JP5509969B2 - Measuring apparatus and measuring method - Google Patents

Description

  The present invention relates to a measuring apparatus and a measuring method for measuring a current value generated between electrodes by applying a potential to an electrode coated with a reagent containing glucose dehydrogenase and potassium ferricyanide having a flavin compound as a coenzyme.

  In recent years, the number of diabetic patients is increasing in each country. As a treatment for diabetes, for example, there is insulin therapy. Insulin is known as a drug that controls blood glucose levels and is administered to diabetic patients as a therapeutic agent for diabetes. The need to administer insulin is determined based on the blood glucose level of the diabetic patient. For this reason, it is important for a diabetic patient to grasp a blood glucose level. The blood glucose level is the glucose concentration in the blood. A simple blood glucose measuring device has been developed for the purpose of allowing a diabetic patient to easily measure his blood glucose level.

  As the blood glucose measurement device described above, one using a biosensor is known (Patent Documents 1 to 4). The biosensor has an electrode on which an enzyme that reacts with blood sugar is fixed. Glucose oxidase (hereinafter sometimes abbreviated as “GOD”) and glucose dehydrogenase (hereinafter sometimes abbreviated as “GDH”) are known as enzymes used for blood glucose level measurement. . In a biosensor, an electrode on which an enzyme is immobilized is called a working electrode, and an electrode that supplies electrons into a sample is called a counter electrode.

  When blood as a sample is introduced into the biosensor and GOD of the working electrode reacts with glucose in the blood, glucose is decomposed into gluconic acid and hydrogen peroxide, and the hydrogen peroxide is decomposed into water and electrons. The electrons generated in this way are transmitted to the working electrode. On the other hand, electrons are supplied from the counter electrode into the blood. In this way, a current flows between the working electrode and the counter electrode due to the reaction between GOD and glucose. Then, based on the flowing current value, the glucose concentration in the blood, that is, the blood glucose level is calculated. In addition, a substance that transmits electrons may be fixed to the working electrode. This material is referred to as an electron mediator. Examples of the electron mediator include organic compounds such as potassium ferricyanide (potassium hexacyanoate (III)), hexaammineruthenium and quinone derivatives, or organic-metal complexes.

  While GOD has the advantage of high specificity for glucose and excellent thermal stability, there is a known problem that dissolved oxygen in blood affects the measured value. GDH has the advantage that it is not affected by dissolved oxygen, but has low stability and poor substrate specificity, so it is known that it reacts with sugars other than glucose, such as maltose and lactose. Yes. In response to such problems, glucose dehydrogenase (hereinafter sometimes abbreviated as “FAD-GDH”) using a flavin compound as a coenzyme has excellent thermal stability and is not easily affected by maltose or the like. (Patent document 5).

  In the blood glucose measurement device described above, attempts have been made to improve the measurement range and measurement accuracy by changing the potential applied between the working electrode and the counter electrode (Patent Documents 6 and 7).

JP 2009-97877 A JP 2005-43280 A JP-A-2005-37335 JP 2002-107325 A JP 2009-195250 A Japanese Patent No. 3487064 Japanese Patent No. 3528521

  In recent years, it has been known that blood glucose measurement is affected by pralidoxime methyl iodide (hereinafter sometimes abbreviated as “PAM”). PAM is administered to humans by intravenous injection or the like as an antidote for organophosphate poisoning. When PAM is present in the blood, the blood glucose level tends to be higher than the actual blood glucose level. As a result, insulin may be administered in an excessive amount based on the blood glucose level affected by the PAM that is higher than the true blood glucose level, thereby causing hypoglycemic symptoms.

  As a result of intensive studies, the present inventors have found that potassium ferricyanide used as an optimal mediator for FAD-GDH may be affected by PAM, and have completed the present invention. That is, the object of the present invention is to suppress the influence of other sugars such as dissolved oxygen and maltose on the measurement value by the measurement system of the reagent containing FAD-GDH and potassium ferricyanide, and the PAM is the measurement value. An object of the present invention is to provide a blood glucose measurement means for suppressing the influence on the blood glucose.

  The present invention relates to a measuring apparatus in which a biosensor is detachably provided on an apparatus main body. The biosensor includes a first electrode, a second electrode, a sample space into which a specimen is introduced so as to be in contact with the first electrode and the second electrode, and a reagent. The measuring apparatus includes voltage applying means electrically connected to the first electrode and the second electrode, and measuring means for measuring a current value generated between the first electrode and the second electrode. . The reagent contains at least glucose dehydrogenase using flavin compound as a coenzyme and potassium ferricyanide. Each component of the reagent is applied to at least one of the first electrode and the second electrode. The voltage applying means applies a voltage of 200 mV or less between the first electrode and the second electrode for a predetermined time. The measuring means includes a current value generated between the first electrode and the second electrode when the voltage applying means applies a voltage of 200 mV or less between the first electrode and the second electrode. Measure.

  The measuring device is for electrochemically detecting glucose in the specimen. Examples of the specimen include mainly liquids such as blood, saliva, and urine. When the specimen is brought into contact with the first electrode and the second electrode in the sample space, the specimen, FAD-GDH, and potassium ferricyanide are mixed, and glucose causes an enzyme reaction. Due to the potential applied between the first electrode and the second electrode, the charge generated during the enzymatic reaction flows between the first electrode and the second electrode via potassium ferricyanide, so that glucose is electrically It can be detected.

  It is preferable that the voltage applied by the voltage applying unit between the first electrode and the second electrode for a predetermined time is in a range of 100 to 200 mV.

  The first electrode functions as a working electrode and is preferably made of carbon.

  Further, the present invention provides a first electrode, a second electrode, a sample space into which a specimen is introduced so as to be in contact with the first electrode and the second electrode, and at least one of the first electrode and the second electrode. A measuring method comprising a biosensor having a coated reagent, wherein at least glucose dehydrogenase and potassium ferricyanide having a flavin compound as a coenzyme are used as the reagent, the first electrode and the first electrode It may be understood as a measurement method in which a voltage of 200 mV or less is applied between the two electrodes for a certain period of time, and a current value generated between the first electrode and the second electrode is measured.

  According to the present invention, by using FAD-GDH, other sugars such as dissolved oxygen and maltose are suppressed from affecting the measurement value, and are applied between the first electrode and the second electrode. By setting the potential to 200 mV or less, the PAM is suppressed from affecting the measurement value.

FIG. 1 is a perspective view showing an appearance of a blood glucose measurement device 10 according to an embodiment of the present invention. FIG. 2 is an exploded perspective view of the biosensor 11.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings as appropriate. Each embodiment described below is only an example of the present invention, and it is needless to say that the embodiment of the present invention can be changed as appropriate without departing from the gist of the present invention.

[Blood glucose measuring device 10]
As shown in FIG. 1, the blood glucose measurement device 10 includes a biosensor 11 and a device body 12. The biosensor 11 and the apparatus main body 12 are electrically connected by inserting the biosensor 11 into the connection portion 13 of the apparatus main body 12. The biosensor 11 is replaced for each blood glucose measurement.

[Apparatus body 12]
As shown in FIG. 1, the apparatus main body 12 is an electronic apparatus in which an electronic component is accommodated in a housing 50. A liquid crystal display 51 and operation keys 52, 53, and 54 are disposed on the front side of the housing 50. The operation keys 52, 53, and 54 are for generating corresponding commands based on user operations. The liquid crystal display 51 displays the state of the apparatus main body 12, measurement results, error display, and the like. Although not shown in each figure, a control board as a voltage applying unit and a measuring unit is built in the housing 50. The control board is configured as an arithmetic device having a CPU, a ROM, a RAM, and the like, and is electrically connected to the liquid crystal display 51 and the operation keys 52, 53, and 54. In response to input from the operation keys 52, 53, and 54, the control board operates a program stored in advance in the ROM. A power source for applying a potential to the biosensor 11 is built in the housing 50. The control board applies a predetermined potential to the biosensor 11 from the power source. The power supply and the control board constitute voltage application means. In addition, the control board calculates a blood glucose level from the current value flowing through the biosensor 11 in response to a predetermined potential. The calculated blood glucose level is displayed on the liquid crystal display 51.

[Biosensor 11]
As shown in FIG. 1, the biosensor 11 has an elongated sheet shape. The biosensor 11 is attached to the apparatus main body 12 by inserting one end of the biosensor 11 in the longitudinal direction 101 into the connection portion 13 of the apparatus main body 12. Further, when the biosensor 11 is pulled out in the longitudinal direction 101, the biosensor 11 is removed from the apparatus main body 10.

  Since the front and back of the biosensor 11 are relative, any of them may be front or back. In this embodiment, the side that appears in FIG. 1 is referred to as the front, and the side that does not appear in FIG. 1 is referred to as the back. That is, the biosensor 11 has a sheet shape in which the front surface 21 and the back surface 22 form front and back surfaces.

  As shown in FIG. 2, the biosensor 11 mainly includes a first substrate 23, a first electrode 24, a spacer 25, a second electrode 26, a third electrode 27, and a second substrate 28. The first substrate 23, the spacer 25, the first electrode 24, the second electrode 26, the third electrode 27, and the second substrate 28 are stacked in this order from the upper side in FIG. It is configured.

[First substrate 23]
As shown in FIG. 2, the first substrate 23 is a sheet that is substantially the same shape as the biosensor 11 in plan view and is slightly shorter than the second substrate 24 in the longitudinal direction 101. The first substrate 23 is made of an electrically insulating material. Examples of the electrically insulating material include polyesters such as polyethylene terephthalate (PET) and polymethyl methacrylate (PMMA), fluororesin, polycarbonate, and glass.

  One surface of the first substrate 23 constitutes the surface 21. On the surface 21 of the first substrate 23, a pair of colored portions 30 and 31 are formed at both ends in the direction 102. The colored portions 30 and 31 are color-coded with the surface 21. The coloring portions 30 and 31 are for facilitating visual recognition of the position of a sample introduction port 41 to be described later. Therefore, the coloring portions 30 and 31 are arranged immediately above the sample introduction port 41. In the first substrate 23, each component of the reagent is fixed in a region 42 corresponding to the space 40. Details of each component of the reagent will be described later.

[Second substrate 28]
As shown in FIG. 2, the second substrate 28 is a sheet having substantially the same shape as the biosensor 11 in plan view. The second substrate 28 is made of an electrically insulating material. Examples of the electrically insulating material include polyesters such as polyethylene terephthalate (PET) and polymethyl methacrylate (PMMA), fluororesin, polycarbonate, and glass.

  One surface of the second substrate 28 constitutes the back surface 22. A first electrode 24, a second electrode 26, and a third electrode 27 are provided on the surface 34 opposite to the back surface 22. In the second substrate 28, one end side in the longitudinal direction 101 inserted into the connection portion 13 of the apparatus main body 12 is not opposed to the first substrate 23. Although not shown in each drawing, the colored portion similar to the colored portions 30 and 31 is formed on the back surface 22 of the second substrate 28.

[First electrode 24]
As shown in FIG. 2, the first electrode 24 is provided on the surface 34 of the second substrate 28 opposite to the back surface 22. The first electrode 24 extends in the longitudinal direction 101 on the surface 34 of the second substrate 28, and has a width of about 1/3 of the second substrate 28 in the direction 102. In the surface 34, the 1st electrode 24 is arrange | positioned in the electrically unconnected with the 2nd electrode 26 and the 3rd electrode 27 which are mentioned later. Examples of the material of the first electrode 24 include carbon. By using carbon for the first electrode 24, the resistance value of the first electrode 24 made of carbon becomes lower than the resistance value of the electrode using silver / silver chloride, gold, palladium, platinum, etc. Even with a small response current due to a weak potential, the blood glucose level measurement sensitivity is easily maintained. The first electrode 24 is laminated on the surface 34 with respect to the first substrate 23 by a method such as screen printing, inkjet, sputtering, vacuum deposition, sol-gel method, cluster beam deposition, or PLD. In the first electrode 24, the end corresponding to the connection portion 13 side of the apparatus main body 12 is exposed without facing the first substrate 23. This end is a connection terminal 33 that is electrically connected to the apparatus main body 12.

[Second electrode 26]
As shown in FIG. 2, the second electrode 26 is provided on the surface 34 opposite to the back surface 22 of the second substrate 28. The second electrode 26 extends in the longitudinal direction 101 on the surface 34 of the second substrate 28, and has a width of about 1/3 of the second substrate 28 in the direction 102. In the surface 34, the 2nd electrode 26 is arrange | positioned in the electrical connection with the 1st electrode 24 and the 3rd electrode 27 mentioned later. Examples of the material for the second electrode 26 include carbon, silver / silver chloride, gold, palladium, and platinum. In the second electrode 26, an end corresponding to the connection portion 13 side of the apparatus main body 12 is exposed without facing the first substrate 23. This end is a connection terminal 37 that is electrically connected to the apparatus main body 12.

[Third electrode 27]
As shown in FIG. 2, the third electrode 27 is provided on the surface 34 of the second substrate 28 opposite to the back surface 22. The third electrode 27 extends in the longitudinal direction 101 on the surface 34 of the second substrate 28, and has a width of about 1/3 of the second substrate 28 in the direction 102. On the surface 34, the third electrode 27 is disposed in an electrically unconnected manner with the first electrode 24 and the second electrode 26. Examples of the material for the third electrode 27 include carbon, silver / silver chloride, gold, palladium, and platinum. The third electrode 27 is an electrode for detecting whether blood is introduced into the space 40. In the third electrode 27, the end corresponding to the connection portion 13 side of the apparatus main body 12 is exposed without facing the first substrate 23. This end is a connection terminal 39 that is electrically connected to the apparatus main body 12.

[Spacer 25]
As shown in FIG. 2, the spacer 25 is a sheet having substantially the same shape as the biosensor 11 in plan view. As the spacer 25, a double-sided tape having electrical insulation is preferably used. The spacer 25 has a space 40 extending in the direction 102 at a position corresponding to the coloring portions 30 and 31. That is, the spacer 25 is composed of two sheets separated by the space 40 with respect to the longitudinal direction 101. The space 40 forms a sample space corresponding to the thickness of the spacer 25 between the region 38 and the region 42. That is, the space 40 becomes the sample space. In the space 40, a part of the first electrode 24, a part of the second electrode 26, and a part of the third electrode 27 are exposed. In the first electrode 24, each component of the reagent is fixed in a region 38 corresponding to the space 40. Details of each component of the reagent will be described later.

  As shown in FIG. 1, the space 40 is opened at the end of the biosensor 11, and this opening serves as the sample introduction port 41. Although not shown in FIG. 1, the space 40 is also opened at a position facing the sample introduction port 41. When the sample inlet 41 is exposed to blood, blood flows into the space 40 by capillary action.

[Components of Reagent Fixed to Regions 38 and 42]
Reagents immobilized in the regions 38 and 42 include FAD-GDH, potassium ferricyanide, a water-soluble polymer, a surfactant, and a buffer solution. The reagent may further contain other components. Each component of the reagent may be selectively fixed to the regions 38 and 42, but as in this embodiment, the first electrode 24 functions as a working electrode and the second electrode 26 functions as a counter electrode. In this case, the FAD-GDH and potassium ferricyanide are preferably fixed to the region 38 of the first electrode 24. Moreover, a well-known method is employ | adopted as the method of apply | coating each component of a reagent to the area | regions 38 and 42, respectively. Examples of such known methods include a dipping method, a screen printing method, an offset printing method, and an ink jet printing method.

[Measurement method of blood sugar]
Hereinafter, a blood glucose measurement method using the blood glucose measurement device 10 will be described.

  The user inserts the biosensor 11 into the connection portion 13 of the apparatus main body 12 when measuring blood glucose. The user introduces blood collected from a finger or the like into the space 40. When blood contacts the first electrode 24 and the third electrode 27 in the space 40, the conductive state between the first electrode 24 and the third electrode 27 changes. The arithmetic unit constituted by the control board determines that blood is introduced into the space 40 due to the change in the conductive state.

  The voltage applying means constituted by the control board and the power supply waits for 10 seconds after judging that blood has been introduced into the blood 40, and thereafter, the connection terminal 33 of the first electrode 24 and the connection terminal of the second electrode 26 A predetermined potential is applied to 37 for 10 seconds. The predetermined potential is set within a range of 100 to 200 mV. Thereby, the 1st electrode 24 becomes a working electrode and the 2nd electrode 26 becomes a counter electrode. In the space 40, D-glucono-1,5-lactone and ferrocyanide ions are generated from ferricyanide electrolyzed by glucose and potassium ferricyanide in the blood by FAD-GDH fixed to the second electrode 26. . Oxidation response current flows between the first electrode 24 and the second electrode 26 by oxidizing the ferrocyan ion at the first electrode 24. Based on the oxidation response current flowing between the first electrode 24 and the second electrode 26 in this way, the measuring means constituted by the control substrate calculates the glucose concentration and displays the result on the liquid crystal display 51.

[Operational effects of this embodiment]
According to this embodiment, by using FAD-GDH as an enzyme that reacts with glucose, other sugars such as dissolved oxygen and maltose are suppressed from affecting the measurement value, and the first electrode 24 and the second electrode When the potential applied between the electrodes 26 is 200 mV or less, the PAM is suppressed from affecting the measurement value.

  Examples of the present invention will be described below.

[Biosensor]
As the biosensor, the biosensor 11 having the same configuration as that of the above-described embodiment was used. Carbon was used as a material for the first electrode 24, the second electrode 26 and the third electrode 27. Region 38 of biosensor 11 includes 2.31 w / v% FAD-GDH, 7.10 w / v% potassium ferricyanide, 4.61 w / v% polyvinylpyrrolidone, 1.68 w / v% tetradecyltrimethylammonium, and 2 0.88 μL of a reagent containing 70 w / v% MES was applied and dried. In the region 42 of the biosensor 11, 0.44 μL of a reagent containing 0.14 w / v% Torton X-100 was applied and dried.

[Example 1]
Using 0.3 μL each of MES buffer solution added so that PAM was 0.0 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, After the specimen is detected, the potential between the first electrode 24 and the second electrode 26 is maintained at 0 mV for 10 seconds, and then the potential between the first electrode 24 and the second electrode 26 is maintained for 10 seconds. The response current was measured 2 seconds after 150 mV was applied while maintaining 150 mV. This measurement was repeated three times (n = 3). Table 1 shows the average of three measurement results.

[Example 2]
Using 0.3 μL each of MES buffer solution added so that PAM was 0.0 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, After the specimen is detected, the potential between the first electrode 24 and the second electrode 26 is maintained at 0 mV for 10 seconds, and then the potential between the first electrode 24 and the second electrode 26 is maintained for 10 seconds. The response current was measured 2 seconds after 200 mV was applied while maintaining 200 mV. This measurement was repeated three times (n = 3). Table 1 shows the average of three measurement results.

[Comparative Example 1]
Using 0.3 μL each of MES buffer solution added so that PAM was 0.0 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, After the specimen is detected, the potential between the first electrode 24 and the second electrode 26 is maintained at 0 mV for 10 seconds, and then the potential between the first electrode 24 and the second electrode 26 is maintained for 10 seconds. The response current was measured at 2 seconds after applying 250 mV while maintaining the voltage at 250 mV. This measurement was repeated three times (n = 3). Table 1 shows the average of three measurement results.

[Comparative Example 2]
Using 0.3 μL each of MES buffer solution added so that PAM was 0.0 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, After the specimen is detected, the potential between the first electrode 24 and the second electrode 26 is maintained at 0 mV for 10 seconds, and then the potential between the first electrode 24 and the second electrode 26 is maintained for 10 seconds. The response current was measured after 2 seconds after applying 300 mV while maintaining 300 mV. This measurement was repeated three times (n = 3). Table 1 shows the average of three measurement results.

  As shown in Table 1, in Examples 1 and 2, there was an increase in the measured value (μA) of the sample to which PAM was added relative to the measured value (current value; μA) of the sample to which PAM was not added. It was 20.4% or less. In contrast, in Comparative Example 1, when the amount of PAM added was 1.5 mg / mL or more, the measured value (μA) increased by 35% or more. In Comparative Example 2, even when the amount of PAM added was 0.5 mg / mL, the measured value (μA) increased nearly 35%, and when the amount of PAM added was 1.0 mg / mL or more, the measured value (ΜA) increased by 45% or more. Thereby, in Examples 1 and 2, it was confirmed that the measured value (μA) is hardly affected by PAM.

24 ... 1st electrode 26 ... 2nd electrode 40 ... space (sample space)

Claims (5)

A measuring device comprising a biosensor and a device main body to which the biosensor can be attached and detached ,
The biosensor includes a first electrode, a second electrode, a sample space into which a specimen is introduced so as to be in contact with the first electrode and the second electrode, and a reagent.
The measuring apparatus includes voltage applying means electrically connected to the first electrode and the second electrode, and measuring means for measuring a current value generated between the first electrode and the second electrode. ,
The reagent contains at least glucose dehydrogenase and potassium ferricyanide having a flavin compound as a coenzyme,
Each component of the reagent is applied to at least one of the first electrode and the second electrode,
The voltage applying means applies a voltage of 200 mV or less between the first electrode and the second electrode for a certain period of time,
The measuring means includes a current value generated between the first electrode and the second electrode when the voltage applying means applies a voltage of 200 mV or less between the first electrode and the second electrode. Measuring device that measures. The voltage application means applies a voltage of 100 to 200 mV between the first electrode and the second electrode for a certain period of time,
The measuring means is generated between the first electrode and the second electrode when the voltage applying means applies a voltage of 100 to 200 mV or less between the first electrode and the second electrode. The measuring apparatus according to claim 1, which measures a current value.   The measuring apparatus according to claim 1, wherein the first electrode functions as a working electrode and is made of carbon. A first electrode, a second electrode, a sample space in which a specimen is introduced so as to be in contact with the first electrode and the second electrode, a reagent applied to at least one of the first electrode or the second electrode, A measuring method using a measuring device including a biosensor having
As the reagent, at least, glucose dehydrogenase and potassium ferricyanide having a flavin compound as a coenzyme are used,
A measurement method of measuring a current value generated between the first electrode and the second electrode by applying a voltage of 200 mV or less between the first electrode and the second electrode for a predetermined time.   The voltage of 100-200mV is applied between the said 1st electrode and the said 2nd electrode for a fixed time, and the electric current value produced between the said 1st electrode and the said 2nd electrode is measured. Measuring method.

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