JPH05264498A - Reactor type sensor utilizing oxygen - Google Patents

Abstract

PURPOSE:To enable the amount of immobilization of oxygen to be constant and at the same time to be change arbitrarily, achieve analysis with a constant sensitivity and with a high sensitivity, and achieve a long-term and stable use. CONSTITUTION:Alcohol oxidase with 100-1000 units/1ml is immobilized to chitosan beads (carrier for immobilizing oxygen) and then it is filled into a reactor 10. By detecting the amount of hydrogen peroxide or the amount of oxygen which is generated due to enzyme decomposition reaction within the reactor by an electrochemical detector 18, alcohol within a sample is analyzed with a certain sensitivity and a high sensitivity. Also, by immobilizing ascorbic acid oxidase with 50-5000 units/1ml into the chitosan beads, vitamin C within the sample is analyzed similarly. The reactor 10 can be attached to and detached from a sample channel freely.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a reactor type sensor using an enzyme, especially alcohol in the food field, medical field, etc.
The present invention relates to the configuration of a biosensor used for analyzing vitamin C and the like.

[0002]

2. Description of the Related Art Conventionally, for example, alcohol in food,
As a method for analyzing vitamin C and the like, there are a gas chromatographic method, a liquid chromatograph method, etc., but a sample containing multiple components such as food requires a complicated sample (test liquid) pretreatment operation. Therefore, the analysis cannot be done easily and quickly. Therefore, in recent years, a sensor using an enzyme has been used in which an enzyme is chemically immobilized on a polymer membrane and the amount of hydrogen peroxide or the amount of oxygen when the enzyme reacts with a sample and decomposes is measured.

FIG. 9 shows the structure of a conventional sensor using an enzyme. As shown in the figure, an inner cylinder 3 is screwed and fixed in an outer cylinder 1 via a coil spring 2. An oxygen electrode (cathode) 4 made of platinum or the like is attached to the tip of the inner cylinder 3, and a polymer film 6 in which an enzyme is immobilized via a Teflon film 5 so as to come into contact with the electrode 4. Are attached by the rubber ring 7. A hydrogen peroxide electrode (anode) may be used instead of the oxygen electrode 4.

According to the above construction, the amount of oxygen generated when alcohol is decomposed by the enzyme of the polymer film 6 can be detected as the oxidation current of the oxygen electrode 4, and this oxidation current can be taken out from the terminal 8 in the figure. Makes it possible to analyze alcohol. Sensors that use such enzymes utilize the substrate specificity of the enzyme, so that sample pretreatment can be performed easily even for substances with complex multi-component mixed systems such as food, and rapid analysis can be performed. The advantage is that

[0005]

However, as described above, in the sensor in which the polymer membrane 6 on which the enzyme is immobilized is attached to the oxygen electrode 4 (or hydrogen peroxide electrode), the attached state of the polymer membrane 6 is There was a problem that the sensitivity was different depending on the type, and skill was required to perform stable analysis. That is, depending on how to attach the enzyme-immobilized polymer membrane,
The thickness of the polymer film may change or the adhesion to the oxygen electrode 4 may deteriorate. Therefore, in this case, the amount of immobilized enzyme changes, and the detection sensitivity also changes.

Further, in the above-mentioned conventional sensor, since the area of the polymer film 6 that can contact the oxygen electrode 4 of a predetermined size is determined, the amount of immobilized enzyme is limited, and detection is performed with high sensitivity. However, there is a problem that it cannot be used for a long period in a stable state.

The present invention has been made in order to solve the above-mentioned problems, and its object is to make the amount of enzyme immobilized constant and to be able to change it arbitrarily, with a constant sensitivity and a high sensitivity. An object of the present invention is to provide a reactor-type sensor using an enzyme that can be analyzed and can be used stably for a long period of time.

[0008]

In order to achieve the above object, a reactor type sensor utilizing an enzyme according to the invention of the first aspect is a reactor filled with a carrier for immobilizing an enzyme, and the reactor is passed through this reactor. An electrochemical detector for electrically detecting the amount of hydrogen peroxide or the amount of oxygen in a sample, wherein an alcohol-degrading enzyme is immobilized on the enzyme immobilization carrier to analyze alcohol. .. According to the second aspect of the invention, chitosan beads are used as the enzyme immobilization carrier, and 100 ml per 1 ml of the chitosan beads.
˜1000 units of alcohol oxidase are immobilized. Here, 1 unit is the amount of enzyme that produces 1 μmol of aldehyde or hydrogen peroxide in 1 minute at a temperature of 37 ° C. using methanol as a substrate. The invention of claim 3 has a reactor filled with a carrier for enzyme immobilization, and an electrochemical detector for electrically detecting the amount of oxygen in a sample that has passed through the reactor. It is characterized by immobilizing ascorbic acid-degrading enzyme on a carrier for analysis of vitamin C. The invention of claim 4 uses chitosan beads as the carrier for immobilizing the enzyme, and 50 to 5 per 1 ml of the chitosan beads.
000 units of ascorbate oxidase are immobilized. Here, 1 unit means pH
5.6, the amount of enzyme that oxidizes 1 μmol of L-ascorbic acid per minute at a temperature of 30 ° C. A fifth aspect of the present invention is characterized in that the reactor is detachably attached to the sample channel.

[0009]

According to the above construction, when analyzing alcohol, for example, chitosan beads are treated with alcohol degrading enzyme 1
Immobilized in the range of 00-1000 units / 1 ml,
The chitosan beads are loaded into the reactor. Then, when the sample flows into this reactor, the alcohol is decomposed by an enzyme under oxygen, and aldehyde and hydrogen peroxide are generated. Therefore, the amount of alcohol can be specified by detecting the consumed oxygen amount by the oxygen electrode or by detecting the generated hydrogen peroxide amount by the hydrogen peroxide electrode.

When analyzing vitamin C, for example, chitosan beads containing ascorbic acid degrading enzyme in an amount of 50 to 5 are used.
Immobilized in the range of 000 units / 1 ml, the chitosan beads are loaded into the reactor. Then, when the sample flows into this reactor, ascorbic acid is decomposed by an enzyme under oxygen, and dehydroascorbic acid and water are generated. Therefore, the amount of vitamin C can be specified by detecting the amount of oxygen consumed by the oxygen electrode.

In the above, if the reactor is detachable from the flow channel, the amount of immobilized enzyme can be adjusted according to the characteristics of the sample, or the type of enzyme can be changed according to different samples. This can be easily done with a sensor on the stand.

[0012]

EXAMPLE FIG. 1 shows the structure of a reactor type sensor using the enzyme according to the first example. The illustrated reactor 10 has a cylindrical shape with an inner diameter of 2 mm and a length of 50 mm, and has both ends at both ends. A filter 11 is provided. The reactor 10 is filled with granular chitosan beads. The chitosan beads are deacetylated natural polymer chitin, and the surface has a porous shape with a pore size of 0.1 to 0.2 μm. ing. In the embodiment, the diameter is 0.1 m
Chitosan beads having a size of about m (0.1 mm or less, 0.1 mm or more and about several mm may be used) are used, and alcohol oxidase, which is an enzyme, is immobilized on the chitosan beads by the following operation. That is, in the embodiment, 1
2.5% after washing and dehydrating ml chitosan beads
Add 10 ml of the glutaraldehyde solution of 2
Shake for hours. After washing with water and dehydration, the pH value is 7.
In step 8, 1 ml of 0.1 mol phosphate buffer is added, alcohol oxidase in the range of 100 to 1000 units is added per 1 ml of chitosan beads, and the mixture is shaken at about 30 ° C. for 2 hours for immobilization. In addition, in the above, 1 unit is an enzyme amount which produces | generates 1 micromol aldehyde or hydrogen peroxide in 1 minute at the temperature of 37 degreeC using methanol as a substrate.

In front of the reactor 10, a sample injection section 12 having a sample injection port 12a and a metering pump 1 are provided in this order.
3, a container 15 for storing 0.1 mol of the phosphate buffer 14 at pH 7.0, and an air vent pipe 16 for introducing air into the container 15 are connected. This air distribution pipe 16
The amount of oxygen in the solution is made constant by sending air into the phosphate buffer solution 14, and the metering pump 13 sends the phosphate buffer solution 14 to the reactor 10 side at a flow rate of 1 ml / min.

On the other hand, Pt is provided in the latter stage of the reactor 10.
An electrochemical detector 18 provided with a hydrogen peroxide electrode made of (platinum) or the like is provided to measure the amount of hydrogen peroxide in the sample supplied from the reactor 10. That is, the hydrogen peroxide electrode 1 in the electrochemical detector 18
The amount of hydrogen peroxide is specified by detecting the reduction current of the redox reaction by the enzyme. of course,
The oxygen amount can also be detected by the oxygen electrode. In this case, the consumed oxygen amount is obtained by subtracting the detected oxygen amount from the oxygen amount in the buffer solution 14.

The first embodiment has the above-mentioned structure, and when the measurement is performed, the phosphate buffer 14 is supplied by the pump 13 at a flow rate of 1 ml / min through the sample injection part 12 to the reactor 1.
At the same time when the sample is supplied to 0, the sample is injected from the sample inlet 12a. Then, in the reactor 10, the following reaction of Chemical Formula 1 occurs.

[0016]

[Chemical 1]

Therefore, the electrochemical detector 18 uses the hydrogen peroxide H ₂ O of the above chemical formula 1 from the effluent of the reactor 10.
The amount of ₂ generated will be measured, and this will measure the alcohol concentration.

FIG. 2 shows a graph in which the optimum amount of alcohol oxidase immobilized on 1 ml of chitosan beads was determined. According to the experiment, 100 units to 100 units were shown.
It was confirmed that the enzyme activity was high when the immobilized amount was 0 unit. Therefore, alcohol can be analyzed with stable sensitivity by setting the immobilized amount in the range of 100 to 1000 units per 1 ml of chitosan beads. Also, the amount of this chitosan bead is the same as that of reactor 1
By converting the size of 0, it can be arbitrarily changed and the sensitivity can be adjusted.

FIG. 3 shows a diagram comparing the sensitivities of the reactor type sensor of the first embodiment and the conventional polymer membrane type sensor. This diagram is obtained by examining ethanol. According to it, it is understood that the higher the ethanol concentration is, the more markedly the sensitivity is decreased in the polymer film type sensor as compared with the reactor type sensor. Also, FIG.
Shows a diagram comparing the two in terms of long-term stability. When refrigerated during storage, the polymer membrane type sensor has a higher immobilized enzyme activity (%) than the reactor type sensor. The result was an early decline.

Further, in the present invention, the reactor 10 is detachably attached to the flow path, and this structure will be described with reference to FIG. In FIG. 5A, the luer lock type receiving portions 21 and 22 are rotatably attached to both ends of the reactor vessel 20, while the connecting portion 23 and the electrochemical detector 18 side are provided in the flow path on the sample injection portion 12 side. Connection part 2 in the flow path
4 is attached. Then, the connecting portions 23, 2
Two protrusions 25 are formed on each of the grooves 4, and on the other hand, the receiving portions 21 and 22 are each formed with a groove 26 for guiding the protrusion 25 therein, as shown in FIG.
6 is connected to a spiral cut formed inside. Therefore, while fitting the protrusion 25 into the groove 26,
When the projections 3, 24 are rotated, the projections 25 are received by the receiving portions 21, 22.
Guided by the spiral cut, the tip portions of the connecting portions 23 and 24 are firmly fixed to both ends of the reactor vessel 20 in a close contact state.

As described above, since the reactor 10 is detachably attached to the flow path, it is possible to arbitrarily attach different reactors 10 to the sensor. Therefore, for example, it is easy to adjust the sensitivity by using the reactor 10 in which the amount of chitosan beads on which the enzyme is immobilized is changed, and to analyze different substances by using the reactor 10 on which different kinds of enzymes are immobilized. You can do it.

Next, the second aspect of the present invention for analyzing vitamin C
An example will be described. Also in the case of the second example, ascorbate oxidase, which is an enzyme, is immobilized on the chitosan beads that are composed of the substantially same members as in FIG. 1 and are filled in the reactor 10. That is, FIG. 6 shows a graph of the optimum amount of ascorbate oxidase immobilized on 1 ml of chitosan beads, and according to the experiment, the enzyme activity becomes high at the immobilized amount of 50 units to 5000 units. It is confirmed. Therefore, 50 to 500 per 1 ml of chitosan beads in the reactor 10
Ascorbate oxidase is immobilized in the range of 0 unit, which enables stable analysis of vitamin C. In addition, 1 unit is pH
5.6, the amount of enzyme that oxidizes 1 μmol of L-ascorbic acid per minute at a temperature of 30 ° C.

In the case of the second embodiment, 0.1 mol of phosphate buffer solution 14 having a pH of 5.6 is used as the buffer solution, and an oxygen electrode is used as the electrochemical detector 18. There is. At the above flow rate, the phosphate buffer solution 14 is supplied to the sample injection part 1
When the sample containing Vitamin C (L-ascorbic acid) is supplied to the reactor 10 via the sample injection port 2 and is injected from the sample injection port 12a, a reaction according to the following chemical formula 2 occurs.

[0024]

[Chemical 2]

Therefore, the electrochemical detector 18 measures the amount of oxygen O ₂ (oxidation current of redox reaction) from the effluent of the reactor 10 and subtracts it from the amount of oxygen in the buffer solution 14 to obtain the chemical formula 2 The amount of oxygen O ₂ used can be detected, and the concentration of vitamin C (L-ascorbic acid) can be measured.

FIG. 7 shows a diagram comparing the sensitivities of the reactor type sensor of the second embodiment and the conventional polymer membrane type sensor. According to this, the higher the concentration of vitamin C is, It is understood that the polymer membrane type sensor has a marked decrease in sensitivity as compared with the reactor type sensor. In addition, Fig. 8 shows a diagram comparing the two in terms of long-term stability. When it is kept at room temperature during storage, the polymer membrane type sensor is more immobilized than the reactor type sensor. This results in a decrease in enzyme activity (%).

As described above, according to the present invention, the reactor 10 is filled with chitosan beads in which alcohol oxidase which is an alcohol degrading enzyme and ascorbate oxidase which is an ascorbic acid degrading enzyme are immobilized in a unit amount within a predetermined range, By exchanging the reactor 10, it is possible to analyze alcohol and vitamin C in foods or samples.

In the above embodiment, an example was shown in which the enzyme was immobilized on chitosan beads, but beads made of other substance of mucopolysaccharide can be used as the carrier for immobilizing the enzyme. Further, any substance having an amino group on the surface of the carrier and capable of binding to the amino group of the enzyme via glutaraldehyde can be used as the enzyme-immobilized carrier, and need not be spherical.

[0029]

As described above, according to the constitution relating to the alcohol analysis of the present invention, 100 to 1000 units / 1 are added to the enzyme immobilization carrier such as chitosan beads.
Immobilized ml of alcohol oxidase was filled in the reactor, and the amount of hydrogen peroxide or oxygen generated by the decomposition reaction of the sample by the enzyme in the reactor was electrically detected, so that the sensitivity was stable. Thus, alcohol in foods and samples can be analyzed, and detection can be performed with high sensitivity.

Further, according to the constitution relating to the vitamin C analysis of the present invention, for example, 50 to 5000 units / ml of ascorbate oxidase is immobilized on chitosan beads, which is charged into the reactor, and oxygen generated in the reactor is charged. Since the amount is electrically detected, it is possible to analyze vitamin C in foods and the like with stable sensitivity, and to perform detection with high sensitivity.

Further, by making the reactor detachable with respect to the sample flow channel in the above, it is possible to arbitrarily attach reactors having different sensitivities and reactors for analyzing different substances, and to facilitate substance analysis. There is an advantage that.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of a reactor type sensor using an enzyme according to a first embodiment of the present invention.

FIG. 2 is a graph showing the relationship between the immobilized amount of alcohol oxidase and the enzyme activity in the first example.

FIG. 3 is a graph chart comparing the sensitivities in ethanol analysis in the reactor type sensor and the polymer membrane type sensor of the first embodiment.

FIG. 4 is a graph comparing long-term stability between the sensor of the first embodiment and a conventional polymer film sensor.

5A and 5B are views showing a structure for attaching and detaching a reactor in an embodiment, FIG. 5A is an overall perspective view, and FIG. 5B is a view showing an insertion portion of a connection receiving portion.

FIG. 6 is a graph showing the relationship between the immobilized amount of ascorbate oxidase and the enzyme activity in the second example.

FIG. 7 is a graph diagram comparing the sensitivities in vitamin C analysis of the reactor type sensor and the polymer membrane type sensor of the second embodiment.

FIG. 8 is a graph comparing long-term stability between the sensor of the second embodiment and a conventional polymer film sensor.

FIG. 9 is a cross-sectional view showing a configuration of a conventional polymer film type sensor.

[Explanation of symbols]

4 ... Oxygen electrode, 6 ... Polymer membrane, 10 ... Reactor, 11 ... Filter, 12 ... Sample injection part,
13 ... Pump, 14 ... Buffer solution, 20 ... Reactor container 21, 22 ... Receiving part, 23, 24 ...
Connection.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 27/28 321 Z 7235-2J 27/327

Claims (5)

[Claims] 1. An enzyme comprising a reactor filled with a carrier for immobilizing an enzyme and an electrochemical detector for electrically detecting the amount of hydrogen peroxide or the amount of oxygen in a sample that has passed through the reactor. A reactor-type sensor using an enzyme in which an alcohol-degrading enzyme is immobilized on a carrier for immobilization to analyze alcohol. 2. Chito acid beads are used as the enzyme immobilization carrier, and 100 to 1 per 1 ml of the chito acid beads.
000 units of alcohol oxidase are immobilized, The reactor type sensor using the enzyme according to claim 1 above. 3. A carrier packed with a carrier for immobilizing an enzyme, and an electrochemical detector for electrically detecting the amount of oxygen in a sample that has passed through the reactor. A reactor type sensor using an enzyme in which ascorbic acid degrading enzyme is immobilized and vitamin C is analyzed. 4. Chitosan beads are used as the enzyme-immobilizing carrier, and 50 to 1 ml of the chitosan beads are used.
A reactor-type sensor utilizing the enzyme according to claim 3, wherein 5000 units of ascorbate oxidase are immobilized. 5. The first reactor characterized in that the reactor is detachably attached to a sample flow channel.
Alternatively, a reactor-type sensor using the enzyme according to claim 3.