JP2007292740A - Microflow type biosensor and use thereof for detecting or quantitating rare sugar - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor for detecting and/or detecting or quantitating rare sugar in food or a drink. <P>SOLUTION: The biosensor features a two-line type switching valve system microflow, comprising a first line for fitting a column for immobilizing an enzyme for generating a substrate to be measured by the direct reaction with a specific constituent contained in a sample to be measured, a second line for composing an electrode system including a substance having a function for converting the generated substrate to be measured to another compound, and a switching valve for switching a liquid feed to the first line or the second line. In the biosensor, the specific constituent is D-psicose, the substrate to be measured is D-fructose, an enzyme for generating the D-fructose by the direct reaction with the D-psicose is D-tagatose-3-epimerase (DTE), and a substance having a function for converting the generated D-fructose to another compound is D-fructosedehydrogenase (DFDH). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a biosensor for detecting or quantifying a rare sugar characterized by a microflow of a two-line switching valve system having an electrode and a bi-enzyme system, and use of the biosensor. More specifically, the present invention relates to a microflow biotechnology used for detecting or quantifying a specific component (rare sugar) contained in a biological sample such as blood or sweat, or a sample such as a raw material or product in the pharmaceutical / food industry. It relates to the use of sensors and biosensors.

Explanation of technical terms MFIA: Abbreviation for Micro Flow Injection Analysis.
DTE: Abbreviation for D-Tagatose-3-Epimerase. Epimerize the hydroxyl group at the 3-position of D-tagatose. It is an enzyme found by the present inventor Ken Mori.
DFDH: Abbreviation for D-Fructose Dehydrogenase. It is an enzyme that produces 5-keto-D-fructose from D-fructose in the presence of an acceptor.
CPE: Abbreviation for carbon paste electrode. This is an electrode prepared by attaching a mixture of enzyme and carbon paste to the tip of the electrode.
Izumoring: This is a ring-shaped system that links the molecular structure of all monosaccharides, including monosaccharides (rare sugars) that exist in trace amounts in the natural world, covering more than 50 types, and the production enzymes.

  In recent years, various types of biosensors utilizing specific catalytic action of enzymes have been developed for the purpose of realizing simple quantification of body fluid components by ordinary people. As an electrochemical glucose determination method, a method using glucose oxidase (GOD) in combination with an oxygen electrode or a hydrogen peroxide electrode is generally well known (for example, Non-Patent Document 1). GOD selectively oxidizes glucose as a substrate to gluconolactone using oxygen as an electron carrier. In the presence of oxygen, oxygen is reduced to hydrogen peroxide during the oxidation reaction of glucose by GOD. The amount of decrease in oxygen is measured with an oxygen electrode, or the amount of increase in hydrogen peroxide is measured with a hydrogen peroxide electrode. Since the decrease amount of oxygen and the increase amount of hydrogen peroxide are proportional to the content of glucose in the sample solution, glucose can be quantified from the decrease amount of oxygen or the increase amount of hydrogen peroxide. In the above method, the measurement result is disadvantageous in that it is affected by the concentration of oxygen contained in the sample solution, and measurement is impossible when oxygen is not present in the sample solution.

Therefore, glucose sensors of the type that use organic compounds such as potassium ferricyanide, ferrocene derivatives, and quinone derivatives or metal complexes as electron carriers, instead of using oxygen as an electron carrier, have been developed. In this type of sensor, the reduced form of the electron carrier generated as a result of the enzyme reaction is oxidized on the electrode, and the concentration of glucose contained in the sample liquid can be determined from the amount of oxidation current. When such an organic compound or metal complex is used as an electron carrier instead of oxygen, the electron carrier is supported on the electrode together with GOD in an accurate amount and in a stable state to form a reagent layer. Is possible. Furthermore, since the reagent layer can be integrated with the electrode system in a state close to a dry state, disposable glucose sensors based on these techniques have attracted much attention in recent years. A typical example is a biosensor disclosed in Patent Document 1. As an example of a biosensor, an electrode system consisting of a counter electrode and a measuring electrode is formed by printing a conductive carbon paste by screen printing on an insulating substrate made of polyethylene terephthalate (PET) and heating and drying the glucose sensor. To do. Next, the electrode system is partially covered, and an insulating paste is printed in the same manner as described above so as to leave 1 mm ^{2 serving} as an electrochemically acting portion of each electrode, and heat treatment is performed to form an insulating layer. To do. An aqueous solution of CMC (carboxymethylcellulose) is applied so as to cover the surface of this electrode system, and further, a solution in which GOD and potassium ferricyanide as an electron acceptor are dissolved as an oxidoreductase in the CMC aqueous solution is dropped, and the solution is 15 at 40 degrees. In the above, an enzyme reaction layer is formed by heat-drying, and the above two aqueous solutions are formed by further dropping a mixture solution of CMC, GOD and potassium ferricyanide without drying the CMC aqueous solution first developed on the electrode. By mixing and drying, a reaction layer made of the above mixture is formed on the electrode system. In a disposable glucose sensor, the glucose concentration can be easily measured with a measuring instrument simply by introducing a sample solution into a sensor detachably connected to the measuring instrument. In the measurement method using the glucose sensor as described above, the glucose concentration in the sample can be obtained in about 30 seconds with a response current of the order of 1 to 10 μA / cm ² .

Patent Document 2 has the following description of a fructose sensor as an example of a biosensor. A silver paste is printed on an insulating substrate made of PET by screen printing to form a lead, and further, an electrode system (measuring electrode, counter electrode) made of a conductive carbon paste containing a resin binder and an insulating paste are printed by a printing method. An insulating layer was formed. The insulating layer has a constant area of the exposed portion of the measurement electrode and the counter electrode, and partially covers the lead. After preparing the electrode part, a 0.5 wt% aqueous solution of CMC was dropped onto the electrode system and dried to form a CMC layer. Next, an aqueous solution of fructose dehydrogenase (EC 1.1.99.11; hereinafter abbreviated as FDH) was developed as an enzyme so as to cover the CMC layer and dried to form a CMC-FDH layer (first layer). In this case, CMC and FDH are in the form of a thin film having a thickness of several microns in a partially mixed state. Further, a 0.5 wt% ethanol solution of polyvinyl pyrrolidone (hereinafter abbreviated as PVP) was dropped so as to completely cover the CMC-FDH layer and dried to form a PVP layer (second layer). By providing a PVP layer, when a liquid such as fruit juice containing a solid component such as pulp is used as a sample solution, the solid component is filtered by the PVP layer and can prevent adsorption to the electrode surface. As a result, a decrease in sensor response can be prevented. Furthermore, the storage characteristics of the sensor can be significantly improved by separating the enzyme and potassium ferricyanide. On this PVP layer, potassium ferricyanide-lecithin layer (third layer) is prepared by dropping and drying a crystal of potassium ferricyanide as an electron acceptor in a 0.5 wt% toluene solution of lecithin as a dispersant. Formed. A fructose sensor reaction layer was formed as described above. A phosphate buffer solution (pH = 4.5) was dropped between the reaction layer and the tip of the substrate corresponding to the sample supply hole, and dried to form a hydrogen ion concentration control layer. The enzyme used in the fructose sensor has a maximum enzyme activity at pH = 4.5 (37 ° C.). Since the fructose standard solution is almost neutral, when the sample solution reaches the hydrogen ion concentration control layer and the pH of the sample solution reaches 4.5, the enzyme activity can be maximized. Further, when the sample solution is acid or alkaline, the pH of the sample solution can be made close to a value at which FDH shows the maximum activity. Fructose standard solution 3 μl as a sample solution is supplied to the fructose sensor from the sample supply hole, and after 2 minutes, a pulse voltage of +0.5 V is applied to the measurement electrode in the anode direction based on the counter electrode, and the current value after 5 seconds is measured. did. When the sample liquid having a desirable pH reaches the reaction layer through the hydrogen ion concentration control layer, the potassium ferricyanide-lecithin layer, the PVP layer, and the CMC-FDH layer are dissolved in the sample liquid in order. Fructose in the sample solution is oxidized by FDH, and potassium ferricyanide is reduced to potassium ferrocyanide by the transferred electrons. Next, by applying the pulse voltage, an oxidation current of the generated potassium ferrocyanide is obtained, and this current value corresponds to the concentration of fructose as a substrate.
In another embodiment, an electrode system is formed by screen printing on an insulating substrate made of PET. A mixed aqueous solution in which FDH and potassium ferricyanide were dissolved in 0.5% by weight of CMC was dropped onto this electrode system, and dried in a warm air dryer at 40 ° C. for 10 minutes to form a reaction layer. A manufacturing process can be simplified by dropping and drying a mixed solution of a hydrophilic polymer, an enzyme and an electron acceptor at a time. The temperature range at the time of drying is suitably 20 ° C. to 80 ° C. because the enzyme is not deactivated and can be dried in a short time.
Furthermore, in another aspect, an electrode portion was formed on an insulating substrate made of PET. Next, a solution in which potassium ferricyanide is dissolved in a phosphate buffer (pH = 4.5) is dropped between the electrode system and the tip of the substrate, which is a sample supply hole, and heated and dried to control the hydrogen ion concentration containing potassium ferricyanide. A layer was formed. The time required for drying varies depending on the heating temperature, and as a result, the crystal grain size of potassium ferricyanide can be controlled. When the drying time is shortened, the crystal grain size is reduced, and the dissolution rate in the sample solution is increased. Therefore, it is possible to increase the sensor response speed. When potassium ferricyanide coexists with the enzyme in the reaction layer, the temperature of the heat drying cannot be freely set because the activity of the enzyme is reduced by heating. Further, including potassium ferricyanide in the hydrogen ion concentration control layer separates the enzyme and potassium ferricyanide, and can significantly improve the storage characteristics of the sensor.

At Kagawa University, the discovery of a novel enzyme, D-tagatose 3-epimerase (DTE), which converts natural monosaccharides that exist in large quantities in nature into rare sugars, links the relationship between all monosaccharides, their molecular structures, and the resulting enzymes. The “Ismoring” is structured in the same way. D-psicose is one of the monosaccharides called rare sugars. Conventionally, since this sugar was difficult to obtain because it could not be mass-produced, there has been little research on its physiological activity and pharmacological activity. Recently, Ken Imorimori et al., Faculty of Agriculture, Kagawa University, developed a mass production method using an enzyme, and its biological activity has been clarified.
D-psicose has attracted attention as a sugar that does not promote fat synthesis and does not accumulate body fat, particularly intraperitoneal fat, compared to monosaccharides such as D-glucose and D-fructose (non-patented). Reference 2). It has also been reported that the effective energy value of D-psicose is almost zero (Non-patent Document 3). There is a high possibility that D-psicose is mass-produced as a complex crystalline saccharide containing D-fructose (Patent Document 3), and is expected to be used as a sweetener.

Regarding the physiological activity and pharmacological activity of D-psicose, Patent Document 4 discloses that glucose is digested and decomposed and absorbed from the intestine, so that the influence of ketohexose belonging to a rare sugar on glucose absorption is described in D- Examined using psicose and intestinal tract, D-psicose supports the action of moderately suppressing blood glucose level in animals (rats), D-psicose has hypoglycemic and hyperglycemic effects during hyperglycemia, and D -It was reported that psicose confirmed that glycation of protein hardly occurred. Patent Document 1 reveals that an insulin secretion stimulating action from pancreatic β cells of a ketohexose belonging to a rare sugar and an insulin secretion enhancing action of a ketohexose belonging to a rare sugar in a hyperglycemic state. The ketohexose belonging to a rare sugar is Expected to be a substance with an unprecedented new mechanism of action, and ketohexose belonging to rare sugars may promote insulin secretion and improve blood glucose levels in diabetic patients with clinically hyperglycemia. What is expected is that ketohexose belonging to rare sugars administered enterally suppresses absorption of glucose, and that ketohexose belonging to rare sugars does not affect glucose metabolism is postprandial hyperglycemia in diabetes Expected to be a useful substance for the prevention or treatment of diabetes, In addition, ketohexose belonging to rare sugars also has a preventive effect on arteriosclerosis, which is strongly related to diabetes and its complications. Since the main cause of diabetes is arteriosclerotic disease, it belongs to rare sugars that also have an arteriosclerotic inhibitory effect. Ketohexose is expected to be a revolutionary anti-diabetic drug for improving blood glucose level and preventing arteriosclerosis, and ketohexose belonging to rare sugars is expected to have therapeutic effects and anti-obesity. It is shown that it is expected to be a supplement.
In addition, as a result of experiments on the effect of D-psicose on blood glucose elevation when starch or sucrose was orally administered to rats, the increase in blood glucose caused by oral loading of starch or sucrose was simultaneously administered orally to D-psicose. Has been confirmed to be significantly suppressed (Patent Document 4).

In addition, D-allose, which is one of aldoses, inhibits active oxygen production in in vitro experiments using leukocytes, while D-psicose, which is one of ketoses, exhibits similar effects on scavenging of active oxygen and MCP. -1 secretion inhibitory action and the like have been found (Patent Document 5). Blood glucose refers to D-glucose present in the blood. From the chemical structure, D-glucose is an aldose and belongs to the same group as D-allose. Therefore, D-allose can be expected to have an effect of suppressing an increase in blood glucose as in the case of D-psicose.

  The establishment of technology for mass production of D-psicose, which is the basic raw material for the production of all rare sugars, has made it possible to produce rare sugars that have been difficult to obtain. D-psicose can be further developed into the production of new rare sugars such as D-allose by enzymatic reaction. As various physiologically active actions of rare sugars are clarified, the development of measuring methods has been demanded. However, these rare sugars are substances that we have finally obtained in recent years and have not yet established a measurement method. In order to search for pharmacological effects and new functions of rare sugars, and to accurately evaluate in vivo functionality, we have developed a sensor for measuring rare sugars that can quickly and easily measure and monitor rare sugars. Expected.

Japanese Patent No. 2517153 Japanese Patent No. 2671693 JP 2001-11090 A International Publication Number WO03 / 097820 JP 2005-213227 A Shuichi Suzuki “Biosensor” Kodansha) MatsuoT, et al., Asia Pacific J. Clin. Nutr. 10, 233-237, 2001 Matsuo T, et al., J. Nutr. Sci. Vitaminol48, 77-80, 2002

Rare sugars are monosaccharides that are very few in nature, and there are about 50 kinds. At Kagawa University, the discovery of DTE, which converts natural monosaccharides into rare sugars, established “Izumoring” (Fig. 7), which links all hexoses, their molecular structures, and the production enzymes. It is expected that a new bioindustry will be created from Kagawa using this Izumoring. Among these rare sugars, D-psicose has various physiological activities such as cancer growth inhibitory action and diabetes prevention action, and has attracted attention as a novel physiologically active substance.
As mentioned above, Kagawa University has succeeded in producing the world's first rare sugars from abundantly existing sugars and has clarified their various functions. However, since the measurement method has not been established and monitoring in vivo is difficult, the progress of applied research and development is currently delayed. The determination requires labor and time. Therefore, the present invention aims to construct a novel biosensor for measuring rare sugars for the purpose of quickly and highly sensitively measuring and monitoring D-psicose, which is a basic raw material for rare sugar production. In other words, development of a new biosensor for the measurement of rare sugars that can detect rare sugars quickly and with high sensitivity that can monitor various effects such as involvement in information transmission in vivo and pharmacological effects. And it was aimed at practical use.

  Assuming measurement methods using existing glucose oxidase, it is difficult to selectively measure traces of rare sugars in living bodies (in blood), drinking water, and foods due to the influence of a large excess of mixed glucose. Therefore, it is necessary to develop a measurement system using a reaction system that can selectively detect rare sugars. In addition, as the name suggests, the amount of rare sugars in the environment is rare (a trace amount). Therefore, investigation of conditions under which contaminants do not directly affect the enzyme immobilized oxygen electrode tip, sensor detection, etc. It is necessary to devise a technique for increasing the limit value and a technique for promoting a conversion reaction (reversible reaction with different ratios) for converting to an existing sugar to a detectable level.

  In the conventional analytical element, if there is a sample of several microliters or more, the substrate concentration in the sample can be easily obtained. However, reliable measurement may be difficult for a very small amount of sample of less than a few microliters. Therefore, an object of the present invention is to provide an electrochemical analysis element that enables reliable measurement of a very small amount of sample.

As is apparent from the above description, a rapid, accurate, easy and convenient analytical instrument tool is required to determine trace amounts of rare sugars in pharmaceuticals and foods. An object of the present invention is to provide a biosensor for detecting and / or detecting or quantifying rare sugars in foods or beverages.
Yet another object of the present invention is to provide a method for using a biosensor as an analytical tool in the detection and / or detection of rare sugars in foods.

The gist of the present invention is the biosensor according to any one of the following (1) to (11).
(1) First line equipped with a column to which an enzyme that generates a substrate to be measured is immobilized by direct reaction with a specific component contained in a sample to be measured, and converts the generated substrate to be measured into another compound. A biosensor characterized by a microflow of a two-line type switching valve system comprising a second line constituting an electrode system including a substance having a function, and a switching valve for switching liquid feeding to the first line and the second line.
(2) The biosensor according to (1), including an injection port for injecting a sample to be measured toward the first line.
(3) A buffer A for the first line is connected to the injection port via the pump 1 and the switching valve 1 or to the second line via another switching valve 3, and the buffer for the second line. The biosensor according to (1) or (2), wherein the container of the liquid B is connected to the second line via the pump 2 and the switching valves 2 and 3 or to the injection port via the other switching valve 4.
(4) The biosensor according to (1), (2), or (3), wherein the buffer A and the buffer B include an electron carrier.
(5) The electrode system includes an working electrode and a counter electrode arranged in the micro flow cell, and the micro flow cell is an electrode system to which a reaction solution including a substrate to be measured, a buffer solution, and an electron carrier is fed. The biosensor according to any one of (1) to (4).
(6) The biosensor according to (5), wherein a substance having a function of converting the generated substrate to be measured into another compound is provided on or near the working electrode.
(7) The specific component is D-psicose, the substrate to be measured is D-fructose, and the enzyme that produces D-fructose by direct reaction with D-psicose is D-tagatose-3-epimerase (DTE). The biosensor according to any one of (1) to (6), wherein the substance having a function of converting the produced D-fructose into another compound is D-fructose dehydrogenase (DFDH).
(8) The biosensor according to (7), comprising a first line equipped with a DTE-immobilized column and a second line equipped with a DFDH-immobilized carbon paste electrode (CPE).
(9) The biosensor according to (8), wherein a line equipped with a column to which an enzyme that converts D-allose to D-psicose is immobilized is provided before the first line.
(10) The biosensor according to (8) or (9), wherein a membrane showing selective permeability is mounted on the CPE surface.
(11) The sample to be measured is injected into the first line equipped with a DTE-immobilized column and reacted under the optimum conditions for DTE, and then the switching valve is operated at the same time as the reaction is completed, and the optimum conditions for DFDH. The second line is set to have a higher ionic strength than the buffer solution of the first line, and this causes the buffer solution of the second line to enter the reaction solution. By mixing a small amount, the pH changes to the optimum value of DFDH, and then the sample is measured by measuring the amount of D-fructose in the reaction solution on the surface of the DFDH-immobilized carbon paste electrode mounted on the second line. The biosensor as set forth in (7), (8), (9) or (10), wherein the amount of D-psicose therein is measured.

The gist of the present invention is the following (12) rare sugar detection or quantification method.
(12) A biological sample selected from the group consisting of serum, plasma, blood, sweat, urine and cells using the biosensor according to any one of (1) to (11), a raw material or a product in the pharmaceutical / food industry A method for detecting or quantifying rare sugars that detects or quantifies a specific component contained in a sample.

  As described above, according to the present invention, an electrochemical analysis capable of quickly and easily quantifying a specific component contained in a biological sample such as blood and urine, and a small amount of sample such as a raw material or product in the food industry. An element can be obtained. Therefore, according to the present invention, it is possible to monitor a variety of effects such as detection of trace amounts of rare sugars, involvement in information transmission in vivo, and pharmacological effects. A sensor can be provided.

By adopting a two-line type switching valve type MFIA, a system capable of continuous reaction of two enzymes (enzyme 1 and enzyme 2) with completely different enzyme characteristics (different optimum pHs) was constructed. Further, this makes it possible to react under optimum conditions in each reaction stage in the sensor, and the detection limit value of the sensor can be significantly improved. In addition, it is important to use a sensor element that accurately discriminates slight differences in sugar, but we have confirmed that the DFDH used in this sensor does not react at all to naturally occurring sugars or rare sugars. By using this enzyme and DTE, it is possible to detect and monitor rare sugars in blood that contains a large amount of glucose, and to detect rare sugars in beverages and dietary supplements that contain a large amount of sugar. It can be measured with high accuracy. It is possible to provide a sensor capable of measuring and monitoring a rare sugar that has no example in Japan and overseas.
Therefore, the present invention can provide a biosensor for detecting and / or detecting or quantifying rare sugars in foods or beverages. In addition, it is possible to provide a method for using a biosensor as an analytical tool in detecting and / or detecting rare sugars in foods.

  The present invention provides a first line equipped with a column to which an enzyme (enzyme 1) that generates a substrate to be measured by direct reaction with a specific component contained in a sample to be measured is attached, It consists of an electrode system consisting of a working electrode and a counter electrode that includes a substance (enzyme 2) that has the function of converting to a compound of the above, and during this conversion reaction, the electrons received by the electron acceptor are measured as a current response corresponding to a specific component. A biosensor characterized by a two-line switching valve system comprising a switching valve for switching between the first line and the second line, wherein the reaction solution of the second line and the first line are injected into the second line.

  Regarding the specific component contained in the sample to be measured, the reaction in the first line is a reaction in which the specific component is converted into the measurement target substrate by the action of the enzyme (enzyme 1) that generates the measurement target substrate. In other words, the first line is equipped with a column (reactor) on which an enzyme (enzyme 1) that generates the substrate to be measured is immobilized, and the sample passes through this line over a certain period of time. The reaction between the enzyme that generates the substrate and the specific component contained in the sample proceeds. The sample to be measured is a biological sample selected from the group consisting of serum, plasma, blood, sweat, urine, cells, raw materials or products in the pharmaceutical / food industry, and the specific component contained in the sample to be measured , A specific component (rare sugar) contained in a biological sample selected from the group consisting of serum, plasma, blood, sweat, urine and cells, and a raw material or product sample in the pharmaceutical / food industry.

Next, the reaction solution in the first line is injected into the second line. The substrate to be measured generated by the reaction is measured as a current response in the second line composed of an electrode system including a substance (enzyme 2) having a function of converting to another compound. The substrate to be measured at this time is generated from a specific component, that is, it can be confirmed as a current response corresponding to the specific component.

As shown in FIG. 7, Izumoring of monosaccharides with 6 carbon atoms (hexoses) for rare sugars as specific components contained in the sample to be measured is a total of monosaccharides with 6 carbon atoms (hexoses). There are 34 types, 16 types of aldose, 8 types of ketose, and 10 types of sugar alcohol. These sugars can be converted by oxidoreductase reaction, aldose isomerase reaction, and aldose reductase reaction, and it is known in studies including the study of the present inventors Ken Mori. D-Tagatose-3-Epimerase is an enzyme that epimerizes the 3-position of all ketoses, and D-fructose, D-psicose, and L-sorbose, which could not be synthesized and connected so far. It was found to be a unique enzyme with a very broad substrate specificity that acts on L-tagatose, D-tagatose and D-sorbose, L-psicose and L-fructose. With the discovery of this DTE, all monosaccharides were linked in a ring shape, and the structuring of monosaccharide knowledge was completed and named Izumoring.
The biosensor of the present invention uses the above-mentioned relationship between D-fructose and D-psicose, which focuses on D-psicose, which is a basic raw material for producing rare sugars. Variations using multiple reactions leading to D-psicose are possible. For example, for D-allose, D-allose and D-psicose on Izumoring are very close together and they are converted by L-rhamnose isomerase. In other words, by examining the optimum conditions with L-rhamnose isomerase, it is converted to D-psicose, then converted to D-fructose, and injected into the D-fructose sensor (second line), the current corresponding to D-allose. The response is confirmed.

In order to measure a small amount of rare sugars in the presence of a large amount of monosaccharides existing in nature in the two-line type switching valve type micro flow, DTE (D-tagatose-3-epimerase) as enzyme 1 and as enzyme 2 A complex enzyme reaction system of DFDH (D-fructose dehydrogenase) is employed. In DTE, the optimum pH for the reaction to proceed is near neutral, whereas in DFDH, the optimum pH is in the vicinity of production, so it is impossible to allow each enzyme reaction to proceed simultaneously in the same buffer. It is. For example, in the vicinity of pH 5 where the DFDH enzyme reaction proceeds, the DTE enzyme reaction hardly proceeds. The DTE used for the first reaction and the DFDH used for the second reaction have completely different enzyme properties (different optimum pH), so it is impossible to co-immobilize and react the two enzymes. Thus, when the properties of each enzyme are remarkably different, it is necessary to employ a two-line type flow path that allows each enzyme to react separately.
In addition, a switching valve system is employed in which each buffer solution can be switched at an optimal timing and ratio. As a solution to these problems, the present invention is an MFIA system that employs a two-line switching valve.

  DTE is an enzyme found by the present inventor Ken Mori and can be measured only by the fusion of the detailed enzyme characteristics of the enzyme and the sensor. There has never been a sensor system tem that can measure rare sugars and sensor system tems that can monitor behavior in vivo. The enzymatic reaction in the second line that constitutes the electrode system that includes the substance (enzyme) that has the function of converting the substrate to be measured into another compound smoothly progresses, so that the substrate can be rapidly and rapidly increased. Accurate measurement is possible. The substance having the function of converting the substrate into another compound should of course not return the substrate to the original specific component or convert it into a compound that adversely affects the enzyme reaction. . In addition, the substance itself should not adversely affect the enzyme reaction.

  Regarding the D-fructose sensor constituting the second flow path (second line), as a method for avoiding the influence of contaminants such as glucose present in blood or food at a high concentration, for example, D-fructose dehydrogenase (DFDH) ) Is used. DFDH does not react at all with existing monosaccharides other than D-fructose and does not show any activity against all the rare sugars available at Kagawa University by the present inventors. It is an extremely high enzyme, and the reliability of the current response value obtained from the constructed sensor is extremely high (FIG. 4). Further, when constructing a sensor that interweaves an electrochemical method and a biological component, for example, a carbon paste electrode is employed in order to switch to an electrical signal so as not to impair the function of the biological component. Carbon paste and enzyme are mixed in an optimal amount ratio, and this is filled into a micro-processed cylinder. A conductive core is attached to the end of the tube, and a dialysis membrane is attached to the end immersed in the solution on the opposite side to form a carbon paste electrode.

  The measurement sample is injected into the first line equipped with a DTE-immobilized column, and is efficiently converted to D-fructose under the optimum reaction conditions of DTE. At the same time as the reaction is completed, the switching valve is switched, and the solution is sent to the second line filled with a buffer solution optimal for DFDH. The second line is equipped with a microflow cell equipped with a DFDH-immobilized carbon paste electrode, and D-fructose converted from D-psicose can be measured under the optimum conditions of DFDH. The microflow cell is a cell having a capacity of several μl to several tens of μl, in which a total of four electrodes, a reference electrode, a counter electrode, and a working electrode (two) are embedded. One working electrode (carbon paste electrode) employs a carbon paste electrode on which no enzyme is immobilized. By measuring the current value of this electrode at the same time, it is a contaminant that shows reactivity to the carbon paste electrode itself. Can be subtracted as background.

  Furthermore, by changing the liquid feeding speed of the first line and the second line, the measurement range can be manipulated, and various samples from a low concentration to a high concentration can be handled. In other words, when the substance to be measured is present in the sample to be measured at a high concentration, the reaction efficiency of “Enzyme 1” attached to the first line is lowered by increasing the flow rate of the first line, and the reaction generation You can reduce things. If the reaction product is fed to the second line in a state where there is little reaction product, the measurement limit value on the high concentration side of “Enzyme 2” attached to the second line is not exceeded, and thus measurement is possible. Further, since the MFIA system is a micro flow channel, consumption of the measurement sample can be minimized, and continuous measurement can be performed in a short time.

Regarding buffer A and buffer B, a solution in which a buffering component is dissolved and the pH is stabilized is referred to as a buffer solution, and a buffering component is referred to as a buffering agent.
When the specific component is D-psicose and the substrate to be measured is D-fructose, D-tagatose-3-epimerase (DTE) and D-fructose dehydrogenase (DFDH) are used. A specific component is a buffer so that D-psicose and D-tagatose-3-epimerase (DTE) react efficiently, and a pH buffer so that D-fructose and D-fructose dehydrogenase (DFDH) react efficiently. It is preferable to add.
When using a pH buffer, it is necessary to consider the optimum pH of D-fructose dehydrogenase (DFDH). Examples of the pH buffer include, in addition to a buffer based on a combination of phosphates used in the examples described later, one or more of phosphate, acetate, borate, citrate, phthalate, and glycine. Can be used. There is no problem with the composition of the buffer as long as the pH matches. It is important to optimize their ratio for the two buffers used.
In addition, the surfactant used as necessary may be any surfactant that does not impair the effects of the present invention. For example, n-octyl-β-D-thioglucoside, polyethylene glycol monododecyl ether, sodium cholate, dodecyl-β -Maltoside, sucrose monolaurate, sodium deoxycholate, sodium taurodeoxycholate, N, N-bis (3-D-gluconamidopropyl) deoxycholamide and polyoxyethylene (10) octylphenyl ether It is done. When lipids are used, for example, phospholipids such as lecithin, phosphatidylcholine, and phosphatidylethanolamine are preferably used, and amphiphilic lipids are particularly preferably used.

In the biosensor of the present invention, the reduced form of the electron carrier generated as a result of the enzyme reaction is oxidized on the electrode, and the concentration of fructose contained in the sample solution can be determined from the amount of oxidation current (FIG. 1).
FIG. 1 is a reaction path for measuring rare sugars in the present invention. D-psicose is reversibly converted to D-fructose by DTE (D-tagatose-3-epimerase) (D-psicose: D-fructose = 1: 3). Next, D-fructose converted by DTE is converted to 5-keto-D-fructose by DFDH (D-fructose dehydrogenase). During this reaction, D-psicose can be measured by electrochemically measuring electrons received by an electron carrier (acceptor).
Moreover, since D-allose is converted to D-psicose by LRI (L-rhamnose isomerase), it is possible to measure D-allose by employing a three-enzyme reaction pathway.
Electron carriers include potassium ferricyanide, metal complexes such as osmium-tris (bipyridinium) and ferrocene derivatives, quinone derivatives such as p-benzoquinone, phenazinium derivatives such as phenazine methosulfate, phenothiazinium derivatives such as methylene blue, nicotinamide Examples include adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. These electron carriers are preferably used in a form dissolved in buffer A and buffer B so as to always exist around the electrodes. In the examples, potassium ferricyanide was used at a concentration of 0.1M. Even at a lower concentration (for example, about 25 mM), it functions sufficiently as a mediator. It can be said that it is common for each sensor to vary the concentration within such a range. Further, the electron carrier may be in a form bonded to a polymer backbone, or a form in which a part or all of itself forms a polymer chain (see Patent Document 2). One or more of these electron carriers are used.

  As a method for measuring the oxidation current, there are a two-electrode method using only a working electrode and a counter electrode, and a three-electrode method using these electrodes and a reference electrode. Of these, more accurate measurement is possible using the three-electrode method.

Hereinafter, the present invention will be described in more detail with reference to examples. The structure of the sensor of the present invention will be described with reference to FIG. 6, but the present invention is not limited to these.
The sensor system pump of FIG. 6 uses two Tosoh HPLC pumps. The injector is made of Leodyne, a stainless steel line, but there is no problem with the peak tube. The column used was a cut Teflon tube, but a GL Science empty column (stainless steel, ready-made) can be used. The materials are not limited to these. In the embodiment, a diameter of 2.1 mM and a length of 6 cm are employed. The flow cell, carbon paste electrode, reference electrode, counter electrode, carbon paste, electrochemical measuring device, and software all use BAS. DFDH is manufactured by Toyobo (Toyobo). The pump and the switching valve may be of any size and performance suitable for the purpose. By properly switching, sensitivity and accuracy are greatly improved. By changing the ratio of the flow rate to this, the reaction time of each enzyme can be secured and the sensitivity can be further increased.

[Development purpose]
To construct a new biosensor for measuring rare sugars for the purpose of measuring and monitoring D-psicose, which is a basic raw material for rare sugar production, quickly and with high sensitivity.

[Method]
The reaction strategy of this sensor is shown in FIG. This sensor adopts a two-line switching valve type MFIA system with quick and simple measurement of D-psicose and continuous measurement in mind.
D-psicose is reversibly converted to D-fructose by DTE (D-psicose: D-fructose = 1: 3). For this reason, if D-fructose converted by DTE can be measured efficiently and with high sensitivity, it is possible to measure D-psicose.
DFDH is an enzyme that converts D-fructose to 5-keto-D-fructose in the presence of an acceptor, but has high substrate specificity and does not exhibit any activity on monosaccharides other than D-fructose. Furthermore, the inventors have shown that DFDH does not show activity in all rare sugars that can be produced at Kagawa University.

  FIG. 2 shows the results of confirming the influence of D-psicose in the electrochemical measurement of D-fructose as an example. 25 mg of carbon paste and 10 mg of DFDH were mixed and covered with the electrode tip. However, it goes without saying that the present invention is not limited to this. It is only necessary that DFDH acts on the electrode tip and the acceptor can exchange electrons. Three electrodes using the carbon paste electrode, the reference electrode, and the counter electrode were immersed in 2 ml of the solution, and the solution was stirred at a constant rotational speed. A voltage of 400 mV was applied to the working electrode with reference to the counter electrode. The current value that flowed when a certain amount (final concentration was set to 0.2 mM in this figure) was added thereto was measured. When D-glucose and D-psicose were added, no change was observed in the current value. Only when D-fructose was added, the response current was confirmed specifically. This showed that D-psicose had no effect on the electrochemical measurement of D-fructose.

FIG. 3 shows a case where D-psicose and DTE are reacted in a batch reaction for a certain time, and then D-fructose is electrochemically measured.
The reaction was fast and stable. The second injected psicose was obtained by measuring D-fructose without reacting with DTE, and no electrode response was observed. In addition, the current response was not confirmed in the case where only DTE was injected last. This shows that DTE and psicose have no direct electrode activity.

FIG. 4 confirms the influence of various rare sugars in the measurement of D-fructose. When each sugar other than fructose was injected, no current response was confirmed. In addition, since no current response was confirmed in all the sugars used when using the carbon paste electrode without enzyme, it was shown that each monosaccharide does not show activity on the electrode itself. .
It was confirmed that this sensor can measure D-fructose with high accuracy and high sensitivity by using an enzyme with high substrate specificity, which is the best of biosensors.

When using two enzyme reactions, it must be noted that both enzyme properties and the reaction pathway of the sensor have a significant effect on sensitivity and accuracy. It has been clarified that DTE used for the first reaction of this sensor and DFDH used for the second reaction have greatly different enzyme characteristics (particularly optimum pH). Therefore, the most efficient method of co-immobilizing both enzymes on one electrode cannot be used.
In view of this, this sensor employs a two-line type switching valve type flow path (central part of FIG. 6). A sample to be measured is injected from the first line equipped with a DTE-immobilized column and allowed to react efficiently under the optimum conditions for DTE. Next, simultaneously with the completion of the reaction, the switching valve is operated, and the reaction solution is sent to the second line set to the optimum conditions for DFDH. The ionic strength of the second line is set to be higher than that of the buffer solution in the first line. This allows the pH to reach the optimum value for DFDH simply by mixing a small amount of the buffer solution in the second line into the reaction solution. Change.
Further, by utilizing the slight difference in flow rate between the first line and the second line, the reaction solution can be more efficiently changed to a pH optimum for DFDH. Thereafter, the amount of D-psicose in the sample can be measured by measuring the amount of D-fructose in the reaction solution on the surface of the DFDH-immobilized CPE (carbon paste electrode) mounted on the second line.
Moreover, the influence of a polymeric contaminant is prevented by mounting | wearing the CPE surface with the film | membrane which shows selective permeability. The measurement principle of this sensor system is not only the measurement of D-psicose, but also an innovative flow path that can be used to measure various substances using two enzymes with different properties. System.

[Application development]
Developing highly versatile sensors can contribute to accelerating rare sugar research. The MFIA system proposed in the present invention can be sufficiently applied to a microchip flow biosensor by further developing the flow path system. At the stage of commercialization of microchip flow biosensors, we will investigate the development of a low-cost simple sensor using the capillary effect. The use of sensors that can be easily and highly sensitively measured in a short time in companies and various research institutions can sufficiently contribute to the discovery of new functions of rare sugars.
Furthermore, paying attention to the relationship between monosaccharides including rare sugars shown by Izumoring, we will examine the measurement of other rare sugars. By further improving the D-psicose sensor constructed according to the present invention, a sensor for measuring D-allose, one of the rare sugars, is developed. D-allose and D-psicose on Izumoring are very close together and they are converted by L-rhamnose isomerase. That is, it is possible to construct a D-allose sensor by examining the optimum conditions between this sensor and L-rhamnose isomerase. D-allose is extremely difficult to measure by HPLC and cannot be measured by the influence of contaminants, but the D-allose sensor, which is an improved D-psicose sensor, is easily sensitive to the amount in a short time. Therefore, it is a sensor with very high development value.

  FIG. 5 shows whether D-allose can be electrochemically measured using the reaction pathway shown in FIG. First, LRI and DTE were simultaneously reacted with D-allose for a certain time, and D-fructose generated in the reaction solution was electrochemically measured. As a result, a current response due to the generation of D-fructose by continuous enzyme reaction from D-allose was confirmed. D-allose + LRI + DTE (x2) shows the injection of twice the amount of the solution, and it can be seen that the current response is doubled. It has been shown that D-allose is measured by a continuous reaction of three enzymes.

[Clinical application research]
Applied research using this sensor will be developed as follows. After preparing a calibration curve for the D-psicose measurement biosensor, an addition / recovery experiment is performed using blood collected from the mouse and D-psicose. This confirms the sensor response in the presence of contaminants in the blood.
Next, as an in vitro analysis, human peripheral blood lymphocytes and D-psicose were cultured in RPMI 1640 medium for 24 hours, and the effect of D-psicose on human lymphoma-derived U937 cells in the culture supernatant and the amount of D-psicose remaining. The correlation is monitored using this sensor. In addition, regarding various biological activities caused by the addition of D-psicose confirmed so far, the influence on the cell metabolism is confirmed by monitoring changes in the amount of D-psicose inside and outside the cell. For the U937 cells subjected to the above treatment, basic knowledge about subsequent cell growth and changes in sensitivity to other monosaccharides is obtained.
As an in-vivo analysis, D-psicose or D-allose is administered to a healthy mouse, blood is collected for a certain period of time, and the time course of blood rare sugars is monitored using this sensor. Next, using a tumor-bearing mouse model using sarcoma 180 tumor cells, etc., the amount of rare sugar in the blood was measured by the same treatment as above, and when anti-tumor of D-psicose was confirmed Consider the optimal concentration of activity. At that time, the antitumor effect and residual rare sugar amount when various antitumor agents are administered orally in addition to ip and iv will be monitored and their correlation will be investigated.

  Furthermore, we will also investigate lymphokines that may be released into the lymphocyte culture supernatant by immunological techniques using specific antibodies, and investigate the relationship with changes in rare sugars. At the same time, we will clarify the effective optimal concentration of rare sugars and experimental conditions such as lymphocyte culture time and addition time. In addition, we will clarify the correlation between the increase in cytotoxicity of lymphocyte culture supernatant and the concentration of rare sugars in combination with antibiotics such as actinomycin D. This study provides valuable insights into rare sugar behavior and cellular responses in vivo. By further encouraging this research, it is expected to be developed as a novel pharmacological effect of rare sugars such as D-psicose and as a remedy for intractable diseases, and as clinical application and health food. I want to connect with the possibility of industrialization.

  FIG. 6 constructed a sensor system reaction path based on the results of FIGS. A double pump type flow path that employs a switching valve is designed. When adapting to other rare sugars, simultaneous analysis is possible instantaneously by increasing the number of switching valves. In addition, it is possible to develop a simple measurement type disposable chip type biosensor by making the dotted line part into a chip or a micro flow path, and it is also effective as an assay system for development.

D-psicose was actually measured by flow injection using a sensor constructed according to the sensor system diagram of FIG. The data in the previous example was measured by the batch method, whereas the two-line type switching valve type MFIA was adopted in this example. As detailed below, we obtained results demonstrating that the enzyme reaction pathway of the sensor proceeds properly. The results of studying various conditions (flow velocity, ratio of two buffers, injection amount, etc.) of the sensor system in FIG. 6 and the calibration curve when D-psicose is sensed last are the current data. Regarding various conditions of the sensor, although it is one of the cases, this condition is the condition that can attain the most sensitivity at present.
For example, with respect to the buffer, the composition is not necessarily a phosphate buffer and a citrate buffer. As long as the optimum pH for each enzyme is met, there is no problem with buffers having other compositions, such as Good buffer, Tris-HCl buffer, and acetate buffer. It can be seen that the injection amount also functions as a sensor if a calibration curve created with various injection amounts is prepared for each sample.
By using several enzymes with completely different properties and incorporating them into a system with different ionic strengths and flow rates, these enzymes can be continuously reacted, making it quicker and easier than ever before. A sensor for measuring rare sugars that could not be detected was constructed. It can be said that an unprecedented sensor has been constructed not only as a rare sugar measurement biosensor but also as a normal sensor.

The left figure A of FIG. 8 shows buffer-A (pump 1, citrate buffer, square display) set to the optimum pH of D-fructose dehydrogenase (DFDH) immobilized on the electrode in the double pump, and D-tagatose. It shows the current response value (circle display) when the ratio of buffer-B (pump 2, phosphate buffer, triangle display) set to the optimum pH of -3-epimerase (DTE) is changed. The total flow rate of pump 1 and pump 2 is set to 0.2 ml / min. 50 μl of 5 mM D-psicose is injected. The current response value was the best when buffer-A: buffer-B = 0.08 ml / min: 0.12 ml / min. Even if the composition of the buffer is not this composition, there is no problem even if it is used as long as the pH matches. It is important to optimize their ratio for the two buffers used.
The right figure B shows the injection amount. This is the result of changing the amount to be injected from 5 μl to 100 μl with the absolute amount being 500 μmol and the amount dissolved. Under this sensor condition, 50 μl had the best current response value. If the sample to be measured is small or dark, it can be measured without problems by creating a calibration curve with an injection amount that matches the sample conditions. 50 μl was adopted as a setting that can satisfy the three conditions of quick, simple and high sensitivity.

The left figure A of FIG. 9 shows the current response value when the size of the gasket of the flow cell is changed variously. A flow cell equipped with DFDH is manufactured by sandwiching a gasket between two plastic molds. By varying the size of the gasket, the volume inside the flow cell can be manipulated. In this case, when a gasket having a thickness of 750 μm was selected, the highest current response value was shown. The reaction cannot be performed without a certain volume inside the flow cell. That is, the result is that the waste liquid moves forward before the enzyme reaction on the carbon paste enzyme electrode proceeds. All are injected with 50 μl of 10 mM.
The right figure B has shown the electric current response value when changing the flow velocity of a sensor system variously. All are injected with 50 μl of 10 mM. The slower the flow rate, the better the reaction efficiency and the higher current response value, but it takes much time. For example, at 0.05 ml / min, it takes about 20 minutes per sample. For this reason, a flow rate at which measurement is completed in about 5 minutes per sample is usually selected.
In this sensor system, we decided to adopt the setting of 0.2 ml / ml.

  FIG. 10 shows values obtained by variously changing the pH of the two types of buffers. For buffer A, a good current response value was obtained in a slightly alkaline condition from around neutrality. Therefore, pH 7.5 was adopted as buffer A. Buffer B showed the best current response value from pH 4.0 to around pH 5.0. DFDH has the most active pH of 4.0, but the stability is most stable around pH 5, so that buffer B employs pH 5.0. Although data were not collected, DFDH felt that the enzyme stability was increased by immobilization.

  FIG. 11 shows a current response curve when a calibration curve is created by optimizing the conventional sensor system conditions. It can be confirmed that it is quite stable. Although a calibration curve is drawn in FIG. 12, the correlation coefficient is obtained up to 0.999 (B). It can be seen that the measurement accuracy is very good.

In FIG. 12A, when measured to a high concentration (50 mM), a slight curve is obtained, but the correlation coefficient is 0.987 even in this state, which is a sufficiently measurable range as a sensor. By creating this curve formula, it is possible to measure the sample more accurately. Since the correlation coefficient triple 9 is obtained up to 3.1 mM, perfect measurement is possible. In repeated measurement (N), error bars are obtained by measuring three times.
The measurement is completed in about 5 minutes per sample. The detection sensitivity is improved by optimizing the column oven, buffer oven, mediator, and the like. The calibration limit value at the present time was 0.09 mM, but it can be measured up to about 10 μM if the system conditions are further optimized.

Commercial drinking water was measured using the D-psicose sensor of Example 2 (sensor system of FIG. 6 in which various measurement conditions were optimized for D-psicose measurement), which is one of rare sugar sensors.
D-psicose is not present in any of the distilled water used as the sample and the beverages A, B, and C. The test was performed by adding 10 mM D-psicose to each sample, all of which were measurable. The results are shown in Table 1.

This is an example demonstrating that all the rare sugars constituting “Izumoring” can be measured using a plurality of rare sugar-related enzymes.
A D-allose sensor was constructed using the reaction pathway of rare sugar measurement in FIG. That is, the D-allose sensor uses the measurement principle of the D-allose sensor shown in FIG. D-allose is converted to D-psicose by using L-rhamnose isomerase. From here on, D-allose can be measured by adopting the same measurement principle as the D-psicose sensor.
Add 2 μl of enzyme (LRI, solution prepared at 20 mg / ml) to D-allose solution (20 mM phosphate buffer, pH 7.5) prepared at each concentration, and incubate at 35 ° C. for 30 minutes This was measured using the sensor system of Example 2. The measurement results are shown in FIG. The sensor current response was confirmed depending on the concentration of D-allose, and it was confirmed that this measurement principle was functioning.
This is preliminary data for positioning the sensor system. As a flow-based continuous measurement biosensing system, it is possible to construct a D-allose sensor or other rare sugar measurement sensor system. By adding a rare sugar-related enzyme to the sensor system of Example 1, It can be seen that rare sugars can be measured in the same manner. That is, it can be seen that by measuring a plurality of rare sugar-related enzymes according to the measurement target, it is possible to measure all the rare sugars that constitute “Ismoring”. It is possible to function as a useful sensor by performing a flow change program, studying temperature conditions, optimizing a micro flow path, and setting a column switching system (program).

  Among rare sugars, D-psicose, which is the target of this sensor system, suppresses the absorption of sugar from the intestinal tract to suppress the increase in blood glucose level, suppresses the growth of cancer, prevents diabetes, prevents arteriosclerosis, It is one of the rare sugars that have various physiological activities such as fat synthesis inhibiting action and is expected to be put to practical use, but its practical use is delayed because its measurement system has not been established. When a new substance is adopted as a food additive or pharmaceutical, its measurement system must be shown, but the completion of this sensor can contribute to the rapid application of D-psicose to the industry. In addition, since the development of this sensor enables monitoring of rare sugars in vivo, it can be expected that new knowledge about the detailed relationship between the behavior of rare sugars and biological activity in vivo will be obtained. Furthermore, the sensor system using the reaction pathway devised in this study can be applied to other rare sugars by using “Izumoring”, which is a new elucidation of the functions of rare sugars whose physiological activity is unknown. It can be applied as a simple tool development. As a practical application, it is expected that a simple sensor using the latest measurement device of the blood glucose level sensor that is the most proven in biosensors can be realized with high throughput. By developing a system that measures trace components in the environment that has not been focused on so far, such as this research, not only in the medical field such as cytoinformatics and pathology, but also in glycobiology and environmental science. It is expected to bring a lot of new knowledge to related fields such as.

It is drawing explaining the reaction path | route of the rare sugar measurement in this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is drawing which shows what confirmed the influence of D-psicose in the electrochemical measurement of D-fructose as Example 1. FIG. It is drawing which shows what D-psicose and DTE were made to react by batch reaction for a fixed time, and D-fructose was measured electrochemically after that. It is a figure which shows what confirmed the influence of various rare sugars in D-fructose measurement. 2 is a graph showing whether D-allose can be electrochemically measured using the reaction pathway shown in FIG. 1. 1 is a diagram illustrating a biosensor characterized by a microflow of a two-line switching valve system of the present invention. It is a drawing of “Izumoring” (registered trademark) that links all hexoses, their molecular structure, and the production enzyme. A is when the ratio of buffer A (square display) set to the optimum pH of DFDH immobilized on the electrode and buffer B (triangle display) set to the optimum pH of DTE in the double pump is changed. It is drawing which showed the electric current response value (circle display). B is a drawing showing the amount of injection. A is a current response value when the size of the gasket of the flow cell is changed variously, and B is a drawing showing the current response value when the flow rate of the sensor system is changed variously. It is drawing which shows the value which changed pH of two types of buffers variously. It is drawing which shows the current response curve when a calibration curve is created by optimizing sensor system conditions. It is drawing which has drawn a calibration curve by optimizing sensor system conditions. It is drawing which shows the response curve of a D-allose sensor.

Claims (12)

  First line equipped with a column to which an enzyme that generates a substrate to be measured is immobilized by direct reaction with a specific component contained in the sample to be measured, and the function to convert the generated substrate to be measured into another compound A biosensor characterized by a microflow of a two-line type switching valve system comprising a second line constituting an electrode system including a substance, and a switching valve for switching liquid feeding to the first line and the second line.   The biosensor according to claim 1, further comprising an injection port for injecting a sample to be measured toward the first line.   A container for the buffer A for the first line is connected to the injection port via the pump 1 and the switching valve 1 or to the second line via another switching valve 3, and the buffer B for the second line The biosensor according to claim 1 or 2, wherein the container is connected to the second line via the pump 2 and the switching valves 2 and 3 or to the injection port via the other switching valve 4.   The biosensor according to claim 1, 2 or 3, wherein the buffer A and the buffer B include an electron carrier.   The electrode system includes a working electrode and a counter electrode arranged in a microflow cell, and the microflow cell is an electrode system to which a reaction solution including a substrate to be measured, a buffer solution, and an electron carrier is fed. The biosensor according to any one of 4 to 4.   The biosensor according to claim 5, wherein a substance having a function of converting the generated substrate to be measured into another compound is provided on or near the working electrode.   The specific component is D-psicose, the substrate to be measured is D-fructose, the enzyme that produces D-fructose by direct reaction with D-psicose is D-tagatose-3-epimerase (DTE), and The biosensor according to any one of claims 1 to 6, wherein the substance having a function of converting the produced D-fructose into another compound is D-fructose dehydrogenase (DFDH).   The biosensor according to claim 7, comprising a first line equipped with a DTE-immobilized column and a second line equipped with a DFDH-immobilized carbon paste electrode (CPE). The biosensor according to claim 8, wherein a line equipped with a column on which an enzyme that converts D-allose to D-psicose is immobilized is provided before the first line. The biosensor according to claim 8 or 9, wherein a membrane showing selective permeability is attached to the CPE surface.   The sample to be measured was injected into the first line equipped with a DTE-immobilized column and reacted under the optimum conditions for DTE, and then the switching valve was operated at the same time as the reaction was completed, and the optimum conditions for DFDH were set. The reaction solution is sent to the second line, and the second line is set to have a higher ionic strength than the buffer solution in the first line, which causes a small amount of buffer solution in the second line to be mixed in the reaction solution. The pH changes to the optimum value of DFDH, and then the amount of D-fructose in the reaction solution is measured on the surface of the DFDH-immobilized carbon paste electrode mounted on the second line. The biosensor according to claim 7, 8, 9 or 10, which measures the amount of psicose. It is contained in a biological sample selected from the group consisting of serum, plasma, blood, sweat, urine and cells, a raw material or a product sample in the pharmaceutical and food industry using the biosensor according to any one of claims 1 to 11. A method for detecting or quantifying rare sugars that detects or quantifies specific components.