JP7466170B2 - Urine substance detection material and its use - Google Patents

Description

The present invention relates to a material for detecting substances in urine and its use.

Due to the aging population and changes in lifestyle, the cost of treating diseases is becoming a heavy burden on society. In order to improve the quality of life of patients and reduce treatment costs, it is important to predict diseases before they develop.

For example, the number of diabetes patients is rapidly increasing worldwide. Diabetes is not limited to humans, but can also develop in pet cats and dogs. To predict the onset of diabetes, it is standard practice to periodically measure blood glucose levels.

The inventors have previously developed technology that can continuously measure glucose concentrations in the body (see, for example, Patent Document 1 and Non-Patent Document 1).

JP 2018-116043 A

Shibata H., et al., Injectable hydrogel microbeads for fluorescence-based in vivo continuous glucose monitoring., 107(42), 17894-17898, 2010.

However, methods for analyzing chemical substances contained in blood, etc., to predict the onset of disease require complicated operations and are expensive. Therefore, the present invention aims to provide a technology that can predict the onset of disease inexpensively and easily.

［式（ＩＶ）中、「ＰＥＧ」は、ポリエチレングリコール鎖を表す。］
［９］前記四分岐型ポリマーが、末端に４個の求核性官能基を有する第１のポリマーと、末端に４個の求電子性官能基を有する第２のポリマーからなる、［４］～［８］のいずれかに記載の尿中物質検出材。
［１０］前記求核性官能基が、ヒドロキシル基、アミノ基及びチオール基からなる群より選択され、前記求電子性官能基が、カルボキシル基、Ｎ－ヒドロキシ－スクシンイミジル（ＮＨＳ）基、スルホスクシンイミジル基、マレイミジル基、フタルイミジル基、及びニトロフェニル基からなる群より選択される、［９］に記載の尿中物質検出材。
［１１］前記第１のポリマーが下記式（Ｉ）で表される構造を有し、前記第２のポリマーが下記式（ＩＩ）で表される構造を有する、［９］又は［１０］に記載の尿中物質検出材。

［式（Ｉ）中、各ｍは、それぞれ同一又は異なる１０～３００であり、各Ｘは、Ｒ^１－ＮＨ_２又はＲ^１－ＳＨであり、Ｒ^１はＣ_１－Ｃ_７アルキレン基である。］

［式（ＩＩ）中、各ｎは、それぞれ同一又は異なる１０～３００であり、各Ｙは、－ＣＯ－Ｒ^２－ＣＯＯ－ＮＨＳ又はＲ^２－マレイミジル基であり、Ｒ^２はＣ_１－Ｃ_７アルキレン基である。］
［１２］哺乳動物が排出した尿を［１］～［１１］のいずれかに記載の尿中物質検出材に接触させることと、尿中物質検出材に励起光を照射して蛍光を検出することと、を含み、検出した前記蛍光が対照と比較して有意に高いこと又は有意に低いことが、疾患に罹患していることを示す、疾患予測方法。
［１３］尿中物質がグルコースである、［１２］に記載の疾患予測方法。 The present invention includes the following aspects.
[1] A material for detecting a substance in urine, comprising a porous support and a fluorescent gel for detecting a substance in urine fixed in the porous support.
[2] The urinary substance detection material according to [1], wherein the porous support is a polymeric porous body or a ceramic porous body.
[3] The urinary substance detection material described in [1] or [2], wherein the urinary substance is one or more selected from the group consisting of glucose, urea, ammonia, creatine, oxygen, uric acid, amino acids, proteins, sulfuric acid, phosphorus, oxalic acid, ions, blood cells, bilirubin, urobilinogen, and ketone bodies.
[4] A urinary substance detection material described in any of [1] to [3], wherein the urinary substance is glucose, the fluorescent gel for detecting urinary substances has a three-dimensional mesh structure in which a plurality of four-branched polymers having a polyethylene glycol backbone are crosslinked at their ends, and a fluorescent dye unit that exhibits a fluorescent response upon binding to glucose is fixed in the three-dimensional mesh structure by a covalent bond.
[5] The fluorescent dye unit has a fluorophore and a quencher, and in the absence of glucose, the fluorophore is quenched by the quencher, but when the quencher binds to glucose, the fluorophore emits light, thereby showing a fluorescent response.
[6] The urinary substance detection material according to [5], wherein the fluorophore has anthracene and the quencher has arylboronic acid.
[7] A urinary substance detection material described in any one of [4] to [6], wherein the fluorescent dye unit has one or more amino groups at an end and is fixed in the three-dimensional mesh structure by a covalent bond between the amino group and the four-branched polymer.
[8] The urinary substance detection material according to any one of [4] to [7], wherein the fluorescent dye unit is represented by the following formula (III) or (IV):

[In formula (IV), "PEG" represents a polyethylene glycol chain.]
[9] A urinary substance detection material described in any of [4] to [8], wherein the four-branched polymer consists of a first polymer having four nucleophilic functional groups at its terminals and a second polymer having four electrophilic functional groups at its terminals.
[10] The urinary substance detection material described in [9], wherein the nucleophilic functional group is selected from the group consisting of a hydroxyl group, an amino group, and a thiol group, and the electrophilic functional group is selected from the group consisting of a carboxyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a maleimidyl group, a phthalimidyl group, and a nitrophenyl group.
[11] The urinary substance detection material according to [9] or [10], wherein the first polymer has a structure represented by the following formula (I) and the second polymer has a structure represented by the following formula (II):

[In formula (I), each m is the same or different and is 10 to 300, each X is R ¹ -NH ₂ or R ¹ -SH, and R ¹ is a C ₁ -C ₇ alkylene group.]

[In formula (II), each n is the same or different and is an integer from 10 to 300, each Y is -CO- ^R2 -COO-NHS or an ^R2 -maleimidyl group, and ^R2 is a _C1 - _C7 alkylene group.]
[12] A disease prediction method comprising: bringing urine excreted by a mammal into contact with a urinary substance detection material described in any one of [1] to [11]; and detecting fluorescence by irradiating the urinary substance detection material with excitation light; wherein the detected fluorescence being significantly higher or significantly lower than that of a control indicates the presence of a disease.
[13] The disease prediction method described in [12], wherein the urinary substance is glucose.

The present invention provides a technology that can predict the onset of disease inexpensively and easily.

This is a photograph of the fluorescence that occurs when a glucose solution is soaked into a urinary substance detection material. 1 is a graph showing the relationship between the concentration of an aqueous glucose solution and the fluorescence intensity of a urinary substance detection material. This shows the results of measuring blood glucose concentration over time in diabetic model rats. This shows the results of measuring urinary glucose concentration over time in diabetic model rats. This is a photograph showing the fluorescence of a urinary substance detection material that was soaked in urine from a diabetic model rat and was not allowed to dry. This is a photograph taken after the fluorescence of a urinary substance detection material that had been soaked in urine from a diabetic model rat and then dried.

[Urine substance detection material]
In one embodiment, the present invention provides a urinary substance detection material comprising a porous support and a fluorescent gel for detecting a substance in urine immobilized in the porous support. The urinary substance detection material may also be referred to as a urinary substance detection device.

The porous support of this embodiment is not particularly limited as long as it has a large number of fine pores, and examples of such support include polymeric porous bodies, ceramic porous bodies, and members with fine pores cut into them.

The pore size of the porous support in this embodiment is preferably 10 nm to 100 μm, more preferably 100 nm to 50 μm, and even more preferably 500 nm to 20 μm.

Examples of materials for the polymeric porous body include synthetic polymers such as synthetic resins, silicone resins, synthetic fibers, and synthetic rubber; semi-synthetic polymers such as cellulose derivatives and chitin derivatives; and natural polymers such as silica and natural rubber. The polymeric porous body may be made of one type of material, or two or more types of materials.

Examples of materials for the ceramic porous body include metal oxides such as aluminum oxide, magnesium oxide, and zirconium oxide; phosphates such as calcium phosphate and hydroxyapatite; and carbides such as silicon carbide. The ceramic porous body may be made of one type of material, or two or more types of materials.

The member with the micro-holes cut may be, for example, a member cut with a micro-drill, or a member in which micro-holes are formed by reacting a chemical substance with the member. The member used to manufacture the member with the micro-holes cut may be any member that can retain its shape, and examples of such members include rigid bodies, elastic bodies, and plastic bodies.

The shape of the porous support in this embodiment can be, for example, a porous membrane, a foam, a mesh, or a combination of these.

Examples of foamed porous supports include those in which air bubbles are dispersed in the above-mentioned materials, those in which air bubbles are generated from a foaming agent kneaded into the above-mentioned materials, and those in which the above-mentioned materials in which fillers are dispersed are stretched and foamed.

The mesh-like porous support may be the same as the foamed porous support, and examples thereof include a support obtained by extracting inorganic salts with water from the above-mentioned material in which inorganic salt particles have been kneaded, or a support obtained by removing the solvent from the above-mentioned material mixed with a solvent.

The porous membrane-like porous support may be, for example, a foamed porous support or a mesh-like porous support, which is thin. The porous membrane-like porous support may be a membrane that has been irradiated with a heavy ion beam or the like and then chemically ground (by track etching). The porous membrane-like porous support may be a membrane that has been chemically ground using a mask or the like having a large number of micropores to form a large number of micropore arrays on the membrane.
More specifically, examples of the porous support in the form of a porous membrane include a nitrocellulose membrane, a cellulose acetate membrane, a polyethylene membrane, or a polycarbonate membrane, a polyethylene terephthalate membrane, a nylon membrane, a hydrophilic polytetrafluoroethylene membrane, and the like, which do not have hydroxyl groups.

Once a fluorescent gel for detecting substances in urine that is not supported by a porous support dries and loses moisture, it becomes difficult for moisture to penetrate. In contrast, a fluorescent gel for detecting substances in urine that is fixed to a porous support with a pore size within the above-mentioned range will rapidly absorb moisture and swell in a short time when it comes into contact with moisture again, even if it dries and loses moisture.

Therefore, if the porous support containing the gel for detecting substances in urine is stored in a dry state and urine is dropped onto the porous support, substances in urine can be detected quickly.

Examples of substances in urine include glucose, urea, ammonia, creatine, oxygen, uric acid, amino acids, proteins, sulfuric acid, phosphorus, oxalic acid, ions, blood cells, bilirubin, urobilinogen, ketone bodies, etc. The substances in urine to be detected may be one type or two or more types.

A gel for detecting substances in urine that is fixed to a porous support maintains a more stable shape and has improved mechanical strength than a gel for detecting substances in urine that is not fixed to a porous support.

The fluorescent gel for detecting substances in urine fixed to the urine substance detection material of this embodiment can absorb liquid containing glucose. When the fluorescent gel for detecting substances in urine takes in glucose, the fluorescence intensity changes when irradiated with excitation light of a specific wavelength.

As described later in the Examples, the glucose concentration in the urine of a rat can be measured by measuring the change in fluorescence intensity using the urinary substance detection material of this embodiment. The urinary substance detection material of this embodiment can measure the glucose concentration contained in the urine of a mammal, for example.

The types of animals whose urinary substance concentrations can be measured using the urinary substance detection material of this embodiment are not particularly limited, and examples include humans, dogs, cats, rats, mice, ferrets, rabbits, monkeys, cows, goats, pigs, sheep, etc.

The polymers constituting the fluorescent gel for detecting substances in urine of this embodiment form a homogeneous three-dimensional mesh structure by crosslinking each other at the ends of the polymers. The polymer constituting the gel may have, for example, a polyethylene glycol skeleton with an ether chain branched into four. Such a gel forms a hydrogel by containing a large amount of water.

The urinary substance may be glucose, and the fluorescent dye unit that exhibits a fluorescent response upon binding to glucose may be immobilized by a covalent bond in the three-dimensional network structure of the above-mentioned polymer.

The fluorescent dye unit preferably has a fluorophore and a quencher. Here, the fluorophore is a moiety containing a molecule that emits fluorescence when excited at a specific wavelength. The quencher is a moiety containing an electron acceptor that can reduce the emission from the fluorophore by interacting with the fluorophore, and the quenching effect is eliminated by binding to a substance in urine, causing the fluorophore to emit light again.

The above-mentioned fluorescent dye unit may be one in which the fluorescence is quenched by the quencher in the absence of glucose, but the fluorophore emits fluorescence when the quencher binds to glucose. This mechanism increases the fluorescence intensity in the presence of urinary substances containing glucose.

In the above-mentioned fluorescent dye unit, the combination of the fluorophore and the quencher is not particularly limited as long as it shows a fluorescent response by binding to a substance in urine. For example, when detecting glucose in urine, the fluorophore may have anthracene and the quencher may have an arylboronic acid.

The fluorescent dye unit is preferably immobilized in the polymer by a covalent bond. The fluorescent dye unit may have one or more amino groups at its terminal, and may be immobilized in the gel structure by a covalent bond between the amino group and the four-branched polymer.

The fluorescent dye unit may be a compound represented by the following formula (III) or (IV):

（式（ＩＶ）中、「ＰＥＧ」は、ポリエチレングリコール鎖を表す。）

(In formula (IV), "PEG" represents a polyethylene glycol chain.)

In the compound represented by formula (III) above, the central anthracene is a fluorophore, and the two arylboronic acids on either side of it are quenchers.

As shown in the equilibrium equation below, in the absence of glucose, the fluorescence of anthracene is quenched by the arylboronic acid moiety. When glucose binds to the arylboronic acid moiety, the electrostatic interaction between the nitrogen and boron atoms in the molecule suppresses electron transfer to the anthracene moiety, resulting in the quenching effect of the arylboronic acid moiety being eliminated and fluorescence being emitted from anthracene. Through this mechanism, the presence of glucose can be detected as a change in fluorescence intensity.

Arylboronic acid recognizes the hydroxyl group of glucose. When the fluorescent dye unit is a compound represented by formula (III) or (IV) above, it is preferable that the porous support does not have a hydroxyl group.

The gel for detecting substances in urine may be one in which the ends of the four polyethylene glycol branches in each of the multiple polymer components are cross-linked to form a uniform mesh-structure network.

Gels consisting of a four-branched polyethylene glycol backbone are generally known as Tetra-PEG gels, and a mesh-structure network is constructed by an AB-type cross-end coupling reaction between two types of four-branched polymers, each of which has an electrophilic functional group such as an active ester structure and a nucleophilic functional group such as an amino group at its terminal.

The four-branched polymer constituting the fluorescent gel for detecting substances in urine may be composed of a first polymer having a nucleophilic functional group at its end and a second polymer having an electrophilic functional group at its end.

The ends of the first polymer and the second polymer form a meshwork network through a covalent cross-linking reaction (AB type cross-end coupling reaction), forming a gel.

The nucleophilic functional group present at the end of the first polymer is preferably a hydroxyl group, an amino group, or a thiol group, more preferably an amino group or a thiol group.

As the nucleophilic functional group present at the end of the first polymer, other nucleophilic functional groups can be used as long as they can crosslink with the second polymer to form a gel. The nucleophilic functional groups may be the same or different, but it is preferable that they are the same. By using the same functional groups, the reactivity with the nucleophilic functional group that is the subject of the crosslinking reaction becomes uniform, making it easier to obtain a high-strength gel with a uniform three-dimensional structure.

The electrophilic functional group present at the end of the second polymer is preferably a functional group selected from the group consisting of a carboxyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a maleimidyl group, a phthalimidyl group, and a nitrophenyl group. More preferably, it is an NHS group or a maleimidyl group.

The electrophilic functional group present at the end of the second polymer may be another active ester group having electrophilicity, and a person skilled in the art can appropriately use a known active ester group. The functional groups may be the same or different, but it is preferable that they are the same. By using the same functional groups, the reactivity with the nucleophilic functional group that is the target of the crosslinking reaction becomes uniform, making it easier to obtain a high-strength gel with a uniform three-dimensional structure.

Instead of the crosslinking reaction by combining the above-mentioned nucleophilic functional group and electrophilic functional group present at the end of the polymer, the so-called click reaction can be used to crosslink the polymers together. For example, the above-mentioned polymers can be crosslinked together to form a gel by using a click reaction by combining an azide group and an alkyne group. Since this click reaction is a 1,3-dipolar cycloaddition reaction, both the azide group and the alkyne group act both nucleophilically and electrophilically.

The first polymer used in the fluorescent gel for detecting substances in urine may be a four-branched polymer having a polyethylene glycol backbone represented by the following general formula (I):

［式（Ｉ）中、各ｍは、それぞれ同一又は異なる１０～３００であり、各Ｘは、Ｒ^１－ＮＨ_２又はＲ^１－ＳＨであり、Ｒ^１はＣ_１－Ｃ_７アルキレン基である。］

[In formula (I), each m is the same or different and is 10 to 300, each X is R ¹ -NH ₂ or R ¹ -SH, and R ¹ is a C ₁ -C ₇ alkylene group.]

Here, the term "C ₁ -C ₇ alkylene group" refers to an alkylene group having from 1 to 7 carbon atoms which may have branches, and refers to a linear C ₁ -C ₇ alkylene group or a C ₂ -C ₇ alkylene group having one or more branches (having from 2 to 7 carbon atoms including the branches). Examples of C ₁ -C ₇ alkylene groups are a methylene group, an ethylene group, a propylene group, and a butylene group. Examples of C ₁ -C ₇ alkylene groups include -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -CH(CH ₃ )-, -(CH ₂ ) ₃ -, -(CH(CH ₃ )) ₂ -, -(CH ₂ ) ₂ -CH(CH ₃ )-, -(CH ₂ ) ₃ -CH(CH ₃ )-, -(CH ₂ ) ₂ -CH(C ₂ H ₅ )-, -(CH ₂ ) ₆ -, -(CH ₂ ) ₂ -C(C ₂ H ₅ ) ₂ -, and -(CH ₂ ) ₃ C(CH ₃ ) ₂ CH ₂ -, and the like.

In the present specification, the alkylene group may have one or more optional substituents. Examples of the substituents include, but are not limited to, an alkoxy group, a halogen atom (which may be a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, an acyl group, or an aryl group. When the alkylene group has two or more substituents, they may be the same or different.

In the above formula (I), each m may be the same or different, but the closer the value of each m is, the more uniform the three-dimensional structure can be and the higher the strength will be. Therefore, in order to obtain a gel with high strength, it is preferable that they are the same. If the value of each m is too high, the strength of the gel will be weak, and if the value of each m is too low, it will be difficult to form a gel due to steric hindrance of the compound. In addition, from the viewpoint of making the voids in the gel a space of a size suitable for taking up glucose, as mentioned above, it is desirable that m is 10 to 300, preferably 25 to 75.

The second polymer used in the fluorescent gel for detecting substances in urine may be a four-branched polymer having a polyethylene glycol backbone represented by the following general formula (II):

［式（ＩＩ）中、各ｎは、それぞれ同一又は異なる１０～３００であり、各Ｙは、－ＣＯ－Ｒ^２－ＣＯＯ－ＮＨＳ又はＲ^２－マレイミジル基であり、Ｒ^２はＣ_１－Ｃ_７アルキレン基である。］

[In formula (II), each n is the same or different and is an integer from 10 to 300, each Y is -CO- ^R2 -COO-NHS or an ^R2 -maleimidyl group, and ^R2 is a _C1 - _C7 alkylene group.]

As with n in formula (I) above, each n in formula (II) may be the same or different, but the closer the values of n are, the more uniform the three-dimensional structure can be and the higher the strength will be. Therefore, to obtain a gel with high strength, it is preferable that they are the same. If the value of n is too high, the strength of the gel will be weak, and if the value of n is too low, it will be difficult to form a gel due to steric hindrance of the compound. In addition, from the viewpoint of making the voids in the gel a space of a size suitable for taking up glucose, as mentioned above, it is desirable that n is 10 to 300, preferably 25 to 75.

The fluorescent gel for detecting substances in urine can be prepared by a gel preparation method known in the art using the above-mentioned four-branched polymer and fluorescent dye unit. For example, a method can be used in which a mixed solution containing a four-branched polymer (first polymer) having a nucleophilic functional group at its end and a fluorescent dye unit having the same nucleophilic functional group at its end is mixed with a solution containing a four-branched polymer (second polymer) having an electrophilic functional group at its end. This causes a crosslinking reaction of the amide bonds between the four-branched polymer components, forming a hydrogel structure with a uniform mesh structure, and a gel in which the fluorescent dye unit is immobilized by a covalent bond can be produced.

In order to maintain an appropriate pH condition during the crosslinking reaction, it is preferable that the solution containing these polymer components contains an appropriate buffer solution. Examples of buffer solutions that can be used include phosphate buffer solution (PBS), citrate buffer solution, citrate-phosphate buffer solution, acetate buffer solution, borate buffer solution, tartrate buffer solution, Tris buffer solution, Tris-HCl buffer solution, phosphate buffered saline, citrate-phosphate buffered saline, potassium chloride-sodium hydroxide buffer solution, disodium hydrogen phosphate-sodium hydroxide buffer solution, sodium bicarbonate-sodium hydroxide buffer solution, boric acid-potassium chloride-sodium hydroxide buffer solution, potassium dihydrogen phosphate-sodium hydroxide buffer solution, and potassium hydrogen phthalate-sodium hydroxide buffer solution.

The mixing step of the polymer solutions can be carried out, for example, using a two-liquid mixing syringe. The temperature of the two liquids during mixing is not particularly limited, and may be any temperature at which the polymer components are dissolved and each liquid has fluidity. If the temperature is too low, the compound is difficult to dissolve, or the fluidity of the solution is reduced, making it difficult to mix uniformly. On the other hand, if the temperature is too high, it becomes difficult to control the reactivity between the four-branched polymers. Therefore, the temperature of the solution during mixing can be 1°C to 100°C, preferably 5°C to 50°C, and more preferably 10°C to 30°C. The temperatures of the two liquids may be different, but it is preferable that the temperatures are the same because the two liquids are more easily mixed.

[Disease prediction method]
In one embodiment, the present invention provides a disease prediction method comprising: contacting urine excreted by a mammal with a urinary substance detection material; and detecting fluorescence by irradiating the urinary substance detection material with excitation light, wherein the detected fluorescence being significantly higher or significantly lower than a control indicates the presence of a disease.

The substance in urine to be detected may be glucose. As described later in the Examples, the glucose concentration in urine can be measured by contacting the urine of a mammal with the urinary substance detection material of the present invention and then measuring the fluorescence intensity of the urinary substance detection material.

As described later in the Examples, in diabetic model rats, urinary glucose concentrations rise significantly before blood glucose concentrations rise to levels that are considered to be diabetes.

In other words, by using the urinary substance detection material of the present invention to measure the glucose concentration in urine, the onset of diabetes can be easily predicted.

The types of mammals for which diabetes can be predicted are not particularly limited, and examples include humans, dogs, cats, rats, mice, ferrets, rabbits, monkeys, cows, goats, pigs, sheep, etc.

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Preparation of urinary substance detection material)
A gel precursor solution A was prepared containing 6% by mass of a gel precursor (TAPEG) represented by the following formula (V) and 0.01 to 0.5% by mass of a glucose-responsive dye represented by the above formula (III). A gel precursor solution B was also prepared containing 6% by mass of a gel precursor (TNPEG) represented by the following formula (VI). Next, a solution obtained by mixing the gel precursor solution A and the gel precursor solution B was immediately dropped onto the nitrocellulose membrane. Next, the obtained nitrocellulose membrane was placed in a container and left to stand for 24 hours to prevent drying. Next, the nitrocellulose membrane after standing was washed with PBS buffer to remove unreacted substances on the nitrocellulose membrane.

[Experimental Example 2]
The fluorescence response of glucose was evaluated using the urinary substance detection material obtained in Experimental Example 1. Glucose solutions of 0 to 1000 mg/dL were added to the gel, and the fluorescence intensity at each concentration was measured (excitation wavelength: 405 nm, detection wavelength: 490 nm). The results are shown in Figures 1 and 2.

Figure 1 shows a photograph of the fluorescence obtained by irradiating excitation light onto a urinary substance detection material onto which glucose of various concentrations has been dropped. Figure 2 is a graph showing the fluorescence intensity of the urinary substance detection material onto which glucose of various concentrations has been dropped.

As a result, for all urinary substance detection materials, an increase in fluorescence intensity was observed as the glucose concentration increased. Even for repeated measurements, the fluorescence intensity was uniquely determined for each glucose concentration.

[Experimental Example 3]
The fluorescence response to glucose contained in the urine of a rat diabetic model administered with streptozocin was evaluated using the urinary substance detection material obtained in Experimental Example 1. Streptozocin was intraperitoneally administered (15 mg/kg) ten times in total every day.

Streptozocin is known to be toxic to pancreatic β cells. In rats administered streptozocin, insulin secretion from pancreatic β cells is impaired, leading to increased glucose levels in the blood and the onset of diabetes-like symptoms.

The glucose concentrations in the blood and urine of three rats with diabetes mellitus were quantified after administration of streptozocin. The results are shown in Figures 3 and 4.

Figure 3 shows the results of quantifying blood glucose concentrations in three diabetic rat models. As a result, it was confirmed that blood glucose concentrations rose to a level that was deemed diabetic 6 to 8 days after the start of streptozocin administration.

Figure 4 shows the results of quantifying the glucose concentration in the urine of the three rat diabetic models mentioned above. The results showed that the glucose concentration in the urine increased significantly 5 to 6 days after the start of streptozocin administration.

In both rats, it was found that the glucose concentration in urine rose significantly before the glucose concentration in blood rose to a level that was judged to be diabetes. In other words, diabetes can be predicted by detecting a significant increase in the glucose concentration in urine.

Furthermore, urine from the three rats mentioned above was dropped onto the urinary substance detection material of the present invention, and the fluorescence was observed. The results are shown in Figures 5 and 6. Figure 5 is a photograph of the fluorescence of the urinary substance detection material taken after urine was dropped onto it and before it dried. Figure 6 is a photograph of the fluorescence of the urinary substance detection material taken after urine was dropped onto it and it dried.

As a result, it was revealed that the urinary substance detection material of the present invention can detect an increase in the glucose concentration in the urine of rat 1 5 and 7 days after the start of streptozocin administration. In other words, it was revealed that the use of the urinary substance detection material of the present invention can predict an increase in the glucose concentration in the blood.

It has also been found that the urinary substance detection material of the present invention emits strong fluorescence even when the absorbed water content of the urine is lost due to drying. Therefore, by using the urinary substance detection material of the present invention, glucose in urine can be detected even if time has passed since contact with urine.

[Experimental Example 4]
Similarly to Experimental Example 1, a solution obtained by mixing gel precursor solution A and gel precursor solution B was dropped onto a polyethylene terephthalate film, a polycarbonate film, a nylon film, and a hydrophilic polytetrafluoroethylene film to prepare urinary substance detection materials. Using these urinary substance detection materials, an experiment similar to that of Experimental Example 2 was carried out.

As a result, it was observed that the fluorescence intensity of these urinary substance detection materials increased with increasing glucose concentration, similar to the urinary substance detection material prepared in Experimental Example 1. Even after repeated measurements, the fluorescence intensity was uniquely determined for each glucose concentration.

The present invention provides a technology that can predict the onset of disease inexpensively and easily.

Claims (10)

A porous support and a fluorescent gel for detecting a substance in urine immobilized in the porous support.
the urinary substance is glucose,
The fluorescent gel for detecting substances in urine has a three-dimensional mesh structure in which multiple four-branched polymers having a polyethylene glycol backbone are cross-linked at their ends, and fluorescent dye units that exhibit a fluorescent response when bound to glucose are fixed in the three-dimensional mesh structure by covalent bonds. The urinary substance detection material according to claim 1, wherein the porous support is a polymeric porous body or a ceramic porous body. the fluorescent dye unit comprises a fluorophore and a quencher,
The urinary substance detection material according to claim 1 or 2, wherein the fluorophore is quenched by the quencher in the absence of glucose, but exhibits a fluorescent response by causing the fluorophore to emit light when the quencher binds to glucose. The material for detecting substances in urine according to claim 3, wherein the fluorophore has anthracene and the quencher has arylboronic acid. The urinary substance detection material according to any one of claims 1 to 4, wherein the fluorescent dye unit has one or more amino groups at its terminal and is immobilized in the three-dimensional network structure by a covalent bond between the amino group and the four-branched polymer. The urinary substance detection material according to any one of claims 1 to 5, wherein the fluorescent dye unit is represented by the following formula (III) or (IV):

[In formula (IV), "PEG" represents a polyethylene glycol chain.] The urinary substance detection material according to any one of claims 1 to 6, wherein the four-branched polymer is composed of a first polymer having four nucleophilic functional groups at its terminals and a second polymer having four electrophilic functional groups at its terminals. the nucleophilic functional group is selected from the group consisting of a hydroxyl group, an amino group, and a thiol group;
The urinary substance detection material according to claim 7, wherein the electrophilic functional group is selected from the group consisting of a carboxyl group, an N-hydroxy-succinimidyl (NHS) group, a sulfosuccinimidyl group, a maleimidyl group, a phthalimidyl group, and a nitrophenyl group. The urinary substance detection material according to claim 7 or 8, wherein the first polymer has a structure represented by the following formula (I), and the second polymer has a structure represented by the following formula (II):

[In formula (I), each m is the same or different and is 10 to 300, each X is R ¹ -NH ₂ or R ¹ -SH, and R ¹ is a C ₁ -C ₇ alkylene group.]

[In formula (II), each n is the same or different and is an integer from 10 to 300, each Y is -CO- ^R2 -COO-NHS or an ^R2 -maleimidyl group, and ^R2 is a _C1 - _C7 alkylene group.] Contacting urine excreted by a mammal with the urinary substance detection material according to any one of claims 1 to 9;
Irradiating the urine substance detection material with excitation light and detecting fluorescence,
the urinary substance is glucose,
A significantly higher or lower fluorescence detected compared to a control is indicative of disease.
Methods for disease prediction.

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