JP4504289B2 - Biosensor - Google Patents

Description

  The present invention relates to a biosensor that analyzes a specific component in blood after blood is separated by centrifugal force.

  In recent years, the number of patients with hypertension, diabetes, and hyperlipidemia has increased due to lifestyle changes. These are called lifestyle-related diseases and are risk factors for arteriosclerosis. National and local governments aim to modify lifestyle habits to reduce stroke mortality, ischemic heart disease mortality, and total cardiovascular mortality. However, even if these diseases are found by a medical examination, the patient himself often fails to treat and worsens the medical condition because there is no subjective symptom.

  Life management is an important treatment method for lifestyle-related diseases, and medical workers such as doctors, nurses, and pharmacists also provide various guidance for treatment. However, in practice, it is often the case that people are busy and negligent in their daily lives and cannot fully manage themselves. Furthermore, it is extremely difficult and impossible to confirm whether the medical staff actually performs the guidance contents in the daily life of the patient. Against this background, patients must continue to practice pharmacotherapy, diet therapy, and exercise therapy with their strong will. Therefore, in addition to the examinations performed at medical institutions, it is expected that it is effective to grasp the pathological condition with their own hands even at home. This is because the patient himself examines and reviews the improvement of lifestyle habits and future issues from the obtained test results, and feels fulfillment when the treatment goal is achieved, strengthening self-management behavior Because it is done. This can lead to the maintenance and improvement of treatment consciousness, and is expected to have great results in the treatment and prevention of lifestyle-related diseases. In view of this, development of testing equipment for home use has been actively conducted so that patients can perform their own tests at home.

  In a blood test in the clinical diagnostic field, a specific component in the blood is analyzed from the collected blood to grasp the disease state, treatment recovery state, etc. of the subject. As a test object, it is common to separate the collected blood into components and analyze only one sample containing the analyte. In blood biochemical tests, serum components are often targeted. Blood clotting occurs when fresh blood is left, so it can be measured by extracting the serum component excluding blood cell components and blood clotting factors after leaving it for 30 minutes or more to coagulate well to make a blood clot. Many. This separation is usually performed by centrifugal separation utilizing a specific gravity difference. In this method, a whole blood sample is put in a test tube or a blood collection tube and allowed to stand at room temperature for 30 minutes or more, and then a blood cell component and a blood coagulation factor are precipitated by centrifugal force to obtain supernatant serum. At this time, a coagulation accelerator may be used. In addition, plasma components including fibrinogen that are not present in serum components may be extracted by mixing the collected blood with an anticoagulant such as heparin and then performing centrifugation.

  For the purpose of facilitating this separation operation, a blood separation agent having a specific gravity intermediate between both components to be separated is used particularly in centrifugation. When blood is centrifuged into both components in the presence of such a blood separating agent, the blood separating agent is located between the two components due to the difference in specific gravity and becomes a septum that separates both components. The component or plasma component can easily be completely separated from the underlying sediment component. Therefore, only the components necessary as a blood sample can be completely taken out and accurately inspected, and the inspection accuracy is improved.

  As a blood separation tube for performing this blood separation, the labor for transferring the collected blood to another tube and the blood loss at that time can be eliminated, so that the blood collection tube is often used as it is. The blood collection tube is a test tube that can be sealed, and a small amount of blood separating agent is prefilled inside the blood collection tube.

This blood separating agent is in a gel form, has an intermediate specific gravity of 1.038 to 1.052 between plasma and blood cell components, a kinematic viscosity of 3000 to 6000 mm ² / s capable of maintaining the shape, and can be filled into a container such as a blood collection tube. This gel-like blood separating agent is prepared, for example, by blending a tackifier, an inorganic filler for adjusting specific gravity, and a thixotropic agent, using polybutene having a specific gravity of 0.87 as a hydrophobic liquid resin as a main material. ing.

  According to Patent Document 1, a vacuum blood collection tube whose inside is decompressed is disclosed. In the blood collection method, first, a blood collection needle attached to a blood collection device is punctured into a blood vessel. Next, the puncture needle formed at the rear end of the blood collection needle is inserted into a sealing member made of natural rubber, and the puncture needle is inserted into the vacuum blood collection tube. Then, blood flows into the vacuum blood collection tube. When blood collection is complete, remove the blood collection device from the blood collection tube. The sealing member has a function of sealing the puncture hole and maintaining airtightness inside the blood collection tube. That is, the collected blood can be transported while maintaining airtightness without being exposed to the atmosphere until it is transported to an automatic analyzer or the like.

  As a typical example of a testing device used for home testing, there is a self blood glucose measuring device that measures the glucose concentration (blood glucose level) in blood. A self-blood glucose measuring machine that is widely used nowadays uses a small amount of blood sample that the subject himself punctures a fingertip or arm and bleeds.

  To be precise, the blood glucose level is the glucose concentration in the serum. The most common method for measuring glucose concentration is a method using an enzyme electrode, and the whole blood is supplied to a biosensor for measurement.

In the enzyme reaction tank inside the biosensor, the current corresponding to the glucose concentration in the serum is measured by the amperometry method without hemolyzing the blood cells. That is, in this case, the concentration of a specific component in the serum is measured without separating the blood cell component. However, as described above, generally, the blood cell component and the blood coagulation component are separated, and the serum component is separated. In many cases, the analysis is performed after extracting only. Extraction technology of serum or plasma components is indispensable for expanding the examination items at home as well as blood glucose levels.
JP-A-6-189944 (page 3-5, FIG. 1)

  Home blood analyzers have been developed to support treatment of lifestyle-related diseases. The method by which a patient can collect blood at home is limited to a method of obtaining a very small amount of blood by puncturing a fingertip or an arm, and the obtained blood is 50 microliters or less. Further, in the method of inserting blood frequently used in medical practice into a vacuum blood collection tube and extracting the supernatant after centrifugation, a blood volume of at least 2 milliliters is required. That is, there is a problem that the method of separating blood components using a vacuum blood collection tube cannot be applied to a home blood analyzer.

  In the case of a self blood glucose meter, whole blood can be supplied to a biosensor to measure the glucose concentration in serum. However, when examining items other than the glucose concentration, it is generally impossible to inspect blood cells without hemolysis.

Thus, a method of extracting a serum or plasma component by sucking a small amount of blood (whole blood), separating blood cell components by centrifugation, and the like as a biosensor of a self blood glucose measuring device is conceivable. As the conditions for the centrifugation, the rotation speed is often 3000 min ⁻¹ or more, and it is often rotated with a centrifuge for 5 minutes. At this time, even if the collected blood is sucked up by the biosensor, if it is exposed to the atmosphere, blood coagulation will occur and blood clots cannot be made and analyzed accurately, or the water in the blood will evaporate There was a problem that the concentration could not be measured accurately.

Therefore, the biosensor of the present invention employs the following configuration.
That is, the biosensor of the present invention is a biosensor having a separation cavity that separates components according to specific gravity by centrifugal force, and a sealed gel having a specific gravity smaller than that of a plasma component or serum component is previously placed in a flow path or separation cavity. It is characterized in that the open end, which is a blood entrance / exit, is blocked by a sealed gel when filled and subjected to centrifugal force.

  The biosensor of the present invention preferably includes a suction cavity that sucks blood by capillary action and temporarily stores it, and the blood stored in the suction cavity is preferably guided to the separation cavity by centrifugal force.

  Furthermore, in the biosensor of the present invention, the sealed gel is more preferably insoluble or non-volatile with respect to blood.

(Function)
The biosensor sucks blood into the suction cavity. The biosensor is attached to the turntable of the analyzer, and the biosensor is rotated to apply centrifugal force. In the separation cavity, the components are separated according to the specific gravity. Since the inside of the biosensor is filled with a sealed gel in advance, the blood cell component, the plasma component, and the sealed gel having a large specific gravity are arranged in this order. The open end is blocked by a sealing gel so that the blood does not touch the atmosphere.

The biosensor of the present invention analyzes one sample containing an analyte by separating the components by centrifugation immediately after collecting a small amount of blood that has been stabbed into a fingertip or arm and collecting a small amount of blood. . Even when the centrifugation is performed at a high speed for a long time, the collected blood does not come into contact with the atmosphere, and the open end is blocked by the sealed gel. As a result, the water in the blood does not evaporate, so that the change in the blood concentration to be analyzed can be suppressed. And even if it is a simple analytical instrument used at home, the biosensor which can be analyzed correctly can be provided.
In addition, hydrogen peroxide is generated from an analyte such as glucose by an oxidase such as glucose oxidase, a reaction between the dye precursor and hydrogen peroxide occurs, and the generated dye is optically quantified. There is a method for measuring a specific analyte. Some of the dyes thus produced may be unstable compounds that decompose due to oxygen in the air or dissolved oxygen in the reaction solution.
In addition, there is a method of obtaining an amount of an analysis target substance from an electrochemical response when an analysis target substance is oxidized by an oxidase and a reduced form of an electron mediator generated thereby is oxidized by an electrode. At this time, the enzyme reaction is performed for a certain period of time, the reductant of the electron mediator is accumulated during that time, and the reductant of the electron mediator accumulated after a certain period of time is oxidized at the electrode to cause a large electrochemical response. There is a method for measuring a target substance with high sensitivity. However, there is a case where an error is given to the measurement due to the influence of the oxidation reaction by oxygen in the air or dissolved oxygen in the reaction solution.

  However, in the present invention, immediately after the blood has moved by applying a centrifugal force, the open end is closed by the sealing gel so that the blood does not come into contact with oxygen in the air. As a result, the oxidation / reduction reaction for the measurement of the analyte can be accurately measured, starting immediately after blood movement.

Further, the collected blood is not exposed to the atmosphere, and the open end is blocked by the sealed gel, so that the plasma component can be analyzed without coagulating the collected blood.
In other words, plasma components can be analyzed without coagulation without mixing anticoagulants with blood. This makes it possible to analyze blood coagulation factors contained in plasma components. In the conventional analysis of plasma components, since the anticoagulant was mixed with the collected blood and then centrifuged, the extracted plasma component contained the anticoagulant. Furthermore, the type of anticoagulant to be mixed needs to be properly used depending on the item to be analyzed and the analysis method, but the troublesomeness regarding the selection of the anticoagulant is eliminated.

  Alternatively, after the collected blood is left in the atmosphere for a while, the serum component can be extracted by centrifugation to analyze the serum component. Of course, by adding a blood coagulation promoter and centrifuging, serum components from which coagulation factors have been reliably removed can also be analyzed.

  In addition, since the separation cavity or flow path of the biosensor is filled with a sealed gel in advance, a special reagent for preventing blood coagulation and a special operation for preventing moisture evaporation are used when used at home testing. Do not need. That is, it is not necessary to add an anticoagulant or close the sealing cap, and the operation for inspection can be simplified. Of course, it is not necessary to transfer the extracted components to another container after centrifugation, and the inspection apparatus only needs to have a simple centrifugal function, so that a system suitable for home use can be provided.

Example 1
FIG. 2 is an external view showing an embodiment of the biosensor of the present invention. The biosensor 1 has a structure in which a lower plate 20 and an electrode substrate 10 are bonded together. The biosensor 1 has a suction port 21 on one side and an air port 22 on the other side. A gap is provided between the suction port 21 and the air port 22 at a portion sandwiched between the lower plate 20 and the electrode substrate 10. This gap is a suction cavity 23, which is a space for sucking up blood and temporarily storing it. Blood is sucked up from the suction port 21 by capillary action and fills the suction cavity 23. The air that has been in the suction cavity 23 is extracted from the air port 22. A connector window 26 is provided on the back surface of the biosensor 1, and a connector terminal 12 is disposed behind the connector window 26, although not shown. The connector terminal 12 is a metal terminal for contact provided on the electrode substrate 10 in order to perform an electrochemical measurement by being electrically connected to the analyzer.

  FIG. 3 is an exploded perspective view of the biosensor 1. The lower plate 20 is made of transparent plastic, and has a suction cavity 23, a separation cavity 24, and a channel for blood conveyance in the flow path 25. The suction cavity 23 shows a state where blood 4 is sucked up. The separation cavity 24 is a space sandwiched between the groove dug in the lower plate 20 and the electrode substrate 10, and the upper and lower surfaces and the side surfaces are closed, and has an open end that allows the blood 4 and the sealed gel 3 to flow only from the flow path 25. It has a structure. The channel 25 is filled with the sealed gel 3. The blood 4 is guided by the centrifugal force from the suction cavity 23 through the flow path 25 to the separation cavity 24, and the sealed gel 3 is also guided to the separation cavity 24. At this time, the air in the separation cavity 24 is pushed out of the separation cavity 24 through the open end. Further, a connector window 26 is opened for electrical connection with the connector terminal 12 from the back surface. The electrode substrate 10 is, for example, a polyimide film FPC. The connector terminal 12 is disposed on the upper surface of the connector window 26, and the electrode for electrochemical measurement is disposed on the upper surface of the separation cavity 24. The working electrode and the counter electrode are gold electrodes, and an enzyme reaction layer containing an oxidoreductase and an electron mediator is formed on the surface of the working electrode. The reference electrode is a silver-silver chloride electrode. For example, in the case of a glucose biosensor, glucose oxidase is used as the oxidoreductase, and potassium ferricyanide is used as the electron mediator.

Next, the operation of the biosensor 1 from when the blood obtained by puncture is aspirated until it is analyzed by the analyzer 2 having a centrifugal function will be described with reference to FIGS. 2 and 3. First, a puncture needle is inserted into a fingertip that is a blood collection site, and blood exceeding the blood volume necessary for analysis is drawn out. The amount of blood necessary for analysis is, for example, about 10 μL, and the volume of the suction cavity 23 is the same as the necessary amount of blood in order to suck up the necessary amount of blood. Then, blood (whole blood) is brought into contact with the suction port 21 of the biosensor 1. The blood 4 is sucked by capillary action and fills the suction cavity 23 from the suction port 21 to the air port 22. At this time, the space formed by the flow path 25 and the separation cavity 24 is a closed space. For this reason, the air inside the flow path 25 and the separation cavity 24 does not escape, and the blood 4 does not flow in the direction of the flow path 25. The sucked blood 4 can be confirmed from the back surface of the transparent plastic lower plate 20. If the blood cannot be sufficiently sucked and the suction cavity 23 is not filled, blood is again drawn from the puncture site of the fingertip and additionally sucked up from the suction port 21.

Next, the structure of the analyzer 2 with a centrifugal function to which the biosensor 1 is attached will be briefly described with reference to FIG. However, FIG. 4 shows only the schematic structure of the centrifugal mechanism part of the analyzer 2, and the electrochemical measurement part, the display part, the operation switch part, etc. are omitted. The centrifugal mechanism unit of the analyzer 2 includes a turntable 30, a motor 31, and a connector spring 32. The turntable 30 is a mount that attaches the biosensor 1 and rotates at high speed. The motor 31 is a drive motor for rotating the turntable 30 and the biosensor 1, and for example, a stepping motor is used. In addition to controlling the rotation speed to be constant, the stepping motor can gradually increase or decrease the rotation speed, and can also control the stop position. The connector spring 32 is a contact that electrically connects the connector terminal 12 of the biosensor 1 and the electrochemical measurement unit of the analyzer. Here, when analyzing with the biosensor 1, it attaches to the turntable 30 of the analyzer 2 with a centrifugal function so that the suction cavity 23 is on the rotation center side and the separation cavity 24 is on the rotation outer periphery side. When the operation switch of the analyzer 2 is pressed, the turntable 30 starts rotating. The rotation speed gradually increases, and the maximum rotation speed is, for example, 3000 min ⁻¹ and continues for 5 minutes. When the rotation radius from the center of rotation to the tip of the separation cavity 24 is 10 cm, the centrifugal force at the tip of the separation cavity 24 is approximately 1000 times the gravity.

  FIG. 1 is an exploded perspective view showing a state in which the biosensor 1 is centrifuged. The movement process of blood 4 and sealed gel 3 in the internal cavity of biosensor 1 will be described with reference to FIGS. 3 and 1. First, in a state before blood 4 is collected and centrifugal force is applied, the sealed gel 3 is in the flow path 25 and the blood 4 fills the suction cavity 23 as shown in FIG. Thereafter, the turntable 30 to which the biosensor 1 is attached is rotated to apply centrifugal force. Then, the blood 4 in the suction cavity 23 is guided to the separation cavity 24 through the flow path 25 by centrifugal force. Then, the sealed gel 3 filled in the flow path 25 also moves to the separation cavity 24. Blood 4 begins to be centrifuged into blood cell component 41 and plasma component 42 by specific gravity. The specific gravity of the plasma component 42 is smaller than that of the blood cell component 41, and the specific gravity of the sealed gel 3 is smaller than that of the plasma component 42. Therefore, in the separation cavity 24, the blood cell component 41, the plasma component 42, and the sealed gel 3 move in this order from the farthest radius of rotation. The rotation time is approximately in this order even for about 10 seconds. That is, the collected blood (blood cell component 41 and plasma component 42) is in a state in which the open end, which is the entrance to the flow path 25, is closed with the sealing gel 3 so that the blood does not touch the atmosphere. As a result, the water in the blood does not evaporate and does not coagulate. Furthermore, since the plasma component 42 is blocked by the sealed gel 3 so as not to come into contact with oxygen in the air, an oxidation reaction due to oxygen in the air does not occur. The enzyme reaction layer 11 is arranged at a position where the plasma component 42 of the separation cavity 24 is extracted. Further, in this embodiment, the sealed gel 3 is filled in close contact with the flow path 25 in advance, but it is only required that it can be peeled off and moved from the filling position after the centrifugal force is applied. Therefore, the sealed gel 3 may be filled in advance at the position behind the separation cavity 24, and the blood 4 may be moved to the filling position so that the sealed gel 3 floats on the blood 4. .

Next, operation | movement of the analyzer 2 is demonstrated using FIG.4 and FIG.1. In the rotational movement of the turntable 30, the blood 4 moves to the separation cavity 24 in about 10 seconds, and can be substantially separated into the blood cell component 41 and the plasma component 42. If the turntable 30 is further rotated for 5 minutes, the separation accuracy of the blood cell component 41 and the plasma component 42 is improved. When the rotation for the centrifugal separation is finished, the turntable 30 to which the biosensor 1 is attached is controlled to stop at the stop position. The connector spring 32 of the analyzer 2 is connected to the connector terminal 12 of the biosensor 1. Then, an enzyme reaction occurs in the enzyme reaction layer 11 and the electrochemical measurement of the biosensor 1 is performed.

  Here, as described above, the characteristic of the sealed gel 3 is smaller than the specific gravity of the plasma component 42. A preferable characteristic of the sealed gel 3 is that the sealed gel 3 is insoluble in blood. If the components of the sealed gel 3 are dissolved in the blood, it is difficult to accurately analyze the components in the blood. Moreover, it is also mentioned as a preferable characteristic that a chemical reaction is not caused to blood. If the sealed gel 3 causes a chemical reaction to blood or an inhibitory reaction at the time of electrochemical measurement, it is difficult to accurately analyze components in the blood. Furthermore, it is one of the preferable characteristics that the sealing gel 3 is non-volatile. If the sealing gel 3 has high volatility, the sealing gel 3 disappears during the rotation of the centrifugal separator for 5 minutes, and the open end that is the entrance to the flow path 25 cannot be blocked. If this happens, the water in the blood will evaporate and the blood will clot. Furthermore, by contacting oxygen in the air, an oxidation reaction not caused by the measurement principle occurs, which hinders accurate measurement. Even if the volatility is not so high, since the inside of the biosensor 1 is filled with the sealed gel 3 in advance and stored, if the sealed gel 3 is not volatilized during storage, the sealed gel 3 Since it does not perform the function as, it is preferably non-volatile.

(Example 2)
5 and 6 are schematic views showing another embodiment of the biosensor of the present invention. FIG. 5 is a schematic view before centrifugation, and FIG. 6 is a schematic view after centrifugation. The top view of the lower plate 20 is shown without showing the electrode substrate 10. The suction cavity 23, the flow path 25, the separation cavity 24, the sealed gel 3, the blood cell component 41, and the plasma component 42 shown here have the same functions as those shown in FIG. This will be described with reference to the schematic diagram of FIG. 5 showing the state before centrifugation. The separation gel 43 is a gel for separating the blood cell component 41 and the plasma component 42, and has an intermediate specific gravity of 1.038 to 1.052 between the plasma and blood cell components, for example, 1.045. The separation gel 43 is filled in the separation cavity 24 in advance in the manufacturing process of the biosensor 1. When the blood 4 is collected, the blood 4 fills the suction cavity 23. In addition, although the sealing gel 3 showed the schematic diagram with which the isolation | separation cavity 24 was filled, you may fill the flow path 25 similarly to Example 1. FIG.

  FIG. 6 will be described with reference to a schematic diagram showing a state after centrifugation. When the biosensor 1 is attached to the turntable 30 of the analyzer 2 and rotated, the blood 4 in the suction cavity 23 is guided to the separation cavity 24 through the flow path 25 by centrifugal force. When the blood 4 flows in, the sealing gel 3 and the separation gel 43 filled in the separation cavity 24 rise. And it isolate | separates by specific gravity, and arranges in order of the blood cell component 41, the separation gel 43, the plasma component 42, and the sealing gel 3 from the one where a rotation radius is far.

  In the above embodiment, the biosensor for extracting and analyzing the plasma component is shown, but the present invention is not limited to the plasma component. The present invention can also be applied to a biosensor that precipitates and separates blood cell components and blood coagulation components and extracts only the serum components for analysis. In this case, a coagulation promoter may be inserted in advance into the biosensor.

As described above, the sealed gel 3 having a specific gravity smaller than that of the plasma component or serum component is filled in the biosensor 1 in advance. After the blood 4 is collected in the suction cavity 23 of the biosensor 1, the biosensor 1 is attached to the turntable 30 of the centrifuge mechanism and centrifuged. Then, in the separation cavity 24, the blood cell component 41 (separation gel 43), the plasma component 42, and the sealed gel 3 move in this order from the farthest radius of rotation. That is, the open end which is the entrance of the blood 4 or the blood cell component 41 and the plasma component 42 is blocked by the sealed gel 3 so that the collected blood 4 does not touch the atmosphere.

  After a small amount of blood is collected and the blood is separated by centrifugal force, it can be applied to a biosensor in which one sample containing an analyte is analyzed. In particular, in the case of an analysis target that cannot be accurately analyzed when the collected blood is exposed to the atmosphere, highly reliable analysis is possible. In addition, a simple analysis system can be provided in the case of home inspection rather than a medical facility.

It is a disassembled perspective view which shows the state centrifuged with the biosensor of this invention. It is the external view which looked at the biosensor of this invention from diagonally upward and downward. It is a disassembled perspective view of the biosensor of this invention. It is a schematic structure figure of the centrifuge mechanism part which attached the biosensor of the present invention. It is a schematic diagram before centrifugation of the biosensor of the present invention. It is the schematic diagram after centrifugation of the biosensor of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Biosensor 2 Analyzer 3 Sealed gel 4 Blood 10 Electrode substrate 11 Enzyme reaction layer 12 Connector terminal 20 Lower plate 21 Suction port 23 Suction cavity 24 Separation cavity 30 Turntable 41 Blood cell component 42 Plasma component 43 Separation gel

Claims (3)

A biosensor having a separation cavity that separates components according to specific gravity by centrifugal force,
A closed gel having a specific gravity smaller than that of a plasma component or a serum component is previously filled in a flow path or the separation cavity, and when a centrifugal force is applied, an open end that is a blood entrance / exit is blocked by the sealed gel. Biosensor. The biosensor according to claim 1, further comprising a suction cavity that sucks blood by capillary action and temporarily stores the blood, and the blood stored in the suction cavity is guided to the separation cavity by centrifugal force. The biosensor according to claim 1, wherein the sealing gel is insoluble or non-volatile with respect to blood.

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