KR20210027879A - Composition for detecting glucose using alginate and diagnostic kit using the same - Google Patents

Abstract

The present invention relates to a composition for detecting sugar in a sample, wherein the composition comprises glucose oxidase, peroxidase, alginate, and a color reagent. In a method for detecting sugar through color change confirmation using an existing enzymatic reaction, the composition containing alginate together with an enzyme detects even a lower concentration of glucose through a clear color change than an existing method for diagnosing diabetes using the enzyme reaction, so that the composition can be provided in a diagnostic reagent and a diagnostic kit capable of diagnosing diabetes simply and non-invasively by effectively detecting a low concentration of glucose in tears.

Description

Composition for detecting glucose using alginate and a kit for diagnosing diabetes using the same

The present invention relates to a composition for detecting sugar and a use thereof for diagnosing diabetes by measuring sugar in tears.

Diabetes is a disease in which glucose present in the blood is excreted through urine. It is one of the most common non-infectious chronic diseases in modern people and one of the chronic degenerative diseases that cannot be fundamentally cured.

In addition, diabetes is a metabolic disorder syndrome characterized by hyperglycemia caused by insulin action and secretion of insulin. It can be caused by a variety of causes.

Diabetes is a serious disease causing complications such as retina, kidney, nerve, cardiovascular system, etc. by causing hyperglycemia to persist, and not only sugar metabolism, but also lipid or protein metabolism, and a lifespan of 5-10 years due to various complications. It can be shortened, and it is currently the fourth disease among the causes of death in Korea.

Due to the recent rapid economic development and dietary changes, the prevalence of diabetes has increased year by year, reaching about 5-10% in Korea. While the number of diabetic patients is increasing rapidly, it is difficult to prevent such diabetes in advance. How to diagnose and manage consistently is important.

The most commonly used diabetes test kit is a method of self-diagnosis using blood from the tip of a finger using a blood collection needle, and this method has the disadvantage of injuring the body during each test and accompanying pain. Accordingly, there is a need to develop a diabetic diagnosis method capable of minimizing patient pain through a non-invasive method and measuring accurate glucose levels.

Republic of Korea Patent Publication No. 10-2017-0002318 (published on January 6, 2017)

An object of the present invention is to provide a composition for diagnosing diabetes by measuring the level of sugar in tears by a non-invasive method in order to solve the problems of the existing diabetic diagnosis method.

The present invention provides a composition for detecting sugar in a non-invasive biological sample comprising a glucose oxidase, peroxidase, alginate, and a color developing reagent.

The present invention comprises the steps of contacting a sample separated from an individual with a composition for detecting sugar; And it provides a method of detecting a sugar from a sample comprising the step of measuring the color change value of the composition.

The present invention comprises the steps of contacting the composition of any one of claims 1 to 5 with a sample isolated from an individual; And measuring a color change value of the composition in contact with the sample.

In addition, the present invention provides a diagnostic kit for diabetes comprising the composition for detecting the sugar.

According to the present invention, in the conventional method for detecting sugar through color change using an enzymatic reaction, a composition containing alginate together with an enzyme detects a lower concentration of glucose through a clear color change than a conventional method for diagnosing diabetes using an enzymatic reaction. As it is confirmed, the composition may be provided as a diagnostic reagent and a diagnostic kit capable of diagnosing diabetes in a simple and non-invasive manner by effectively detecting a low concentration of glucose in tears.

1 is a schematic diagram showing the main enzyme reaction for glucose measurement.
2 is a result of confirming the color change by glucose concentration twice (a and b) using a basic enzymatic reaction.
3 is a result of confirming the color change by glucose concentration twice (a and b) through the addition of alginate.
4 is a result of confirming the color change of glucose detection twice (a and b) at various alginate concentrations.
5 is a result of confirming the color change of chitosan and alginate twice (a and b) according to the glucose concentration.

Hereinafter, the present invention will be described in more detail.

Since abnormal sugar levels in the body cause diabetes, metabolic diseases, and chronic diseases, in order to provide a more convenient and non-invasive sugar level measurement method for patients with metabolic diseases who need continuous sugar level monitoring, the present inventors The present invention was completed by confirming a method for minimizing the pain of patients by measuring sugar levels in tears, which are samples containing various biomaterials including and have high accessibility.

The present invention can provide a composition for detecting sugar in a non-invasive biological sample comprising a glucose oxidase, peroxidase, alginate, and a color developing reagent.

The glucose oxidase may react with glucose under oxygen conditions in the presence of glucose in a non-invasive biological sample to produce gluconic acid and hydrogen peroxide.

The peroxidase may react with hydrogen peroxide generated by a reaction of glucose and glucose oxidase to induce a color change of a color developing reagent.

The glucose oxidase and peroxidase may be included in a 4:1 concentration ratio.

The alginate may be included in 2 to 5% (w/v), more preferably 5% (w/v), but is not limited thereto.

The non-invasive biological sample may be urine, tears, or saliva, more preferably tears, but is not limited thereto.

In the present invention, the color developing reagent is a reagent that exhibits a specific color by a chemical reaction with a sample material. TMB (3,3',5,5'-Tetramethylbenzidine), potassium iodide and 4AAP (4-Aminoantipyrine) ) And phenol or HBA (4-Hydroxybenzoic acid), but more preferably TMB (3,3',5,5'-Tetramethylbenzidine), but is not limited thereto.

The present invention comprises the steps of contacting a sample isolated from an individual with a composition under any of the above conditions; And it may provide a method of detecting a sugar from a sample comprising the step of measuring the color change value of the composition.

The sample separated from the individual may be urine, tears, or saliva, more preferably tears, but is not limited thereto.

The present invention comprises the steps of contacting a sample isolated from an individual with the composition for detecting sugar; And measuring a color change value of the composition contacted with the sample.

In addition, the present invention can provide a diagnostic kit for diabetes comprising the composition for detecting the sugar.

In the present invention, "diagnosis" refers to determining the susceptibility of an individual to a specific disease or disorder, determining whether or not an individual has a specific disease or disease, and determining the prognosis of an individual suffering from a specific disease or disorder. To do, or therametrics (eg, monitoring the condition of an individual to provide information about treatment efficacy).

In the present invention, the diagnostic kit may be paper, cotton, nitrocellulose, cellulose, cellulose acetate, polyethylene, or the like, but more preferably paper, and any material capable of absorbing liquid is not limited thereto.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

<Reference Example>

The following reference examples are intended to provide reference examples commonly applied to each of the examples according to the present invention.

1. Solution preparation

The solution used in the experiment was prepared by the following method.

① Chitosan solution: A solution was prepared by dissolving 0.5% (w/v) chitosan in 2% acetic acid solvent.

② Alginate solution: A solution was prepared by dissolving 2% (w/v) of alginate in distilled water.

③ TMB (3,3',5,5'-Tetramethylbenzidine) solution: A 15 mM TMB solution was prepared by dissolving 0.018 g of TMB in 5 ml of methanol.

④ Glucose oxidase solution: 　 6 mg of glucose oxidase was dissolved in 1 ml of pH 7.4 PBS to prepare a 120 U/ml glucose oxidase solution.

⑤ Horseradish peroxidase solution: Dissolve 1 mg of peroxidase in 1 ml of pH 7.4 PBS to make 200 U/ml, and this 200 U/ml was diluted to make 30 U/ml.

<Example 1> Paper Selection for Glucose Measurement

The basic principle for measuring sugar is a reaction between glucose and glucose oxidase as shown in FIG. 1, and a reaction between hydrogen peroxide and peroxidase, which is a product of the reaction, and a color developing reagent (Dye).

First, before observing the color change by glucose concentration using only basic enzymes, among the three candidate papers to be used in the kit development, the ability to easily absorb tears is the characteristic of the paper's fine pore size and water absorption. Check the speed.

As a result, as shown in Table 1, filter paper No. 41 of Whatman, which has a large pore size and a short time taken to absorb 100 ml of water, was selected.

Fine pore size (μm) Water absorption rate of 100ml (sec/100ml) No. One 11 No. One 150 No. 40 8 No. 40 340 No. 41 20-25 No. 41 54

<Example 2> Confirmation of the effect of detecting changes in glucose color using alginate

1. Check the color change by glucose concentration using enzyme

Using glucose oxidase and peroxidase, which are conventional glucose measurement methods, and TMB (3,3',5,5'-Tetramethylbenzidine) as a color developing reagent, check whether the color of the color developing reagent (TMB) changes according to the glucose concentration. I did.

After the Whatman's filter paper selected in the previous experiment was cut into 6 mm × 6 mm, 4.5 μl of a 15 mM TMB solution was dropped onto the cut paper into 6 mm × 6 mm and dried at room temperature for about 10 minutes.

After 10 minutes, 4.5 μl of a 30 U/ml peroxidase solution was dropped on paper and dried at room temperature for about 10 minutes. Finally, 4.5 μl of a 120 U/ml glucose oxidase solution was dropped on paper and dried at room temperature for about 10 minutes to prepare a detection pad.

Glucose solutions of 0, 0.1, 0.25, 0.375, 0.5, 0.625, 0.75 and 1 mM were dropped on the prepared detection pad by 5 μl and left for 2 minutes for color development.

After 2 minutes, the color of the center part of the reagent pad whose color changed after photographing the reagent pad showing the color development reaction was expressed as R, G, B values using the RGB color table and analyzed.

As a result, as the glucose concentration increased as shown in FIG. 2, as shown in FIG. 2, the color change clearly appeared, but it was difficult to distinguish the color difference at a low concentration of 0.1 nM.

2. Check the color change by glucose concentration added with alginate

As in the previous experiment, as a result of confirming the color change by glucose concentration using the conventional enzyme reaction, it was confirmed that the color change due to the low concentration of glucose was difficult to confirm.

To solve this problem, alginate with enzyme immobilization ability was used to determine the effect on the color change by glucose concentration.

Whatman's filter paper was cut into 6 mm×6 mm, and alginate was dissolved in distilled water at a concentration of 2% and 5% to prepare a solution, and 6 μl was dropped on the cut paper and dried at room temperature for about 15 minutes.

After that, 4.5 μl of a 15 mM TMB solution was dropped onto the cut paper and dried at room temperature for about 10 minutes, and 4.5 μl of a 30 U/ml peroxidase solution was dropped on the paper and dried at room temperature for about 10 minutes.

Finally, 4.5 μl of a 120 U/ml glucose oxidase solution was dropped on paper and dried at room temperature for 10 minutes to prepare a sugar detection pad.

5 μl of a glucose solution of 0, 0.1, 0.25, 0.375, 0.5, 0.625, 0.75 and 1 mM was dropped on the sugar detection pad, left for about 2 minutes, and then the detection pad showing color change was activated, and the RGB color table was used. The results were confirmed.

As a result, as shown in FIG. 3, the color change of the alginate-added detection pad was clearly observed compared to the conventional glucose measurement method (most of the R value difference was 10 or more), and a clear color change was also observed for low-concentration glucose.

3. Check the color change according to the alginate concentration

From the previous experimental results, alginate, which has the ability to immobilize enzymes and makes color change distinct, was selected as an auxiliary material of the sugar measurement kit.In order to confirm the optimal alginate concentration of alginate, the glucose concentration was fixed at 0.5 mM and the concentration of alginate The color change was confirmed.

Cut Whatman's filter paper into 6 mm × 6 mm, use distilled water to make alginate solutions of concentrations 1, 2, 3, 4, 5, and 6%, and add 6 μl of the alginate solution to the paper cut into 6 mm × 6 mm. Dropped and dried at room temperature for about 15 minutes.

After drying, 4.5 μl of a 15 mM TMB solution was dropped onto the cut paper and dried at room temperature for about 10 minutes, and 4.5 μl of a 30 U/ml peroxidase solution was dropped on the paper and dried at room temperature for 10 minutes.

Finally, 4.5 μl of a 120 U/ml glucose oxidase solution was dropped on paper and dried at room temperature for 10 minutes to prepare a sugar detection pad.

5 μl of a 5 mM glucose solution was dropped on the prepared detection pad, left for about 2 minutes, and then the detection pad showing a color change was activated, and the color change result was confirmed using an RGB color table.

As a result, when comparing the R values as shown in FIG. 4, the color change did not appear as the alginate concentration was changed. However, as the alginate concentration was increased, it was found that it was not easily dissolved in water at room temperature, and the viscosity increased, so that the paper did not spread and became clumped, and accordingly, a problem in that the color development was not uniform was confirmed.

As it was confirmed from the above results that alginate at a high concentration was unsuitable, it was confirmed that 2% alginate, which is well soluble in water and not high in viscosity, is most suitable.

<Example 3> Comparison of glucose detection efficiency of chitosan and alginate

In order to compare the sugar detection efficiency of chitosan and the alginate of the present invention reported in the past, color change was confirmed by using various concentrations of glucose in a sugar detection pad prepared by adding chitosan and alginate, respectively.

Chitosan solution was prepared by dissolving 0.5% (w/v) chitosan in 2% acetic acid solvent by a conventionally reported method, and the alginate solution was 6 mm × 6 after preparing the alginate solution of 2% concentration confirmed in the previous experiment. 6 μl of chitosan or alginate solution was dropped on Whatman's filter paper cut into mm and dried at room temperature for about 15 minutes.

After drying, 4.5 μl of a 15 mM TMB solution was dropped on paper and dried at room temperature for about 10 minutes, and 4.5 μl of a 30 U/ml peroxidase solution was dropped on paper and dried at room temperature for 10 minutes.

Finally, 4.5 μl of a 120 U/ml glucose oxidase solution was dropped on paper and dried at room temperature for 10 minutes to prepare a sugar detection pad.

5 μl of a 5 mM glucose solution was dropped on the prepared detection pad, left for about 2 minutes, and then the detection pad showing a color change was activated, and the color change result was confirmed using an RGB color table.

As a result, as shown in Fig. 5 and Table 2, the difference in the R value of the detection pad to which alginate was added was large in most of the glucose range. It was confirmed that alginate is more suitable for sugar detection compared to chitosan, which has been studied in the past.

As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (9)

A composition for detecting sugar in a non-invasive biological sample comprising a glucose oxidase, peroxidase, alginate, and a color developing reagent. The composition for detecting sugar according to claim 1, wherein the glucose oxidase reacts with glucose in the presence of glucose in a non-invasive biological sample to produce gluconic acid and hydrogen peroxide. The composition for detecting sugar according to claim 1, wherein the peroxidase reacts with hydrogen peroxide produced by a reaction of glucose and glucose oxidase to induce a color change of a color developing reagent. The composition for detecting sugar according to claim 1, wherein the glucose oxidase and peroxidase are contained in a 4:1 concentration ratio. The composition for detecting sugar according to claim 1, wherein the alginate is contained in an amount of 2 to 5% (w/v). The composition for detecting sugar according to claim 1, wherein the non-invasive biological sample is a tear. Contacting the sample isolated from the subject with the composition of any one of claims 1 to 5; And
A method of detecting sugar from a sample comprising the step of measuring a color change value of the composition in contact with the sample. Contacting the sample isolated from the subject with the composition of any one of claims 1 to 5; And
A method of providing information on diabetes diagnosis comprising the step of measuring a color change value of the composition in contact with the sample. Diabetes diagnostic kit comprising the composition of any one of claims 1 to 5.

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