JP6610025B2 - Biosensor - Google Patents

Description

  The present invention relates to a biosensor using an electrochemical method.

  Various biosensors using electrochemical methods are known, such as glucose sensors such as blood glucose level sensors.

  For example, when a specimen (blood or the like) is introduced into a cavity (a space formed by a groove formed in a spacer) of a biosensor, a component (substrate) contained in the specimen reduces the mediator via an enzyme. Here, when a predetermined voltage is applied to the electrode, the reduced mediator is reversely oxidized by an electrochemical reaction. By measuring the oxidation current generated at this time, the amount of the component of interest can be detected.

  Such a biosensor generally includes two or more electrodes including at least a working electrode and a counter electrode. After a spacer for forming a cavity is bonded on the electrode, an enzyme, It has a structure in which a reagent layer containing a mediator or the like is formed and a cover is attached.

  Here, as conventional biosensors, for example, in Patent Document 1 (Japanese Patent No. 4627912) and Patent Document 2 (Japanese Patent Laid-Open No. 10-282037), the cavity width in the plan view and the diameter of the air hole are approximately the same. A biosensor is disclosed.

Japanese Patent No. 4627912 JP-A-10-282037

  However, the present inventors have found that variations in measured values are likely to occur in a biosensor in which the cavity width and the diameter of the air hole in the plan view are similar. Note that this occurs, for example, due to turbulent flow in the flow of the specimen (sample liquid) in the cavity near the air hole, and accompanying turbulent flow on the working electrode and the counter electrode detecting the electrochemical reaction. It is estimated that one of the causes is that the It curve is disturbed due to the fluctuation of the specimen.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a biosensor capable of measuring with higher accuracy than before.

[1]
A biosensor for quantifying a substrate contained in a sample solution,
An insulating substrate;
An electrode layer including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A reagent layer containing an enzyme that reacts with the substrate and a mediator formed on a part of the surface of the electrode layer opposite to the insulating substrate;
A spacer having a notch for forming a cavity for guiding the sample liquid to the reagent layer, and a spacer disposed on the electrode layer so that the reagent layer is located inside the notch;
A cover provided on a surface opposite to the insulating substrate of the spacer so as to cover at least the notch,
The cover has an air hole communicating with the cavity,
In plan view, the ratio of the area of the air hole to the area of the back part, which is a part on the back side of the reagent layer, of the cavity is 0.3 or less.
[2]
In plan view, the air hole is located in the vicinity of the innermost edge, which is the innermost edge of the innermost part of the cavity, and the width of the inner part of the cavity is directed toward the air hole. The biosensor according to [1], which is narrowed.
[3]
The biosensor according to [2], wherein the shape of the innermost edge of the cavity is an arc shape in a plan view.
[4]
The biosensor according to any one of [1] to [3], wherein a width of a reagent portion that is a portion corresponding to the reagent layer of the cavity is wider than a width of a portion other than the reagent portion of the cavity.

  According to the present invention, it is possible to provide a biosensor capable of measuring with higher accuracy than before.

3 is a schematic top view illustrating a configuration of a cavity in the biosensor of Embodiment 1. FIG. FIG. 3 is a schematic top view for explaining a usage state of the biosensor of the first embodiment. It is an upper surface schematic diagram which shows the structure of the cavity in the biosensor of the reference form 1. It is an upper surface schematic diagram for demonstrating the use condition of the biosensor of the reference form 1. FIG. It is an upper surface schematic diagram which shows an example of a structure of the cavity in the conventional biosensor. 4 is a graph showing a relationship between a CV value of a glucose measurement current and an area ratio of air holes in Test Example 1. 6 is a graph showing a CV value of a glucose measurement current in Test Example 2. 10 is a graph showing a CV value of a glucose measurement current in Test Example 3. 1 is an exploded perspective view showing a configuration of a biosensor of Embodiment 1. FIG. 6 is a perspective view for explaining an example of a manufacturing process of the biosensor of Embodiment 1. FIG. It is another perspective view for demonstrating an example of the manufacturing process of the biosensor of Embodiment 1. FIG. It is another perspective view for demonstrating an example of the manufacturing process of the biosensor of Embodiment 1. FIG. It is another perspective view for demonstrating an example of the manufacturing process of the biosensor of Embodiment 1. FIG. It is another perspective view for demonstrating an example of the manufacturing process of the biosensor of Embodiment 1. FIG.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, the same reference numerals represent the same or corresponding parts. In addition, dimensional relationships such as length, width, thickness, and depth are changed as appropriate for clarity and simplification of the drawings, and do not represent actual dimensional relationships. Each embodiment is an exemplification, and needless to say, partial replacement or combination of configurations shown in different embodiments is possible.

[Embodiment 1]
Referring to FIG. 9, the biosensor of the present embodiment is a biosensor for quantifying a substrate contained in a sample solution, and includes an insulating substrate 1, an electrode layer, a reagent layer 3, and a spacer 4. And a cover 5.

The electrode layer includes a working electrode 21 and a counter electrode 22 provided on one surface of the insulating substrate 1.
The reagent layer 3 is formed on a part of the surface of the electrode layer opposite to the insulating substrate 1 and includes at least an enzyme that reacts with a substrate and a mediator.

  The spacer 4 has a notch 42 for forming a cavity 41 for guiding the sample liquid to the reagent layer 3 and is arranged on the electrode layer so that the reagent layer 3 is located inside the notch 42 (cavity 41). Is done.

  The cover 5 is provided on the surface of the spacer 4 opposite to the insulating substrate 1 so as to cover at least the notch 42. The cover 5 has an air hole 5 a communicating with the cavity 41.

  The biosensor of the present embodiment is characterized in that the ratio of the area of the air hole 5a to the area of the inner part, which is the part on the inner side of the reagent layer 3 of the cavity 41, is 0.3 or less in plan view. The ratio of the area of the air hole 5a to the area of the back part, which is the part on the back side of the reagent layer 3 of the cavity 41, is more preferably 0.001 to 0.2.

  Thereby, the dispersion | variation in the measured value of a biosensor can be reduced. This is presumed to be because, for example, the occurrence of turbulence in the flow of the specimen (sample liquid) in the cavity 41 near the air hole 5a is suppressed. Therefore, by adopting such a structure, a biosensor capable of measuring with high accuracy can be provided.

  In the present embodiment, the air hole 5a is located in the vicinity of the innermost edge, which is the innermost edge of the innermost part of the cavity 41, and the width of the innermost part of the cavity 41 is the direction of the air hole 5a in plan view. It is preferable that it becomes narrow toward. Specifically, the shape of the innermost edge portion of the cavity 41 is more preferably an arc shape in plan view.

  FIG. 1 is a schematic top view showing the configuration of the cavity 41 in the biosensor of this embodiment. In the present application, the “back part” of the cavity 41 is a part on the back side from the reagent layer 3 from the inlet 41 c side of the cavity 41, and can also be called a rear end part. Specifically, referring to FIG. 1, in plan view, the reagent layer 3 (reagent part 41 a) passes through the position farthest from the inlet 41 c of the cavity 41 and passes through the width direction of the cavity 41 (perpendicular to the depth direction). This is a portion (back portion 41b) surrounded by the straight line A extending in the direction), the innermost edge portion B, and the side walls C1 and C2 of the cavity 41.

  In the present specification, the “plan view” means a plan view as viewed from the stacking direction of the substrate, the spacer 4 and the cover 5. Further, “the innermost edge” is the innermost edge of the innermost part of the cavity 41, and in FIG. 1, means a semicircular arc-shaped wall part continuous with the side walls C 1 and C 2.

  FIG. 3 is a schematic top view showing the configuration of the cavity 41 in the biosensor of Reference Embodiment 1. As shown in FIG. 4, when the shape of the innermost edge of the cavity 41 is a straight line as in the first embodiment shown in FIG. In the vicinity 7 of the back edge, bubble scum (air inclusion) is generated.

  This is because the flow of the sample solution 6 introduced from the inlet 41c of the cavity 41 is temporarily expanded in the vicinity of the glucose measurement electrode (reagent layer 3) of the cavity 41, and air is entrained at this time, It is thought that it reaches the back edge. In this way, when bubble smear occurs at the time of introduction of the sample liquid 6, due to the fluctuation of the sample liquid 6 caused by the shock due to the generation of bubble smear on the working electrode 21 and the counter electrode 22 detecting the electrochemical reaction, I− It has been found that the t-curve is disturbed and the measurement accuracy is reduced.

  On the other hand, in the present embodiment, by making the shape of the innermost edge of the cavity 41 into an arc shape, the air entrained when the sample liquid 6 is introduced moves along the innermost edge of the arc shape, It is collected locally in the vicinity of the air hole 5a arranged in the vicinity of the innermost edge, and can be released to the outside through the air hole 5a (FIG. 2).

  However, the innermost edge portion does not necessarily have an arc shape. For example, the innermost edge portion may have two sides of a triangle or a stepped shape, and the air hole 5a is formed at the inner portion of the cavity 41 in a plan view. A similar effect can be achieved if the innermost edge, which is the innermost edge, is located in the vicinity of the innermost edge and the width of the cavity 41 is narrower toward the air hole 5a.

  In the present embodiment, it is preferable that the width of the reagent part 41 a that is a part corresponding to the reagent layer 3 of the cavity 41 is wider than the width of the part other than the reagent part 41 a of the cavity 41.

  FIG. 5 shows an exemplary configuration of a conventional biosensor. As shown in FIG. 5, in the conventional biosensor, the width of the reagent part 41a that is a part corresponding to the reagent layer 3 of the cavity 41 is approximately the same as the width of the part other than the reagent part 41a of the cavity 41. It was common. In this case, in general, in order to increase the measurement accuracy of the biosensor, it is desirable to increase the area of the reagent layer 3 that causes an electrochemical reaction in the cavity 41. However, if the area of the reagent layer 3 is increased, There was a problem that the volume increased and the amount of specimen (sample solution) required for measurement increased.

  In contrast, the width of the reagent part 41a, which is a part corresponding to the reagent layer 3 of the cavity 41, is wider than the width of the part other than the reagent part 41a of the cavity, so that the reagent layer 3 necessary for the measurement reaction While ensuring the size (reagent amount), the volume of the cavity 41 can be reduced to the minimum necessary to minimize the amount of sample necessary for measurement. Therefore, for example, when the specimen is blood, it is possible to reduce the load on the patient during blood collection.

  In the present embodiment, examples of the substrate (analyte) include glucose (blood glucose), lactic acid, cholesterol, alcohol, sarcosine, fructosylamine, pyruvic acid, lactic acid, and hydroxybutyric acid.

  The material of the insulating substrate 1 is not particularly limited, and examples thereof include a plastic material such as a PET (polyethylene terephthalate) film, a photosensitive material, paper, glass, ceramic, or a biodegradable material. These materials are also used as materials for the spacer 4 and the cover 5.

  The electrode layer provided on the insulating substrate 1 includes at least a working electrode 21 and a counter electrode 22. In addition to the working electrode 21 and the counter electrode 22, the electrode layer may include a reference electrode serving as a potential reference when measuring the electrode potential, and a detection electrode for detecting that the sample is supplied to the cavity 41.

  Materials for these electrodes (working electrode, counter electrode, reference electrode, detection electrode, etc.) include platinum, gold, palladium and other precious metals, carbon, copper, aluminum, nickel, titanium, ITO (Indium Tin Oxide) ), ZnO (zinc oxide) and the like. For the electrode layer, for example, a conductive layer made of the above-mentioned material is formed on one surface of the insulating substrate 1 using screen printing or sputtering vapor deposition, and further, a pattern is formed using laser processing, photolithography, or the like. Can be produced.

  Examples of the enzyme include glucose oxidase, glucose dehydrogenase, alcohol oxidase, alcohol dehydrogenase, lactate oxidase, lactate dehydrogenase, cholesterol esterase, cholesterol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, hydroxybutyrate dehydrogenase, creatininase , Creatinase and DNA polymerase. Various biosensors can be produced by selecting these enzymes according to the substance to be detected (glucose, alcohol, lactic acid, cholesterol, sarcosine, fructosylamine, pyruvic acid, hydroxybutyric acid, etc.).

  For example, glucose oxidase or glucose dehydrogenase can be used to make a glucose sensor that detects glucose in a blood sample, and alcohol oxidase or alcohol dehydrogenase can be used to make an alcohol sensor that detects ethanol in a blood sample. For example, a lactic acid sensor for detecting lactic acid in a blood sample can be produced, and a total cholesterol sensor can be produced by using a mixture of cholesterol esterase and cholesterol oxidase.

  The mediator is a compound (electron carrier) that mediates electron transfer between the working electrode 21 and the counter electrode 22, and is preferably a substance that itself performs a redox reaction. As the mediator, for example, potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes, ruthenium complexes and the like can be used.

  The reagent layer 3 preferably contains a hydrophilic polymer. In this case, the reagent layer 3 can be easily fixed to the surface of the electrode layer. The hydrophilic polymer also functions as a filtering agent for filtering impurities (such as blood cells in blood) in the sample solution.

  The hydrophilic polymer is not particularly limited, for example, carbonyl group, acyl group, carboxyl group, aldehyde group, sulfo group, sulfonyl group, sulfoxide group, tosyl group, nitro group, nitroso group, ester group, keto group, Examples thereof include hydrophilic polymers having a ketene group. Examples of the hydrophilic polymer having a carboxyl group include carboxymethyl cellulose and carboxymethyl ethyl cellulose, and carboxymethyl cellulose (CMC) is preferable.

  Further, the reagent layer 3 may contain a hydrophilizing agent for promoting the introduction of the specimen. Examples of the hydrophilizing agent include surfactants such as Triton X100, Tween 20, sodium bis (2-ethylhexyl) sulfosuccinate, and phospholipids such as lecithin. The hydrophilizing agent may be mixed with the above reagent and dropped, or may be further dropped from above the reagent layer. Moreover, you may form a hydrophilizing agent in the below-mentioned cover.

  The material of the cover 5 is preferably an insulating material. For example, a plastic such as a PET film, a photosensitive material, paper, glass, ceramic, or a biodegradable material can be used. The cover 5 preferably has an air hole 5 a communicating with the cavity 41 formed by the spacer 4. This is because the sample is sucked toward the air hole 5a by the capillary phenomenon, so that the sample can be easily introduced into the cavity 41.

(Biosensor manufacturing method)
An example of the manufacturing method of the biosensor of this embodiment will be described with reference to FIGS. FIG. 9 is an exploded perspective view showing the configuration of the biosensor of this embodiment. FIGS. 10-14 is a figure for demonstrating an example of the manufacturing process of the biosensor of this embodiment, Comprising: Each shows a different process. In this embodiment, a plurality of biosensors can be manufactured simultaneously.

  First, referring to FIG. 10, an electrode layer (working electrode 21 and counter electrode 22) is formed on each of a plurality of insulating substrates 1. Specifically, a conductive layer is formed on one surface of the insulating substrate 1 by screen printing or sputtering deposition, and the formed conductive layer is subjected to patterning by laser processing or photolithography to thereby form an electrode layer. Form. In addition to the working electrode 21 and the counter electrode 22, the above-described reference electrode, detection electrode, and the like may be formed. Further, the electrode layer and the surface of the insulating substrate 1 may be subjected to plasma treatment.

  Next, referring to FIG. 11, a part of the electrode layer (working electrode 21 and counter electrode 22) on the side opposite to insulating substrate 1, and an electrode layer in a region where the electrode layer of insulating substrate 1 is not formed The spacer 4 having the notch 42 is bonded to a part of the surface on the side.

  Next, referring to FIG. 12, the reagent solution is dropped on the electrode layer (working electrode 21 and counter electrode 22) opposite to the insulating substrate 1 (inside the notch 42), and the reagent solution is dried to dry the reagent. Layer 3 is formed. In addition, for example, the CMC solution, the enzyme solution, and the mediator solution may be dropped in this order and then dried to form the reagent layer 3. Alternatively, the solution containing the enzyme, mediator, and CMC may be dropped and then dried. The reagent layer 3 may be formed.

  Next, referring to FIG. 13, the cover 5 having the air holes 5 a is laminated on the spacer 4 so as to cover at least the notch portion 42, whereby the cavity for guiding the sample solution to the reagent layer 3. 41 is formed. The air hole 5 a is provided on the opposite side of the opening of the cavity 41 so as to communicate with the inside of the cavity 41.

  Next, the biosensor having the cavity 41 is obtained by dividing the biosensor assembly substrate formed by the above steps (FIGS. 14 and 9).

(How to use biosensor)
The biosensor (biochip) of the present invention is used by being mounted on a measuring instrument. In other words, a sample (blood or the like) is supplied to the cavity 41 of the biosensor mounted on the measuring instrument, and a reducing substance is generated by a reaction between a substance to be measured (such as glucose) in the sample, an enzyme, and a mediator. A voltage is applied between the working electrode 21 and the counter electrode 22 by a measuring device electrically connected to the working electrode 21 and the counter electrode 22 of the biosensor, and an oxidation current obtained by oxidizing this reducing substance is obtained. By measuring, the measurement target substance contained in the sample is quantified.

  Hereinafter, an example of a method for using the biosensor of the present invention will be described. First, blood is brought into contact with the distal end portion (inlet 41c) of the cavity 41, and the blood is introduced into the cavity 41 using capillary action. Then, a voltage is applied between the working electrode 21 and the counter electrode 22, and the current value is measured at a constant timing. The applied voltage is, for example, 0.3V. When blood is introduced into the cavity 41, the analyte in the blood reduces the mediator via the enzyme. The current that flows when a voltage is applied between the working electrode 21 and the counter electrode 22 has a correlation with the reductant concentration of the mediator, that is, the analyte concentration.

  Next, the current value after a certain time has elapsed from the voltage application is measured. For example, the current value after 3 to 5 seconds is measured. Using this current value, the analyte concentration can be determined from a calibration curve obtained in advance.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

[Test Example 1]
Basically, a biosensor having the configuration described in the first embodiment was manufactured. The reagent layer was formed by dropping the CMC solution, the enzyme solution, and the mediator solution in this order and then drying them. A mixed solution of glucose dehydrogenase and methylcellulose was used as the enzyme solution, and a mixed solution of potassium ferricyanide and hydroxypropylmethylcellulose was used as the mediator solution. The material of the insulating substrate 1 and the spacer is polyethylene terephthalate. The thickness of the spacer is 185 μm. In addition, five types of biosensors were produced in which the ratio of the area of the air holes to the area of the back of the cavity in plan view was changed. The width of the reagent layer in plan view was 1 mm, and the width of the inner part was 1.2 mm.

(Measurement of glucose concentration)
A glucose aqueous solution having a predetermined concentration was measured using the biosensor (n = 10). A voltage of 0.3 V was applied between the working electrode and the counter electrode, and the value of glucose response current (glucose measurement current) was measured. The CV value of the glucose measurement current obtained from the measurement results is shown in FIG.

  As shown in FIG. 6, in the plan view, the ratio of the area of the air hole to the area of the back part of the cavity (the part on the back side of the reagent layer) is set to 0.3 or less, so that the CV value is 3% or less. It can be seen that a highly accurate biosensor with little variation for each measurement can be obtained.

[Test Example 2]
A biosensor having a conventional structure as shown in FIG. 5 and biosensors of Reference Embodiment 1 and Embodiment 1 were produced. The value of glucose measurement current was measured in the same manner as in Test Example 1. FIG. 7 shows the accuracy (cv value) of 1 glucose measurement current for each biosensor.

  From the results of FIG. 7, it can be seen that the variation in the glucose measurement current of Embodiment 1 is equivalent to that of the conventional structure, whereas the variation of Reference Embodiment 1 is larger than that of the conventional structure. The measurement accuracy of the reference form 1 is lower than that of the conventional structure because the flow of the sample introduced from the cavity tip temporarily spreads at the reagent formation site of the cavity, and the entrained air flows to the cavity rear end. It is speculated that due to the impact, the fluctuation of the specimen occurred on the working electrode and the counter electrode detecting the electrochemical reaction, the It curve was disturbed, and the variation of the measured current between the sensor chips increased. The

  On the other hand, in Embodiment 1, by making the shape of the innermost edge of the cavity into an arc shape, the sample introduced from the tip of the cavity temporarily expands the flow path at the reagent layer portion of the cavity, The entrained air moves along the innermost edge and is locally collected in the vicinity of the air hole arranged at the rear end of the cavity, and the release of air from the air hole to the outside is promoted. For this reason, it is considered that the occurrence of fluctuations in the specimen accompanying the occurrence of bubble flaws (air inclusion), which was a problem in Reference Form 1, could be suppressed.

[Test Example 3]
For Reference Form 1 (FIG. 3), the cv value of the glucose measurement current was determined in the same manner as in Test Example 1 when the depth (length) of the depth of the cavity was changed to 250, 500, and 750 μm. In addition, the width of the reagent layer in plan view was 1.2 mm, and the width of the inner part was 1 mm. The results are shown in FIG.

  As shown in FIG. 8, it can be seen that the accuracy of the glucose measurement current is improved with respect to the reference form 1 by increasing the depth of the cavity. This is because the clearance between the electrode part for detecting the electrochemical reaction and the innermost edge part of the cavity increases as the size of the inner part of the cavity increases, and along with the occurrence of bubble scratches in the vicinity of the innermost edge part. This is presumably because the influence of the fluctuation of the sample received by the glucose measuring electrode is reduced.

  That is, similarly in the biosensor of the present invention such as the first embodiment, it is considered that the measurement accuracy can be improved by lengthening the depth of the cavity. However, if the depth of the depth is made longer, the cavity volume increases. However, as the increase in the cavity volume leads to an increase in the amount of specimen necessary for the measurement as described above, the depth of the depth is made longer than necessary. That is not desirable.

  DESCRIPTION OF SYMBOLS 1 Insulating substrate, 21 Working electrode, 22 Counter electrode, 3 Reagent layer, 4 Spacer, 41 Cavity, 41a Reagent part, 41b Back part, 41c Inlet, 42 Notch part, 5 Cover, 5a Air hole, 6 Sample liquid, 7 Near the back edge.

Claims (3)

A biosensor for quantifying a substrate contained in a sample solution,
An insulating substrate;
An electrode layer including a working electrode and a counter electrode provided on one surface of the insulating substrate;
A reagent layer containing an enzyme that reacts with the substrate and a mediator formed on a part of the surface of the electrode layer opposite to the insulating substrate;
A spacer having a notch for forming a cavity for guiding the sample liquid to the reagent layer, and a spacer disposed on the electrode layer so that the reagent layer is located inside the notch;
A cover provided on a surface opposite to the insulating substrate of the spacer so as to cover at least the notch,
The cover has an air hole communicating with the cavity,
In the plan view state, and are ratios of 0.1 or less of the area of the air hole to the area of the inner part is a part of the inner side of the reagent layer of the cavity,
In plan view, the air hole is located in the vicinity of the innermost edge, which is the innermost edge of the innermost part of the cavity, and the width of the inner part of the cavity is directed toward the air hole. A biosensor characterized by being narrowed . In plan view, the shape of the innermost edge of the cavity is a circular arc shape The biosensor of claim 1. The biosensor according to claim 1 or 2 , wherein a width of a reagent portion that is a portion corresponding to the reagent layer of the cavity is wider than a width of a portion other than the reagent portion of the cavity.

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