Patent Grant
8871457
The disclosure relates to a method for immobilizing glucose oxidase on a self-assembled monolayer.
A biosensor is used to detect or quantify a target substance contained in a sample. Some of biosensors comprise glucose oxidase to detect or quantify glucose.
When a sample containing glucose is supplied to the biosensor comprising glucose oxidase, the glucose is decomposed into gluconolactone and hydrogen peroxide by the glucose oxidase. At least one of the gluconolactone and hydrogen peroxide is detected or quantified, to detect or quantify the glucose contained in the sample.
Patent Literature 1 discloses a prior biosensor comprising enzyme.
According to the description regarding FIG. 7 of Patent Literature 1, the biosensor is used for screening of biomoleculer activity. The biosensor comprises a monolayer 7, an affinity tag 8, an adaptor molecule 9, and a protein 10. The monolayer 7 is composed of a self-assembled monolayer represented by chemical formula: X—R—Y (see Page 24 lines 23-26, Page 25 lines 3-20, Page 25 line 27-Page 26 line 13, and Page 26 lines 14-22 of Patent Literature 1). Examples of X, R, and Y are HS—, an alkane, and a carboxyl group, respectively (see Page 25 lines 3-20, Page 25 lines 27-Page 26 line 13, and Page 28 lines 21-23 of Patent Literature 1).
In order to improve the detection sensitivity or the quantification accuracy of glucose, it is required to increase an amount of glucose oxidase to be immobilized on the biosensor.
The present inventor has discovered that the amount of the immobilized glucose oxidase per unit area was increased significantly by binding one molecule amino acid to a self-assembled monolayer and then immobilizing glucose oxidase. The invention has been provided on the basis of the discovery.
Thus, provided herein are a method for increasing an amount of glucose oxidase to be immobilized on the self-assembled monolayer, and a sensor with the glucose oxidase immobilized in accordance with the same method.
The following items (A1) to (C6) solve the above problem(s).
(A1) A method for immobilizing a glucose oxidase on a self-assembled monolayer, comprising the following steps (a) and (b) in this order:
a step (a) of preparing a substrate comprising one molecule of an amino acid and the self-assembled monolayer,
wherein the one molecule of the amino acid is bound to the self-assembled monolayer through a peptide bond represented by the following chemical formula (I):
(wherein R represents side chain of the one molecule of the amino acid)
the one molecule of the amino acid is selected from the twenty kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, tyrosine, glycine, asparagine, methionine, serine, tryptophan, leucine, glutamine, alanine, isoleucine, threonine, proline, glutamate, aspartate, arginine, and valine, and
a step (b) of supplying the glucose oxidase to the substrate to form a peptide bond represented by the following chemical formula (II) as a result of reaction between the carboxyl group of the one molecule of the amino acid and the amino group of the glucose oxidase
(wherein R represents side chain of the one molecule of the amino acid).
(A2) The method according to (A1), wherein the step (a) comprises the following steps (a1) and (a2):
a step (a1) of preparing a substrate comprising a self-assembled monolayer on the surface thereof, the self-assembled monolayer having a carboxyl acid at one end, and
a step (a2) of supplying the one molecule of the amino acid to form a peptide bond represented by the chemical formula (I) as a result of reaction between the carboxyl group of the one end of the self-assembled monolayer and the amino group of the one molecule of the amino acid.
(A3) The method according to (A1), further comprising the following step (ab) between the step (a) and the step (b):
a step (ab) of activating the carboxyl group of the one molecule of the amino acid with a mixture of N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
(A4) The method according to claim (A2), further comprising the following step (a1a) between the step (a1) and the step (a2):
a step (a1a) of activating the carboxyl group of the self-assembled monolayer with a mixture of N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
(A5) The method according to (A1), wherein the chemical formula (II) is represented by the following chemical formula (III).
(wherein R represents side chain of the one molecule of the amino acid)
(A6) The method according to (A1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, valine, argnine, and proline.
(A7) The method according to (A1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, and valine.
(A8) The method according to (A1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine.
(A9) The method according to (A1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, and alanine.
(B1) A sensor comprising a self-assembled monolayer, one molecule of an amino acid, and a glucose oxidase, wherein,
the one molecule of the amino acid is interposed between the self-assembled monolayer and the glucose oxidase,
the glucose oxidase is bound to the self-assembled monolayer through two peptide bonds represented by the following chemical formula (II),
(wherein R represents side chain of the one molecule of the amino acid)
the one molecule of the amino acid is selected from the twenty kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, tyrosine, glycine, asparagine, methionine, serine, tryptophan, leucine, glutamine, alanine, isoleucine, threonine, proline, glutamate, aspartate, argnine, and valine.
(B2) The sensor according to (B1), wherein the chemical formula (II) is represented by the following chemical formula (III).
(wherein R represents side chain of the one molecule of the amino acid)
(B3) The sensor according to (B1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, valine, argnine, and proline.
(B4) The sensor according to (B1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, and valine.
(B5) The sensor according to (B1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine.
(B6) The sensor according to (B1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, and alanine.
(C1) A method for detecting or quantifying glucose contained in a sample with a sensor, comprising the following steps (a) to (c) in this order, a step (a) of preparing the sensor comprising a self-assembled monolayer, one molecule of an amino acid, and a glucose oxidase, wherein,
the one molecule of the amino acid is interposed between the self-assembled monolayer and the glucose oxidase,
the glucose oxidase is bound to the self-assembled monolayer through two peptide bonds represented by the following chemical formula (II),
(wherein R represents side chain of the one molecule of the amino acid)
the one molecule of the amino acid is selected from the twenty kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, tyrosine, glycine, asparagine, methionine, serine, tryptophan, leucine, glutamine, alanine, isoleucine, threonine, proline, glutamate, aspartate, argnine, arginine, and valine,
a step (b) of supplying the sample to the sensor to generate gluconolactone and hydrogen peroxide due to decomposition of the glucose by the glucose oxidase,
a step (c) of detecting or quantifying at least one of the gluconolactone and the hydrogen peroxide to detect or quantify the glucose contained in the sample.
(C2) The method according to (C1), wherein the chemical formula (II) is represented by the following chemical formula (III).
(wherein R represents side chain of the one molecule of the amino acid)
(C3) The method according to (C1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, valine, argnine, and proline.
(C4) The method according to (C1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, and valine.
(C5) The method according to (C1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine.
(C6) The method according to (C1), wherein the one molecule of the amino acid is selected from the group consisting of cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, and alanine.
The exemplary embodiments achieve significant increase of the amount of the glucose oxidase to be immobilized per unit area.
An exemplary embodiment is described below with reference to
Preferably, a substrate 1 is a gold substrate. An example of the gold substrate is a substrate having gold uniformly on its surface. Specifically, the gold substrate may be a substrate having a gold film formed with a sputtering method on the surface of glass, plastic, or SiO2.
First, the substrate 1 is immersed into a solvent containing an alkanethiol. Preferably, the substrate is washed before immersed. The alkanethiol has a carboxyl group at the end thereof. It is preferable that the alkanethiol has carbon number within the range from six to eighteen. Thus, a self-assembled monolayer 2 is formed on the substrate 1.
The preferred concentration of the alkanethiol is approximately 1 mM to 10 mM. The solvent is not limited as long as it dissolves the alkanethiol. Examples of the preferred solvent are ethanol, dimethyl sulfoxide (hereinafter, referred to as “DMSO”), and dioxane. The preferred immersing period is approximately 12 to 48 hours.
Next, an amino acid 3 is supplied to the self-assembled monolayer 2. The carboxyl group (−COOH), which locates at the top end of the self-assembled monolayer 2, reacts with an amino group (—NH2) of the amino acid 3 to form a peptide bond represented by the following the chemical formula (I).
(wherein R represents side chain of the one molecule of the amino acid)
In the chemical formula (I), one molecule of the amino acid 3 binds to the self-assembled monolayer 2.
The amino acid 3 is selected from twenty kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, tyrosine, glycine, asparagine, methionine, serine, tryptophan, leucine, glutamine, alanine, isoleucine, threonine, proline, glutamate, aspartate, argnine, and valine. Namely, in the chemical formula (I), R is the side chain of these twenty kinds of amino acids.
When the amino acid 3 is supplied to the self-assembled monolayer 2, two or more kinds of amino acids may be supplied simultaneously. Namely, when a solution containing the amino acid 3 is supplied to the self-assembled monolayer 2, the solution may contain not less than two kinds of the amino acids 3. In light of uniform bind of the glucose oxidase to the amino acid 3, which is described later, it is preferred that the solution contains a sole kind of amino acid.
Subsequently, glucose oxidase 4 is supplied. The 5′-terminal amino group of the glucose oxidase 4 reacts with the carboxyl group of the amino acid 3. The amino group of the lysine contained in the glucose oxidase also reacts with the carboxyl group of the amino acid 3. Thus, two peptide bonds represented by the following chemical formula (II) are formed to obtain a sensor.
(wherein R represents side chain of the one molecule of the amino acid)
One molecule of the glucose oxidase 4 has only one 5′-terminal, whereas the one molecule of the glucose oxidase 4 has many lysine groups. Therefore, almost all of the chemical formula (II) is represented more specifically by the following chemical formula (III).
(wherein R represents side chain of the one molecule of the amino acid)
The obtained sensor is used for detecting or quantifying the glucose contained in the sample.
Specifically, glucose is detected or quantified with an electric mediator or peroxidase.
First, the detection or quantification with an electric mediator is described below.
An electric mediator (Oxidant) is added to a sample expected to contain glucose. An example of the electric mediator (Oxidant) is potassium ferricyanide. Subsequently, the sample is supplied to the sensor.
If the sample contains glucose, the glucose oxidase (GOD) converts glucose into gluconic acid, as shown in the following chemical formula (IV). Simultaneously, the electric mediator (Oxidant) is converted into the electric mediator (Reductant). In the chemical formula (IV), potassium ferricyanide is exemplified as the electric mediator (Oxidant).
As shown in the following chemical formula (V), the amount of the resultant electric mediator (Reductant) is measured electrochemically to detect or quantify the glucose.
[Fe(CN)6]−4→[Fe(CN)6]−3+e− (V)
Next, the detection or quantification with peroxidase is described below.
A sample expected to contain glucose is supplied to the sensor in the presence of oxygen and water. If the sample contains glucose, the glucose oxidase (GOD) converts glucose into gluconic acid, as shown in the following chemical formula (VI). Simultaneously, hydrogen peroxide is generated.
The generated hydrogen peroxide is mixed with a chromogen reagent containing peroxidase. An example of the chromogen is 4-aminoantipyrine, diammonium 2,2′-azinobis[3-ethyl-2,3-dihydrobenzothiazole-6-sulphonate], 3,3′,5,5′-tetramethylbenzidine, and 3,3′-diaminobenzidine. After mixing, the chromogen development occurs to detect the glucose. The degree of the chromogen development reveals the concentration of the glucose.
The following examples and a comparative example describe the technologies in more detail.
As shown in
[Preparation of a Sample Solution]
A sample solution of 16-Mercaptohexadecanoic acid with final concentration of 10 mM was prepared. The solvent thereof was ethanol.
[Formation of a Self-Assembled Monolayer]
A gold substrate (available from GE healthcare company, BR-1004-05) with gold vapor-deposited on glass was used as a substrate 1. The substrate 1 was washed for ten minutes with a piranha solution containing concentrated sulfuric acid and 30% hydrogen peroxide water. The volume ratio of the concentrated sulfuric acid to the 30% hydrogen peroxide water contained in the piranha solution was 3:1.
Subsequently, the gold substrate was immersed in the sample solution for 18 hours to form a self-assembled monolayer on the surface of the gold substrate. Finally, the substrate 1 was washed with pure water and dried.
[Immobilization of Glucose Oxidase]
Glucose oxidase was bound to the carboxyl acid group located at the top end of the 16-Mercaptohexadecanoic acid which formed the self-assembled monolayer to immobilize the glucose oxidase.
Specifically, the carboxyl acid group located at the top end of the 16-Mercaptohexadecanoic acid was activated with use of 35 microliters of a mixture of 0.1M NHS (N-Hydroxysuccinimide) and 0.4M EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). Subsequently, thirty-five microliters of glucose oxidase (40 ug/ml) was added at the flow rate of five microliters/minute. Thus, the carboxyl acid of the 16-Mercaptohexadecanoic acid as coupled with the amino group of the glucose oxidase.
Experiment was conducted similarly to the comparative example except that glycine was supplied as the one molecule of the amino acid between the formation of the self-assembled monolayer and the immobilization of the glucose oxidase. The procedure and the results are described below.
[Immobilization of Amino Acid (Glycine)]
Glycine was bound with the carboxyl group located at the top end of the 16-Mercaptohexadecanoic acid which formed the self-assembled monolayer 2 to immobilize the glycine.
Specifically, after the carboxyl group was activated similarly to the comparative example, thirty-five microliters of 0.1M glycine (pH: 8.9) was added at the flow rate of 5 microliters/minute. Thus, the carboxyl group of 16-Mercaptohexadecanoic acid was coupled with the amino group of the glycine.
[Immobilization of Glucose Oxidase]
Subsequently, glucose oxidase was bound to the carboxyl group of the glycine to immobilize glucose oxidase. Specifically, after the carboxyl group of the glycine was activated similarly to the above, thirty-five microliters of glucose oxidase (concentration: 250 micrograms/ml) was added at the flow rate of 5 microliters/minute. Thus, the carboxyl group was coupled with the 5′-terminal amino acid of the glucose oxidase or the amino group of the lysine contained in the glucose oxidase.
[Comparison of the Immobilization Amounts]
The immobilization amounts in the example 1 and in the comparative example were measured with use of an SPR device, Biacore 3000 (available from GE healthcare company).
The term “immobilization amount” means the amount of the glucose oxidase immobilized per unit area.
The ratio of the immobilization amount measured in the example 1 to the immobilization amount measured in the comparative example was approximately 30.32:1.
Threonine, methionine, isoleucine, proline, serine, glutamine, asparagine, phenylalanine, tryptophan, cysteine, histidine, alanine, lysine, leucine, glutamate, valine, aspartate, argnine, and tyrosine were used instead of glycine to measure the respective immobilization amounts similarly to the example 1. These amino acids are twenty kinds of natural amino acid. Table 1 shows the measured immobilization amounts.
A skilled person would understand the following matters from the table 1.
When the twenty kinds of amino acids was used, the immobilization amounts increase, compared to the comparative example. Furthermore, the immobilization amount changes depending on the employed amino acid.
Cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, valine, argnine, and proline are preferred, because each measured immobilization amounts are five or more in a case where one amino acid selected from these amino acids is supplied.
Cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, tyrosine, glutamate, isoleucine, threonine, aspartate, tryptophan, and valine are preferred, because each measured immobilization amounts are ten or more in a case where one amino acid selected from these amino acids is supplied.
Cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, alanine, asparagine, leucine, and tyrosine are more preferred, because each measured immobilization amounts are the average value (21.6) or more in a case where one amino acid selected from these amino acids is supplied.
Cysteine, lysine, histidine, phenylalanine, glycine, methionine, serine, and alanine, are most preferred, because each measured immobilization amounts are 1.2 times greater than the average value (21.6) in a case where one amino acid selected from these amino acids is supplied.
The technologies achieve significant increase of the amount of the glucose oxidase to be immobilized per unit area. This allows the sensitivity or the accuracy of the biosensor to be improved. The biosensor may be used for an inspection or a diagnosis which requires the detection or the quantification of the glucose contained in the living sample derived from a patient at a clinical practice.
| Number | Date | Country | Kind |
|---|---|---|---|
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This is a continuation of International Application No. PCT/JP2011/004127, with an international filing date of Jul. 21, 2011, which claims priority of Japanese Patent Application No. 2010-234314, filed on Oct. 19, 2010, the contents of which are hereby incorporated by reference.
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| Child | 13627811 | US |
