Method for measuring serum glucose concentration

Abstract

The invention relates to the technical field of blood glucose measurement, in particular to a method for measuring serum glucose concentration. The method comprises the following steps: firstly, respectively preparing a glucose oxidase solution, a glucose standard solution, a ferrous sulfate solution, a potassium thiocyanate solution and a sulfuric acid solution, then adding a collected serum specimen and the glucose oxidase solution into a colorimetric tube for incubation, and then adding the ferrous sulfate solution and the sulfuric acid solution for further reaction; and finally, adding a potassium thiocyanate solution to fully react, and measuring the absorbance A so as to calculate the concentration of glucose in the serum. The method is simple in operation process and low in cost, and used reagents are safe, harmless and long in storage time; the light sensitivity is good, the lowest detection limit is 0.008 mmol / L, and the clinical blood glucose detection requirement is met; the method is good in repeatability and strong in anti-interference capability, the determination wavelength is 460nm, the influence of the natural color of serum is avoided, and the interference caused by coexisting substances is effectively overcome by taking enzyme blank as a contrast.

Description

Technical Field

[0001] The present invention relates to the technical field of blood sugar determination, and in particular to a method for determining serum glucose concentration. Background Art

[0002] Glucose is closely related to human life activities. Blood sugar specifically refers to the glucose in the blood. Diabetes, hyperpituitarism, hyperadrenocortical function, meningitis, pancreatic cancer, etc. cause increased blood sugar, while hunger, excessive insulin secretion, hypothyroidism, acute liver damage, primary liver cancer, etc. cause decreased blood sugar. Therefore, blood sugar levels are an important indicator for measuring clinically related diseases. The blood sugar level of healthy people is in the range of 3.89 to 6.11 mmol / L in a day. It is very important to develop a safe, convenient, efficient, accurate, and economical method for measuring blood sugar concentration. Traditional methods for measuring glucose concentration include high-performance liquid chromatography, ion chromatography, spectrophotometry, biosensor method, fluorescence method, etc.; potassium iodide is used as a conversion substance, and serum glucose concentration is determined based on the rhodamine series fluorescence quenching method, with a minimum detection limit of 1.0×10 -8 mol / L, however, the process is complicated and the intermediate uncertainties increase.

[0003] The phenol-4-aminoantipyrine method (double enzyme method) is a universal classic method for glucose detection. It is widely used in the fields of medical care, food inspection and quarantine, etc. The methodological principle is carried out in two steps: glucose oxidase (GOD) catalyzes the oxidation of glucose to produce H2O2. Under the catalysis of peroxidase, H2O2 further reacts with 4-aminoantipyrine / phenol to develop a color reaction. The absorbance of the color developing solution at a wavelength of 500nm in the visible light region determines the glucose concentration in the blood sample.

[0004]

[0005] The kit uses two enzymes, which increases the cost and makes the reagents difficult to store. 4-Aminoantipyrine is an aromatic primary amine with acute toxicity and teratogenicity, a small molar absorption coefficient, poor photosensitivity, and a limited storage period. Phenol appears in the form of a third catalyst, which is reducing and more easily oxidized by air. Six reagents are used in the entire process, making the operation cumbersome and increasing system errors.

[0006] Therefore, providing a method for simply measuring serum glucose concentration is of great significance in the technical field of blood glucose measurement. Summary of the invention

[0007] Based on the above content, the present invention provides a complexation colorimetric method for simply measuring the colorimetric blood glucose concentration.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for measuring serum glucose concentration, comprising the following steps:

[0010] S1: Prepare solutions: prepare glucose oxidase solution, glucose standard solution, ferrous sulfate solution, potassium thiocyanate solution and sulfuric acid solution respectively;

[0011] S2: Establish a glucose working standard curve;

[0012] S3: Collect serum samples: serum samples include clinical serum and quality control serum;

[0013] S4: Analysis and detection: The serum specimen and the glucose oxidase solution are mixed and incubated, and then the ferrous sulfate solution and the sulfuric acid solution are added. After the reaction at room temperature, the potassium thiocyanate solution is added, the volume is fixed, and distilled water is used as a reference. The absorbance value A at a wavelength of 460nm is measured, and the enzyme blank is deducted;

[0014] S5: Calculate the glucose concentration in the serum: Compare the absorbance value measured in step S4 with the working curve established in step S2. Calculate the glucose concentration in serum, where A is the absorbance at 460 nm; K is the slope of the glucose working curve equation, K = 2.4228 L / mmol·cm; V is the total volume of the reaction system; and v is the sample volume.

[0015] In a preferred embodiment of the present invention, the enzyme activity of the glucose oxidase solution is 15KU / L; the concentration of the glucose standard solution is 0.01mol / L; the concentration of the ferrous sulfate solution is 0.01mol / L; the concentration of the potassium thiocyanate solution is 0.1mol / L; and the concentration of the sulfuric acid solution is 0.5mol / L.

[0016] In a preferred embodiment of the present invention, in step S2, the absorbance values ​​of glucose reaction solutions of different concentrations at a wavelength of 460 nm are measured by a spectrophotometer, a glucose working curve is established based on the correspondence between the absorbance values ​​and the glucose concentrations, and the slope K of the glucose working curve equation is calculated.

[0017] In a preferred embodiment of the present invention, the concentration of the glucose reaction solution is 0 to 0.25 mmol / L.

[0018] In a preferred embodiment of the present invention, in step S4, the incubation is performed in a 10 mL colorimetric tube.

[0019] In a preferred embodiment of the present invention, in step S4, the incubation temperature is 37° C. and the time is 15 min.

[0020] In a preferred embodiment of the present invention, in step S4, the reaction time at room temperature is 5 minutes.

[0021] In a preferred embodiment of the present invention, the volume ratio of the serum sample to the glucose oxidase solution, ferrous sulfate solution, sulfuric acid solution and potassium thiocyanate solution is 0.5:0.2:1:0.4:1; and the serum sample accounts for 5% of the volume of the container used for incubation.

[0022] In a preferred embodiment of the present invention, in the process of measuring the absorbance value A in step S4, the optical path d is 1 cm, and the wavelength λ of the incident light is 460 nm.

[0023] The measurement wavelength is selected as 460nm, which can avoid the influence of serum color, and the enzyme blank is used as a control to effectively overcome the interference caused by coexisting substances.

[0024] The present invention discloses the following technical effects:

[0025] In the method of the present invention, glucose oxidase is used to catalyze the oxidation of glucose in serum to produce H2O2, and H2O2 reacts with Fe in a sulfuric acid medium. 2+ Oxidation-reduction reaction occurs to generate Fe 3+ , Fe 3+ With SCN - The generated complex has maximum absorption at 460nm, and the absorbance is positively correlated with the glucose concentration. The absorbance is measured by a UV-visible spectrophotometer to calculate the glucose content in the serum. The method of the present invention has a simple operation process, low cost, safe and harmless reagents, long storage time, and is suitable for the development of commercial kits; it has good light sensitivity, and the minimum detection limit is 0.008mmol / L, which meets the requirements of clinical blood glucose detection; it has good reproducibility, strong anti-interference ability, good selectivity, can effectively eliminate the interference of dissolved oxygen and serum coexisting substances, and is not affected by the true color of serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The complexes [Fe(SCN) n ] 3-n Absorption curve;

[0028] Figure 2 is the glucose working curve in the present invention;

[0029] Figure 3 This is a scanning diagram of the ultraviolet spectrum of the quality control serum in the present invention. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Potassium thiocyanate (KSCN), also known as potassium thiocyanate, is Fe 3+ A commonly used detection reagent, which turns blood red after addition. 2+ When present (C6H 12 O6 / Fe 2+ When the glucose oxidation product H2O2 under the action of glucose oxidase can convert part of Fe 2+ Oxidized to Fe 3+ , Fe 3+ / SCN - The complex has a maximum absorption at 460nm, and the absorbance is positively correlated with the glucose concentration. 3+ / SCN- The color development principle has established a method for accurate glucose detection and is used for different types of serum blood glucose testing.

[0036] In order to avoid the problems existing in the prior art such as complex process, high cost, instability, toxicity, large system error and susceptibility to the true color of serum, the present invention provides a coordination colorimetric method for simply determining the concentration of colorimetric blood glucose.

[0037] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0038] The instruments and reagents used in the examples are: UV1102 ultraviolet-visible spectrophotometer (Shanghai Tianmei Scientific Instrument Co., Ltd.); glucose oxidase standard (50 U / mg, Sigma); quality control serum (clinical biochemistry quality control product, 5 mL / bottle, Jiangxi Tekang Technology Co., Ltd.); glucose (analytical grade, Tianjin Tianli Chemical Reagent Co., Ltd.); ferrous sulfate heptahydrate (analytical grade, Tianjin Tianli Chemical Reagent Co., Ltd.); potassium thiocyanate (analytical grade, Tianjin Dengfeng Chemical Co., Ltd.).

[0039] The present invention utilizes a dual enzyme method to measure the glucose concentration. Specifically, the dual enzyme method is a glucose oxidase-peroxidase method, and the specific process is as follows:

[0040] (1) Reagent preparation

[0041] Glucose oxidase-peroxidase reagent: usually in the form of a kit, containing glucose oxidase, peroxidase, 4-aminoantipyrine, phenol and other ingredients. It needs to be prepared and diluted according to the instructions before use.

[0042] Standard glucose solution: Accurately weigh a certain amount of glucose standard, dissolve it in distilled water and make up to a certain volume to prepare standard solutions of different concentrations, such as 0.5, 1.0, 2.0, 5.0, 10.0 mmol / L, etc.

[0043] (2) Standard curve drawing

[0044] Take several clean test tubes, add 0.1 mL of standard glucose solution of different concentrations to each of them, then add appropriate amount of glucose oxidase-peroxidase reagent in sequence, and make up to a certain volume with distilled water, such as 3.0 mL.

[0045] After thorough mixing, incubate the mixture in a 37°C water bath for 15-30 minutes.

[0046] After the reaction was completed, the absorbance of each tube was measured at a wavelength of 505 nm using distilled water as a blank control.

[0047] Draw a standard curve with glucose concentration as the horizontal axis and absorbance value as the vertical axis.

[0048] (3) Sample measurement

[0049] For liquid samples, such as serum, plasma, urine, etc., a certain amount of sample can be directly aspirated and added to a test tube; for solid samples, pretreatment such as extraction and dilution is required to make it a solution suitable for measurement, and then an appropriate amount is taken for measurement.

[0050] Following the same operating steps as for drawing the standard curve, glucose oxidase-peroxidase reagent was added to react and measure the absorbance value.

[0051] According to the absorbance value of the sample, the corresponding glucose concentration is found on the standard curve, or the glucose content in the sample is calculated by the regression equation of the standard curve.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] This embodiment provides a method for measuring serum glucose concentration, which specifically comprises the following steps:

[0055] S1: Prepare solution:

[0056] Preparation of 15KU / L glucose oxidase: weigh 30 mg of glucose oxidase standard, dissolve in 10 mL of double distilled water, and store at 4°C;

[0057] Prepare 0.01 mol / L glucose standard solution: weigh 198 mg of glucose monohydrate solid and dissolve it in 100 mL of double distilled water;

[0058] Prepare 0.01 mol / L ferrous sulfate solution: weigh 0.2780 g of ferrous sulfate heptahydrate solid, put it directly into a small reagent bottle, add 100 mL of water, shake to dissolve, add one zinc particle, and store it for a long time;

[0059] Prepare 0.1 mol / L potassium thiocyanate solution: weigh 0.9718 g of potassium thiocyanate solid, dissolve and transfer into a 100 mL volumetric flask, and dilute to volume;

[0060] Prepare 0.5 mol / L sulfuric acid solution: accurately pipette 2.7 mL of concentrated sulfuric acid into 100 mL of ultrapure water and mix well;

[0061] S2: Establish a glucose working standard curve:

[0062] The absorbance values ​​of glucose reaction solutions of different concentrations at a selected wavelength of 460 nm were measured by a spectrophotometer, and a glucose working curve was established based on the corresponding relationship between the absorbance value and the glucose concentration, and the slope K (L / mmol·cm) of the glucose working curve equation was calculated;

[0063] S3: Collect serum samples: serum samples include clinical serum and quality control serum;

[0064] S4: Analysis and detection: add 0.50mL of serum specimen and 0.20mL of 15KU / L glucose oxidase solution into a 10mL colorimetric tube, incubate at 37°C for 15min, then add 1.00mL of ferrous sulfate solution and 0.40mL of sulfuric acid solution in S1, react at room temperature for 5min, add 1.00mL of potassium thiocyanate solution in S1, make up to volume with distilled water, and use distilled water as a reference, measure the absorbance value at the wavelength (460nm) selected in step S2, and deduct the enzyme blank; wherein the optical path d in the process of measuring the absorbance value A is 1cm, and the incident light wavelength λ is 460nm;

[0065] Enzyme blank (no glucose oxidase added) refers to adding ferrous sulfate solution and sulfuric acid solution to serum specimens, reacting at room temperature, adding potassium thiocyanate solution, fixing the volume, using distilled water as a reference, and measuring the absorbance value A0 at a wavelength of 460nm;

[0066] S5: Calculate the glucose concentration in the serum: Compare the absorbance value measured in step S4 with the working curve established in step S2. Calculate the glucose concentration in serum, where A is the absorbance of the reaction solution (excluding the blank effect); K is the slope of the glucose working curve equation (glucose apparent millimolar absorption coefficient, L / mmol·cm), K = 2.4228 L / mmol·cm; V is the total volume of the reaction system; v is the sample volume.

[0067] The determination principle of this embodiment is: glucose oxidase catalyzes the oxidation of glucose to produce H2O2, and H2O2 reacts with Fe in a sulfuric acid medium. 2+ Oxidation-reduction reaction occurs to generate Fe 3+ , Fe 3+ With SCN - A blood-red coordination compound is generated, and the reaction equation is as follows:

[0068]

[0069] Effect Example 1

[0070] Take 6 10mL colorimetric tubes, add 0.00, 0.05, 0.10, 0.15, 0.20, 0.25mL of 0.01mol / L glucose standard solution in sequence, complete the enzyme-catalyzed oxidation reaction and complex formation reaction according to the method of Example 1, distilled water is used to make the volume, and full wavelength scanning is performed one by one. The scanning results are shown in Figure 1 .

[0071] Read the absorbance A at 460nm. Draw the glucose working curve with the actual glucose concentration C (mmol / L) in the reaction solution as the horizontal axis and A as the vertical axis, see Figure 2 .

[0072] Depend on Figure 1 and Figure 2 It can be seen that at a wavelength of 460nm, there is a good linear relationship between the absorbance and the glucose concentration of the reaction solution. The effective detection range of glucose is 0.01-0.25mmol / L. The glucose blank solution was measured in parallel 11 times, and the standard deviation was calculated. The glucose detection limit D was calculated by dividing 3 times the standard deviation by the slope of the curve. D=0.008mmol / L, and the slope of the glucose working curve is 2.4228. The apparent millimolar absorption coefficient of glucose K=2.4228L / mmol·cm-1 is derived, which represents the absorbance corresponding to the complete reaction of 1mmol / L glucose.

[0073] Effect Example 2

[0074] Serum contains amino acids, vitamins, inorganic substances, lipids, nucleic acid derivatives, hormones, etc. The above amino acids and lipids are themselves yellowish and clear. In order to investigate their interference, the following experiment was performed: 0.5 mL of quality control serum was taken, diluted to 10 mL with distilled water, and full wavelength scanning was performed in the wavelength range of 200-700 nm, see Figure 3 , the original quality control serum has only one absorption peak at 280nm, and the absorbance at 460nm is 0. Therefore, when measuring the absorbance at 460nm, there is no interference with the original color of the serum.

[0075] Effect Example 3

[0076] Take 0.25mL of 0.01mol / L glucose standard solution, and measure the absorbance A (optical path d is 1cm, incident light wavelength λ is 460nm) with a UV-visible spectrophotometer according to the method of Example 1, and repeat 6 times to calculate the average value and relative standard deviation RSD. At this time, the glucose concentration in the reaction solution is 0.25mmol / L. The relative error RE is calculated based on the measured value. The results are shown in Table 1.

[0077] Table 1 Glucose determination precision test

[0078]

[0079]

[0080] The average glucose reaction concentration is 9.848mmol / L. As can be seen from the above table, the relative standard deviation RSD = 0.06%, indicating that the ultraviolet spectrophotometric method for determining glucose has good accuracy. Compared with the true value, the relative error is 1.25%, which meets the requirements of optical analysis.

[0081] Effect Example 4

[0082] The serum samples collected in this effect example were from the Affiliated Hospital of Gansu Medical College, and were recorded as blood sample 1, blood sample 2, and blood sample 3, respectively. 0.50 mL of serum was collected in a colorimetric tube, and the absorbance A was determined by a UV-visible spectrophotometer according to the method for determining serum glucose concentration in Example 1 (optical path d was 1 cm, and the incident light wavelength λ was 460 nm), and a spike recovery experiment was performed. The results are shown in Table 2.

[0083] Table 2 Human serum glucose analysis results (n=6)

[0084]

[0085] As can be seen from the above table, the blood glucose measurement value of a healthy human body is between 4.59 and 5.32 mmol / L, which is in line with the normal blood glucose concentration range (3.89-6.11 mmol / L). The highest recovery rate reaches 98.67%, indicating that the method of the present invention has high accuracy.

[0086] Effect Example 5

[0087] 0.05, 0.10, 0.15, 0.20, and 0.25 mL of quality control serum (Jiangxi Tekang Technology Co., Ltd., clinical biochemical quality control product, 5 mL / bottle) were taken respectively, and the glucose concentration was measured by the method for measuring serum glucose concentration in Example 1 (KSCN colorimetric method), and the measurement was repeated 5 times in parallel to calculate the mean value (x), standard deviation (s) and coefficient of variation (RSD). The results are shown in Table 3.

[0088] Comparative Example 1

[0089] 0.05, 0.10, 0.15, 0.20, and 0.25 mL of quality control serum (Jiangxi Tekang Technology Co., Ltd., clinical biochemical quality control product, 5 mL / bottle) were taken respectively, and the glucose concentration was measured by double enzyme method. The measurement was repeated 5 times in parallel, and the mean (x), standard deviation (s) and coefficient of variation (RSD) were calculated. The results are shown in Table 3.

[0090] Table 3 Comparison of KSCN colorimetric method and dual enzyme method (n=5)

[0091]

[0092] As shown in Table 3, the measured values ​​of the two methods are consistent and close to the labeled amount of 6mmol / L; the RSD of the KSCN colorimetric method is smaller and the process is more convenient.

[0093] From the above results, it can be seen that the complexation colorimetric method for simply determining the colorimetric blood sugar concentration of the present invention uses glucose oxidase to catalyze the oxidation of glucose in the serum to produce H2O2, and H2O2 reacts with Fe in a sulfuric acid medium. 2+ Oxidation-reduction reaction occurs to generate Fe 3+ , Fe 3+ With SCN - A blood-red coordination compound is generated, and the absorbance is measured by an ultraviolet-visible spectrophotometer to calculate the content of glucose in the serum. The method of the present invention has a simple operation process, low cost, safe and harmless reagents, good photosensitivity, long storage time, and the method of the present invention is efficient, accurate, reproducible, economical and applicable, and is suitable for clinical blood sugar detection.

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for measuring serum glucose concentration, characterized in that: The following steps are involved: S1: Prepare solutions: prepare glucose oxidase solution, glucose standard solution, ferrous sulfate solution, potassium thiocyanate solution and sulfuric acid solution respectively; S2: Establish a glucose working standard curve; S3: Collect serum samples: serum samples include clinical serum and quality control serum; S4: Analysis and detection: the serum specimen and the glucose oxidase solution are mixed and incubated, and then the ferrous sulfate solution and the sulfuric acid solution are added. After the reaction at room temperature, the potassium thiocyanate solution is added, the volume is fixed, and distilled water is used as a reference. The absorbance value A at a wavelength of 460 nm is measured; S5: Calculate the glucose concentration in the serum: Compare the absorbance value measured in step S4 with the working curve established in step S2 to obtain C= A × V The glucose concentration in serum was calculated according to K v, where A is the absorbance at 460 nm; K is the slope of the glucose working curve equation, K = 2.4228 L / mmol·cm; V is the total volume of the reaction system; and v is the sample volume.

2. The method for measuring serum glucose concentration according to claim 1, characterized in that: The enzyme activity of the glucose oxidase solution is 15KU / L; the concentration of the glucose standard solution is 0.01mol / L; the concentration of the ferrous sulfate solution is 0.01mol / L; the concentration of the potassium thiocyanate solution is 0.1mol / L; and the concentration of the sulfuric acid solution is 0.5mol / L.

3. The method for measuring serum glucose concentration according to claim 1, characterized in that: In step S2, the absorbance values ​​of glucose reaction solutions of different concentrations at a wavelength of 460 nm are measured by a spectrophotometer, a glucose working curve is established based on the corresponding relationship between the absorbance value and the glucose concentration, and the slope K of the glucose working curve equation is calculated.

4. The method for measuring serum glucose concentration according to claim 3, characterized in that: The concentration of the glucose reaction solution is 0-0.25 mmol / L.

5. The method for measuring serum glucose concentration according to claim 1, characterized in that: In step S4, the incubation is performed in a 10 mL colorimetric tube.

6. The method for measuring serum glucose concentration according to claim 1, characterized in that: In step S4, the incubation temperature is 37° C. and the incubation time is 15 min.

7. The method for measuring serum glucose concentration according to claim 1, characterized in that: In step S4, the reaction time at room temperature is 5 minutes.

8. The method for measuring serum glucose concentration according to claim 1, characterized in that: The volume ratio of the serum sample to the glucose oxidase solution, ferrous sulfate solution, sulfuric acid solution and potassium thiocyanate solution is 0.5:0.2:1:0.4:1; the serum sample accounts for 5% of the volume of the container used for incubation.

9. The method for measuring serum glucose concentration according to claim 1, characterized in that: In the process of measuring the absorbance value A in step S4, the optical path d is 1 cm, and the wavelength λ of the incident light is 460 nm.

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