阅读说明：本技术 一种尿酸生物传感器的检测配方 (Detection formula of uric acid biosensor ) 是由 梁明龙 伍寿胜 刘勤 李中 于 2020-06-18 设计创作，主要内容包括：本发明公开了尿酸生物传感器的检测配方,所述配方包括磷酸盐、羧甲基纤维素、海藻糖、铁氰化钾、甘油、聚乙二醇200和非反应活性组分；在该配方中,磷酸的浓度为0.01%～10%W/V；羧甲基纤维素的浓度为0.01%～10%W/V；海藻糖的浓度为0.01%～10%W/V；铁氰化钾的浓度为0.01%～20%W/V；甘油的浓度为0.5%～10%W/V；聚乙二醇200的浓度为1%～8%W/V；非反应活性组分的浓度为0.1%～70%W/V。本检测配方可以使尿酸生物传感器反应层变得疏松,从而使样品中的尿酸能够更好地渗透进入反应层,增加反应速度,从而提高检测的灵敏度,检测结果的线性也得到了显著提高。(The invention discloses a detection formula of a uric acid biosensor, which comprises phosphate, carboxymethyl cellulose, trehalose, potassium ferricyanide, glycerol, polyethylene glycol 200 and non-reactive active components; in the formula, the concentration of the phosphoric acid is 0.01-10% W/V; the concentration of the carboxymethyl cellulose is 0.01 to 10 percent W/V; the concentration of the trehalose is 0.01-10% W/V; the concentration of the potassium ferricyanide is 0.01-20% W/V; the concentration of the glycerol is 0.5 to 10 percent W/V; the concentration of the polyethylene glycol 200 is 1-8% W/V; the concentration of the non-reactive component is 0.1-70% W/V. The detection formula can loosen the reaction layer of the uric acid biosensor, so that uric acid in a sample can better permeate into the reaction layer, the reaction speed is increased, the detection sensitivity is improved, and the linearity of a detection result is obviously improved.)

1. The detection formula of the uric acid biosensor is characterized by comprising phosphate, carboxymethyl cellulose, trehalose, potassium ferricyanide, glycerol, polyethylene glycol 200 and non-reactive active components;

in the formula, the concentration of the phosphoric acid is 0.01-10% W/V;

the concentration of the carboxymethyl cellulose is 0.01 to 10 percent W/V;

the concentration of the trehalose is 0.01-10% W/V;

the concentration of the potassium ferricyanide is 0.01-20% W/V;

the concentration of the glycerol is 0.5 to 10 percent W/V;

the concentration of the polyethylene glycol 200 is 1-8% W/V;

the concentration of the non-reactive component is 0.1-70% W/V.

2. The detection formula of uric acid biosensor according to claim 1, characterized in that the concentration of phosphate is 0.1% -5% W/V.

3. The detection formula of uric acid biosensor according to claim 1, wherein the concentration of carboxymethyl cellulose is 0.1% -5% W/V.

4. The detection formula of uric acid biosensor according to claim 1, wherein the concentration of trehalose is 0.1% -5% W/V.

5. The detection formula of uric acid biosensor according to claim 1, wherein the concentration of potassium ferricyanide is 0.1% -15% W/V.

6. The detection formula of uric acid biosensor according to claim 1, wherein the concentration of glycerol is 1% -5% W/V.

7. The detection formula of uric acid biosensor according to claim 1, characterized in that the pH of the detection formula is in the range of 5-8, and the pH is adjusted by adding a buffer.

8. The detection formulation of uric acid biosensor according to claim 7, characterized in that the buffer is phosphate buffer.

9. The detection formulation of uric acid biosensor according to claim 7 or 8, wherein the concentration of the buffer solution is 1% -3% W/V.

Technical Field

The invention relates to the technical field of biosensors, in particular to a detection formula of a uric acid biosensor.

Background

Uric acid is the end product of purine metabolism in the human body and has no physiological function in the human body. The main sources of purines in the human body are dietary, nucleoprotein decomposition and de novo synthesis. Normally, it is excreted mainly from the kidney, and the rest is excreted from the body through the intestines, skin, hair, etc. Due to the fact that modern people have tense and irregular lives and three meals are abnormal and lack of sufficient rest and proper exercise, people can easily suffer from gout caused by over-fatigue, overeating or drinking. Gout attacks may be hyperuricemic, and therefore, accurate detection of uric acid levels is essential to prevent gout attacks.

At present, methods for detecting uric acid in blood and urine mainly comprise a high performance liquid chromatography method, an electrochemical method, a photochemical colorimetric method and a chemiluminescence method. Among these methods, the direct electrochemical method has advantages of rapidness, simplicity, high sensitivity, etc., and is mainly based on detecting an electroactive substance for biological recognition or chemical reaction, a driving force is provided for an electroactive electron transfer reaction by fixing the potential of a working electrode, the change of current with time is detected, the current directly measures the electron transfer speed, reflects the speed of biomolecule recognition, and is proportional to the concentration of the detected substance.

The uric acid biosensor is an electric current type electrochemical biosensor for analyzing the concentration of uric acid by using an electronic mediator and a reaction system. The biosensor comprises: the detection device comprises an electric insulating substrate, a sample guide groove, a working electrode, a reference electrode and a reaction area, wherein the reaction area is coated with a reaction layer, and human body fluid can be guided into the reaction layer by utilizing the siphonage of the sample guide groove to act with a mediator and the like placed in the reaction layer, so that the detection of an object to be detected is carried out. The uric acid biosensor has high sensitivity and wide linear range, so that the uric acid concentration in biological liquid such as blood and urine can be rapidly detected. The high sensitivity of the device enables the uric acid analysis to apply lower working voltage, and avoids the wrong analysis result caused by some interfering substances.

The uric acid content in human blood and urine is very low, so that the uric acid biosensor in the prior art still has low test sensitivity, so that the test reproducibility is poor, and the detection result is not accurate enough.

Disclosure of Invention

In view of the above, the invention provides a detection formula of a uric acid biosensor aiming at the defects of the prior art, the preparation of the reagent of the formula is simple, and the reagent prepared by the formula is coated on the reaction layer of the uric acid biosensor, so that the sensitivity of testing low-concentration uric acid in blood and urine can be improved.

The technical scheme for realizing the purpose of the invention is as follows:

a detection formula of a uric acid biosensor comprises phosphate, carboxymethyl cellulose, trehalose, potassium ferricyanide, glycerol, polyethylene glycol 200 and non-reactive components;

in the formula, the concentration of the phosphate is 0.01-10% W/V (mass concentration ratio);

the concentration of the carboxymethyl cellulose is 0.01 to 10 percent W/V;

the concentration of the trehalose is 0.01-10% W/V;

the concentration of the potassium ferricyanide is 0.01-20% W/V;

the concentration of the glycerol is 0.5 to 10 percent W/V;

the concentration of the polyethylene glycol 200 is 1-8% W/V;

the concentration of the non-reactive component is 0.1-70% W/V.

Preferably, the concentration of phosphate is 0.1% to 5% W/V.

Preferably, the concentration of carboxymethyl cellulose is 0.1% to 5% W/V.

Preferably, the trehalose concentration is 0.1% to 5% W/V.

Preferably, the concentration of potassium ferricyanide is 0.1% to 15% W/V.

Preferably, the concentration of glycerol is 1% to 5% W/V.

Further, the pH range of the detection formula is 5-8, and the pH is adjusted by adding a buffer solution.

Preferably, the buffer is a phosphate buffer.

Further, the concentration of the buffer solution is 1-3% W/V.

The glycerol and the polyethylene glycol 200 have good biocompatibility, are nontoxic, can be adsorbed or retained on the surface of the electrode, improve the looseness of the reaction layer, increase the permeability of the detected substance, enable the detected substance to rapidly react with the reaction layer, improve the corresponding current density and the like, increase the reaction sensitivity, and improve the linearity of the detection result.

Compared with the prior art, the detection formula in the uric acid biosensor provided by the invention has the following advantages and remarkable progress:

the glycerin and the polyethylene glycol 200 are added into the formula of the biosensor, so that the reaction layer of the uric acid biosensor becomes loose, uric acid in a sample can better permeate into the reaction layer, the reaction speed is increased, the detection sensitivity is improved, and the linearity of a detection result is also obviously improved.

Drawings

FIG. 1 is a linear relationship graph of uric acid concentration and current in the examples.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiments of the present invention is described below in a clear and complete manner, and the described embodiments are only a part of the embodiments of the present invention, and do not represent all the embodiments. Other embodiments, which can be obtained by one skilled in the art without inventive step based on the embodiments of the present invention, shall fall within the scope of the present invention.

The invention provides a detection formula of a uric acid biosensor, which comprises phosphate, carboxymethyl cellulose, trehalose, potassium ferricyanide, glycerol, polyethylene glycol 200 and non-reactive active components;

in the formula, the concentration of phosphate is 0.01-10% W/V;

the concentration of the carboxymethyl cellulose is 0.01 to 10 percent W/V;

the concentration of the trehalose is 0.01-10% W/V;

the concentration of the potassium ferricyanide is 0.01-20% W/V;

the concentration of the glycerol is 0.5-10% W/V;

the concentration of the polyethylene glycol 200 is 1-8% W/V;

the concentration of the non-reactive component is 0.1-70% W/V.

The concentration of any one of the above-mentioned components is not the concentration of the solution of the component itself, but the concentration in the formulation after mixing with other components to constitute the formulation, or it is understood that the formulation includes a solvent and each component is dissolved in the solvent.

Preferably, in practice, when phosphate is used, it may be sodium phosphate or other salts such as potassium phosphate. In some embodiments, the phosphate concentration ranges from 1% to 3% W/V.

In some embodiments, the preferred concentration of carboxymethyl cellulose is 0.1% to 5% W/V.

In some embodiments, the preferred concentration of trehalose is 0.1% to 5% W/V.

In some embodiments, the preferred concentration of potassium ferricyanide is 0.1% to 15% W/V.

In some embodiments, the preferred concentration of glycerol is 1% to 5% W/V.

The pH value of the formula is preferably within a range of 5-8, so that the optimal effect of phosphoric acid and phosphate is ensured, and the best inhibition effect on reduction of potassium ferricyanide is achieved. The method can be realized by adding a buffer solution, the pH buffer solution can be any substance capable of adjusting the pH of the test solution to be in a range of 5-8, and a phosphate buffer solution is preferred.

Non-reactive components include, but are not limited to, sodium carboxymethylcellulose, hydroxyethyl fiber, bovine serum albumin, gelatin, gum arabic, and the like, and in some embodiments, the concentration of non-reactive components ranges from 20% to 50%.

Description of the experiment:

a. example 1: the formula of adding 4% W/V polyethylene glycol 200 substance is used as an improved formula, the rest components are composed of buffer solution, carboxymethyl cellulose, trehalose, potassium ferricyanide and non-reactive active components, the formula is used as a reaction formula, and the reaction formula is printed on an electrode plate to manufacture the improved biosensor.

b. Example 2: the formula with 3% W/V glycerol as improved formula, the rest components including buffer solution, carboxymethyl cellulose, trehalose, potassium ferricyanide and non-reactive active components is printed on the electrode plate to produce the improved biosensor.

c. Example 3: the formula of adding two substances of 5% W/V polyethylene glycol 200 and 2% W/V glycerin is used as an improved formula, the rest components consist of buffer solution, carboxymethyl cellulose, trehalose, potassium ferricyanide and non-reactive active components, and the formula is used as a reaction formula and is printed on an electrode plate to manufacture the improved biosensor.

The order of addition of the polyethylene glycol 200 and glycerol is not limited herein.

d. Comparative example 1: the formula without two substances of polyethylene glycol 200 and glycerol is used as a reference formula, the buffer solution is used for replacing the formula, and the rest components of carboxymethyl cellulose, trehalose, potassium ferricyanide and non-reactive active components are used as the reaction formula, and the formula is printed on an electrode plate to manufacture the reference biosensor.

This example illustrates the linear relationship between uric acid concentration and current, and the calibration curve method constructed using the uric acid biosensor prepared in example 1, example 2, and example 3.

Collecting fresh venous blood and urine, adjusting the hematocrit ratio to 40%, and preparing blood samples with uric acid concentrations of 180 [ mu ] mol/L, 375 [ mu ] mol/L, 500 [ mu ] mol/L, 650 [ mu ] mol/L and 900 [ mu ] mol/L respectively. Placing the uric acid biosensor prepared in example 1, example 2 and example 3 in a matched tester, and applying a 300mV working voltage to two ends of a working electrode and a reference electrode; blood and urine are taken and added into a sampling port, the blood and urine automatically absorb samples through the siphonage of a sample guide groove, the blood and urine enter a reaction area to react with a reagent in a reaction membrane to form current, the current corresponding to the concentration of uric acid can be quickly detected on an electrochemical biosensor detection system, and the linear relation between the concentration of uric acid and the current is shown in figure 1. After calibration by the method, the obtained linear curve is input into a storage device of a matched tester, and the uric acid concentration of an unknown sample can be directly read from the matched tester.

This comparative example illustrates a linear relationship between uric acid concentration and electric current, and a method of constructing a calibration curve using the uric acid biosensor prepared in comparative example 1. The calibration curve was constructed in the same manner as in example 2, except that the calibration biosensor used was the uric acid biosensor prepared in comparative example 1. The linear relationship between uric acid concentration and current is shown in FIG. 1.

As can be seen from fig. 1, the slopes of the curves obtained by calibration of the uric acid biosensors prepared in examples 1, 2 and 3 are all larger than the slope of the curve obtained by calibration of the uric acid biosensor prepared in comparative example 1, wherein the slope obtained in example 3 is the largest. Therefore, the reaction formula with the addition of the polyethylene glycol 200 and the glycerol is improved linearly, and the detection sensitivity is also improved.

This example illustrates the use of the uric acid biosensor manufactured in example 3 and a test set containing the linear curve obtained by calibration according to the method of example 3, and the use of the uric acid biosensor manufactured in comparative example 1 and a test set containing the linear curve obtained by calibration according to the method of comparative example 1 to test the accuracy of blood samples.

Collecting fresh venous blood and urine, wherein the hematocrit ratio of the blood needs to be adjusted to 40%, and prepared uric acid concentrations are 275 mu mol/L, 450 mu mol/L and 750 mu mol/L respectively. Placing the uric acid biosensor prepared in example 3 in a matched tester, and applying a 300mV working voltage to two ends of a working electrode and a reference electrode; blood and urine are taken and added to a sampling port, the blood and urine automatically absorb about 2 muL of samples through a siphon sample guide groove, the samples enter a reaction area and react with a reagent in a reaction membrane to form current, the current corresponding to the concentration of uric acid can be quickly detected on an electrochemical biosensor detection system, and a recording instrument displays a result. Each blood sample was tested 20 times and CV values were calculated. Similarly, the uric acid biosensor prepared in comparative example 1 was placed in a matched tester and a 300mV working voltage was applied across the working electrode and the reference electrode; blood and urine are taken and added to a sampling port, the blood and urine automatically absorb about 2 muL of samples through a siphon sample guide groove, the samples enter a reaction area and react with a reagent in a reaction membrane to form current, the current corresponding to the concentration of uric acid can be quickly detected on an electrochemical biosensor detection system, and a recording instrument displays a result. Each blood sample was tested 20 times and CV values were calculated. CV of different blood samples measured by the uric acid biosensor and the matching tester manufactured in example 3 and comparative example 1 are shown in Table 1.

Table 1, example 3 and comparative examples of uric acid biosensor and associated tester for measuring CV values of different blood samples

As can be seen from the data in table 1, when blood samples with different uric acid concentrations were measured using the uric acid biosensor manufactured in example 3 and the calibration curve constructed in comparative example 1, and the uric acid biosensors manufactured by both methods were used to test the same blood sample, the variation coefficient of the biosensor test using glycerin and polyethylene glycol 200 in example 3 was smaller than that of the biosensor test using no glycerin and polyethylene glycol 200 in comparative example 1, the variation coefficient of the biosensor manufactured in example 3 was 1.95% to 3.05%, and the variation coefficient of the biosensor manufactured in comparative example 1 was 4.12% to 5.82%. Therefore, the biosensor of glycerol and polyethylene glycol 200 in example 3 of the present invention has high accuracy.

Therefore, the uric acid biosensor provided by the invention has the advantages that the glycerol and the polyethylene glycol 200 are added into the reaction liquid, so that the detection sensitivity is greatly improved, the uric acid biosensor can be used for quickly, simply and conveniently detecting the low-concentration uric acid content, and the sensitivity is high, accurate and reliable.