JP2006314211A - Analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analyzer enabling hydrogen peroxide in a sample to be removed easily and ensuring hydrogen peroxide formed by the subsequent enzyme reaction to be detected in high accuracy. <P>SOLUTION: The analyzer includes a mechanism functioning to feed an aqueous solution at a constant flow and a mechanism functioning to inject the aqueous solution with a given amount of a substance to be assayed. In this analyzer, downstream of the injection mechanism, there is provided an enzyme-immobilized form functioning to form hydrogen peroxide by catalyzing a redox reaction of the substance in the aqueous solution in the vicinity of a hydrogen peroxide electrode and its upstream side. This analyzer works to calculate the concentration of the substance by detecting the concentration change of hydrogen peroxide in association with enzyme reaction at the hydrogen peroxide electrode, wherein a column packed with kaolinite-based ceramics subjected to baking or sintering treatment after undergoing hydrothermal treatment is inserted in between the injection mechanism and the enzyme-immobilized form. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention discloses a flow type analyzer having a mechanism for quantifying the concentration of a substance by detecting hydrogen peroxide, which can easily and inexpensively remove interference components and enables high precision analysis. Is.

  Analytical apparatuses using electrochemical detectors are frequently used because the apparatus configuration is simple and high sensitivity and high accuracy analysis is possible. In particular, the method of analyzing hydrogen peroxide in a sample by anodic polarization of a solid electrode such as platinum, carbon, gold, etc. is easy to downsize, has a fast response speed, and provides very high sensitivity. There are advantages. Thus, many biosensors utilizing the specificity of the enzyme have been proposed in combination with an enzyme-immobilized body that catalyzes a reaction that generates hydrogen peroxide.

  However, hydrogen peroxide may be generated along with the disproportionation reaction of active oxygen in the living body and oxidation of the reducing substance, and there is a problem that a trace amount of hydrogen peroxide causes a positive error.

  Conventionally, it is known that unnecessary hydrogen peroxide is removed by an enzyme reaction such as catalase, but this method has a drawback that it causes an increase in analysis cost due to the use of the enzyme and takes time.

  In addition, it has been proposed to use a combination of a hydrogen peroxide electrode that does not involve an enzyme reaction and an electrode that detects hydrogen peroxide generated by the enzyme reaction, and to eliminate the response of hydrogen peroxide alone by arithmetic processing. As the apparatus configuration becomes complicated, there is a problem that an error is increased when a minute amount of substance is quantified.

  In the conventional technique, when a substance is quantified by hydrogen peroxide detection, the hydrogen peroxide contained in the sample itself becomes an interfering component, but this is easily removed and the hydrogen peroxide generated by the subsequent enzymatic reaction is increased. It was difficult to detect with accuracy. The present invention solves this problem.

  The present invention includes a mechanism for feeding an aqueous solution at a constant flow rate and a mechanism for injecting a certain amount of a substance to be measured into the aqueous solution, and a hydrogen peroxide electrode and a hydrogen peroxide electrode in the vicinity of the injection mechanism. An enzyme-immobilized body that catalyzes the oxidation-reduction reaction of substances contained in an aqueous solution and generates hydrogen peroxide. The measurement is performed by detecting the change in the hydrogen peroxide concentration accompanying the enzyme reaction with the hydrogen peroxide electrode. In the apparatus for calculating the concentration of the target substance, a column filled with kaolinite-based ceramics that has been subjected to baking or sintering treatment after hydrothermal treatment is inserted between the injection mechanism and the hydrogen peroxide-producing enzyme-immobilized body. A featured analytical device is disclosed.

  Moreover, it is desirable to fix an enzyme that does not participate in the hydrogen peroxide generation reaction to the kaolinite-based ceramics, and to dispose the hydrogen peroxide-producing enzyme immobilized body downstream of the immobilized body.

  The kaolinite-based ceramic that decomposes hydrogen peroxide can be produced by subjecting the kaolinite-based ceramic raw material to baking or sintering after hydrothermal treatment.

  The present invention further discloses the use of kaolinite ceramics as a carrier for immobilizing enzymes that are not involved in the hydrogen peroxide production reaction.

  By disposing the kaolinite ceramics upstream of the immobilized enzyme involved in the hydrogen peroxide production reaction, hydrogen peroxide contained in the sample can be removed.

  According to the present invention, hydrogen peroxide contained in a sample can be easily removed, and hydrogen peroxide generated by a subsequent enzyme reaction can be detected with high accuracy.

  As a result of investigating a rapid and reproducible method for removing hydrogen peroxide in the sample, the problem can be solved by bringing the sample into contact with the calcined ceramic after firing or sintering after the hydrothermal reaction. As a result, the present invention has been completed.

  Processing such as increasing the specific surface area by chemically treating clay minerals has long been performed. In recent years, so-called hydrothermal reaction treatment, in which surface properties are further modified by heating water at high pressure to cause various reactions in water near the critical state, has come to be used frequently.

  Examples of raw materials for producing kaolinite ceramics include natural kaolin, kaolinite, dickite, nacrite, halloyite, and refractory bricks.

When a raw material such as natural kaolin is subjected to a hydrothermal reaction treatment under acidic conditions, the specific surface area is generally increased. Furthermore, the granulated, sintered or fired product after hydrothermal reaction is called kaolinite ceramics. It is known that by increasing the specific surface area, it can be used to immobilize biocatalysts such as enzymes, but it is not known that kaolinite-based ceramics can efficiently decompose hydrogen peroxide.
As a method for immobilizing an enzyme that is not involved in the hydrogen peroxide production reaction to kaolinite-based ceramics, or as a method for immobilizing an enzyme to a carrier other than kaolinite-based ceramics that produces hydrogen peroxide, a physical adsorption method, Known methods for immobilizing proteins such as ionic bond method, inclusion method, and covalent bond method can be used. Among them, the covalent bond method is preferable because of its long-term stability. As a method for covalently binding a protein, various methods such as a method using a compound having an aldehyde group such as formaldehyde, glyoxal, and glutaraldehyde, a method using a polyfunctional acylating agent, a method of crosslinking a sulfhydryl group, and the like are used. it can.

  Since the silanol group is exposed on the surface of the kaolinite-based ceramics, when a silanic group is reacted with the silane coupling agent and an amino group is introduced at the terminal, an enzyme or the like can be easily obtained using a polyfunctional aldehyde. A biocatalyst can be immobilized.

  The hydrogen peroxide-producing enzyme-immobilized body can be immobilized in a film shape and placed on an electrode made of platinum, gold, carbon, etc., or a polymer such as cellulose, agarose, chitin, carrageenan, polyacrylamide, etc. It can also be used by immobilizing on a known insoluble carrier such as organic material, general porous glass, inorganic material such as ceramics, etc., and filling the column reactor with the carrier.

  The enzyme that can be used for the immobilized hydrogen peroxide-producing enzyme may be any enzyme that generates hydrogen peroxide, and examples thereof include oxidoreductases that generate hydrogen peroxide such as glucose oxidase and fructosyl amino acid oxidase.

  As an example of kaolinite ceramics as an enzyme immobilization carrier, Toyonite 200 (manufactured by Toyo Denka Kogyo Co., Ltd.) can be exemplified, and Toyonite 200 immobilized with an enzyme not involved in the hydrogen peroxide production reaction is packed into a column reactor and used. be able to.

  Enzymes that can be used for the kaolinite-based ceramic immobilized body may be enzymes that do not participate in the hydrogen peroxide production reaction. Proteolytic enzymes such as papain and proteinase K, hydrogen peroxide such as maltose phosphorylase, invertase, and β-galactosidase. An enzyme that does not participate in the production reaction can be exemplified. These enzymes may be used alone or in combination of two or more.

  A combination of enzymes suitable for detection of the target substance is selected, and these enzyme-immobilized bodies may be incorporated into the measuring apparatus of FIG. The buffer solution is sent from the buffer solution tank (1) by the pump (2). Insert the needle (6) into the sample (5), close the valve (9), open the valve (10), and pull the syringe pump (11) to pull the sample into the metering loop (4) of the metering valve (3). Pull in. Next, the metering valve (3) is switched, and the specimen accumulated in the loop (4) is pushed out by the buffer solution. Excess specimen once opens the valve (9) and closes the valve (10), draws the cleaning liquid (8) into the syringe pump (11), then closes the valve (9) and opens the valve (10) to push out the cleaning liquid. As a result, it is pushed out to the waste pot (7) and stored in the waste liquid bottle (20). The injected sample follows the flow of the buffer solution, passes through the mixing pipe (13) installed in the thermostatic chamber (12), is mixed with the temperature adjustment and the buffer solution, and fixed with kaolinite ceramic enzyme. The hydrogen peroxide is detected by the hydrogen peroxide electrode (15). Further, the waste liquid passes through the back pressure coil (18) and accumulates in the waste liquid bottle (19).

  When two or more kinds of immobilized enzymes are used, the buffer tank (1) uses a buffer having an optimum pH for two or more kinds of enzyme reactions. As the buffer solution, phosphoric acid, acetic acid, citric acid and the like may be adjusted to the optimum pH for the enzyme reaction. Various salts and antibacterial agents may be added to the buffer solution.

  The kaolinite-based ceramic enzyme-immobilized column (25) and the hydrogen peroxide-producing enzyme-immobilized column (14) may be installed in a thermostatic bath, and the enzyme reaction may be performed at a temperature optimal for the enzyme reaction.

By selecting a combination of enzymes used for the kaolinite-based ceramic enzyme immobilization column (25) and the hydrogen peroxide-producing enzyme immobilization column (14), various substances can be detected.
Measurement of lactose β-galactosidase is immobilized on the kaolinite ceramic enzyme immobilization column (25), and glucose oxidase is immobilized on the hydrogen peroxide-producing enzyme immobilization column (14). When the sample is injected by incorporating the immobilized body into the measurement apparatus of FIG. 1, lactose in the sample is converted to glucose by β-galactosidase, and the glucose produced by glucose oxidase is converted to hydrogen peroxide and detected.
Measurement of sucrose Invertase and mutarotase are immobilized on the kaolinite ceramic enzyme immobilization column (25), and glucose oxidase is immobilized on the hydrogen peroxide-producing enzyme immobilization column (14). Similar to (1), sucrose in the sample may be converted to hydrogen peroxide and detected.
・ Measurement of glycated amino acid in glycated protein Fruc that can immobilize proteolytic enzyme in kaolinite ceramic enzyme immobilization column (25) and convert glycated amino acid to hydrogen peroxide in hydrogen peroxide producing enzyme immobilization column (14) Tosyl amino acid oxidase is immobilized. These immobilized columns are incorporated in the same manner as in (1), and after the sample is decomposed into amino acids and glycated amino acids by proteolytic enzymes, the generated glycated amino acids are converted to hydrogen peroxide by fructosyl amino acid oxidase and detected. That's fine.

    Among glycated proteins, glycated hemoglobin is widely used as an indicator for long-term blood glucose control in diabetic patients. Glycated hemoglobin is a type of glycated protein in which sugar is non-enzymatically bound to hemoglobin. Among them, a fraction called HbA1c is an aldimine (HbA1c) in which glucose forms a Schiff base at the N-terminal valine residue of hemoglobin A. Instability type) and further undergoes Amadori transition to produce a ketoamine compound. There are a variety of methods for measuring glycated hemoglobin. As described above, the enzymatic method is to detect the amount of glycated amino acid generated using a glycated amino acid oxidase or the like after the glycated amino acid has been cut out from the glycated protein by some technique. It is.

    When measuring glycated amino acids in glycated hemoglobin, the sample is blood cells, whole blood, etc., so hydrogen peroxide can be generated with the disproportionation of active oxygen in the living body and oxidation of reducing substances There is a problem that a small amount of hydrogen peroxide that does not originate from the immobilized enzyme reaction causes a positive error. Accordingly, when a trace amount of hydrogen peroxide is decomposed by bringing the sample into contact with the kaolinite ceramic carrier, the glycated amino acid can be measured with higher accuracy.

    Further, in the measurement of glycated amino acid in glycated hemoglobin, when the sample is contaminated with a hydrogen peroxide electrode, such as blood cells or whole blood, the sensitivity is significantly reduced. ) And a hydrogen peroxide-producing enzyme-immobilized body (14), it is preferable to incorporate a separation mechanism such as a semipermeable membrane to prevent contamination continuously.

  As an analyzer incorporating such a mechanism, the analyzer shown in FIG. 2 can be considered. Buffer B is sent from the buffer tank (21) by the pump (22), the needle (6) is inserted into the sample (5), the valve (9) is closed, the valve (10) is opened, and the syringe pump By pulling (11), the specimen is drawn into the metering loop (4) of the metering valve (3). Next, the metering valve (3) is switched, and the specimen accumulated in the loop (4) is pushed out by the buffer solution. Excess specimen once opens the valve (9) and closes the valve (10), draws the cleaning liquid (8) into the syringe pump (11), then closes the valve (9) and opens the valve (10) to push out the cleaning liquid. As a result, it is pushed out to the waste pot (7) and stored in the waste liquid bottle (20). The injected sample passes through the kaolinite ceramic enzyme immobilization column (25) of the proteolytic enzyme arranged in the thermostatic bath (12) along the flow of the buffer B, and from the protein in the sample to the fructosyl amino acid. Is generated. The sample on which the protease has acted is transported to a dialysis module (26) installed further downstream, where only a low molecular component in the sample is detected in a regenerated cellulose membrane having a film thickness of 20 μm and a molecular weight fraction of 12000 to 14000. (14, 15, 16, 17). The fructosyl amino acid detection mechanism may be composed of only (14, 15) (for example, a combination of fructosyl amino acid and hydrogen peroxide electrode), and (16, 17) is arbitrary.

  Since the buffer A is sent from the buffer tank (1) by the pump (2), the low molecular component in the sample dialyzed by the dialysis module (26) is the first hydrogen peroxide generation immobilized enzyme column. (14) Only hydrogen peroxide generated by the first immobilized enzyme after passing through the hydrogen peroxide electrode (15) is detected. Furthermore, only the hydrogen peroxide produced by the second immobilized enzyme is detected after passing through the second hydrogen peroxide producing / immobilizing enzyme column (16) and the hydrogen peroxide electrode (17). The polymer component in the sample that has not passed through the dialysis membrane of the dialysis module (26) accumulates in the waste liquid bottle (19).

  Among glycated proteins, when measuring glycated amino acids of glycated hemoglobin, especially fructosyl valine at the N-terminus of hemoglobin β chain, the kaolinite ceramic enzyme immobilization column (25) is glycated at the N terminus from glycated hemoglobin or a fragment thereof. It is preferable to immobilize a protease having a large action of generating an amino acid, and examples include Aspergillus proteases, and alkaline protease derived from Aspergillus oryzae is more preferable. The amount of alkaline protease derived from Aspergillus oryzae is 1 to 20 mg / column, more preferably 5 to 10 mg / column. The hydrogen peroxide-producing enzyme-immobilized column (14, 16) is contained in hemoglobin when lysine in which ε-amino group is glycated is liberated from glycated albumin contained in serum or in hemoglobin. Since lysine saccharification products may interfere, fructosyl amino acid oxidase that does not act on glycated amino acids in which the ε-amino group is glycated but acts on glycated amino acids in which the α-amino group is glycated is preferable. As an enzyme having such an action, a fructosyl amino acid oxidase derived from Corynebacterium can be exemplified.

  Examples of the first and second hydrogen peroxide producing enzymes include a combination of fructosyl amino acid oxidase and amino acid oxidase (oxidase of any amino acid produced together with fructosyl amino acid by the action of protease).

  Hemoglobin, which is a protein, is decomposed by the proteolytic enzyme of the kaolinite-based ceramic enzyme immobilized body to generate glycated amino acids, and hydrogen peroxide that is an obstacle in the sample is decomposed. The generated glycated amino acid may be generated by fructosyl amino acid oxidase on a hydrogen peroxide-producing immobilized body, and hydrogen peroxide may be detected by an electrochemical method such as amperometry.

  The semipermeable membrane used in the low molecular component separation mechanism (26) in the sample after protease degradation is difficult or non-permeable for pentapeptides, hexapeptides and larger peptides that are thought to coexist in the process of protease degradation. What is necessary is just to be able to permeate fructosyl valine and amino acids. Examples of the membrane used include dialysis membranes made of regenerated cellulose, acetyl cellulose, polyvinylidene fluoride, and the like. The molecular weight fraction of the dialysis membrane is displayed as the minimum molecular weight that permeates when equilibrium dialysis is performed. On the other hand, when used for analytical purposes, separation is often based on the difference in the initial rate of dialysis, and the molecular weight desired for fractionation does not always match the performance display of the dialysis membrane. For the purpose of the present invention, those having a fractional molecular weight of 300 to 500,000 can be used. More preferably, it is 1,000 or more and 100,000 or less, and more preferably 10,000 or more and 20,000 or less.

  When processing a sample containing glycated hemoglobin, the sample is mixed with a surfactant-containing buffer, the mixed sample is brought into contact with a carrier on which Aspergillus proteolytic enzymes are immobilized, and the sample is decomposed together with the buffer solution. It is a desirable form to obtain a processed sample.

  A surfactant has two functions when hemoglobin is decomposed into an amino acid and a glycated amino acid by a protease. Hemolysis and hemoglobin molecular structure change. Most of hemoglobin is present in erythrocytes, and hemoglobin comes out of erythrocytes in the presence of an appropriate concentration of surfactant. In addition, hemoglobin is usually present in a folded state, but is present in a relaxed state in an appropriate concentration of surfactant, and it is presumed that degradation by a protease is facilitated. Surfactants include nonionic polyoxyethylene alkyl ethers [for example, polyoxyethylene (10) octylphenyl ether (Triton X-100), polyoxyethylene (23) lauryl ether, etc.] and polyoxyethylene sorbitan fatty acids Esters [eg, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), etc.], anionic polyoxyethylene alkyl ethers and alkyl sulfates [sodium dodecyl sulfate (SDS ) Etc.]], cationic and zwitterionic, but anionic surfactants are desirable because they have the two effects described above. The concentration is 0.01 to 10%, preferably 0.05 to 5%.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is of course not limited thereto.
Example 1
(1) Manufacture of fructosyl amino acid oxidase-immobilized column 150 mg of refractory brick (80-100 mesh) was thoroughly dried, immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, and then thoroughly washed with toluene. And dry. The carrier thus treated with aminosilanization is immersed in 5% glutaraldehyde for 1 hour, then thoroughly washed with distilled water, and finally replaced with 100 mM sodium phosphate buffer at pH 7.0, and this buffer is removed as much as possible. 400 μl of a solution prepared by dissolving fructosyl amino acid oxidase (manufactured by Kikkoman) at a concentration of 18 units / ml in a pH 7.0, 100 mM sodium phosphate buffer solution was brought into contact with this formylated refractory brick and left at 0 to 4 ° C. for 1 day. And fix. This enzyme-immobilized carrier is packed in a column having an inner diameter of 3.5 mm and a length of 30 mm to obtain a fructosyl amino acid oxidase-immobilized column.
(2) Production of Umamizyme G immobilized column (Toyonite) 300 mg of Toyonite 200 (average particle size 170 μm, porous, manufactured by Toyo Denka Kogyo Co., Ltd.), a carrier made of kaolinite ceramics, was aminosilanized in the same manner as in (1). Formylize. This formylated toyonite is brought into contact with 800 μl of a solution prepared by dissolving Umamizyme G (manufactured by Amano Enzyme) at a concentration of 100 mg / ml in a pH 7.0, 100 mM sodium phosphate buffer, and allowed to stand at 0 to 4 ° C. for 1 day for immobilization. To do. This enzyme-immobilized carrier is packed in a column having an inner diameter of 3.5 mm and a length of 30 mm to form an equinezyme G-immobilized column (toyonite).
(3) Production of hydrogen peroxide electrode The side surface of a platinum wire having a diameter of 2 mm is covered with heat-shrinkable Teflon (registered trademark), and one end of the wire is smoothly finished with a file and No. 1500 emery paper. Using this platinum wire as a working electrode, a 1 cm square platinum plate as a counter electrode, and a saturated calomel electrode as a reference electrode, electrolytic treatment is performed in 0.1 M sulfuric acid at +2.0 V for 10 minutes. Thereafter, the platinum wire is thoroughly washed with water, dried at 40 ° C. for 10 minutes, immersed in an anhydrous toluene solution of 10% γ-aminopropyltriethoxysilane for 1 hour, and then washed. 20 mg of bovine serum albumin (manufactured by Sigma, Fraction V) is dissolved in 1 ml of distilled water, and glutaraldehyde is added to 0.2% therein. This mixed solution is quickly put on 5 μl of the previously prepared platinum wire and dried and cured at 40 ° C. for 15 minutes. This is a hydrogen peroxide electrode.

An Ag / AgCl reference electrode was used as the reference electrode, and a conductive pipe was used as the counter electrode.
(4) Measuring apparatus FIG. 2 is a flow type fructosyl amino acid measuring apparatus incorporating an equinezyme G immobilized column and a dialysis module. The kaolinite-based ceramic enzyme-immobilized column (25) of FIG. 2 is arranged with an equinezyme G-immobilized column (toyonite), and a first hydrogen peroxide-producing enzyme-immobilized column (14) with a fructosyl amino acid oxidase-immobilized column. did. In the present embodiment, the second hydrogen peroxide production-immobilized enzyme column (16) and the hydrogen peroxide electrode (17) are not arranged. Buffer B is sent from the buffer tank (21) by the pump (22), and 100 μl of sample is injected using the metering valve (3). The injected sample is carried along the flow of the buffer B to the equinezyme G-immobilized column (toyonite) (25) arranged in the thermostatic bath (12), and glycated amino acid is generated by the action of equinezyme G. . The sample on which the Umamizyme G acted is transported to the dialysis module (26) installed further downstream by the flow of the buffer B, and the low molecular component in the sample is formed with a regenerated cellulose membrane having a thickness of 20 μm and a molecular weight fraction of 12000 to 14000 Only leads to the fructosyl amino acid detection mechanism (14, 15). Since the buffer A is sent from the buffer tank (1) by the pump (2), the low molecular components in the sample dialyzed by the dialysis module (26) are the fructosyl amino acid oxidase immobilized column (14) and Hydrogen peroxide generated from fructosyl amino acids in the sample is detected after passing through the hydrogen peroxide electrode (15).

The buffer solution to be flowed to the apparatus has a buffer A containing 100 mM phosphoric acid, 50 mM potassium chloride, 1 mM sodium azide, pH 8.0, and a flow rate of 0.8 ml / min. Buffer B contains 50 mM phosphate, 0.1% sodium dodecyl sulfate, has a pH of 8.0 and a flow rate of 0.8 ml / min.
The temperature of the thermostatic bath was 37 ° C.
(5) Response of equine zyme G-immobilized column (toyonite) to hydrogen peroxide 100 μl of each solution of 2, 5, 10 μM hydrogen peroxide and 20, 50, 100 μM fructosylglycine was injected with the measuring device of (4). The detected value was obtained.

  The obtained calibration curve is shown in FIG. 3, and a calibration curve of the following formula was obtained. However, Y is a detected value (nA) and X is a concentration (μM) in a sample. R is a correlation coefficient.

Hydrogen peroxide calibration curve Y = 0.000X + 0.000
Fructosylglycine calibration curve Y = 0.190X + 0.004 r = 0.9999
Good linearity was obtained for fructosylglycine, but no response to hydrogen peroxide was obtained. Toyonite can decompose hydrogen peroxide.
Comparative Example 1
(1) Production of fructosyl amino acid oxidase-immobilized column A fructosyl amino acid oxidase (manufactured by Kikkoman) immobilized column was produced in the same manner as in Example 1.
(2) Manufacture of Umamizyme G immobilized column (refractory brick) In the same manner as in (1), 150 mg of refractory brick is formylated. This formylated refractory brick was brought into contact with 350 μl of a solution prepared by dissolving Umamizyme G (manufactured by Amano Enzyme) at a concentration of 30 mg / ml in a pH 7.0, 100 mM sodium phosphate buffer, and left at 0 to 4 ° C. for 1 day to fix. Turn into. This enzyme-immobilized carrier is packed in a column having an inner diameter of 3.5 mm and a length of 30 mm to form an equinezyme G-immobilized column (refractory brick) (25).
(3) Method for Producing Hydrogen Peroxide Electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1.
(4) Measuring device The same measuring device as in Example 1 was used, and the equinezyme G immobilized column (refractory brick) prepared in (2) was incorporated into the equinezyme G immobilized column (toyonite).
(5) Response of equinezyme G-immobilized column (refractory brick) to hydrogen peroxide 100 μl each of solutions of 2, 5, 10 μM hydrogen peroxide and 20, 50, 100 μM fructosylglycine with the measuring device of (4) The detection value was obtained by injection.

  The obtained calibration curve is shown in FIG. 4, and a calibration curve of the following formula was obtained. However, Y is a detected value (nA) and X is a concentration (μM) in a sample. R is a correlation coefficient.

Hydrogen peroxide calibration curve Y = 0.115X-0.030 r = 0.9997
Fructosylglycine calibration curve Y = 0.167X-0.045 r = 0.9997
The linearity was good and, unlike toyonite, a response to hydrogen peroxide was obtained.
Example 2
(1) Production of fructosyl amino acid oxidase-immobilized column A fructosyl amino acid oxidase (manufactured by Kikkoman) immobilized column was prepared in the same manner as in Example 1.
(2) Manufacture of equinezyme G-immobilized column (toyonite) In the same manner as in Example 1, equinezyme G (manufactured by Amano Enzyme) was immobilized on toyonite to produce an equinezyme G-immobilized column (toyonite).
(3) Method for Producing Hydrogen Peroxide Electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1.
(4) Measuring device Using the measuring device shown in FIG. 2, the second hydrogen peroxide production-immobilized enzyme column (16) and the hydrogen peroxide electrode (17) are not arranged, and the kaolinite ceramic enzyme-immobilized column ( 25), an equinezyme G-immobilized column (toyonite), a fructosyl amino acid oxidase-immobilized column, and a hydrogen peroxide electrode (15) were disposed on the first hydrogen peroxide-producing / immobilized enzyme column (14).
(5) Measurement of glycated hemoglobin using a protease-immobilized column (toyonite) It was shown in Example 1 that kaolinite-based ceramics (toyonite) can decompose hydrogen peroxide, and Comparative Example 2 shows that in the sample when hemoglobin is decomposed. Toyonite is applied to measure glycated hemoglobin because it has been shown that hydrogen peroxide presents positive interference.

  An equinezyme G-immobilized column (toyonite) is incorporated into the kaolinite-based ceramics-immobilized enzyme column (25) of the measuring device of (4), and 100 μl of hemoglobin hemolyzed blood is injected. Hemoglobin is decomposed to fructosyl amino acids and amino acids by the protease column (25), and hydrogen peroxide in the hemolyzed blood is decomposed by toyonite. The generated low molecular components such as fructosyl amino acids and amino acids are guided to a detection system through a semipermeable membrane. The dialyzed fructosyl L-valine generates hydrogen peroxide with a fructosyl amino acid oxidase fixed column (14), and the generated hydrogen peroxide is detected with a hydrogen peroxide electrode (15).

  The hemoglobin solution was prepared by weighing a predetermined amount of human hemoglobin (manufactured by Sigma) so that the hemoglobin concentration was 30 mg / ml and dissolving it in 20 mM phosphate buffer pH 8.0 containing 2% sodium dodecyl sulfate.

  When the fructosyl valine concentration in the 30 mg / ml human hemoglobin solution was measured with the measuring device of (4), the fructosyl valine was 15.1 μM.

By applying toyonite to the protease-immobilized carrier, the concentration of fructosyl amino acid in the sample could be accurately measured, excluding the interference of hydrogen peroxide and electrode active material in the sample.
Comparative Example 2
(1) Production of fructosyl amino acid oxidase-immobilized column A fructosyl amino acid oxidase (manufactured by Kikkoman) immobilized column was prepared in the same manner as in Example 1.
(2) Method for Producing Hydrogen Peroxide Electrode A hydrogen peroxide electrode was produced in the same manner as in Example 1.
(3) Measuring device Using the measuring device shown in FIG. 2, the kaolinite-based ceramic enzyme-immobilized column (25) and the first hydrogen peroxide-immobilized enzyme column (14) are not arranged, and the second hydrogen peroxide is generated. A fructosyl amino acid oxidase immobilization column and a hydrogen peroxide electrode (15, 17) were arranged on the enzyme immobilization column (16).
(4) Peroxide generated from standard hemoglobin protease treatment solution Human hemoglobin solution is treated with protease at a constant temperature for a predetermined time, and 100 μl of protease treatment solution is injected with the measuring device of (3). The low-molecular components in the protease treatment solution are guided to the detection system through the semipermeable membrane, the hydrogen peroxide in the sample is the hydrogen peroxide electrode (15) of the first channel, and the fructosyl amino acid is the fructose of the second channel. It is detected with a tosylamino acid oxidase immobilization column (16) and a hydrogen peroxide electrode (17).

  A human hemoglobin protease treatment solution was prepared by preparing human hemoglobin (manufactured by Sigma) so as to have a hemoglobin concentration of 30 mg / ml in the same manner as in Example 2, followed by 1 mg / ml of the protease Umamizyme G (manufactured by Amano Enzyme). Add to ml and react at 37 ° C. for a predetermined time.

  After reacting for 8, 17, 25, 33, 42, and 51 minutes, hydrogen peroxide and fructosyl amino acids in the sample were detected with the measuring device of (4). The results are shown in FIG. It was found that hydrogen peroxide of about 1 μM can be detected at any reaction time, and hydrogen peroxide generated from hemolyzed hemoglobin and protease is obstructed when a hydrogen peroxide electrode is used to detect fructosyl amino acids. .

Schematic diagram of measuring device Schematic diagram of a measuring device for dialysis Calibration curve obtained in Example 1 Calibration curve obtained in Comparative Example 1 Detection results of hydrogen peroxide and fructosyl amino acid in Comparative Example 2

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Buffer tank 2 Buffer solution pump 3 Metering valve 4 Metering loop 5 Sample tube 6 Sample suction needle 7 Waste pot 8 Washing solution tank
9 Valve 10 Valve 11 Syringe pump 12 Thermostatic bath 13 Mixing pipe 14 First immobilized enzyme column 15 Hydrogen peroxide electrode 16 Second immobilized enzyme column 17 Hydrogen peroxide electrode 18 Back pressure coil 19 Waste liquid bottle 20 Waste liquid bottle 21 Buffer Tank 22 Buffer Solution Pump 23 Damper 24 Constant Temperature Pipe 25 Kaolinite Ceramic Enzyme Immobilized Column 26 Dialysis Module

Claims (5)

Provided with a mechanism for feeding an aqueous solution at a constant flow rate and a mechanism for injecting a constant amount of a substance to be measured into the aqueous solution. The hydrogen peroxide electrode and the hydrogen peroxide electrode are included in the aqueous solution downstream of the injection mechanism. An enzyme-immobilized body that catalyzes the oxidation-reduction reaction of the substance to be produced and generates hydrogen peroxide, and the concentration of the substance to be measured is detected by detecting the change in the concentration of hydrogen peroxide accompanying the enzyme reaction with the hydrogen peroxide electrode. In the apparatus for calculating the analysis, an analysis is characterized in that a column filled with kaolinite ceramics which has been subjected to baking or sintering treatment after hydrothermal treatment is inserted between the injection mechanism and the hydrogen peroxide-producing enzyme immobilization body. apparatus. 2. The analyzer according to claim 1, wherein an enzyme that does not participate in the hydrogen peroxide generation reaction is immobilized on the kaolinite-based ceramics, and the hydrogen peroxide-producing enzyme immobilized body is disposed downstream of the immobilized body. A method for producing kaolinite-based ceramics for decomposing hydrogen peroxide, comprising calcining or sintering a kaolinite-based ceramic raw material after hydrothermal treatment. Use of kaolinite ceramics as a carrier to immobilize enzymes that are not involved in the hydrogen peroxide formation reaction A method for removing hydrogen peroxide contained in a sample, comprising arranging kaolinite ceramics upstream of an immobilized enzyme involved in a hydrogen peroxide production reaction.

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