JP2011053052A - Biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stable, sufficiently sensitive and inexpensive biosensor for measuring a substrate concentration by using an oxidation-reduction enzyme having a NAD(P) as a coenzyme. <P>SOLUTION: The biosensor has two or more electrodes comprising at least a working electrode and a counter electrode on an insulative substrate and a substrate, contacts a composition including at least the NAD or the NADP, a dehydrogenase containing the NAD and/or the NADP as the coenzyme, a phenazine methosulfate derivative and a mediator in a portion or the entire electrode, and measures a substrate concentration of the dehydrogenase. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a biosensor that uses a dehydrogenase having nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme and measures the substrate concentration of the dehydrogenase.

Various methods for quantifying substances serving as substrates of enzymes using various oxidoreductases have been conventionally studied. The principle is that a current is detected by finally passing electrons generated when the substrate is oxidized by an enzyme to an electrode in a voltage application state, and the concentration of the substrate is measured based on the current value. When an oxidoreductase using NAD and / or NADP as a coenzyme is used, the transfer of electrons directly from the reduced NAD (P) to the electrode is inefficient, and a high applied voltage is required to achieve this. .
As a method for efficiently detecting the current value at a lower voltage, there is a method in which a mediator is interposed between the reduced NAD (P) and the electrode, and the reduced NAD (P) is directly converted to the oxidized type on the electrode. Compared with the case, the electron transmission speed is improved. However, even in this case, the rate of electron transfer between the coenzyme and the mediator is not sufficient, and this is the rate-limiting step of the electrochemical reaction on a series of sensors.
As a method for solving this problem, for example, Patent Document 1 discloses a method in which another enzyme that converts reduced NAD (P) to oxidized NAD (P) coexists. Patent Document 2 discloses a method of generating formazan by using phenazine methosulfate or a derivative thereof and a tetrazolium salt, and transferring electrons from the formazan to the electrode under voltage application.
These methods have other problems while the electron transfer speed from the reduced NAD (P) to finally the electrode is remarkably improved. For example, as for the former, diaphorase can be mentioned as a representative example of converting reduced NAD (P) into an oxidized form, but the stability of diaphorase itself is not sufficient. There is concern about the apparent decline in value. The latter also has storage stability problems such as alteration of the tetrazolium salt during storage and a reduction in function as a reducing agent.
In addition, the use of 1-methoxy-5-phenazine methosulfate (mPMS), which is a derivative of phenazine methosulfate, as a mediator that passes between the coenzyme and the electrode, may eliminate both electron transfer rate and stability. is there. However, since this mPMS is remarkably expensive compared to ferricyanide, ferrocene, etc., which are mediators used for general purposes, it is not possible to prescribe it alone in an amount sufficient to obtain a satisfactory electron transfer rate. It was not realistic from an economic point of view.

Japanese Unexamined Patent Publication No. 55-13072 WO00 / 57166

Accordingly, an object of the present invention is to provide a biosensor that measures substrate concentration using an oxidoreductase that uses NAD (P) as a coenzyme, and that is stable and sufficient in sensitivity and inexpensive. That is.

The present inventors have made extensive studies to solve the above problems. As a result, the phenazine methosulfate derivative can be used in combination with other general-purpose mediators, compared with the case where the mediator is used alone, even if the phenazine methosulfate derivative is used in combination with other general-purpose mediators. It has been found that a significantly high electron transfer rate can be achieved, thereby increasing the electrochemical signal, and the present invention has been completed.

In addition, the present invention also increases the electrochemical signal corresponding to a higher concentration of the substrate, so that the measurable concentration range is wider than when the mediator is used alone. It was also seen.

That is, the present invention has the following configuration.
[Claim 1]
An insulating substrate and two or more electrodes comprising at least a working electrode and a counter electrode on the substrate, and a composition containing at least all of the following (1) to (4) in contact with a part or all of the electrode A biosensor for measuring a substrate concentration of dehydrogenase.
(1) NAD or NADP
(2) Dehydrogenase using NAD and / or NADP as a coenzyme (3) Phenazine methosulfate derivative (4) Mediator [Claim 2]
Item 2. The biosensor according to Item 1, wherein the substrate for dehydrogenase is glucose.
[Section 3]
Item 3. The biosensor according to Item 1 or 2, wherein the mediator is a ferricyanide salt.
[Claim 4]
Item 4. The biotechnology according to any one of Items 1 to 3, wherein the content of the phenazine methosulfate derivative in the composition is in a range in which an electrochemical signal sensitivity that is at least twice that of a case where the phenazine methosulfate derivative is not included is obtained. Sensor.
[Section 5]
Item 4. The biosensor according to any one of Items 1 to 3, wherein the content of the phenazine methosulfate derivative in the composition is from 1/4000 to 1/200 of the mediator material.

According to the present invention, there is provided a biosensor for measuring a substrate concentration using an oxidoreductase having NAD (P) as a coenzyme, which can obtain a stable and sufficient electrochemical signal and is inexpensive. be able to.

In addition, according to the present invention, the current value increases correspondingly even with a higher concentration of substrate, so that the measurable concentration range is widened as compared with the case where the mediator is used alone. This is because the bottleneck in the reaction path from the electron extracted from glucose to the electrode has been eliminated, and the concentration range in which the current value can rise in a substrate concentration-dependent manner has expanded to a higher concentration side. Understood.

An example of the electrode used for the biosensor of this invention is shown. The relationship between mPMS concentration and electrochemical signal intensity is shown. The correlation between the substrate concentration and the electrochemical signal intensity in the biosensor of the present invention is shown.

The present invention will be described in detail below.
The biosensor of the present invention forms an electrode composed of at least a working electrode and a counter electrode on an insulating substrate, and at least the entire working electrode of the electrode includes at least NAD or NADP, dehydrogenase, phenazine methosulphate derivative and mediator. It is the biosensor formed by making it contact with the composition which comprises. A carbon electrode may be used as the working electrode, or a metal electrode such as platinum, gold, silver, nickel, or palladium may be used. In the case of a metal electrode, gold is particularly preferred. As a method for forming the electrode on the insulating substrate, specifically, it is desirable to form the electrode on the substrate by a printing technique such as photolithography technique, screen printing, gravure printing, flexographic printing or the like. In addition, examples of the insulating substrate material include silicon, glass, glass epoxy, ceramic, polyvinyl chloride, polyethylene, polypropylene, polyester, and polyimide, but it is more preferable to use a material that is highly resistant to various solvents and chemicals. preferable.
The composition comprising at least NAD or NADP, dehydrogenase, phenazine methosulphate derivative and mediator is usually supported on the working electrode, but if it has a counter electrode and a reference electrode, it may extend over the reference electrode. Good.
Examples of the method of contacting the composition containing an enzyme or the like with the electrode include a method of dropping an aqueous solution of the composition onto the electrode, a method of air-drying after dropping, and a method of immobilizing the composition on the electrode. Examples of the immobilization method include 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, and the like. Typically, a method of blocking glutaraldehyde by immobilizing a composition containing an enzyme or the like on a carbon electrode using glutaraldehyde and then treating with a reagent having an amine group is exemplified.
The reference electrode is not particularly limited, and a common electrode in electrochemical experiments can be applied. Examples thereof include a saturated calomel electrode and silver-silver chloride.

The dehydrogenase described in the present invention is not particularly limited as long as it catalyzes a reaction of oxidizing and / or reducing a substrate using NAD and / or NADP as a coenzyme. For example, formaldehyde dehydrogenase (EC 1.2.1.46) Glucose dehydrogenase (EC 1.1.1.47), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glutamate dehydrogenase (EC 1.4.1.2 or EC 1.4.1) .4), glycerol dehydrogenase (EC 1.1.1.6), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), lactate dehydrogenase (EC 1.1.1.27), leucine dehydrogenase (EC 1.4.1.9), malate dehydrogenase (EC 1.1. .37), but alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2), and the like, without limitation. For example, when the measurement target is glucose, glucose hydrogenase is selected.

Specifically exemplified as the phenazine methosulfate derivative is 1-methoxy-5-phenazine methosulfate (mPMS). In a broad sense, this mPMS also falls within the category of mediators. The mediator described in the present invention is defined as a substance other than a PMS derivative, which can accept electrons from a reduced PMS derivative and donate them to an electrode.

Specific examples of mediators other than mPMS include quinones, cytochromes, viologens, phenazines, phenoxazines, phenothiazines, ferricyanides, ferredoxins, ferrocene and derivatives thereof. Specific examples include benzoquinone / hydroquinone, ferricyanide (potassium or sodium salt), ferricinium / ferrocene, and 2,6-dichlorophenolindophenol, and metal complexes such as osmium, cobalt, and ruthenium may be used. Is possible. Of these, potassium ferricyanide is preferably used.

When using a low water-soluble compound as a mediator, using an organic solvent may impair the stability of the enzyme itself or deactivate the enzyme activity. Therefore, an enzyme modified with a hydrophilic polymer such as polyethylene glycol (PEG) may be used to increase water solubility.

The mediator concentration in the reaction system at the time of sample addition is preferably in the range of about 1 mM to 1 M, more preferably 5 to 500 mM, and still more preferably 10 to 300 mM.

Further, the addition amount of the PMS derivative is preferably a small amount and sufficient to promote the electron transfer rate from the reduced NAD to the mediator.
As an indication of the amount of PMS derivative added enough to promote the electron transfer rate from reduced NAD to mediator, the addition of an electrochemical signal that is twice or more that of when the mediator is used alone is obtained. It is a concentration, and more preferably an addition concentration at which an electrochemical signal of 3 times or more can be obtained as compared with the case where the mediator is used alone. Further, the standard of the addition amount of the PMS derivative sufficient to promote the electron transfer rate from the reduced NAD to the mediator from another viewpoint is preferably 1/4000 or more of the mediator as a substance amount, more preferably 4000 1 to 200/200, more preferably 1/800 to 1/200. Here, the substance amount can be paraphrased as the number of molecules. When the content of the PMS derivative in the composition is 1/4000 of the mediator, there are 4000 mediator molecules per molecule of the PMS derivative. Means that. Further, the content of NAD (P) in the composition is not particularly limited as long as it is an amount capable of stably detecting an electrochemical signal having an intensity dependent on the concentration of the dehydrogenase substrate to be measured. Is equal to or greater than the Michaelis constant (Km) of the dehydrogenase for NAD (P) as the concentration in the solution when the substrate and dehydrogenase react, more preferably, the Michaelis constant (Km) of 5 for the NAD (P) of the dehydrogenase. It is more than 10 times, and most preferably more than 10 times the Michaelis constant (Km) with respect to NAD (P) of the dehydrogenase.

The composition on the electrode may contain various buffering agents in addition to NAD or NADP, dehydrogenase, phenazine methosulfate derivative and mediator. The buffer is preferably a substance having a buffer capacity under a suitable pH condition set in the range of pH 5.0 to 9.0. In addition to phosphate, fumaric acid, maleic acid, glutaric acid, phthalate Examples include various organic acids such as acid and citric acid, and GOOD buffering agents such as MOPS, PIPES, HEPES, MES, and TES, but are not limited thereto. Furthermore, for the purpose of enhancing the storage stability of dehydrogenase, protein such as bovine serum albumin, ovalbumin, sericin, saccharides and amino acids that cannot be a substrate of the dehydrogenase, metal salts such as calcium, magnesium, zinc, and manganese, or EDTA Various chelating agents such as Triton X-100, deoxycholic acid, cholic acid, Tween 20, Brij 35, emulgen and the like may be appropriately included.

The shape of the electrode is not particularly limited, and examples thereof include a circle, an ellipse, and a rectangle. For example, in the case of a circular shape, the radius is preferably 3 mm or less, more preferably 2.5 mm or less, and even more preferably 2 mm or less.

A method of measuring the concentration of a substance of interest using the biosensor shown in the present invention is performed as follows.
An electrode system comprising at least a working electrode and a counter electrode is connected to a device exemplified by a potentiostat / galvanostat and comprises NAD or NADP in contact with the electrode, a dehydrogenase based on the substance of interest, a phenazine methosulfate derivative, and a mediator A sample containing a dehydrogenase substrate to be measured is added to the product. The substrate solution to be measured can be added dropwise to the composition on the electrode using a micropipette or the like, or a liquid channel from the opening to the electrode system is provided, and the sample is electroded through the channel. The liquid may be fed upward. A current value as an electrochemical signal obtained by applying a voltage in a state where the composition on the electrode is in contact with the sample is measured. This current value depends on the substrate concentration contained in the sample, and the substrate concentration can be calculated from the current value based on a calibration curve prepared in advance using a standard solution of the substrate. The strength of the applied voltage is preferably 50 to 1000 mV, more preferably 100 to 500 mV. The type of sample used for the measurement is not particularly limited, and may be a biological sample such as blood, body fluid, urine, as well as an aqueous solution that may contain an enzyme substrate as a component. In the measurement, the reaction temperature may be kept constant as much as possible. Also, the measurement environment temperature is measured, and the temperature-dependent reaction rate is calculated from the correlation equation between the temperature and the electrochemical signal prepared in advance. It is also possible to correct the change.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. Embodiments are also included in the technical scope of the present invention.

  Hereinafter, the present invention will be specifically described by way of examples. Needless to say, the examples described below do not limit the present invention.

A modified glucose dehydrogenase (hereinafter referred to as GDH) derived from a thermoproteus genus hyperthermophilic archaeon was obtained as follows.
The GDH gene shown in SEQ ID NO: 1 is contained in the GDH gene using the Quick Change Multi Site Mutationis Kit (manufactured by Stratagene) using the expression plasmid pBSGDH-2 inserted into the NdeI-BamHI site of the plasmid pBluescriptKSN (+) for Escherichia coli. The expression plasmid pBSTGDH-3 was obtained by introducing a mutation into the specific site. pBSTGDH-3 is a plasmid formed by connecting a DNA molecule corresponding to SEQ ID NO: 3 under the lac promoter. This plasmid was transformed into an E. coli JM109 strain competent cell to obtain a production strain. The transformed strain was 200 ml preculture medium (0.5% yeast extract, 0.25% peptone, 0.5% sodium chloride, 0.5% glucose, 100 μg / ml ampicillin sodium, pH 7) in a 500 ml Sakaguchi flask. 4) and cultured with shaking at 30 ° C. and 180 rpm for 16 hours. The obtained precultured solution was mixed with 6 L of GDH production medium (2.4% yeast extract, 2.4% peptone, 1.25% dihydrogen phosphate, 0.23% phosphate in 10 L jar fermenter. 2 hydrogen 1 potassium, 0.4% glycerol, 0.1% antifoaming agent, 100 μg / ml ampicillin sodium, pH 7.0), the whole amount was charged, aeration rate 2 L / min, stirring speed 310 rpm, tank pressure 0.02 MPa, The culture was aerated and stirred at a temperature of 37 ° C. for 24 hours. The resulting culture is centrifuged to obtain bacterial cells, which are suspended in 1 L of 20 mM potassium phosphate buffer (pH 8.0), and the bacterial cells are removed at an average pressure of 80 MPa using a French press bacterial cell crusher. It was crushed. Further, 152 g of ammonium sulfate per liter was added to this crushed solution for dissolution, followed by heating at 60 ° C. for 1 hour, and further removing the precipitate by centrifugation. This solution was applied to Octyl-Sepharose resin (manufactured by GE Healthcare) buffered with 20 mM potassium phosphate buffer (pH 8.0) containing 15.2% ammonium sulfate to adsorb GDH to the resin. The resin was washed with 20 mM potassium phosphate buffer (pH 8.0) containing 6% ammonium sulfate. Then, the GDH was eluted by passing the buffer through a gradient while changing the ammonium sulfate concentration from 7.6% to 0% and simultaneously the ethylene glycol concentration from 0% to 0.2%. Next, desalting and buffer replacement were performed using G-25 Sepharose resin buffered with 50 mM potassium phosphate buffer (pH 7.0). Finally, the GDH solution was passed through DEAE-Sepharose buffered with 50 mM potassium phosphate buffer (pH 7.0) to adsorb the contaminating protein to the resin, and the permeate was used as purified GDH. The thermoproteus-derived mutant GDH thus prepared has the amino acid sequence shown in SEQ ID NO: 4.

An electrode sensor having a working electrode, a counter electrode, and a reference electrode arranged on an insulating substrate was obtained from Biodevice Technology Co., Ltd. (Nomi City, Ishikawa Prefecture). This electrode sensor has electrodes printed on a 4.0 mm × 17 mm substrate. FIG. 1 illustrates the structure of this sensor. 10 μL of an aqueous solution serving as a reagent layer was dispensed on the working electrode (area: about 1.3 mm 2) of this sensor. The aqueous solution to be the reagent layer includes the following composition.
・ 20 mM oxidized NAD
NAD (P) -dependent glucose dehydrogenase (produced in Example 1)
80 mM potassium ferricyanide 0-0.4 mM 1-methoxy-5-phenazine methosulfate 20 mM potassium phosphate buffer (pH 7.0)
An equal amount of 20 mM D-glucose solution (360 mg / dl) was added and mixed to the solution having the above composition with a micropipette, and a voltage of +300 mV was applied with a potentiostat after 1 minute. That is, the composition in the solution (20 μl) on the electrode after addition of the sample is as follows.
・ 10 mM oxidized NAD
NAD (P) -dependent glucose dehydrogenase (produced in Example 1)
40 mM potassium ferricyanide 0-0.2 mM 1-methoxy-5-phenazine methosulfate 10 mM D-glucose 10 mM potassium phosphate buffer (pH 7.0)
The electrochemical signals (average current value for 5 seconds from 1 second to 6 seconds after voltage application) obtained at each mPMS concentration are shown in Table 1 and FIG. When the mPMS concentration is 0.01 mM, that is, the amount of mPMS as a substance amount is 1/4000 of potassium ferricyanide as a mediator, the signal intensity reaches 3.5 times that without mPMS, the mPMS concentration is 0.05 mM, That is, when the amount of mPMS as a substance amount was 1/800 of potassium ferricyanide as a mediator, the signal intensity reached 5.9 times that when mPMS was not included.

Comparative Example 1

The same operation as in Example 2 was performed using a composition not containing potassium ferricyanide. That is, the composition in the solution (20 μl) on the electrode after adding the D-glucose solution is as follows.
・ 10 mM oxidized NAD
NAD (P) -dependent glucose dehydrogenase (produced in Example 1)
-0-0.2 mM 1-methoxy-5-phenazine methosulfate-10 mM D-glucose-10 mM potassium phosphate buffer (pH 7.0)
The electrochemical signals obtained at each mPMS concentration are shown in Table 1 and FIG. In this range, the electrochemical signal sensitivity when mPMS was used alone as an electron transfer substance was 0.053 to 0.55 times compared to the case where it was combined with a mediator. It can be seen that a series of electrochemical reactions at the electrode can be remarkably promoted by combining with a mediator even if the amount of mPMS alone cannot provide sufficient sensitivity. Moreover, it is understood from the comparison with Example 1 that the increase in signal sensitivity due to the addition of mPMS is not a mere additive effect due to the added mPMS, but a synergistic effect due to the combination with the mediator.

On the working electrode of the same sensor as that used in Example 2, 5 μL of an aqueous solution serving as a reagent layer was dispensed. The aqueous solution to be the reagent layer includes the following composition.
・ 40 mM oxidized NAD
NAD (P) -dependent glucose dehydrogenase (produced in Example 1)
-160 mM potassium ferricyanide-0.4 mM 1-methoxy-5-phenazine methosulfate-40 mM potassium phosphate buffer (pH 7.0)
To the solution having the above composition, 15 μl of D-glucose solution having various concentrations was added and mixed with a micropipette, and after 1 minute, a voltage of +300 mV was applied with a potentiostat, and the response current was measured. That is, the composition in the solution (20 μl) on the electrode after addition of the sample is as follows.
・ 10 mM oxidized NAD
NAD (P) -dependent glucose dehydrogenase (produced in Example 1)
-40 mM potassium ferricyanide-0.1 mM 1-methoxy-5-phenazine methosulfate-5-30 mM D-glucose The response current was measured using a solution having a composition obtained by removing mPMS or potassium ferricyanide from the reagent layer. FIG. 3 is a graph plotting the glucose concentration and the response current value with respect to each reagent composition. It is understood that in the above-mentioned glucose concentration range, a composition in which an appropriate amount of PMS derivative and mediator is combined can obtain a significantly higher electrochemical signal compared to the case where the PMS derivative is absent. It is also understood that the concentration-dependent increase in signal intensity is improved particularly in the region where the substrate concentration exceeds 10 mM compared to the case where the PMS derivative is absent, and the quantifiable substrate concentration range is further expanded.

The present invention can be used as a biosensor for measuring a substrate concentration using an oxidoreductase having NAD (P) as a coenzyme.

Claims (5)

An insulating substrate and two or more electrodes comprising at least a working electrode and a counter electrode on the substrate, and a composition containing at least all of the following (1) to (4) in contact with one or all of the electrodes A biosensor for measuring a substrate concentration of dehydrogenase.

(1) NAD or NADP
(2) Dehydrogenase using NAD and / or NADP as a coenzyme (3) Phenazine methosulfate derivative (4) Mediator The biosensor according to claim 1, wherein the substrate for dehydrogenase is glucose. The biosensor according to claim 1 or 2, wherein the mediator is a ferricyanide salt. The content of the phenazine methosulfate derivative in the composition is in a range in which an electrochemical signal sensitivity that is at least twice as high as that in the case where the phenazine methosulfate derivative is not included can be obtained. Biosensor. The biosensor according to any one of claims 1 to 3, wherein a content of the phenazine methosulfate derivative in the composition as a substance amount is 1/4000 to 1/200 with respect to the mediator substance.