Method for sensing target molecule, buffer solution, and sensor

Abstract

To provide a sensing method of a target molecule advantageous from the viewpoint of promoting an enzyme reaction.SOLUTION: The method for sensing the target molecules includes causing an enzyme reaction in which the target molecules are involved as a substrate S in a buffer solution 1a. The buffer solution 1a contains water, a buffering agent 11, and liquid organic compounds 12. In the buffer solution 1a, the buffering agent 11 is dissolved in water. The liquid organic compound 12 is a liquid organic compound that is miscible with water and promotes an enzyme reaction in which a hydrophobic molecule participates as a substrate.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present disclosure relates to a method for sensing a target molecule, a buffer solution, and a sensor. [Background technology]

[0002] Conventionally, for example, it is known to utilize enzyme reactions for sensing.

[0003] For example, Patent Document 1 explains that a certain host-guest metal-organic framework (MOF) system is attractive for sensing applications, and can be applied to, for example, highly sensitive bioassays and biosensors. MOFs have an inclusion structure that encapsulates biomolecules such as enzymes. According to Patent Document 1, a buffer solution is used to evaluate the physiological activity of MOFs that encapsulate a certain enzyme. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2017-522904 Summary of the Invention [Problem to be solved by the invention]

[0005] The technology described in Patent Document 1 above needs to be reconsidered from the viewpoint of promoting an enzymatic reaction in a buffer solution. Therefore, the present disclosure provides a method for sensing a target molecule that is advantageous from the viewpoint of promoting an enzymatic reaction. [Means for solving the problem]

[0006] The present disclosure provides: The enzyme reaction involves carrying out an enzymatic reaction in a buffer solution in which a target molecule that is a hydrophobic molecule serves as a substrate; The buffer solution comprises water, a buffering agent dissolved in the water, and a liquid organic compound that is miscible with the water and that promotes the enzymatic reaction. A method for sensing a target molecule is provided. [Effects of the Invention]

[0007] The target molecule sensing method of the present disclosure is advantageous in terms of promoting the enzyme reaction. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of a buffer solution according to an embodiment. [Figure 2A] FIG. 2A is a schematic diagram showing an example of the interaction between an enzyme and a hydrophobic molecule in a buffer solution. [Figure 2B] FIG. 2B is a schematic diagram showing an example of interactions between an enzyme, a liquid organic compound, and a hydrophobic molecule in a buffer solution. [Figure 3A] FIG. 3A is a schematic diagram showing the interaction of 11β-hydroxysteroid dehydrogenase (HSD) with cortisol in a buffer solution. [Figure 3B] FIG. 3B is a schematic showing the interactions between 11β-HSD, dimethyl sulfoxide, and cortisol in buffer. [Figure 4] FIG. 4 is a flowchart showing an example of a method for sensing a target molecule according to an embodiment. [Figure 5] FIG. 5 is a diagram schematically illustrating an example of a sensor for a target molecule according to an embodiment. [Figure 6] FIG. 6 is a graph showing the relationship between the rate of change in absorbance of a solution in which an enzyme reaction has occurred and the concentration of dimethyl sulfoxide (DMSO) in the buffer solution.

[0009] (Findings that formed the basis of this disclosure) Since the activity of enzymes in enzymatic reactions is affected by pH fluctuations, it is thought that if an enzymatic reaction occurs in a buffer solution, the pH of the enzyme's environment is less likely to fluctuate and the enzyme's activity is less likely to decrease. However, the reaction rate of enzymatic reactions is often slow. In particular, if the substrate in an enzymatic reaction is a hydrophobic molecule, the substrate is less likely to come into contact with the enzyme, which tends to slow the reaction rate.

[0010] In view of these circumstances, the present inventors have conducted extensive research into whether it is possible to increase the reaction rate of an enzymatic reaction when the substrate in the enzymatic reaction is a hydrophobic molecule. After extensive trial and error, the present inventors have newly discovered that the reaction rate of an enzymatic reaction can be increased by including a specific component in a buffer solution. Based on this new finding, the present inventors have completed the target molecule sensing method of the present disclosure.

[0011] (Embodiments of the present disclosure) Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. Note that the embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component placement and connection configurations, steps, and step sequences shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. Furthermore, among the components in the following embodiments, components that are not recited in the independent claims that represent the highest concepts are described as optional components. Furthermore, the drawings are not necessarily rigorous illustrations. In the drawings, substantially identical components are denoted by the same reference numerals, and redundant descriptions may be omitted or simplified. Furthermore, in this disclosure, terms indicating the relationship between elements, such as "parallel" and "perpendicular," terms indicating the shape of elements, such as "rectangle," and numerical values ​​do not only have strict meanings but also include a substantially equivalent range, for example, a difference of a few percent.

[0012] (Embodiment) Hereinafter, the embodiment will be specifically described with reference to FIGS.

[0013] FIG. 1 is a diagram schematically illustrating an example of a buffer solution according to an embodiment. As shown in FIG. 1, buffer solution 1a includes water (not shown), a buffering agent 11, and a liquid organic compound 12. In buffer solution 1a, buffer solution 11 is dissolved in water. Liquid organic compound 12 is miscible with water and promotes an enzymatic reaction involving a hydrophobic molecule as a substrate. Liquid organic compound 12 is liquid at, for example, 20°C. By including liquid organic compound 12 in buffer solution 1a, for example, when an enzymatic reaction involving a hydrophobic molecule as a substrate is caused to occur in buffer solution 1a, the reaction rate of the enzymatic reaction is likely to be higher than when liquid organic compound 12 is not included.

[0014] Figure 2A is a schematic diagram showing an example of the interaction between an enzyme and a hydrophobic molecule in a buffer solution. As shown in Figure 2A, the surface of enzyme E tends to be hydrophilic, and water molecules tend to gather around enzyme E. On the other hand, repulsive forces tend to occur between substrate S, which is a hydrophobic molecule, and enzyme E, making it difficult for substrate S to come into contact with enzyme E. As a result, an enzymatic reaction involving a hydrophobic molecule as a substrate is unlikely to occur.

[0015] 2B is a schematic diagram showing an example of interactions between an enzyme, a liquid organic compound, and a hydrophobic molecule in a buffer solution. As shown in FIG. 2B, when liquid organic compound 12 is present in addition to hydrophobic molecules such as substrate S and enzyme E, the surface of enzyme E is hydrophobized by liquid organic compound 12, which tends to generate an attractive force between enzyme E and substrate S. This facilitates contact between substrate S and enzyme E, facilitating an enzymatic reaction involving the hydrophobic molecule as a substrate.

[0016] The enzyme E that causes the above enzymatic reaction is not limited to a specific enzyme. The enzyme E is, for example, an enzyme that specifically acts on a substrate S, which is a hydrophobic molecule. The enzyme E is, for example, an oxidoreductase. In this case, an oxide or a reduction product of the substrate S is produced by the enzymatic reaction. The enzyme E may be an oxidase or a dehydrogenase.

[0017] Examples of oxidoreductases are cholesterol esterase, cholesterol oxidase, alcohol oxidase, bilirubin oxidase, and 11β-HSD. When enzyme E is cholesterol esterase or cholesterol oxidase, cholesterol is involved in the enzymatic reaction as a substrate. When enzyme E is alcohol oxidase, alcohol is involved in the enzymatic reaction as a substrate. When enzyme E is bilirubin oxidase, bilirubin is involved in the enzymatic reaction as a substrate. When enzyme E is 11β-HSD, for example, cortisol is involved in the enzymatic reaction as a substrate.

[0018] Enzyme E, for example, is coenzyme-dependent and activated by a coenzyme. Examples of coenzymes include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), pyrroloquinoline quinone (PQQ), and flavin adenine dinucleotide (FAD). The coenzyme is preferably FAD or PQQ. In this case, the coenzyme is bound to the protein that is enzyme E. Alternatively, the coenzyme may constitute a part of the protein. The coenzyme may be NAD or NADP. These coenzymes can function without being bound to the protein that is enzyme E. Enzyme E may be an FAD-dependent oxidase or a dehydrogenase that is NAD-dependent, NADP-dependent, or PQQ-dependent. Enzyme E may also be an enzyme that does not have coenzyme dependence.

[0019] The enzyme E may be present alone, may be supported on a carrier, or may be complexed with other solid materials or gels. For example, the enzyme E may be complexed with a material such as a metal-organic framework (MOF) or chitosan gel. In this case, the enzyme E may be in contact with the surface of these materials or may be disposed inside these materials.

[0020] For example, when enzyme E and MOF are conjugated, the method for preparing the complex between enzyme E and MOF is not limited to a specific method. The complex can be prepared, for example, by a coprecipitation method or a surface modification method. According to the coprecipitation method, MOF is synthesized in a solution containing enzyme E to obtain a complex. According to the surface modification method, a complex is obtained by causing chemical or physical adsorption between enzyme E and MOF. Enzyme E may be encapsulated in MOF.

[0021] The substrate S, which is a hydrophobic molecule, is not limited to a specific molecule as long as it has hydrophobicity and can be involved in the enzymatic reaction by the enzyme E. The substrate S is, for example, a molecule with a solubility of 0.28 g / L or less in water at 20°C.

[0022] The buffering agent 11 in the buffer solution 1a is not limited to a specific buffering agent. For example, the buffering agent 11 can be selected to match the optimal pH of the enzyme E. The buffering agent 11 can be, for example, a Tris-based buffer, a Bis-Tris-based buffer, a phosphate buffer, an acetate buffer, a sodium phosphate buffer, a potassium phosphate buffer, a citrate buffer, a citrate-phosphate buffer, a borate buffer, a tartrate buffer, a phosphate-buffered saline solution, or a McIlvaine buffer. For example, a Tris-based buffer can contain, in addition to trishydroxymethylaminomethane (Tris), sodium chloride, potassium chloride, hydrochloric acid, ethylenediaminetetraacetic acid (EDTA), acetic acid, or boric acid. A Bis-Tris-based buffer can contain, in addition to 2-[Bis-(2-hydroxyethyl)-amino]-2-hydroxymethyl-propane-1,3-diol (Bis-Tris), sodium chloride, potassium chloride, hydrochloric acid, EDTA, acetic acid, or boric acid.

[0023] The liquid organic compound 12 is not limited to a specific liquid organic compound, as long as it can increase the reaction rate of the enzymatic reaction involving the substrate S, which is a hydrophobic molecule, compared to when the buffer solution 1a does not contain the liquid organic compound 12. The liquid organic compound 12 is, for example, a non-electrolyte. As shown in FIG. 2B , the liquid organic compound 12 has, for example, a hydrophilic group 12a and a hydrophobic group 12b. In this case, the hydrophilic group 12a acts on the hydrophilic portion of the enzyme E, while the hydrophobic group 12b is present around the enzyme E, making the surface of the enzyme E more hydrophobic. As a result, the substrate S is more likely to come into contact with the enzyme E, and the enzymatic reaction involving the hydrophobic molecule as a substrate is more likely to occur.

[0024] The hydrophilic group 12a is not limited to a specific hydrophilic group. For example, the hydrophilic group 12a has at least one selected from the group consisting of a hydroxyl group, a sulfinyl group, and a ketone group. In this case, the hydrophilic group 12a is likely to act on the hydrophilic site of the enzyme E, and the hydrophilic group 12a is unlikely to inhibit the enzymatic reaction in which a hydrophobic molecule is involved as a substrate.

[0025] The hydrophobic group 12b is not limited to a specific hydrophobic group. The hydrophobic group 12b is, for example, a hydrocarbon group. The hydrocarbon group may be an alkyl group or an aryl group. The hydrocarbon group has, for example, 1 to 4 carbon atoms. The hydrophobic group 12b is preferably an alkyl group. In this case, the surface of the enzyme E is easily hydrophobized, and the hydrophobic group 12b is less likely to inhibit an enzymatic reaction in which a hydrophobic molecule is involved as a substrate. The hydrophobic group 12b is more preferably a methyl group or an ethyl group.

[0026] The liquid organic compound 12 has a viscosity of, for example, 6.0×10 at 20° C. -8 S / m or more 1.0×10 -3 In this case, the electrical conductivity of the liquid organic compound 12 tends to be close to that of water, and the liquid organic compound 12 is less likely to inhibit enzyme reactions involving hydrophobic molecules as substrates.

[0027] The liquid organic compound 12 preferably contains at least one selected from the group consisting of ethanol, dimethyl sulfoxide (DMSO), and acetone. In this case, the liquid organic compound 12 is easily dispersed in the buffer solution 1a, and the surface of the enzyme E is easily hydrophobized anywhere in the buffer solution 1a. In addition, the liquid organic compound 12 is unlikely to inhibit an enzymatic reaction involving a hydrophobic molecule as a substrate.

[0028] The content of liquid organic compound 12 in buffer solution 1a is not limited to a specific value. The content is adjusted so that the reaction rate of the enzymatic reaction involving a hydrophobic molecule as a substrate is higher than when buffer solution 1a does not contain liquid organic compound 12. The content of liquid organic compound 12 in buffer solution 1a is, for example, 5% by mass or more and 30% by mass or less. This content may be 10% by mass or more, 15% by mass or more, or 20% by mass or more, or may be 28% by mass or less, or 25% by mass or less.

[0029] Figure 3A is a schematic diagram showing the interaction between 11β-HSD and cortisol in a buffer solution. As shown in Figure 3A, the surface of 11β-HSD is hydrophilic, and water molecules tend to gather around 11β-HSD. On the other hand, repulsion tends to occur between cortisol, a hydrophobic molecule, and 11β-HSD, making it difficult for cortisol to come into contact with 11β-HSD. As a result, enzymatic reactions involving cortisol as a substrate are less likely to occur.

[0030] Figure 3B is a schematic diagram showing the interaction of 11β-HSD, dimethyl sulfoxide, and cortisol in a buffer solution. As shown in Figure 3B, when dimethyl sulfoxide is present in addition to the hydrophobic molecules cortisol and 11β-HSD, the surface of 11β-HSD is hydrophobized by dimethyl sulfoxide, which facilitates attraction between 11β-HSD and cortisol. This facilitates access of cortisol to 11β-HSD, facilitating enzymatic reactions involving cortisol as a substrate. For example, the sulfinyl group of dimethyl sulfoxide acts on the hydrophilic sites of 11β-HSD, while the methyl group of dimethyl sulfoxide surrounds 11β-HSD, hydrophobizing the surface of enzyme E.

[0031] The activity of enzyme E can be evaluated by quantifying the reaction rate of the enzymatic reaction involving enzyme E in buffer solution 1a. For example, the activity of enzyme E can be evaluated based on the rate of change in the concentration of substrate S or product that changes with the enzymatic reaction. The concentration of substrate S or product can be measured by optical or electrochemical measurement.

[0032] The use of buffer solution 1a is not limited to a specific application, but for example, buffer solution 1a can be used to provide a method for sensing hydrophobic molecules as target molecules.

[0033] 4 is a flowchart showing an example of a method for sensing a target molecule according to an embodiment. As shown in FIG. 4, this method includes causing an enzymatic reaction involving the target molecule as a substrate S in a buffer solution 1a.

[0034] In step S11, a sample containing a target molecule, substrate S, and enzyme E are added to buffer solution 1a. If necessary, a coenzyme is added in step S11. Next, in step S12, an enzymatic reaction involving the target molecule as substrate S is caused in buffer solution 1a. For example, in step S12, the temperature of buffer solution 1a is adjusted to a temperature suitable for the enzymatic reaction, and buffer solution 1a is stirred. Next, in step S13, an optical change or electrochemical change occurring due to the enzymatic reaction is measured. Next, in step S14, the concentration of the target molecule in the sample is determined based on the measurement results of step S13. For example, the concentration of substrate S or the concentration of the product in the solution in which the enzymatic reaction occurred is calculated from the measurement results of step S13, and the concentration of the target molecule in the sample is further calculated from this calculation result. In this way, the target molecule can be sensed using buffer solution 1a, and for example, the concentration of the target molecule in the sample can be determined. Since the enzymatic reaction is measured in buffer solution 1a using liquid organic compound 12, the target molecule can be sensed in a short period of time.

[0035] In step S13, the measurement of the optical or electrochemical change occurring during the enzymatic reaction is not limited to a specific measurement. For example, if the enzyme E is 11β-HSD and the substrate S is cortisol, NADP acts as a coenzyme on 11β-HSD to activate 11β-HSD. In this case, NADPH is produced during the enzymatic reaction. For example, an electrode is placed inside buffer solution 1a and a voltage is applied to oxidize NADPH. The NADPH produced during the enzymatic reaction is oxidized at the electrode, generating a current. The concentration of NADPH in the solution in which the enzymatic reaction occurred can be calculated by measuring this current. In this case, the oxidation potential may be changed using a mediator. Since the concentration of NADPH in the solution in which the enzymatic reaction occurred correlates with the concentration of cortisol decreased during the enzymatic reaction, cortisol can be sensed as a target molecule. Furthermore, in measuring the optical change, the absorbance of the solution in which the enzymatic reaction occurred may be measured at a wavelength corresponding to the maximum absorbance wavelength of the substance produced during the enzymatic reaction. In this case, the target molecule can be sensed based on the absorbance measurement results. In measuring the optical change, a reducing dye may be used to adjust the wavelength of the object of absorbance measurement.

[0036] FIG. 5 is a schematic diagram illustrating an example of a target molecule sensor according to an embodiment. As shown in FIG. 5, the sensor 3 includes a reaction chamber 31 and a measuring device 32. The reaction chamber 31 is a reaction chamber for causing an enzymatic reaction in a buffer solution 1a, in which a hydrophobic target molecule acts as a substrate S. The reaction chamber 31 can accommodate the buffer solution 1a. Additionally, a sample containing the substrate S and an enzyme E are supplied to the reaction chamber 31. A coenzyme may be supplied to the reaction chamber 31 as needed. The enzyme E may be immobilized in the reaction chamber 31. The measuring device 32 measures an optical change or an electrochemical change that occurs in the enzymatic reaction. This optical change or electrochemical change may occur in a solution in which the enzymatic reaction has occurred. The measuring device 32 measures, for example, a current that occurs in the enzymatic reaction or the absorbance at a predetermined wavelength of the solution in which the enzymatic reaction has occurred. The current may occur when a product produced in the enzymatic reaction is oxidized or reduced by an electrode. In this way, the sensor 3 measures optical or electrochemical changes over time before and after the enzyme reaction. The sensor 3 senses the target molecule based on the measurement results of the optical or electrochemical changes in the measuring device 32.

[0037] (Addendum) From the above description, the following techniques are disclosed. (Technology 1) The enzyme reaction involves carrying out an enzymatic reaction in a buffer solution in which a target molecule that is a hydrophobic molecule serves as a substrate; The buffer solution comprises water, a buffering agent dissolved in the water, and a liquid organic compound that is miscible with the water and that promotes the enzymatic reaction. A method for sensing target molecules. (Technology 2) The enzyme that causes the enzymatic reaction is an oxidoreductase. The method for sensing a target molecule according to technique 1. (Technology 3) The enzyme that causes the enzymatic reaction is activated by a coenzyme. A method for sensing a target molecule according to technique 1 or 2. (Technology 4) The enzyme that causes the enzymatic reaction is 11β-hydroxysteroid dehydrogenase. A method for sensing a target molecule according to any one of techniques 1 to 3. (Technology 5) The liquid organic compound has a hydrophilic group and a hydrophobic group. A method for sensing a target molecule according to any one of techniques 1 to 4. (Technology 6) The hydrophilic group has at least one selected from the group consisting of a hydroxy group, a sulfinyl group, and a ketone group. A method for sensing a target molecule according to technique 5. (Technology 7) The hydrophobic group is an alkyl group. A method for sensing a target molecule according to any one of the preceding claims. (Technology 8) The liquid organic compound includes at least one selected from the group consisting of ethanol, dimethyl sulfoxide, and acetone. A method for sensing a target molecule according to any one of techniques 1 to 7. (Technology 9) The liquid organic compound has a viscosity of 6.0×10 at 20° C. -8 S / m or more 1.0×10 -3 Electrical conductivity of S / m or less A method for sensing a target molecule according to any one of techniques 1 to 8. (Technology 10) The buffer is a buffer contained in a Tris-based buffer, a Bis-Tris-based buffer, a phosphate buffer, an acetate buffer, a sodium phosphate buffer, a potassium phosphate buffer, a citrate buffer, a citrate-phosphate buffer, a borate buffer, a tartrate buffer, a phosphate-buffered saline solution, or a McIlvaine buffer; A method for sensing a target molecule according to any one of techniques 1 to 9. (Technology 11) further comprising measuring an optical or electrochemical change that occurs in association with the enzyme reaction. A method for sensing a target molecule according to any one of techniques 1 to 9. (Technology 12) Water and a buffer dissolved in the water; a liquid organic compound that is miscible with water and promotes an enzymatic reaction involving a hydrophobic molecule as a substrate; buffer solution. (Technology 13) a reaction chamber for causing an enzymatic reaction in a buffer solution involving a target molecule that is a hydrophobic molecule as a substrate; and a measuring device for measuring an optical change or an electrochemical change that occurs in association with the enzyme reaction. Sensor. [Example]

[0038] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0039] <Example> Several buffer solutions containing trishydroxymethylaminomethane (Tris), NaCl, and dimethyl sulfoxide (DMSO) were prepared. In each buffer solution, the Tris concentration was 50 mM and the NaCl concentration was 500 mM. The DMSO concentrations in the buffer solutions were 6.4 wt%, 9.7 wt%, 14.5 wt%, and 21.7 wt%.

[0040] <Comparative Example> A buffer solution according to the comparative example was prepared in the same manner as in the example, except that DMSO was not added.

[0041] (Enzyme activity evaluation) 11β-HSD1 was dissolved in the buffer solution of each Example and Comparative Example at a concentration of 10 μM to obtain an enzyme solution of each Example and Comparative Example. NADP was dissolved in the buffer solution of each Example and Comparative Example at a concentration of 5.0 mM to obtain a coenzyme solution of each Example and Comparative Example. Cortisol was dissolved in the buffer solution of each Example and Comparative Example at a concentration of 500 μM to obtain a substrate solution.

[0042] To 700 μL of the buffer solution of each Example and Comparative Example, 100 μL of the enzyme solution of each Example and Comparative Example and 100 μL of the substrate solution of each Example and Comparative Example were added, respectively, to obtain a raw material solution of each Example and Comparative Example. Next, 100 μL of the coenzyme solution of each Example and Comparative Example was further added to each raw material solution of each Example and Comparative Example to cause an enzyme reaction. As the enzyme reaction proceeded, NADP was thought to be converted to NaDPH. The maximum absorbance wavelength of NaDPH is approximately 340 nm. The absorbance at a wavelength of 340 nm of the solution in which the enzyme reaction had occurred was measured, and the rate of absorbance change [s -1 The greater the rate of change in absorbance, the greater the reaction rate of the enzyme reaction.

[0043] Figure 6 is a graph showing the relationship between the absorbance change rate of a solution in which an enzymatic reaction occurred and the concentration of dimethyl sulfoxide (DMSO) in the buffer solution. In Figure 6, the vertical axis represents the absorbance change rate of a solution in which an enzymatic reaction occurred, and the horizontal axis represents the DMSO concentration in the buffer solution. Figure 6 shows that, within the range of 0 to 30% by mass of DMSO in the buffer solution, the higher the DMSO concentration, the faster the enzymatic reaction rate. It is believed that the presence of DMSO in the buffer solution hydrophobizes the surface of 11β-HSD1, making it easier for the hydrophobic molecule cortisol to react with 11β-HSD1. These results suggest that adding a liquid organic compound such as DMSO to the buffer solution can increase the reaction rate of an enzymatic reaction involving a hydrophobic molecule such as cortisol as a substrate. It was also suggested that the absorbance change rate of a solution in which an enzymatic reaction occurred may decrease when the DMSO concentration in the buffer solution exceeds 30% by mass. [Industrial Applicability]

[0044] The buffer solution of the present disclosure can increase the reaction rate of an enzymatic reaction involving a hydrophobic molecule as a substrate, and can therefore be used for sensing target molecules that are hydrophobic molecules. [Explanation of symbols]

[0045] 1a buffer 3 sensors 11 Buffers 12 Liquid organic compounds 12a Hydrophilic group 12b Hydrophobic group 31 Reaction chamber 32 Measuring instruments E enzyme S substrate

Claims

1. The enzyme reaction involves carrying out an enzymatic reaction in a buffer solution in which a target molecule that is a hydrophobic molecule serves as a substrate; The buffer solution comprises water, a buffering agent dissolved in the water, and a liquid organic compound that is miscible with the water and that promotes the enzymatic reaction. A method for sensing target molecules.

2. The enzyme that causes the enzymatic reaction is an oxidoreductase. The method for sensing a target molecule according to claim 1 .

3. The enzyme that causes the enzymatic reaction is activated by a coenzyme. The method for sensing a target molecule according to claim 1 .

4. The enzyme that causes the enzymatic reaction is 11β-hydroxysteroid dehydrogenase. The method for sensing a target molecule according to claim 1 .

5. The liquid organic compound has a hydrophilic group and a hydrophobic group. The method for sensing a target molecule according to claim 1 .

6. The hydrophilic group has at least one selected from the group consisting of a hydroxy group, a sulfinyl group, and a ketone group. The method for sensing a target molecule according to claim 5 .

7. The hydrophobic group is an alkyl group. The method for sensing a target molecule according to claim 5 .

8. The liquid organic compound includes at least one selected from the group consisting of ethanol, dimethyl sulfoxide, and acetone. The method for sensing a target molecule according to claim 1 .

9. The liquid organic compound has a viscosity of 6.0 × 10 at 20 °C. -8 S / m or more 1.0×10 -3 S / m or less electrical conductivity, The method for sensing a target molecule according to claim 1 .

10. The buffer is a buffer contained in a Tris-based buffer, a Bis-Tris-based buffer, a phosphate buffer, an acetate buffer, a sodium phosphate buffer, a potassium phosphate buffer, a citrate buffer, a citrate-phosphate buffer, a borate buffer, a tartrate buffer, a phosphate-buffered saline solution, or a McIlvaine buffer; The method for sensing a target molecule according to claim 1 .

11. further comprising measuring an optical or electrochemical change that occurs in association with the enzyme reaction. The method for sensing a target molecule according to claim 1 .

12. Water and a buffer dissolved in the water; a liquid organic compound that is miscible with water and promotes an enzymatic reaction involving a hydrophobic molecule as a substrate; buffer solution.

13. a reaction chamber for causing an enzymatic reaction in a buffer solution involving a target molecule that is a hydrophobic molecule as a substrate; and a measuring device for measuring an optical change or an electrochemical change that occurs in association with the enzyme reaction. Sensor.

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