JPH0659236B2 - Method for measuring biological substrate concentration and oxygen sensor used therefor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring a substrate concentration or an enzyme reaction rate in a living body and an enzyme sensor used for the method, and in particular, an illuminant (metal complex) is added to an enzyme system. The present invention relates to a method for measuring the substrate concentration of a living body by coexisting and measuring the luminescence intensity by utilizing the fact that the luminescence of the luminescent material is quenched by an enzyme that changes by an enzymatic reaction, and an enzyme sensor used therefor.

(Prior Art) Conventionally, when measuring a substrate concentration or an enzyme reaction rate, for example, the following reaction is performed.

Therefore, the glucose concentration can be measured by the generation amount of H ₂ O ₂ and gluconolactone and the amount of enzyme consumption, and these measurements are performed with electrodes such as H ₂ O ₂ electrode, pH electrode, and enzyme electrode.

However, in these methods, (1) the pH change by gluconolactone is small.

(2) Oxygen consumption is limited to measuring only oxygen molecules reaching the oxygen electrode, and it is troublesome to measure the consumption. In addition, since it is necessary to pass a small amount of current through the electrodes, it is not preferable for biological system measurement.

(3) The amount of hydrogen peroxide (H ₂ O ₂ ) is H
_Since only ₂ O ₂ is measured, there is a drawback that the H ₂ O ₂ concentration on the interface is rate-determining.

(Problems to be Solved by the Invention) The present invention has completed the present invention as a result of various studies to solve the above-mentioned problems of the prior art, and an object of the present invention is to reduce oxygen consumption during enzyme reaction. Utilizing the fact that the luminescence from the excited state of the enzyme-based coexisting luminescent material is quenched by oxygen without being disturbed by the surrounding interfering substances, and the substrate concentration or the biological substrate concentration is measured by measuring the luminescence intensity at that time. And an enzyme sensor used therefor.

(Means for Solving Problems) That is, according to the present invention, in a solution containing a biological substrate, the biological substrate and dissolved oxygen are reacted under an enzyme, and light in the ultraviolet region or visible light region is immersed in the solution. The method for measuring a biological substrate concentration, which comprises irradiating the luminescent substance, and measuring the biological substrate concentration by the intensity of light emitted from the luminescent substance according to the dissolved oxygen concentration consumed and reduced by the reaction. Further, on the substrate, an enzyme that consumes dissolved oxygen in a solution containing a biological substrate, and a luminescent material that emits light by excitation light in the ultraviolet region or visible light region and that emission is quenched by oxygen are immobilized. It is an enzyme sensor characterized by the following.

That is, in other words, in the present invention, a luminescent material (metal complex) is allowed to coexist in the enzyme system, and the oxygen consumption due to the enzymatic reaction is determined by the change in luminescence intensity due to the reaction between the excited state of the luminescent material and oxygen. It is a method for measuring the concentration of a biological substrate, which is obtained by measuring the substrate concentration or the enzyme reaction rate, and the enzyme sensor used at that time. In this case, the enzyme as the enzyme reaction system is an enzyme reaction substance that consumes oxygen by directly or indirectly reacting with oxygen. Examples of such a reaction system substrate include glucose, uric acid, cholesterol, assimilating sugar, other sugars (sucrose, maltose, galactose), phosphatidylcholine, lactic acid, nicotinamide adenine dinucleotide (NADP), oxalic acid, amino acids, Examples thereof include alcohols, monoamines, ascorbic acid and pyruvic acid.

As conventional methods described above, (1) the generation amount of generated H ₂ O ₂ and gluconolactone enzymatic reactions such as the example shown in the expression, in each electrode method and the glucose concentration than such oxygen consumption (H ₂ O ₂ electrode, pH electrode, oxygen electrode, etc.), in the present invention, a luminescent material (metal complex) is allowed to coexist in the enzyme system, and the oxygen consumption amount due to the enzymatic reaction is compared between the excited state of the luminescent material and oxygen. The oxygen concentration is determined by the change in luminescence intensity due to the reaction, and the coexisting luminescent material (metal complex) is used without disturbing the surrounding chemical substances in the chemical reaction of the enzyme (oxygen consumption directly or indirectly involved in the enzymatic reaction). It is possible to calculate the oxygen concentration at that time, that is, the substrate concentration or the enzymatic reaction rate, by measuring the oxygen concentration by the emission intensity by utilizing the chemical reaction between the excited state and the oxygen.

Next, the illuminant and the enzyme sensor in the present invention will be described.

Luminescent substance: A luminescent substance refers to a compound that emits a photoexcited state or fluorescence or phosphorescence to return to the original ground state. In the present invention, utilizing the fact that the emission intensity decreases by reacting with oxygen in the photoexcited state, the principle is to determine the oxygen concentration by measuring the emission intensity, so that light emission from the photoexcited state In addition, any luminescent material can be used as long as its luminous intensity is reduced by the coexistence of oxygen. Examples of the luminescent material that can be used in the present invention include a metal complex having a ligand of 2.2'-bipyridine or a derivative thereof, such as tris (2.
Examples thereof include 2'-bipyridine) ruthenium complex and other complexes such as iridium, and organic compounds such as tris (o-phenanthroline) ruthenium complex pyrene and its derivatives (pyrenesulfonic acid, pyrene butyric acid, etc.).

Enzyme sensor system: An enzyme or a group of enzymes, a substrate and a luminescent material may coexist in a homogeneous solution. Alternatively, a membrane obtained by immobilizing these in a synthetic or natural polymer membrane can be used. Synthetic polymer membranes that can be used include acrylic acid, methacrylic acid, hydroxyethyl methacrylate, styrene, N-
Examples include homo- or copolymers such as vinylpyrrolidone. The natural polymer film may be a film of silk, cellulose, gelatin or the like. In order to obtain the enzyme sensor membrane used in the present invention, the enzyme and the luminescent material are physically or chemically fixed in these polymer membranes. As a method of physically fixing as a fixing means, it is simply adsorbed on a membrane, or a film is formed from a solution of a membrane component containing a substance to be adsorbed (enzyme, luminescent material, etc.) by a casting method or the like. A film is formed by polymerizing a mixture of monomers with light rays or the like. As a method of chemically fixing, an enzyme or a luminescent substance is introduced into a polymer compound by a covalent bond in advance and then a film is formed, or after the film is formed, an enzyme or a luminescent substance is introduced and bonded by a chemical reaction. . An example of chemically fixing a light-emitting substance to a polymer compound by a covalent bond is the following polymer having a tris (2.2'-bipyridine) ruthenium complex (abbreviated as Ru (bpy) ₃ ²⁺ ) structure as a pendant group. Can be mentioned.

However, X ⁻ is an anion such as Cl ⁻ , ClO ₄ ⁻ , M is 4-methyl-
Monomer units such as 4'-vinyl-2.2'-bipyridine or acrylic acid, methacrylic acid, hydroxyethyl methacrylate, styrene, N-vinylpyrrolidone, X, Y, Z are the molar fractions of each repeating unit X, Y Is selected from the range of 0 to 0.99, and Z is selected from the range of 0.01 to 1. This polymer pendant type luminescent material can be used alone or together with other polymer compounds to form a film, and an enzyme or a group of enzymes can coexist in the film to be used as an enzyme sensor. Since this polymeric light-emitting body is difficult to elute from the membrane, it can be used as a stable enzyme sensor.

Of these membrane materials, silk membrane is compatible with biological components, so
It is extremely suitable for use as a membrane for an enzyme sensor that is directly inserted into a biological system.

Next, the present invention will be described in detail with reference to examples.

Example 1 0.1 M glucose, 50 μM (micromol), tris (2.2'-bipyridine) ruthenium (II) complex chloride in phosphate buffered aqueous solution (pH 7) in a fully transparent quartz cell with a stopper (optical path length 1 cm). .0) Added 3cc.

The cell was set in a spectrofluorometer (FP55OA manufactured by JASCO Corporation), and the emission intensity Io was measured at an excitation light wavelength of 460 nm and an emission side spectroscope wavelength of 605 nm.

Thus, the emission side spectroscope wavelength 605 nm is kept constant, and glucose oxidase 1 mg / ml is placed in the cell.
An aqueous solution having a concentration of 1 to 5 μM is added. Oxygen molecules are consumed by the enzymatic reaction (enzymatic oxidation reaction of glucose) at this time, and the luminescence intensity increases. Therefore, the change over time of the luminescence intensity is measured.

In a system in which glucose and glucose oxidase coexist, oxygen is consumed by the oxidation of glucose and the emission intensity increases. The relationship between oxygen concentration and emission intensity is shown in FIG.
From FIG. 1, the oxygen concentration at any time can be known, and therefore the enzyme reaction rate can also be known. Under these conditions, glucose oxidase 1 μg
The increase in the luminescence intensity per minute per minute is 1.04 times that before the reaction, which corresponds to a change in oxygen concentration of 1.04 mM / min / μg glucose oxidase.

By using a system in which the substrate (here glucose) concentration is present in excess of the oxygen concentration, the activity and reaction rate of the enzyme (here glucose oxidase) can be determined, and conversely, the substrate concentration is sufficiently higher than the oxygen concentration. Under small conditions, it is possible to measure the substrate concentration by measuring the emission intensity.

As described above, first, in a Ru (bpy) ₃ ²⁺ aqueous solution,
When the relative intensity of light emission (605 nm) from the excited state of the Ru complex is plotted against the oxygen concentration in the solution, a straight line as shown in FIG. 1 is obtained, and the oxygen concentration can be obtained from the relative intensity of the light emission from now on.

Example 2 Glucose oxidase was added to a 4% aqueous solution of silk fibroin in an amount of 0.2% relative to silk, and Ru (bpy) ₃ ²⁺ was added at a concentration of 0.1 mM, cast on an acrylic plate and air-dried to a thickness. A silk film of about 1 μm was obtained. First, in order to create a calibration curve, this silk film was immersed in an aqueous solution containing a known concentration of oxygen, and the relationship between oxygen concentration and relative luminescence intensity was determined to obtain the relationship shown in FIG.

An enzyme sensor with such characteristics can
It was immersed in an aqueous solution containing M and saturated with air, excited by 450 nm light, and the emission at 605 nm was measured. The amount of change in oxygen concentration was determined from the amount of decrease in the emission intensity, and therefore the glucose concentration in the system could be quantified.

Example 3 A biosensor capable of measuring glucose concentration was prepared in the same manner as in Example 2 except that 0.2% of invertase was used for silk in addition to glucose oxidase in Example 2. By dipping this enzyme sensor in an aqueous solution containing glucose in an amount of 0.05 to 1 mM and saturated with air, the glucose concentration could be determined from the relationship between the decrease in luminescence intensity and FIG.

Example 4 In Example 2, instead of Ru (bpy) ₃ ²⁺ , in Structural Formula 1, M is acrylic acid, X is Cl ⁻ , X = 0.924, Y = 0.054, Z.
= 0.022 polymer pendant type Ru (bpy) ₃ ²⁺ was added to the silk membrane aqueous solution so that the Ru complex simple substance concentration would be 0.1 mM. Otherwise, a glucose sensor was prepared in the same manner as in Example 2, and its calibration curve was obtained. The same result as in FIG. 2 was obtained. With this glucose sensor, the glucose concentration in the aqueous solution could be measured by the luminescence method.

Example 5 A glucose sensor was produced in the same manner as in Example 2 except that pyrenebutyric acid was used instead of Ru (bpy) ₃ ²⁺ in Example 2. The sensor was dipped in water containing a known concentration of enzyme, excited with 338 nm light, and the relative intensity of 380 nm emission was measured. The relationship shown in FIG. 3 was obtained.

Using this relationship, the glucose concentration in an aqueous solution containing unknown glucose could be quantified. In addition, a microenzyme sensor having a diameter of several μm can be obtained by immobilizing an enzyme and a luminescent material using the tip of an optical fiber such as PMMA or quartz as a substrate. In this example, glucose was used as an example of the biological substrate, but the present invention is not limited to this, and the same applies to the other biological substrates described above. Further, the silk film is not limited to the examples, but a silkworm regenerated silk fibroin film and a silkworm regenerated silk fibron film can be used.

(Effect) As described above, the present invention utilizes the fact that the visible light from the excited state of the coexisting luminescent material is quenched by oxygen by allowing the luminescent material to be present together with oxygen in the enzyme reaction system, and measuring the luminescence intensity at that time. An enzyme sensor for measuring the substrate concentration or the enzyme reaction rate by the method and a method for measuring a biological substrate concentration using the enzyme sensor. Therefore, there is no need to pass an electric current, it is extremely safe for measurement of biological systems, and highly accurate measurement can be performed without being affected by other biological substrates in solution or other interfering ions. It has the effect of.

[Brief description of drawings]

FIG. 1 is a relationship diagram of relative emission intensity with respect to oxygen concentration in Example 1 of the present invention, FIG. 2 is a relationship diagram of relative emission intensity with respect to oxygen concentration of Example 2 of the present invention, and FIG. It is a related figure of relative luminescence intensity to oxygen concentration in a case.

Claims (10)

[Claims] 1. In a solution containing a biological substrate, the biological substrate and the dissolved substrate are reacted under an enzyme, and light in the ultraviolet region or visible light region is irradiated to the luminescent material immersed in the solution, and the reaction is carried out. A method for measuring a biological substrate concentration, which comprises measuring the biological substrate concentration by the intensity of light emitted from the luminescent material according to the dissolved oxygen concentration which is consumed and reduced. 2. The method for measuring the concentration of a biological substrate according to claim 1, wherein the luminescent material emits light by excitation light in the ultraviolet region and the visible light region and the emitted light is quenched by oxygen. 3. The method for measuring the concentration of a biological substrate according to claim 1 or 2, wherein the luminescent material is an organometallic complex compound of a transition metal. 4. The organometallic complex compound, wherein the metal is ruthenium,
The method for measuring the concentration of a biological substrate according to claim 3, wherein the ligand is 2.2'-bipyridine, o-phenanthroline or a derivative thereof. 5. The method for measuring the concentration of a biological substrate according to any one of claims 2 to 4, wherein the luminescent material is a polymer compound containing an organometallic complex compound of a transition metal as one component. 6. An enzyme which consumes dissolved oxygen in a solution containing a biological substrate and a luminescent material which emits light by excitation light in the ultraviolet region or visible light region and whose emission is quenched by oxygen are immobilized on a substrate. An enzyme sensor characterized by the fact that 7. The enzyme sensor according to claim 6, wherein the luminescent material is a transition metal organometallic complex compound. 8. The metal of the organometallic complex compound is ruthenium,
The enzyme sensor according to claim 7, wherein the ligand is 2.2'-bipyridine, o-phenanthroline or a derivative thereof. 9. The enzyme sensor according to any one of claims 6 to 8, wherein the light-emitting body is a polymer compound containing an organometallic complex compound of a transition metal as one component. 10. The enzyme sensor according to any one of claims 6 to 9, wherein the substance for immobilizing the enzyme and the luminescent material is a silk film.