JP5798044B2 - Biosensor - Google Patents

Description

The present invention relates to a biosensor that detects a specific component in a sample solution, and more particularly to a configuration of a detection reagent.

Conventionally, a biosensor for measuring a blood sugar level and the like of a specimen and a manufacturing method thereof have been devised (for example, see Patent Documents 1 to 4). FIG. 1 shows a conventional general biosensor (sensor chip) 1. This biosensor 1 is provided with a working electrode 3 and a counter electrode 4 in parallel proximity on an electrode insulating substrate 2, and a mask sheet 6 having a reaction cell 5 on the electrode insulating substrate 2, working electrode 3 and counter electrode 4. Is applied to the working electrode 3 and the counter electrode 4 in the reaction unit cell 5, and the reaction unit coating solution containing oxidoreductase is applied and dried to form the reaction unit 7 composed of a reagent layer. It was. An electrically insulating spacer sheet 8 and a transparent cover sheet 9 are laminated on the mask sheet 6. According to the biosensor 1, the blood glucose level can be recognized by attaching the sample to the blood glucose level measurement display and taking the sample, and the blood glucose level measurement display displays the blood glucose level.

Here, in general, in the biosensor, the mediator contained in the reagent layer may be reduced by moisture absorption, and the amount of mediator that is actually reduced by the reaction increases, so that the background increases and an error may occur. there were.

For this reason, biosensors have been implemented with measures such as adding a desiccant to keep the inside of individual aluminum packages and resin bottles at low humidity. However, in particular, when a plurality of chips are put in a bottle, the output may change due to the influence of moisture absorption due to repeated opening. Also, aluminum individual packaging increases manufacturing costs.

For this reason, the biosensor which suppresses the output change by moisture absorption and does not have a performance change is desired.

In Japanese Patent Laid-Open No. 2002-207022, the presence of heat and water causes a reduction reaction between an enzyme protein or a part of a hydrophilic polymer contained in a reagent layer and an electron carrier. Current) and the background current value increases with time, and the problem that sensor performance deteriorates is noticeable. Sugar alcohol and metal salts are added as a countermeasure. Take measures to do.

JP-A-2008-261653 discloses that in order to stabilize the solution state of the target protein aqueous solution, sericin and / or a hydrolyzate thereof or the equivalent thereof are allowed to coexist in the aqueous solution in addition to the protein. It is disclosed that generation can be suppressed.

Alternatively, Japanese Patent Application Laid-Open No. 2008-239512 discloses a method for stabilizing an antibody, wherein the antibody coexists with sericin and / or a hydrolyzate thereof, or an equivalent thereof.

  Furthermore, Japanese Patent Application Laid-Open No. 2008-143790 discloses a method for improving protein solubility, characterized in that sericin and / or a hydrolyzate thereof or an equivalent thereof coexists in an aqueous solution.

Furthermore, JP-A-2007-151546 discloses (a) a biomolecule and (b) sericin and / or a hydrolyzate thereof, in order to stabilize a biomolecule, particularly an enzyme or a labeled antibody used in a clinical diagnostic agent. , And the equivalent thereof, and a method for stabilizing a biomolecule is disclosed.

  However, even if these methods are applied as they are to the above-described biosensor for measuring a blood glucose level or the like, a desired improvement effect for stabilization against moisture absorption is not necessarily obtained.

  On the other hand, in Patent 3867959, an electrically insulating substrate, an electrode system having at least a working electrode and a counter electrode provided on the substrate, and formed in contact with or in the vicinity of the electrode system, at least pyrroloquinoline quinone as a coenzyme A glucose sensor comprising a reaction layer containing glucose dehydrogenase is described, wherein the reaction layer contains at least one additive selected from the group consisting of gluconic acid and salts thereof.

  However, even if these methods are applied as they are to the above-described biosensor for measuring a blood glucose level or the like, a desired improvement effect for stabilization against moisture absorption is not necessarily obtained.

International Publication No. 2004/017057 Pamphlet Japanese Patent Publication No.7-114705 Japanese Patent No. 3063442 Japanese Patent No. 3483314 JP 2002-207022 A JP 2008-239512 A JP 2008-143790 A JP 2007-151546 A Japanese Patent No. 3867959

An object of the present invention is to provide a biosensor that suppresses changes in output due to moisture absorption and has no change in performance.

The gist of the present invention is that a substrate made of an insulator, a pair of electrodes provided on the substrate at a predetermined distance from each other, and a specific component that reacts with a specific component formed to be electrically connected to the electrodes. A reaction part having a reagent layer to be supplied, and a supply port for introducing a specimen to the reaction part,
The reaction unit reacts with a specific component in the sample introduced from the supply port, quantitatively analyzes the specific component,
In the reagent layer,
(A) an enzyme (b) a hydrophilic polymer,
(C) To contain a mixture of sericin hydrolyzate and potassium gluconate .

  The hydrophilic polymer may be a sodium salt of carboxymethyl cellulose.

  The reagent layer may include an enzyme and an electron carrier.

  The reagent layer may be composed of a reaction layer containing an enzyme and a layer containing an electron carrier.

  The present invention is also characterized in that, in the biosensor, the enzyme is glucose oxidase.

  Moreover, the present invention is characterized in that, in the biosensor, the enzyme is glucose dehydrogenase.

According to the present invention, there is provided a biosensor that suppresses changes in output due to moisture absorption and has no performance change.

It is a figure which shows a common biosensor, The same figure (a) is a top view, The same figure (b) is an AA line | wire cut part sectional drawing. It is a figure which shows the principal part of the biosensor used for the experiment example in this invention, The same figure (a) is a top view, The same figure (b) is AA cut | disconnected section sectional drawing. It is a figure for demonstrating the method of producing the other biosensor used for the experiment example in this invention, The figure (a) is a top view when forming a 1st layer, The figure (b) It is sectional drawing when forming a 1st layer, The figure (c) is a top view when forming a 2nd layer, The figure (d) is sectional drawing when forming a 2nd layer. . It is a graph which shows the relationship between the glucose concentration and current integral value in the experiment example of this invention. It is a graph which shows the relationship between the glucose concentration and current integral value in the experiment example of this invention. It is a graph which shows the relationship between the glucose concentration and current integral value in the experiment example of this invention. It is a graph which shows the relationship between 30 degreeC, 65% exposure time, and an electric current integral value in the experiment example of this invention. It is a graph which shows the relationship between sodium glutamate addition amount and the current integral value in the experiment example of this invention. It is a graph which shows the relationship between potassium glutamate addition amount and the current integral value in the experiment example of this invention. It is a graph which shows the relationship between potassium gluconate addition amount and change amount in the experiment example of this invention. It is a graph which shows the relationship between CMC1% + potassium gluconate addition amount and change amount in the experiment example of this invention. It is a graph which shows the relationship between the addition amount of sericin and the variation | change_quantity in the experiment example of this invention. It is a graph which shows the relationship between CMC1% + sericin addition amount and change amount in the experiment example of this invention. It is a graph which shows the relationship between potassium gluconate addition amount and change amount in the experiment example of this invention. It is a graph which shows the relationship between the addition amount of sericin and the variation | change_quantity in the experiment example of this invention. It is a graph which shows the relationship between the glucose concentration and current integral value in the experiment example of this invention, and is a graph which shows an initial stage characteristic. It is a graph which shows the relationship between the elapsed days in the experiment example of this invention, and the change of an electric current integrated value, and is a graph which shows the case where glucose concentration is 0 mg / dl. It is a graph which shows the relationship between the elapsed days in the experiment example of this invention, and the change of an integrated current value, and is a graph which shows the case of glucose concentration of 100 mg / dl. It is a graph which shows the relationship between the elapsed days in the experiment example of this invention, and the change of an integrated current value, and is a graph which shows the case of glucose concentration of 100 mg / dl. It is a graph which shows the relationship between the elapsed days in the experiment example of this invention, and the change of an electric current integral value, and is a graph which shows the case where glucose concentration is 300 mg / dl. It is a graph which shows the relationship between the elapsed days in the experiment example of this invention, and the change of an electric current integral value, and is a graph which shows the case where glucose concentration is 300 mg / dl. It is a graph which shows the relationship between the elapsed days in the experiment example of this invention, and the change of an electric current integrated value, and is a graph which shows the case of glucose concentration 500 mg / dl. It is a graph which shows the relationship between the elapsed days in the experiment example of this invention, and the change of an electric current integrated value, and is a graph which shows the case of glucose concentration 500 mg / dl.

The present inventors have found that if the enzyme stabilizer added to maintain the enzyme activity is not added to the reagent layer, the background does not increase due to moisture absorption, and the enzyme protein is not responsible for the background increase. Revealed. However, in the absence of this enzyme stabilizer, the output is reduced due to enzyme inactivation during storage at high temperatures or for long periods.

  Here, the present inventors further examined various additives that are effective for enzyme stabilization and do not increase the background, and as a result, a hydrolyzate of a hydrophilic polymer and sericin, or a hydrophilic polymer and potassium gluconate, Alternatively, it has been found that it is effective to contain a hydrophilic polymer, a hydrolyzate of sericin, and potassium gluconate.

  These will be described with reference to the following experimental examples. In this specification, unless otherwise specified, the concentration or the percentage of other things relative to a certain thing is a weight-based ratio (% by weight).

Production of sensor chip A sensor chip having the structure shown in FIG. 2 was used. In FIG. 2, reference numeral 10 denotes a sensor chip used in this experimental example. The manufacturing method of the sensor chip 10 includes an electrode part forming step in which a working electrode 14 and a counter electrode 16 are provided in parallel and proximity on an electrically insulating substrate 12, and a mask step in which a mask sheet 20 having reaction part cells 18 is thermally bonded. A reaction part forming step for forming a reaction part 22 having an oxidoreductase on the working electrode 14 and the counter electrode 16 in the reaction part cell 18, and an electrically insulating spacer sheet 24 and a transparent cover sheet on the mask sheet 20. And a laminating step of laminating 26.
In addition, the working electrode 14 and the counter electrode 16 were obtained by sputtering platinum on one surface of a polyimide film and coating the other surface with a heat fusion material (ethylene vinyl acetate) into a tape shape. A thing was used. This tape is thermally bonded onto the electrically insulating substrate 12 to form an electrode portion.

The reaction part forming step includes applying a first reaction part coating solution having an oxidoreductase on the working electrode 14 and the counter electrode 16 and drying to form a first layer 28; A second layer forming step of forming a second layer 30 by applying and drying a second reaction part coating solution having a hydrophilic polymer compound and an electron acceptor on the first layer 28; In the first layer formation step, for example, an oxidoreductase is dissolved in water and applied. In the second layer forming step, the hydrophilic polymer compound is dissolved in a solvent that does not dissolve the first layer 28 so that the first layer 28 does not dissolve. As this solvent, ethyl cellosolve was used in this experimental example. By such a reaction part formation step, as shown in FIG. 2, a reaction part 22 composed of a first layer 28 and a second layer 30 which are reagent layers is formed.

Sensor Evaluation Method Using an aqueous glucose solution, the relationship between the glucose concentration and the current integral value was determined. Here, the current integrated value is the voltage scan between -0.1V and + 0.2V by changing the potential between the electrodes from 0V → -0.2V → 0V → + 0.2V at a rate of 50mV / sec after inhaling the measurement sample. Sometimes the current flowing between the electrodes is changed to voltage, and the results of A / D conversion 60 times every 0.1 sec are integrated.

Evaluation method of heat resistance The sensor chip is individually packaged in an aluminum bag together with a desiccant, and the output decrease at a glucose concentration of 300 mg / dl after maintaining at a temperature of 50 ° C. for 7 days is used as an index. Specifically, the following mathematical formula was used.
Concentration decrease after storage at 50 ° C. for 7 days Δ300 = initial concentration 300 mg / dl integrated value−300 mg / dl integrated value decrease after heating (mg / dl) = Δ300 / sensitivity a
* Sensitivity a: slope when the output values of glucose concentrations 0, 100, and 300 mg / dl are linearly approximated, the smaller the amount of decrease, the better the heat resistance.

Method for evaluating moisture resistance The sensor chip is not packaged individually, and the increase in output at a glucose concentration of 0 mg / dl after maintaining at a temperature of 30 ° C. and a humidity of 65% for 16 hours is used as an index. Specifically, the following mathematical formula was used.
Concentration drop after holding temperature 30 ° C and humidity 65% Δ0 = 0mg / dl integrated value after humidification-Initial concentration 0mg / dl integrated value increase (mg / dl) = Δ0 / sensitivity a
* Sensitivity a: slope when the output values of glucose concentrations 0, 100, and 300 mg / dl are linearly approximated. This increase amount is an increase in background, and the smaller the increase amount, the better the moisture resistance.

Formation Method of Reaction Layer After 0.78 μl of an aqueous solution containing 1.8% GOD (glucose oxidase) and an additive is dropped on the electrode, drying is performed at 40 ° C. for 6 minutes to form the first layer. Furthermore, 0.76 μl of a solution obtained by dispersing finely divided potassium ferricyanide (median diameter 3.9 μm) in an ethyl cellosolve solution of 1.3% PVP (polyvinylpyrrolidone) to 27.6%, A second layer was formed by dropping on the first layer 18.

FIG. 4 shows the difference in the relationship between the glucose concentration and the current integral value depending on the presence or absence of the additive. As an additive, sodium glutamate was added at 1.8%, which is the same as the enzyme amount. When there is an additive, there is linearity up to a glucose concentration of 500 mg / dl, but when there is no additive, the linearity is low. The enzyme activity at this time was 4.2 U / chip with an additive and 1.0 U / chip without an additive per sensor chip. Without an additive, the enzyme activity is low, and the sensitivity to glucose concentration is reduced.

Next, the characteristics after heating these sensors at 50 ° C. for 7 days are shown in FIGS. With the additive (FIG. 5), the characteristics after heating at 50 ° C. were not substantially different from the initial values, and the amount of change at a concentration of 300 mg / dl was 9.8 mg / dl. Without additive (FIG. 6), the amount of decrease is as large as 61.3 mg / dl, and the output is greatly reduced by heating during storage.

Further, FIG. 7 shows changes with time in output when these sensors are humidified to 30 ° C. and 65% RH.
With the additive, the output increased with time and was 37.0 mg / dl at 16 hours. Almost no increase was made without the additive, and it was 0.5 mg / dl at 16 hours.

From the above, it was found that the background increase due to moisture absorption was not caused by the enzyme, but by an additive such as a stabilizer contained in the enzyme.

The results of the above evaluation performed by changing the type and concentration of the additive are shown below.

FIG. 8 shows evaluation results of heat resistance and moisture resistance when sodium glutamate addition amount-dependent GOD (glucose oxidase) 1.8% and sodium glutamate are added in the range of 0 to 1.8%. The greater the amount added, the better the heat resistance, but the humidity resistance becomes very poor.

FIG. 9 shows the evaluation results of heat resistance and moisture resistance when GOD (glucose oxidase) 1.8% and potassium glutamate are added in the range of 0 to 1.8% depending on the addition amount of potassium glutamate. The greater the amount added, the better the heat resistance, but the humidity resistance becomes very poor. The result is almost the same as that of sodium glutamate.

FIG. 10 shows the evaluation results of heat resistance and moisture resistance when potassium gluconate addition amount GOD (glucose oxidase) 1.8% and potassium gluconate are added in the range of 0 to 1.8%. It has less effect on moisture resistance than sodium glutamate and potassium glutamate.

FIG. 11 shows the evaluation results of heat resistance and moisture resistance when CMC (carboxymethylcellulose) 1%, potassium gluconate addition amount-dependent GOD (glucose oxidase) 1.8% and potassium gluconate are added. The heat resistance increased to 40 mg / dl by adding CMC alone at 1%. (Point of 0% addition of potassium gluconate) Further, by adding potassium gluconate, the heat resistance is improved even if the addition amount of potassium gluconate is small, so the influence on moisture resistance can be reduced.

Addition Dependency of Sericin Hydrolyzate GOD (glucose oxidase) 1.8% and evaluation results of heat resistance and moisture resistance when adding sericin hydrolyzate are shown in FIG. Although it has little effect on heat resistance, the addition does not deteriorate the moisture resistance.

Dependence of CMC 1% and sericin hydrolyzate on addition amount GOD (glucose oxidase) 1.8%, CMC 1% and sericin hydrolyzate were added, and the evaluation results of heat resistance and moisture resistance are shown in FIG. . The heat resistance increased to 40 mg / dl by adding CMC alone at 1%. Furthermore, heat resistance improves without adding moisture resistance by adding a slight hydrolyzate of sericin.

In the case of simultaneous addition of CMC, potassium gluconate and sericin hydrolyzate In this case, a glucose aqueous solution is used as the sensor evaluation method in the same manner as described above, and the relationship between the glucose concentration and the current integral value is obtained. However, here, as the current integration value, after inhaling the measurement sample, the potential between the electrodes is changed from 0 V → -0.2 V → 0 V → +0.2 V at a rate of 200 mV / sec, and -0.1 V → +0.2 V The current flowing between the electrodes during voltage scanning was changed to voltage, and the result obtained by integrating A / D conversion results 60 times every 0.025 sec was used.

As a method for forming the reaction layer, a sensor chip having the configuration shown in FIG. 3 was used. The working electrode 114 and the counter electrode 116 were formed by patterning by photolithography, which was obtained by directly sputtering nickel on the electrode insulating substrate 110. By dropping 0.15 μl of an aqueous solution containing GOD (glucose oxidase) 3.0% and CMC (carboxymethylcellulose) 0.8% onto the working electrode 114 and the counter electrode 116 and drying at 40 ° C. for 6 minutes, As shown in 3 (a) and (b), the first layer 118 was formed. Further, 0.20 μl of a solution obtained by dispersing finely divided potassium ferricyanide (median diameter 3.9 μm) in an ethyl cellosolve solution of 1.13% HPC (hydroxypropylcellulose) to 12.5% was used. It was dropped on the layer 118 to form the second layer 120 as shown in FIGS. 3 (c) and 3 (d). In this way, the reaction part 122 composed of the first layer 118 and the second layer 120 was formed.

FIG. 14 shows characteristics when CMC 0.8%, potassium gluconate and sericin hydrolyzate are added to the additive in this manner. Even when potassium gluconate is added at 1.8% sericin hydrolyzate, both heat resistance and moisture resistance are good.

When the amount of sericin hydrolyzate added is changed with potassium gluconate 0.3% By the above method, CMC 0.8%, potassium gluconate and sericin hydrolyzate are added to the additive, and the amount of sericin hydrolyzate added FIG. 15 shows the characteristics when the value is changed. Good properties regardless of the amount of sericin hydrolyzate added.

  Hereinafter, the performance change of the biosensor of the present invention when stored at a high temperature for a long time will be described. As an evaluation method, the relationship between the elapsed days and the integrated value of the current when the biosensor of the present invention was stored at a high temperature for a long time was determined.

  For the current integration value, change the potential between the electrodes after inhaling the measurement sample from 0 V → -0.2 V → 0 V → +0.2 V at a rate of 200 mV / sec. The current flowing in was changed to voltage, and the result obtained by integrating A / D conversion 60 times every 0.025 sec was used.

  When GOD (glucose oxidase) is used as the enzyme and potassium gluconate and sericin hydrolyzate are added simultaneously, GDH (glucose dehydrogenase) is used as the enzyme and potassium gluconate and sericin hydrolyzate are added simultaneously If GDH (glucose dehydrogenase) is used as the enzyme, potassium gluconate is added and sericin hydrolyzate is not added, then GDH (glucose dehydrogenase) is used as the enzyme and sericin hydrolyzate is added. When potassium gluconate is not added and the biosensor 1 is not added, put the chip in a bottle that can keep the biosensor 1 at low humidity, store at 70 ° C., measure the output against the aqueous glucose solution, and determine the ratio to the initial output. Plotted.

When GOD (glucose oxidase) is used as an enzyme and hydrolysates of potassium gluconate and sericin are added simultaneously, GOD (glucose oxidase) 3.0% and CMC (carboxymethylcellulose) 0.8%, potassium gluconate By dropping 0.15 μl of an aqueous solution containing 0.3% and 1.8% sericin hydrolyzate onto the working electrode 114 and the counter electrode 116 and drying at 40 ° C. for 6 minutes, FIG. As shown in (b), the first layer 118 was formed. The second layer 120 was formed by the same method as the second layer 120 in FIG.

When GDH (glucose dehydrogenase) is used as the enzyme and hydrolyzate of potassium gluconate and sericin is added simultaneously, GDH (glucose dehydrogenase) 3.0% and CMC (carboxymethylcellulose) 0.8%, glucone 3A by dropping 0.15 μl of an aqueous solution containing 0.3% potassium acid and 1.8% sericin hydrolyzate onto the working electrode 114 and the counter electrode 116 and drying at 40 ° C. for 6 minutes. ) And (b), the first layer 118 was formed. The second layer 120 was formed by the same method as the second layer 120 in FIG.

  When GDH (glucose dehydrogenase) is used as an enzyme and potassium gluconate is added and sericin hydrolyzate is not added, GDH (glucose dehydrogenase) 3.0% and CMC (carboxymethylcellulose) 0.8% As shown in FIGS. 3A and 3B, 0.15 μl of an aqueous solution containing 0.3% potassium gluconate is dropped on the working electrode 114 and the counter electrode 116 and dried at 40 ° C. for 6 minutes. Then, the first layer 118 was formed. The second layer 120 was formed by the same method as the second layer 120 in FIG.

When GDH (glucose dehydrogenase) is used as an enzyme and sericin hydrolyzate is added and potassium gluconate is not added, GDH (glucose dehydrogenase) 3.0% and CMC (carboxymethylcellulose) 0.8% Then, 0.15 μl of an aqueous solution containing 1.8% hydrolyzate of sericin was dropped on the working electrode 114 and the counter electrode 116 and dried at 40 ° C. for 6 minutes to obtain the results shown in FIGS. As shown, a first layer 118 was formed. The second layer 120 was formed by the same method as the second layer 120 in FIG.

  When the glucose concentration was 0 mg / dl, as shown in FIG. 17, the same characteristics were obtained regardless of GOD and GDH. When only sericin hydrolyzate was added, the change in output was small. Although the increase in output is observed only by adding potassium gluconate, the increase in output is suppressed by the simultaneous addition with the hydrolyzate of sericin.

  When the glucose concentrations were 100 mg / dl, 300 mg / dl, and 500 mg / dl, the same characteristics were obtained regardless of GOD and GDH, as shown in FIGS. When only potassium gluconate was added, the initial maintenance characteristics were good, but long-term storage was not possible. When only sericin hydrolyzate was added, the initial decrease in output was large. It was found that long-term stability can be maintained while suppressing initial decrease by simultaneously adding a hydrolyzate of potassium gluconate and sericin.

According to the present invention, there is provided a biosensor that suppresses changes in output due to moisture absorption and has no performance change. For this reason, this invention can be widely utilized for manufacture of various biosensors.

1: Biosensor 2, 12, 110: Electrode insulating substrate 3, 14, 114: Working electrode 4, 16, 116: Counter electrode 5, 18: Reaction part cell 6, 20: Mask sheet 7, 22, 122: Reaction part 8, 24: Spacer sheet 9, 26: Cover sheet 10: Sensor chip 28, 118: First layer 30, 120: Second layer

Claims (6)

A substrate made of an insulator, a pair of electrodes provided on the substrate at a predetermined interval, and a reaction part having a reagent layer formed so as to be electrically connected to the electrodes and reacting with a specific component; A supply port for introducing the specimen to the reaction unit,
The reaction unit reacts with a specific component in the sample introduced from the supply port, quantitatively analyzes the specific component,
In the reagent layer,
(A) an enzyme (b) a hydrophilic polymer,
(C) A biosensor comprising a mixture of sericin hydrolyzate and potassium gluconate .   The biosensor according to claim 1, wherein the hydrophilic polymer is carboxymethyl cellulose.   The biosensor according to claim 1 or 2, wherein the reagent layer includes an enzyme and an electron carrier.   The biosensor according to claim 1, wherein the reagent layer includes a reaction layer containing an enzyme and a layer containing an electron carrier.   The biosensor according to any one of claims 1 to 4, wherein the enzyme is glucose oxidase.   The biosensor according to any one of claims 1 to 4, wherein the enzyme is glucose dehydrogenase.

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