JP6569203B2 - Method for measuring glucose with a glucose sensor - Google Patents

Description

  The present invention relates to a glucose measurement method using a glucose sensor, which relates to a glucose measurement method with reduced temperature-dependent signal intensity fluctuation, and use thereof.

Measurement of blood glucose concentration is indispensable for diabetic patients to perform appropriate blood glucose control. A simple self-blood glucose measurement kit that is used to check blood glucose levels on a daily basis is known, which uses glucose oxidase (hereinafter also referred to as GOD) or glucose dehydrogenase (hereinafter also referred to as GDH). GOD has long been used as an enzyme for measuring blood glucose, but since dissolved oxygen has an effect on the measured value, GDH has become the mainstream in recent years. A glucose sensor using GDH as a raw material measures the glucose concentration in blood using the following reaction of GDH.

D-glucose + electron acceptor (oxidized) →
D-glucono-δ-lactone + electron acceptor (reduced form)

In other words, glucose can be quantified by measuring the flow of electrons generated by oxidizing glucose. GDH used so far for blood glucose measurement includes nicotinamide-dependent type, pyrroloquinoline quinone (hereinafter also referred to as PQQ) -dependent type, flavin adenine dinucleotide (hereinafter also referred to as FAD) due to the difference in coenzyme required for the reaction. Three types of dependency types are known. As a nicotinamide-dependent type, those derived from the genus Bacillus are commercially available, but since they cannot be purified in the state of a holoenzyme containing a coenzyme, nicotinamide adenine dinucleotide (which becomes a coenzyme in producing a sensor) (Hereinafter also referred to as NAD). The problem is that this complexity and NAD that is a coenzyme are expensive. On the other hand, PQQ-dependent GDH can be provided by a holoenzyme and has an advantage of high specific activity and a sufficient response signal to glucose. The point that it reacts with saccharides other than glucose is regarded as a problem. FAD-dependent GDH is spreading as something that can clear these problems.

  Furthermore, a FAD-dependent GDH derived from a fungus family that has eliminated xylose activity, as found in Aspergillus genus GDH (Patent Documents 1 and 2), Penicillium genus GDH (Patent Document 3), etc. Patent applications have been filed (Patent Documents 4 to 6). It is presumed that a blood glucose sensor using such GDH has been intensively studied and developed.

  By the way, in performing glucose determination in the glucose sensor, use in a temperature range of 10 ° C. to 40 ° C. is assumed. In general, a chemical reaction via an enzyme catalyzed by an enzyme shows a behavior in which the reaction rate increases as the temperature increases, and the reaction rate rapidly decreases as the enzyme is deactivated at a temperature exceeding a certain temperature. . Such a temperature-dependent change in enzyme activity can be a factor that causes the quantitative value of glucose to change depending on the outside temperature at the time of measurement. In order to avoid this, for example, a glucose sensor is provided with a thermometer, and a measure for correcting the measured value from the temperature at the time of measurement is made. However, it is pointed out in Non-Patent Document 1 that such a temperature correction function cannot sufficiently eliminate the influence of the environmental temperature on the quantitative value. Therefore, in order to reduce the influence of environmental temperature on the quantitative value of glucose, it is more desirable that the enzyme activity has a small fluctuation in the temperature range of 10 ° C. to 40 ° C. as a characteristic of the enzyme itself.

  From such a viewpoint, various ideas have been made so far. Patent Documents 7 to 9 disclose modified enzymes in which the reaction temperature dependency of the enzyme is improved, and the difference in activity in the low temperature region is reduced compared to the activity at 25 ° C. to 40 ° C. Although the temperature-dependent sensitivity divergence has been resolved to some extent by such modification, it cannot be said that it is complete, and it is necessary to solve the problem from a different viewpoint, which is different from the reaction temperature dependency of the enzyme activity.

Japanese Patent No. 4494978 Japanese Patent No. 4292486 US Pat. No. 7,494,494 Japanese Patent No. 4648893 JP2013-90621A JP2013-116102A JP2011-152129A JP2012-019756 WO2010 / 137487 US Pat. No. 7,871,805

Uno Shiho et al. (2009) Medical Examination Vol. 58 p76-78

An object of the present invention is to provide a glucose measurement method using a glucose sensor, in which a temperature-dependent signal intensity fluctuation is reduced, and glucose having improved measurement sensitivity in a low temperature region used in the method It is to provide a sensor and glucose dehydrogenase for improving measurement sensitivity in a low temperature region on the glucose sensor.

As a result of intensive studies to achieve the above object, the present inventors have applied a viewpoint that is different from the temperature dependence of enzyme activity, more specifically, an enzyme having a low Michaelis constant (Km) for glucose. The inventors have found that the sensitivity in a low temperature region can be improved, and have completed the present invention. From such a viewpoint, it has not been reported so far that the decrease in signal sensitivity in the low temperature region can be suppressed, and the possibility has not been suggested.

That is, the present invention has the following configuration.
Item 1.
A method for measuring glucose using a glucose sensor comprising glucose dehydrogenase having all the following characteristics (A) to (D).
(A) Flavin adenine dinucleotide is used as a coenzyme.
(B) Catalyzing the reaction of converting D-glucose to D-gluconolactone.
(C) The reactivity with respect to maltose is 5% or less with respect to the D-glucose ratio.
(D) The Michaelis constant for D-glucose is 40 mM or less.
Item 2.
A method for measuring glucose by using a glucose sensor comprising an electron transfer substance and glucose dehydrogenase having all the following characteristics (A) to (D) on an electrode comprising at least a working electrode and a counter electrode.
(A) Flavin adenine dinucleotide is used as a coenzyme.
(B) Catalyzing the reaction of converting D-glucose to D-gluconolactone.
(C) The reactivity with respect to maltose is 5% or less with respect to the D-glucose ratio.
(D) The Michaelis constant for D-glucose is 40 mM or less.
Item 3.
Item 3. The glucose measurement method according to Item 1 or 2, wherein the signal intensity obtained under an environment of 5 ° C is 50% or more as compared with the signal intensity obtained under an environment of 25 ° C.

According to the present invention, it is possible to obtain a glucose sensor with reduced temperature-dependent response signal fluctuation in a temperature range of 25 ° C. or lower.

An embodiment of the present invention is a method of measuring glucose using a glucose sensor comprising a glucose dehydrogenase having all the following characteristics.
(A) Flavin adenine dinucleotide is used as a coenzyme.
(B) Catalyzing the reaction of converting D-glucose to D-gluconolactone.
(C) The reactivity with respect to maltose is 5% or less with respect to the D-glucose ratio.
(D) The Michaelis constant for D-glucose is 40 mM or less.

The Michaelis constant in (D) is preferably 35 mM or less, more preferably 15 mM or less, and more preferably 2 mM or less.

The origin of the glucose dehydrogenase used in the above glucose measurement method is not particularly limited, and examples include microorganisms of the genus Penicillium and Mucor. Of these, penicillium italycam, penicillium relassinoechinuratam, mucor pliny, mucor hyalmaris and the like are preferable. Extract DNA encoding glucose dehydrogenase from these microorganisms, or create synthetic DNA based on information such as amino acid sequence of glucose dehydrogenase known in the literature or DNA encoding the glucose dehydrogenase, and A desired glucose dehydrogenase can be obtained by introducing a suitable host vector system and purifying a culture solution obtained by culturing the obtained transformant in a nutrient medium.

Although the form of the glucose sensor used by said glucose measuring method is not specifically limited, An example consists of the following structures. That is, the glucose sensor includes at least an electrode composed of a working electrode and a counter electrode, and may further include a reference electrode, and includes at least the glucose dehydrogenase and the electron transfer substance on the electrodes.
As the electrode, a carbon electrode, a gold electrode, a platinum electrode, or the like is used, and GDH is immobilized on the electrode. As immobilization methods, there are a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc., or an electron mediator At the same time, it may be fixed in a polymer or adsorbed and fixed on an electrode, or these may be used in combination. Typically, the GDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, and then treated with a reagent having an amine group to block glutaraldehyde. Examples of electron mediators that can be used include those that receive electrons from GDH and can donate electrons to the color-developing material and the electrodes. For example, ferricyanide salts, phenazine etsulfate, phenazine methosulfate, phenylenediamine, N, N, N ', N'-tetramethylphenylenediamine, 1-methoxy-phenazine methosulfate, 2,6-dichlorophenolindophenol, 2,5-dimethyl-1,4-benzoquinone, 2,6-dimethyl-1,4-benzoquinone 2,5-dichloro-1,4-benzoquinone, nitrosoaniline, ferrocene derivative, osmium complex, ruthenium complex, cytochrome and the like, but are not limited thereto. Further, in the GDH composition on the electrode, as a stabilizer and / or an activator, a protein such as bovine serum albumin or sericin, a surfactant such as Triton X-100, Tween 20, cholate or deoxycholate , Glycine, serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine and other amino acids, trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and other sugars and / or sugar alcohols, sodium chloride, potassium chloride and other inorganic substances Salts may be added as appropriate, or alternatively, a hydrophilic polymer typified by pullulan, dextran, polyethylene glycol, carboxymethylcellulose, polyvinylpyrrolidone and the like may be included as an excipient.

The glucose concentration can be measured as follows. A sample is added on the composition comprising at least glucose dehydrogenase and an electron transfer substance on the electrode, a constant voltage is applied to the electrode, and the current value is measured. From the measured current value, the glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution. At this time, the obtained current value is affected by the temperature, and the current value is low at a low temperature, and the current value is high at a high temperature. Therefore, it is necessary to calculate the glucose concentration after correcting the temperature at the time of measurement. In the present invention, as an index for improving measurement sensitivity at low temperatures, the current value obtained from a glucose solution having the same concentration in an environment of 5 ° C. and 25 ° C. is measured by the above method, and the current value at 25 ° C. is 100%. It evaluates as a ratio of the electric current value in 5 degreeC at the time of doing. A preferred ratio is preferably 50% or more, more preferably 55% or more, still more preferably 75% or more, and most preferably 80% or more.

Another embodiment of the present invention is a glucose measurement method using the glucose sensor described above, wherein the temperature-dependent signal intensity fluctuation is reduced.
In the present invention, the signal intensity means the height of the current value caused by the oxidation of glucose by GDH, which occurs when a voltage is applied in the glucose sensor.
In the present invention, the signal intensity is compared by comparing the signal intensity obtained in an environment of 5 ° C. with the signal intensity obtained in an environment of 25 ° C.
In the present invention, if the signal intensity obtained in an environment of 5 ° C. is 50% or more as compared with the signal intensity obtained in an environment of 25 ° C., it is determined that the signal intensity fluctuation is reduced.

Another embodiment of the present invention is a glucose sensor used for the glucose measurement method using the glucose sensor or the glucose measurement method using the glucose sensor, wherein the glucose measurement method has a reduced temperature-dependent signal intensity fluctuation. is there.

Another embodiment of the present invention is a glucose dehydrogenase used in the glucose sensor for improving measurement sensitivity in a low temperature region on the glucose sensor.

Example of activity measurement In this specification, FAD-dependent GDH activity measurement is carried out according to the following method unless otherwise specified.
2.9 mL of the reaction solution (0.1 mol / L HEPES, 200 mmol / L D-glucose, 0.55 mmol / L DCPIP, pH 6.5) is placed in a quartz cell, and preheated at 37 ° C. for 5 minutes. Then, 0.1 mL of GDH solution is added and mixed, and the mixture is reacted at 37 ° C. for 5 minutes. During this time, the absorbance at 700 nm is measured. The degree of increase in absorbance per minute (ΔOD _TEST ) is calculated from the linear portion of the absorbance change. In the blind test, a buffer solution is added in place of the GDH solution and mixed, and similarly, incubated at 37 ° C. for 5 minutes, the absorbance at 700 nm is recorded, and the change in absorbance per minute (ΔOD _BLANK ) is calculated. The activity value (U / mL) is calculated by applying these values to the following equation. Here, the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of a substrate is defined as 1 U.

GDH activity (U / mL) = [(ΔOD _TEST− ΔOD _BLANK ) × 3.0 × dilution ratio] / (4.5 × 1.0 × 0.1)

Here, 3.0: Volume after mixing with GDH solution (mL)
4.5: millimolar molecular extinction coefficient of DCPIP (cm ² / micromol)
1.0: Optical path length (cm)
0.1: Volume of GDH solution to be added (mL)
It is.

Example of calculating Michaelis constant (Km) for glucose The method for calculating the Michaelis constant (Km) for a substrate described in the present invention is performed by the following measuring method. That is, five types of reaction liquids were used in which the concentration of D-glucose in the reaction liquid composition described in the above activity measurement examples was 200 mmol / L, 100 mmol / L, 50 mmol / L, 20 mmol / L, 10 mmol / L as measurement solutions. ΔOD (ΔOD _TEST -ΔOD _BLANK ) of the GDH solution (solution adjusted so that the activity value in the above activity measurement example is 0.8 U / ml) using each measurement solution according to the method of the above activity measurement example ). Based on these measured values, the Michaelis constant (Km) is calculated according to the Lineweaver-Burk plot method (both reciprocal plot method).

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.

Example 1 Preparation of FAD-dependent GDH with low Km value for glucose As described in Patent Document 10, each purified enzyme was prepared.
For mucor plinie-derived FAD-dependent GDH (hereinafter referred to as MpGDH), a synthetic DNA having the sequence described in SEQ ID NO: 2 was prepared with reference to the amino acid sequence described in Patent Document 4. In addition, the FAD-dependent GDH (hereinafter referred to as MhGDH) derived from Mucor Himalis prepared a synthetic DNA consisting of the sequence described in SEQ ID NO: 4 with reference to the amino acid sequence described in Patent Document 6. These synthetic DNAs are transformed into a host (Cryptococcus sp. S-2.U-5 strain) described in a patent document (WO2013 / 088081) which has already been disclosed, according to the flow described in the general patent. Went. In addition, pCsUX was used as a vector at the time of transformation, and the genes of SEQ ID NOs: 2 and 4 were fused with the secretion signal of the xylanase derived from Cryptococcus sp. Was inserted into the MluI / SpeI site of the general vector with a stop codon (TAA) added thereto. The outline of the vector pCsUX and the sequence of the xylanase secretion signal are also described in the above patent document. The transformant thus prepared is inoculated into 60 mL of a YM medium in a 200 mL baffled flask, cultured at a temperature of 25 ° C. and a rotation speed of 180 rpm for 2 days, and the total amount of the culture solution is 6 L of production medium (5 % Yeast extract, 2% xylose, 0.02% adecanol) and aerated and agitated culture at 30 ° C. The culture solution was passed through a continuous centrifuge rotor (model number: R10C) set in a high-speed cooling centrifuge (Hitachi High-Tech CR22 type) and rotated at 5000 rpm to sediment the cells, and a culture solution supernatant was obtained. . This solution was hydrolyzed and concentrated using an ultrafiltration membrane having a molecular weight of 50,000 cut so that the solution became 1000 times or more, and finally concentrated to a liquid volume of 2 L. In this solution, ammonium sulfate was dissolved to 0.4 saturation, and adsorbed on Phenyl-Sepharose 6FF HS (manufactured by GE Healthcare) buffered with 50 mM potassium phosphate buffer (pH 6.0) containing 0.4 saturated ammonium sulfate. Gradient elution was performed until the ammonium sulfate concentration reached 0, and fractions having GDH activity were collected. This solution was concentrated to 10 mL using an ultrafiltration membrane with a molecular weight of 50000 cut, passed through Superdex 200 buffered with 50 mM potassium phosphate buffer containing 0.3 M sodium chloride, and subjected to gel filtration to obtain a purified enzyme. . For the above four types of GDH, the Michaelis constant (Km value) was calculated according to the above-mentioned calculation example of the Michaelis constant, and the result was compared with the Km value of a commercially available FAD-dependent GDH (AoGDH, derived from Aspergillus oryzae) Is shown in Table 1.

Preparation of glucose sensor and comparison of temperature dependency of signal intensity A solution (pH = 7.5) having the following composition was prepared as a reagent for measuring glucose.
1 mM citric acid 50 mM potassium ferricyanide 500 U / ml FAD-dependent GDH
0.5% Carboxymethylcellulose 5 μL of the above solution is dropped on the working electrode / counter electrode / reference electrode of a disposable chip (DEP-CHIP, manufactured by Biodevice Technologies) with 3 electrodes, and heated at 50 ° C. for 10 minutes. By doing so, a sensor chip was obtained. This sensor chip was connected to a potentio / galvanostat (HSV-100, manufactured by Hokuto Denko) via a dedicated socket, placed in an environment of 25 ° C., and the composition on the electrode was preincubated at 25 ° C. with 10 mM. A current glucose was monitored by adding 5 μL of a standard glucose solution and applying a voltage of +0.3 V. In addition, these sensor chips are similarly reacted with a glucose solution preincubated at 5 ° C. in an environment of 5 ° C., and the current value is monitored to determine the ratio of the current value at 5 ° C. to the current value at 25 ° C. It was. The results are shown in Table 2.

As shown in the table, the ratio of the signal at 5 ° C. to the current value at 25 ° C. is affected by the Km value of GDH for glucose, and in GDH having a Km value of less than 40 mM, It has been found that it exhibits a higher ratio than the GDH it has, that is, it exhibits a relatively high current value even in a low temperature environment. This means that the glucose sensor is less susceptible to low temperatures.

  The glucose sensor manufactured according to the present invention can be supplied as a reagent for measuring blood glucose level, a blood glucose sensor, and a glucose determination kit.

Claims (5)

A method for measuring glucose using a glucose sensor comprising glucose dehydrogenase having all the following characteristics (A) to (E) :
(A) Catalytic reaction of converting D-glucose into D-gluconolactone using flavin adenine dinucleotide as a coenzyme (C) Reactivity to maltose is 5% or less of D-glucose ratio ( D) Michaelis constant for D-glucose is 15 mM or less
(E) When the current value obtained from the glucose solution of the same concentration at 25 ° C. is 100%, the ratio of the current value at 5 ° C. is 75% or more,
The pH of the reagent solution for measuring glucose is 7.5 and has the following compositions (a) to (d):
(A) Citric acid
(B) Potassium ferricyanide
(C) Glucose dehydrogenase using flavin adenine dinucleotide as a coenzyme
(D) Carboxymethylcellulose The method for measuring glucose according to claim 1, wherein the glucose measurement reagent solution has the following compositions (a) to (d).
(A) 1 mM citric acid
(B) 50 mM potassium ferricyanide
(C) Glucose dehydrogenase using 500 U / ml flavin adenine dinucleotide as a coenzyme
(D) 0.5% carboxymethylcellulose The method for measuring glucose according to claim 1, wherein the composition on the electrode comprises the following (a) to (d).
(A) 5 nmol citric acid
(B) 250 nmol potassium ferricyanide
(C) Glucose dehydrogenase using 2.5 U flavin adenine dinucleotide as a coenzyme
(D) 2.5 μg carboxymethylcellulose The Michaelis constant for D-glucose is 2 mM or less , and the current value ratio at 5 ° C. is 80% or more when the current value obtained from a glucose solution of the same concentration at 25 ° C. is 100%. Item 4. A method for measuring glucose according to any one of Items 1 to 3 . The method for measuring glucose according to any one of claims 1 to 4 , using a glucose sensor having at least an electrode comprising a working electrode and a counter electrode.