
It is useful to measure the PH value at the same time as measuring the enzyme activity. True enzyme activity is most accurately determined by using the experimentally defined relationship between activity and relative signal change as a function of actual PH value.

The measuring device according to the present invention is provided with a photodetector, in which the enzyme substrate is disposed at the end of the optical fiber and comes into contact with the sample, and the output side is connected to the signal evaluation device, and the photodetector reacts with the enzyme substrate. The fluorescence emitted from the product or the reflected excitation light is measured. By using the beam splitter to send a portion of the excitation light to the reference photodetector and using the output signal of this reference photodetector to calibrate the measured output of the photodetector,
It is also possible to remove the influence of fluctuations in the excitation light source. Various measurement techniques can be arranged according to the purpose of measurement and the properties of the enzyme. However, it is common to all measurement systems that the enzyme substrate is arranged at the end of the optical fiber and that the change in spectral characteristics is measured using the optical fiber.

Suitable photodetectors are photomultipliers, phototransistors or photodiodes. Since a photodiode is inexpensive, but it is sensitive only in the visible spectrum region, an enzyme substrate used in this case is preferably one whose decomposition is observed at a wavelength of 450 nm or more, ideally at a wavelength of 500 nm or more. Economical light-emitting diodes can be used as light sources with analysis wavelengths of 460 nm and above.

The light source may be an incandescent lamp, a fluorescent lamp, a light emitting diode or a laser. A dichroic mirror can be used in place of the beam splitter to provide better separation of scattered or reflected excitation light and fluorescence. The optical fiber is preferably a single fiber, but may be a fiber bundle.

It is also possible to use a filter to separate the fluorescence from the reflected excitation light.

In the measuring device according to the present invention, the enzyme substrate is provided by being chemically or physically fixed to the end of the optical fiber. For example, the enzyme substrate is directly fixed to the polished exit end face of the optical fiber. It is also possible to fix the enzyme substrate on the outer peripheral surface of the core by peeling off the coating on the end of the optical fiber. Attenuation of excitation light or generation of fluorescence is caused by so-called evanescent waves. This is an electromagnetic wave that travels along the interface by entering a few nm into the inside of the enzyme substrate, which is a medium with a smaller refractive index than the optical fiber, when total internal reflection occurs at the interface of the optical fiber, and is rapidly attenuated. To do. If a dye is present in the reach of the evanescent wave, the excitation light is absorbed there and also causes fluorescence. The reflected or emitted light returns through the optical fiber and is measured by the photodetector.

Immobilization of the enzyme substrate is performed by various methods. For example, a substrate of the following structure derived from coumarin is suitable for measuring butyl-cholinesterase, and is immobilized on the end of an optical fiber by the following method.

The end face of a quartz optical fiber with a thickness of 200 μm is precisely polished, and the surface is chemically activated by using a 20% sulfuric acid aqueous solution to remove ionic surface stains. Subsequently, the glass surface is treated in a known manner with the glass-deriving reagent aminopropyl-triethoxysilane. This treatment basically involves the active surface of the glass being treated with toluene.
It is carried out by heating in a 10% silane solution at 100 ° C. for 2 hours, washing with acetone, and drying at 120 ° C. In this way, an optical fiber having a free amino group at the end is obtained.

An enzyme substrate is bound to this amino group by a known chemical method. Substrate I described above is immobilized using the peptide coupling reagent ethyl-N '-(3-dimethylaminopropyl) -carbodiimide-hydrochloride. Alternatively,
The substrate may be converted to the corresponding carboxylic acid chloride and then reacted with amino groups on the glass surface.

By contacting the end of the thus obtained optical fiber with a solution containing the enzyme butyrylcholinesterase, 410
A significant increase in light absorption at the analysis wavelength of ~ 430 nm and an increase in fluorescence intensity at 460-470 nm are eventually detected by the measurement device.

In a manner similar to the above example, typical substrates for sulfatase, amylase, protease derived from the same basic molecule are immobilized. Substrates for sulfatase, amylase, and protease are the following structures II and II, respectively.
It has I and IV.

In all cases, the enzymatic activity of the sample results in an increase in absorption at 420 nm and an increase in fluorescence at 460 nm.

The following structure V is a substrate for the enzyme revertase.

It has the structure I-hydroxypyrene-3,
It is an example of an oxygen substrate derived from 6,8-trisulfonate and electrostatically immobilized. This immobilization is due to the interaction between the negatively charged sulfone groups of the substrate and the positively charged surface ammonium groups of the anion exchanger used as carrier.

In yet another embodiment according to the invention, the enzyme substrate can be fixed to a thin carrier film and attached to the end of the optical fiber. Next, a typical preparation of a membrane in which an enzyme substrate is electrostatically immobilized will be described.

The anion exchange membrane is immersed in a solution of 1 g of 1-hydroxypyrene-3,6,8-trisulphonate VI in 50 ml of methanol. After about 2 minutes, the green-fluorescing, yellow-colored, fluorescing membrane is withdrawn from the solution, which is first washed with about 0.06 molar phosphate buffer of PH7 and then with water. After it is dried, it is immersed in a solution of 100 mg of lauric anhydride in dioxane, which converts VI bound to the surface of the membrane into V. This lauric anhydride solution was prepared by dissolving 0.1 g of lauric acid in 1 ml of dioxane and adding 0.1 g of N,
It is made by adding N'-dicyclohexylcarbodiimide and filtering the resulting precipitate. After 2 hours, the membrane is removed from the solution and washed with dry acetone. The film is fluorescent and now bluish-purple and the yellow has disappeared.

When the membrane is attached to the end of an optical fiber and comes into contact with a solution of the enzyme revertase, the substrate is enzymatically converted to a bright green fluorescent yellow hydrolysis product. An increase in absorption at 460 nm or an increase in green fluorescence at 520-535 nm is detected via the optical fiber. This directly measures the enzyme activity in the sample. Degradation of this substrate is caused not only by the enzyme ribase but also by serum albumin. Since the activity of the latter is quite high, this sensor is used for measuring serum albumin in humans, for example in blood.

The latter method is also suitable for producing other immobilized enzyme substrates for hydrolases. It is used not only as a method for preparing enzyme-sensitive membranes, but also for the regeneration of already used sensors.

Degradation of the immobilized enzyme substrate by various hydrolases occurs on the solid surface or substrate of the substrate. Such degradation proceeds more slowly with chemically immobilized substrates than in solutions where the substrate is exposed to the enzyme in all directions around. The slower the rate of hydrolysis, the greater the effect of non-enzymatic substrate hydrolysis on a small scale.

According to the present invention, the presence of a long chain spacer between the surface of the optical fiber or the surface of the carrier film and the enzyme substrate was found to significantly accelerate the rate of enzymatic hydrolysis of the immobilized substrate. It was

As a suitable spacer, a long-chain diamine such as hexamethylenediamine is used. The spacer is directly introduced with the silylating reagent, for example using a "long chain amine".

To prevent dark samples, such as blood, from affecting the optics, it is preferable to cover the end of the optical fiber with a protective cap. This protective cap has large colored particles,
For example, it blocks the entry of red blood cells while allowing the passage of enzymes. The ideal material for this protein-permeable protective cap is cellulose.

Instead of directly immobilizing the enzyme substrate on the end of the optical fiber, it is also preferable to wrap the end of the optical fiber with an oxygen permeable membrane forming a reaction chamber. This membrane contains the enzyme substrate and is impermeable to the cellular components of the sample. The reaction chamber must be permeable to the enzyme to be measured, but at the same time it must prevent the enzyme substrate from diffusing out.

According to the invention, diffusion of the oxygen substrate is prevented by binding the enzyme substrate to the water-soluble or swellable polymer, for example by chemical immobilization. As the water-soluble or swellable polymer used as a carrier for the enzyme substrate in the reaction chamber, dextran, agarose, polyethyleneimine,
There are polyacrylamides or polyalcohols. The carrier polymer must be large enough so that the molecule cannot pass through the protective cap. The immobilized substrate is hydrolyzed by the enzyme, changing its spectral properties. This change is measured as enzyme activity via the optical fiber. Since a considerable amount of the generated dye diffuses through the cellulose membrane, the enzymatic reaction needs to take place much faster than the diffusion.

In the case of the above device, only enzyme substrates whose spectral properties are significantly changed by the enzymatic reaction are used. This is due to the fact that the optical fiber carries signals for both the enzyme substrate and its degradation products. Therefore, in another embodiment, the enzyme substrate is immobilized on the inner peripheral surface of the cylindrical reaction chamber arranged at the end of the optical fiber, and the cells of the sample are attached to the sample-side end face of the reaction chamber. An enzyme-permeable membrane that does not allow the permeation of oxygen is provided, and the reaction product generated by the oxygen reaction is configured to diffuse into the reaction chamber. Due to the separation of the enzyme substrate and its fission products, the production of degradation products is noticeably observed.

Due to the relationship between the maximum exit angle α of excitation light and the diameter and length of the cylindrical reaction chamber, an optical separation is easily obtained, which avoids direct excitation of the enzyme substrate. The cylindrical wall provided with the enzyme substrate on the inner peripheral surface is located outside the region of the excitation light emitted from the optical fiber at the maximum emission angle α, and the enzyme substrate does not generate a signal unless an enzymatic reaction occurs.

When the enzyme to be measured enters the reaction chamber, an enzymatic reaction occurs with the release of dye. The dye disperses in the reaction chamber and its color or fluorescence is detected via the optical fiber. An increase in color or fluorescence intensity is measured as enzyme activity.

The above measuring device has the following basic advantages.

(A) Since the enzyme substrate and its degradation products are spatially separated, there is no overlap in the optical spectra, and a high ineffective value (blank
values) are avoided.

(B) Not only a synthetic enzyme substrate, but also a dye-dyed natural substrate can be used.

The following example illustrates the wide range of applicability of the measuring device according to the invention, the immobilization of which is largely done by the chemical binding of the polymer carrier, the spacer and the natural dye.

(1) By immobilizing 7-oxycoumarin-3-carboxylic acid on the surface of a cellulose membrane or another polymer having an OH group through a spacer (adipic acid), a surface having the following structure is formed.

CO by enzymes in the carboxyesterase group
A dye is generated by the decomposition of the O-bond, and this dye is diffused into the region of the excitation light from the optical fiber and measured.

(2) the 3-cyano-7-hydroxycoumarin CH ₂ -CH ₂
Immobilization on the polymer containing OH via the —CH ₂ —CH ₂ spacer gives the following structure.

The immobilized dye resulting from this process is a synthetic substrate for dealkylase. This decomposes the ether groups to liberate the fluorescent dye 7-hydrocyclmarin-3-carboxylic acid, which is detected via the optical fiber.

(3) The structure of the protease substrate immobilized on the glass surface is shown below.

Not only the synthetic substrate, but also a natural substrate dyed with a dye, such as a protein, is immobilized on the inner surface of the reaction chamber. For example, egg white lysozyme is immobilized on the inner surface of the wall and then dyed with a fluorescent dye such as fluorescein-isothiocyanate. This substrate decomposes on contact with oxygen trypsin.
Fluorescein and lysozyme dyed with fluorescein diffuse into the region of the excitation light exiting the optical fiber and are measured by fluorescence analysis.

Immobilized proteins can be easily prepared,
A commercial product can also be obtained. Saccharides, sugar-like compounds, are also commercially available in immobilized form. For example, immobilized N-acetylglucosamine, lactose, melibiose, N-acetylgalactosamine, fucose, mannose, cellobiose, galactose, and glucose. These may be attached to the inner wall of the reaction chamber and dyed with a dye. The dyes include fluorescein-isothiocyanate, dansyl chloride, fluorescamine, naphthyl isocyanate and europium chelate. The dyed molecules are released by the enzymatic reaction, diffused into the reaction chamber, and detected by the optical fiber.

The wall material surrounding the reaction chamber may be the enzyme substrate itself. For example, amylose belonging to polyglucoside is used, and the inner surface of the wall material is dyed with a fluorescent dye. Other substances, including polypeptides and fatty acid esters, are also used as substrates and wall materials. It goes without saying that only water-insoluble substances are used.

The advantage of using a stained natural enzyme substrate is that it can have the high specificity that the enzyme has for the natural substrate. In combination with fluorescence analysis, the purpose of finding a sensitive and specific measurement method was achieved.

Many enzymes occur in multiple forms, forming subgroups. A typical example is phosphatase. This includes
There are alkaline phosphatases that exhibit maximum activity in alcal solutions and acid phosphatases that exhibit maximum activity in acidic solutions. Clinically important acid phosphatases, for example in PH7.4, cannot be measured unless they at least partially include the activity of alkaline phosphatase.

This problem is avoided by using two enzyme sensitive fiber optic catheters. These catheters must be distinguished in that the enzyme substrates provided at their respective ends have different affinities for each enzyme and have different maximum reaction rates. The affinity of the enzyme for the substrate is given by the Michaelis-Menten constant, and the maximum rate is given by the amount giving the maximum reaction rate.

〔Example〕

Next, the present invention will be described in detail based on the embodiment shown in FIGS. In FIG. 1, the light emitted from the light source 1 is condensed by the collimator lens 2, passes through the filter 3, and then enters the input side end 5 of the optical fiber 6 by the focus lens 4. The wavelength of the light is adjusted using the filter 3 so that the absorption by the dye liberated by the hydrolysis by the enzyme is maximized. A portion of the light is split by the beam splitter 7 and enters the standard photodetector 8. This standard photodetector 8 is used to eliminate the influence of the intensity fluctuation of the light source 1.

The enzyme substrate 9 on which the excitation light enters through the optical fiber 6 is
It is fixed to the exit end 10 of the optical fiber 6. When the enzyme substrate 9 is decomposed by the enzyme, the incident light is gradually absorbed and attenuated by the formation of the dye reaction product 9 '. On the other hand, if the dye is fluorescent, a gradually increasing fluorescence intensity is observed. The reflected excitation light and the fluorescent light are returned by the same optical fiber 6, reflected by the half-mirrored surface 11 of the beam distributor 7 and pass through lenses 12, 13 to reach the photodetector 14. When measuring fluorescence, a second optical filter 15 that does not pass the reflected excitation light is installed in the optical path from the beam distributor 7 to the detector 14, and only the selected fluorescence is detected.

The measurement signal S, which is the output of the photodetector 14, and the standard signal R, which is the output of the standard photodetector 8, are amplified and sent to a display device or evaluation device (not shown).

FIG. 2 shows an enlarged end 10 of an optical fiber 6 in which the core 16 and the sheathing 17 have different refractive indices.
The enzyme substrate 9 is a thin carrier film 18, eg cellulose,
It is fixed to an ion exchange membrane or one made of polyacrylamide, and is attached to the exit side end portion 10 of the optical fiber 6.

As shown in FIG. 3, the enzyme substrate 9 can be attached to the outer peripheral surface 19 of the core 16 at the exit side end 10 of the optical fiber 6.
In this case, the coating 17 on the exit side end 10 of the optical fiber 6 is peeled off. A cap 21 that reflects light is attached to the end surface of the optical fiber 6.

In FIG. 4, a spherical reaction chamber 22 surrounded by a protein permeable membrane 23 made of, for example, cellulose is shown at the outlet end 10.
The optical fiber 6 provided in FIG. The reaction chamber 22 contains an enzyme substrate 9 of a relatively small molecule.
Can diffuse out through the membrane 23. To prevent this diffusion, the enzyme substrate 9 is immobilized on a large molecule of water-soluble polymer. Water-soluble or swellable polymers are suitable as carriers for the enzyme substrates in the reaction chamber 22.

In the embodiment shown in FIG. 5, the reaction product 9'is spatially separated from the original enzyme substrate 9. A reaction space 24 surrounded by a cylindrical body 26 is provided at the exit side end 10 of the optical fiber 6, and the sample side end face thereof is covered with an enzyme-permeable membrane 23 made of, for example, cellulose or porous polycarbonate. There is.
The inner wall 25 of the cylinder 26 is covered with a dye or fluorescent enzyme substrate 9. Unlike the above-mentioned embodiment, the enzyme substrate 9 is not uniformly distributed in the entire reaction space, and is immobilized chemically, electrostatically or physically only on the inner wall 25 of the cylindrical body 26.

When the enzyme to be measured enters the reaction space 24 through the membrane 23 along the arrow 29, an enzymatic reaction takes place, releasing the reaction product 9 '. This reaction product 9'moves freely in the reaction space 24 and diffuses into a region determined by the maximum emission angle α of the excitation light. In this configuration, it is necessary to determine the diameter 27 and the length 28 of the cylindrical body 26 so that the excitation light emitted from the end face of the optical fiber 6 does not hit the enzyme substrate 9 immobilized on the inner wall 25 of the cylindrical body.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of an optical measuring apparatus for enzyme activity according to an embodiment of the present invention, and FIGS. 2 to 5 are optical signals showing various embodiments of an optical fiber sensor used in the measuring apparatus of FIG. It is an expanded sectional view of an end of a fiber. 6 ... Optical fiber, 7 ... Beam distributor, 9 ... Enzyme substrate, 9 '... Reaction product, 10 ... End of optical fiber, 16
... optical fiber core, 20 ... optical fiber end face, 14 ...
… Photodetector, 18 …… Carrier film, 23 …… Enzyme permeable membrane, 24 …… Reaction space, 26 …… Cylindrical body, 25 …… Cylindrical inner wall surface.

Claims (9)

[Claims] 1. A sensor for measuring enzyme activity, wherein an enzyme substrate (9) is arranged at the end (10) of an optical fiber (6) so that it can come into contact with a sample. 2. The method according to claim 1, wherein the enzyme substrate (9) is physically or chemically fixed to the end surface (20) of the optical fiber (6) or the outer peripheral surface (19) of the core (16). Sensor. 3. The method according to claim 1, wherein the enzyme substrate (9) is fixed to a carrier film (18), and the carrier film (18) is attached to the end (10) of the optical fiber (6). The sensor according to item 2. 4. An enzyme substrate (9) fixed to the surface of an optical fiber (6) or the surface of a carrier film (18) through a spacer, as claimed in claim 1, claim 2, or claim 3. Sensor. 5. An optical fiber sensor in which an enzyme substrate (9) is arranged at the end (10) of an optical fiber (6) so as to be in contact with a sample, and its optical output is measured to evaluate a measurement signal (S). And a photodetector (14) for supplying the device, and this detector (14)
An enzyme activity measuring device for measuring the change of fluorescence or excitation light emitted by the enzyme substrate (9) or its reaction product (9 ') via an optical fiber. 6. An enzyme substrate (9) is fixed to an inner wall surface (25) of a cylindrical body (26) forming a reaction space (24) arranged at an end (10) of an optical fiber (6), An enzyme permeable membrane (23) that does not allow the cells of the sample to permeate is provided on the end surface of the cylindrical body (26) on the sample side, and the reaction product (9 ') generated by the enzymatic reaction diffuses into the reaction space (24). The measuring device according to claim 5. 7. Excitation light emitted from an end face (20) of an optical fiber (6) at an angle smaller than a maximum emission angle (α) is applied to an enzyme substrate (9) fixed to an inner wall surface (25) of the cylindrical body. The measuring device according to claim 6, wherein the diameter and the length of the cylindrical body (26) are determined so as not to hit. 8. An optical fiber sensor in which an enzyme substrate is arranged at the end of an optical fiber so that it can come into contact with a sample, and a change in fluorescence or excitation light emitted by the enzyme substrate or a reaction product thereof is detected by the optical fiber. A method for measuring enzyme activity, which is measured via. 9. The pH value of the sample is measured in addition to the enzyme activity,
9. The measuring method according to claim 8, wherein the measured PH value is used for correcting the measured enzyme activity value.