阅读说明：本技术 一种纳米花及其检测葡萄糖浓度的应用 (Nanometer flower and application thereof in detecting glucose concentration ) 是由 赵肃清 杨慧怡 何绮怡 崔锡平 卢明磊 于 2019-08-31 设计创作，主要内容包括：本发明提供一种纳米花及其检测葡萄糖浓度的应用,该纳米花是由血红蛋白、葡萄糖氧化酶以及金属离子反应得到。本发明基于血红蛋白和葡萄糖氧化酶制备的纳米花,稳定性高,耐高温,耐强酸强碱,可在室温条件下储存长达一个月,制备方法简单,操作方便,绿色无污染,得到的纳米花可以直接用于葡萄糖浓度的检测,降低检测的成本。(The invention provides a nanoflower and application thereof in detecting glucose concentration. The nanoflower prepared based on hemoglobin and glucose oxidase has the advantages of high stability, high temperature resistance, strong acid and strong alkali resistance, capability of being stored for up to one month at room temperature, simple preparation method, convenience in operation, greenness and no pollution, and the obtained nanoflower can be directly used for detecting the concentration of glucose, so that the detection cost is reduced.)

1. A nanoflower for detecting glucose concentration is characterized by being obtained by reacting hemoglobin, glucose oxidase and metal ions.

2. The nanoflower of claim 1, wherein the metal ions are selected from Cu²⁺、Ga²⁺At least one of (1).

3. The nanoflower of claim 1, wherein: the metal ions are from salts containing metal ions, and the salts are selected from at least one of copper sulfate, copper chloride, calcium sulfate and calcium chloride.

4. The nanoflower of claim 1, wherein: the hemoglobin can be replaced by horse radish peroxidase.

5. The nanoflower of claim 1, wherein: the dosage ratio of the hemoglobin, the glucose oxidase and the metal ions is 1.0-1.5 mg: 0.5-1.0 mg: 2.0-3.0X 10^-3mmol。

6. The nanoflower of claim 1, wherein: the reaction is carried out in the presence of a phosphate buffer.

7. A carrier on which the nanoflower of any one of claims 1 to 6 is spread, wherein: the carrier is provided with a hydrophobic area and a hydrophilic area, the hydrophobic area isolates the hydrophilic area, and the nano flowers are laid in the hydrophilic area.

8. The carrier of claim 7, wherein: the hydrophobic matrix of the hydrophobic region is selected from paraffin;

and/or the hydrophilic regions of the carrier are in a circular hole array and are separated by the hydrophobic regions;

and/or the hydrophobic region is sequentially paved with 3,3',5,5' -tetramethyl benzidine and the nanoflower;

and/or, the carrier is selected from filter paper.

9. Use of a nanoflower according to any one of claims 1 to 6 or a vector according to any one of claims 7 to 8 for the detection of glucose concentration.

10. A method for detecting glucose concentration based on a nanoflower carrier is characterized by comprising the following steps:

1) providing hemoglobin, glucose oxidase and metal ions, mixing and reacting, and performing post-treatment to obtain nanoflowers, wherein the hemoglobin can be replaced by horseradish peroxidase;

2) providing a carrier, and sequentially paving 3,3',5,5' -tetramethylbenzidine and the nanoflower prepared in the step 1) in a hydrophilic area of the carrier to prepare the carrier paved with the 3,3',5,5' -tetramethylbenzidine and the nanoflower;

3) preparing glucose standard solutions with different concentrations, adding the carrier prepared in the step 2), and drawing a standard curve;

4) detecting the glucose solution to be detected by using the carrier prepared in the step 2) to obtain a detection result.

Technical Field

The invention relates to the technical field of medical chemistry, in particular to a nanoflower and application thereof in detecting glucose concentration.

Background

Serum Glucose (GLU), commonly referred to as blood glucose, is the most reliable indicator of blood glucose concentration for the diagnosis of diabetes and the observation of therapeutic effects. Under normal conditions, the decomposition and synthesis of serum glucose are in dynamic balance under the participation of hormones such as insulin, and the blood sugar is kept relatively stable. The concentration of normal blood glucose in the fasting blood glucose is 3.61-6.11 mM. Fasting plasma glucose concentrations above 7.0mM are termed hyperglycemic. Blood glucose concentrations below 3.61mM are termed hypoglycemia.

The existing method for detecting blood sugar is basically a glucose oxidase method, but free glucose oxidase used in the method is easily affected by high temperature, strong acid, strong alkali and the like to lose activity, so that the detection effect and accuracy of serum glucose are affected, and a solution is needed.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the existing glucose concentration determination method, and provide a nanoflower for detecting the glucose concentration, wherein the nanoflower is high in stability, simple in preparation method, convenient to use, green and pollution-free, can be directly used for detecting the glucose concentration, and reduces the detection cost.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a nanoflower for detecting glucose concentration, which is obtained by reacting hemoglobin, glucose oxidase and metal ions.

Optionally, the metal ion is selected from Cu²⁺、Ga²⁺At least one of (1).

Optionally, the metal ion is derived from a salt containing a metal ion selected from at least one of copper sulfate, copper chloride, calcium sulfate, calcium chloride.

The method uses the nanoflower for detecting the concentration of glucose, has high stability, high temperature resistance, strong acid and strong alkali resistance, can be stored for up to one month at room temperature, has simple and convenient detection process, high sensitivity and low detection limit, and can realize the rapid detection of the glucose in an actual sample.

As an alternative embodiment, the metal ion-containing salt is selected from at least one of copper sulfate, copper chloride, calcium sulfate, and calcium chloride.

As an alternative embodiment, the hemoglobin can be replaced with horseradish peroxidase.

As an alternative embodiment, the dosage ratio of the hemoglobin, the glucose oxidase and the metal ions is 1.0-1.5 mg: 0.5-1.0 mg: 2.0-3.0X 10^-3mmol。

As an alternative embodiment, the reaction is carried out in the presence of a phosphate buffer. More preferably, the reaction is to mix phosphate buffer containing hemoglobin and phosphate buffer containing glucose oxidase, add metal ions, and stand to obtain the nano flower after precipitation. Preferably the standing time is 2-5 days.

As an alternative embodiment, the ratio of the amount of glucose oxidase to the phosphate buffer solution is 0.5-1.0 mg: 3-4 ml.

The second aspect of the invention provides a carrier paved with the above nanoflower, wherein the carrier is provided with a hydrophobic area and a hydrophilic area, the hydrophobic area isolates the hydrophilic area, and the hydrophilic area is paved with the nanoflower. The carrier is used for bearing nano flowers and other substances, and facilitates subsequent glucose concentration detection. The vector can be prepared, stored and taken out in large quantities at a previous stage.

As an alternative embodiment, the hydrophobic matrix of the hydrophobic region is selected from paraffin waxes.

As an alternative embodiment, the hydrophilic regions of the carrier are in an array of circular holes, separated by hydrophobic regions.

As an alternative embodiment, the hydrophilic region is sequentially paved with 3,3',5,5' -tetramethylbenzidine and the nanoflower.

As an alternative embodiment, the support is selected from filter paper.

Of course, in the present invention, a substrate such as an microplate may be used as the carrier, but the cost is high, and the above-mentioned paper chip carrier is preferably used.

The third aspect of the invention provides the application of the nanoflower or the carrier in the detection of the glucose concentration.

The fourth aspect of the invention provides a method for detecting the glucose concentration based on a nanoflower carrier, which comprises the following steps:

1) providing hemoglobin, glucose oxidase and metal ions, mixing and reacting, performing post-treatment to obtain nanoflowers, and storing the nanoflowers in a phosphate buffer solution for later use;

2) providing a carrier, and sequentially paving 3,3',5,5' -tetramethylbenzidine and the nanoflower prepared in the step 1) in a hydrophilic area of the carrier to prepare the carrier paved with the 3,3',5,5' -tetramethylbenzidine and the nanoflower;

3) preparing glucose standard solutions with different concentrations, adding the carrier prepared in the step 2), and drawing a standard curve;

4) detecting the glucose solution to be detected by using the carrier prepared in the step 2) to obtain a detection result.

The detection method has high stability, the prepared nanoflower can keep higher enzyme activity under the condition that the pH value is 1-14, particularly when the pH value is less than or equal to 3 and the pH value is 11-14, the enzyme activity of the nanoflower is higher than that of free enzyme, the thermal stability of the nanoflower is also higher than that of the free enzyme, particularly when the temperature is 30-70 ℃ or 30-60 ℃, the enzyme activity of the nanoflower is higher than that of the free enzyme, the detection method has excellent thermal stability, high sensitivity, a detection limit, simple and convenient detection process, wide market prospect and great application value, and the glucose concentration in an actual sample can be quickly detected.

As an alternative embodiment, in the step 1), the dosage ratio of the hemoglobin, the glucose oxidase and the metal ions is 1.0-1.5 mg: 0.5-1.0 mg: 2.0-3.0X 10^-3mmol。

As an alternative embodiment, in step 1), the metal ion is selected from Cu²⁺、Ga²⁺At least one of (1).

As an alternative embodiment, in the step 1), the metal ion is derived from a salt containing a metal ion, and the salt is at least one selected from copper sulfate, copper chloride, calcium sulfate and calcium chloride.

As an alternative embodiment, in the step 1), the reaction is performed in the presence of a phosphate buffer solution.

As an alternative embodiment, in step 1), the hemoglobin can be replaced with horseradish peroxidase.

As an alternative embodiment, in the step 1), the pH of the phosphate buffer solution is 7.0 to 7.4;

as an alternative embodiment, in step 1), the reaction is carried out at 20-25 ℃, preferably at room temperature, i.e. 23 ℃ ± 2 ℃.

As an alternative embodiment, in step 1), the reaction time is 48 to 72 hours.

As an optional implementation scheme, in the step 1), after the reaction is finished, performing solid-liquid separation, and obtaining a solid, namely the nanoflower.

As an alternative embodiment, in step 1), the obtained solid is dissolved in a phosphate buffer solution for use.

As an alternative embodiment, in step 1), the pH of the phosphate buffer solution in which the resulting solid is dissolved is 7 to 7.5, preferably 7.4.

As an alternative embodiment, in step 1), the ratio of the amount of solid obtained to the phosphate buffer solution is 1 to 1.5 g: 4-5 mL.

As an alternative embodiment, in the step 2), the carrier is selected from filter paper, and specifically may be 3MM filter paper and the like.

As an alternative embodiment, in the step 2), 3',5,5' -tetramethylbenzidine is dissolved in absolute ethanol to obtain a 3,3',5,5' -tetramethylbenzidine solution, and then the solution is spread, or other solvents besides absolute ethanol may be used, so as to uniformly spread 3,3',5,5' -tetramethylbenzidine on the carrier.

As an alternative embodiment, in the step 2), the nanoflower prepared in the step 1) is diluted in a phosphate buffer in advance, and the incubation temperature is 30 to 80 ℃, preferably 30 to 70 ℃, and more preferably 30 to 60 ℃.

As an alternative embodiment, the storage time of the carrier prepared in the step 2) is more than or equal to 20 days, preferably more than or equal to 30 days, and more preferably more than or equal to 34 days.

Compared with the prior art, the invention has the following beneficial effects:

1. the nanoflower prepared based on peroxidase and glucose oxidase has the advantages of good stability, high temperature resistance, strong acid and strong alkali resistance, capability of being stored for up to one month at room temperature, simple preparation method, convenient operation, greenness and no pollution. The obtained nanoflower can be directly used for detecting the concentration of glucose, and the complexity and the cost of detection are effectively reduced.

2. The detection method provided by the invention has the advantages of high sensitivity, low detection limit, simple and convenient detection process, wide market prospect and great application value, and can realize the rapid detection of the glucose concentration in an actual sample.

Drawings

Fig. 1 shows a paper chip in an embodiment of the present invention.

FIG. 2 is a graph showing a standard curve of a paper chip for detecting glucose in an embodiment of the present invention.

FIG. 3 shows a specificity chart of a paper chip for detecting glucose in an embodiment of the present invention.

FIG. 4 is a graph showing the effect of pH of PBS buffer on enzyme activity in examples of the present invention.

FIG. 5 is a graph showing the effect of temperature on enzyme activity in examples of the present invention.

FIG. 6 is a graph showing the effect of storage time of paper chips on enzyme activity in examples of the present invention.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The invention develops a method for detecting the glucose concentration, which has simple and convenient detection process, low detection cost and strong specificity, and the method is successfully prepared by synthesizing nanoflower and paving a paper chip.

In the following examples, glucose oxidase and hemoglobin were purchased from sigma. The preparation method of the phosphate buffer solution comprises the following steps: 0.2g of sodium dihydrogen phosphate, 8.0g of sodium chloride and 2.9g of disodium hydrogen phosphate are accurately weighed, and distilled water is used for fixing the volume to 1000 ml. The phosphate buffer solution had a pH of 7.4.

The concentrations of the reagents in the following examples were those before mixing, unless otherwise specified.

In the following examples, the TMB solution is an absolute ethanol solution in which 3,3',5,5' -tetramethylbenzidine is dissolved.