Method for measuring serum glucose concentration

Abstract

The invention relates to the technical field of blood glucose measurement, and discloses a method for measuring serum glucose concentration, which comprises the following steps: respectively preparing a 15KU / L glucose oxidase solution, a 1mmol / L glucose standard solution, a 0.1 mol / L potassium iodide solution and a 0.5 mol / L sulfuric acid solution, then adding the collected serum specimen and glucose oxidase solution into a colorimetric tube for incubation, and measuring the concentration of glucose in the serum by using the colorimetric tube. And finally, adding a KI solution and a sulfuric acid solution for reaction, and measuring 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 2.72 * 10 <-6 > mol / L, and the clinical blood glucose detection requirement is met; the stability is good, the anti-interference capability is strong, the determination wavelength is 360 nm, the influence of the natural color of serum is avoided, and the interference caused by coexisting substances can be 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] Serum glucose determination is mainly achieved by testing the glucose content in serum, so as to understand whether glucose metabolism is normal. Serum glucose is blood sugar, which mainly refers to the concentration of glucose in the blood. Serum glucose mainly comes from food, such as starch, lactose, maltose, muscle glycogen, sugar cane, etc. After entering the human body, this type of substance will be digested and absorbed by the human body and converted into glucose, which will be stored in the liver or muscles. It will be transported to tissues and organs through the blood for oxidation and decomposition, providing the human body with sufficient energy for life activities. Under normal circumstances, the normal value of serum glucose should be 3.89-6.11mmol / L. If the glucose value is not within this range, it means that glucose metabolism is abnormal. When fasting blood sugar exceeds this range, it is generally diagnosed as diabetes or prediabetes. If the blood sugar value is lower than the normal range, it means excessive consumption.

[0003] 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 measured based on the rhodamine series fluorescence quenching method, with a minimum detection limit of 1.0×10 -8 mol / L, but the process is complicated and the uncertainty factors in the middle increase. At present, the detection method of blood sugar usually uses the kit method, represented by the double enzyme method, which is widely used in the fields of medical health, food inspection and quarantine, etc. Glucose is oxidized by glucose oxidase (GOD) to generate 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 of the blood sample. However, the kit uses two enzymes, which increases the cost and makes it difficult to store. 4-aminoantipyrine belongs to the aromatic primary amine class of substances, which has 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 itself reducing and more easily oxidized by air; it is easily affected by the true color of serum. Six reagents are used in the whole process, the operation process is cumbersome, and the system error increases.

[0004] In view of the above problems, it is urgently necessary for technical personnel in this field to propose a method for measuring serum glucose concentration with a simple testing process, low cost, stability and safety, small system error and no influence from the true color of serum. Summary of the invention

[0005] The present invention aims at the above technical problems and provides a method for measuring serum glucose concentration, which solves the problems in the prior art of complex process, high cost, instability, toxicity, large system error and susceptibility to the influence of serum color.

[0006] A method for measuring serum glucose concentration of the present invention comprises the following steps: S1: Prepare solution: prepare 15KU / L glucose oxidase, 1mmol / L glucose standard solution, 0.1mol / L potassium iodide solution, and 0.5mol / L sulfuric acid solution respectively; S2: Establish a glucose working standard curve: Use a spectrophotometer to measure the absorbance of glucose reaction solutions of different concentrations at a selected wavelength of 360nm, establish a glucose working curve based on the corresponding relationship between the absorbance value and the glucose concentration, and calculate the slope K of the glucose working curve equation; (L / mmol·cm) S3: Collect serum samples: serum samples include clinical serum (for clinical testing) and quality control serum (for experimental teaching); S4: Analysis and detection: Add serum specimen and glucose oxidase reagent into a 10 mL colorimetric tube, incubate at 37°C for 30 min, then add KI solution and sulfuric acid solution in S1, react at 37°C for 20 min, fix the volume, use distilled water as reference, measure the absorbance value at the wavelength (360 nm) selected in step S2, and deduct the enzyme blank; 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 absorbance; K is the slope of the glucose working curve equation, K=11.54L / mmol·cm; V is the total volume of the reaction system; and v is the sample volume.

[0007] Furthermore, the specific steps of preparing 15KU / L glucose oxidase in step S1 are as follows: weighing 30 mg of glucose oxidase standard (50U / mg, Sigma), dissolving it with 10 ml of double distilled water, and storing it at 4°C.

[0008] Furthermore, the specific steps of preparing the 1 mmol / L glucose standard solution in step S1 are to weigh 19.8 mg of glucose monohydrate solid and dissolve it in 100 ml of double distilled water.

[0009] Furthermore, the specific steps of preparing the 0.1 mol / L potassium iodide solution in step S1 are to weigh 1 g of potassium iodide solid, directly put it into a small reagent bottle, add water to the 60 mL mark, shake to dissolve, and prepare it as needed.

[0010] Furthermore, the specific step of preparing the 0.5 mol / L sulfuric acid solution in step S1 is to accurately pipette 2.7 mL of concentrated sulfuric acid into 100 mL of ultrapure water and mix well.

[0011] Furthermore, in step S4, 100 μL of serum specimen and 50 μL of 15KU / L glucose oxidase solution are added to a 10 mL colorimetric tube, incubated at 37°C for 30 min, and then 0.50 mL of KI solution and 2.00 mL of sulfuric acid solution in S1 are added, reacted at 37°C for 20 min, fixed to volume, and distilled water is used as a reference. The absorbance value at the wavelength (360 nm) selected in step S2 is measured, and the enzyme blank is subtracted.

[0012] Furthermore, in the process of measuring the absorbance value A in step S4, the optical path d is 1 cm, and the incident light wavelength λ is 360 nm.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 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 I in a sulfuric acid medium. - The redox reaction generates I2, and the absorbance is measured by 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 2.72×10 -6 mol / L; it has strong anti-interference ability and 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

[0014] Attached Figure 1 This is a spectrum scan of the glucose standard solution in this embodiment; Attached Figure 2 This is the glucose working curve in this embodiment; Attached Figure 3 This is the ultraviolet spectrum scanning diagram of the quality control serum in this embodiment; Attached Figure 4 This is a UV spectrum scan of the quality control serum after adding glucose oxidase in this example. DETAILED DESCRIPTION

[0015] In order to make the purpose and advantages of the present invention clearer, the present invention is further described in detail in conjunction with the following examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The instruments and reagents used in the examples are: UV1102 UV-Vis spectrophotometer (Shanghai Tianmei Scientific Instrument Co., Ltd.); glucose oxidase standard (50U / mg, Sigma); quality control serum (clinical biochemical quality control product, 5ml / bottle, Jiangxi Tekang Technology Co., Ltd.); glucose (analytical grade, Tianjin Tianli Chemical Reagent Co., Ltd.); potassium iodide (analytical grade, Tianjin Tianli Chemical Reagent Co., Ltd.) A method for measuring serum glucose concentration in this embodiment specifically comprises the following steps: S1: Preparation solution: Prepare 15KU / L glucose oxidase: weigh 30 mg of glucose oxidase standard (50U / mg, Sigma), dissolve in 10 ml of double distilled water, and store at 4°C; Prepare 1mmol / L glucose standard solution: weigh 19.8mg of glucose monohydrate solid and dissolve it in 100ml of double distilled water; Prepare 0.1 mol / L potassium iodide solution: weigh 1 g of potassium iodide solid and put it directly into a small reagent bottle. Add water to the 60 mL mark and shake to dissolve. Prepare as needed. 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; S2: Establish a glucose working standard curve: Use a spectrophotometer to measure the absorbance of glucose reaction solutions of different concentrations at a selected wavelength of 360nm, subtract the enzyme blank, and establish a glucose working curve based on the corresponding relationship between the absorbance value and the glucose concentration. Calculate the slope K of the glucose working curve equation; (L / mmol·cm) S3: Collect serum samples: serum samples include clinical serum and quality control serum; S4: Analysis and detection: add 100 μL of serum sample and 50 μL of 15KU / L glucose oxidase solution into a 10 mL colorimetric tube, incubate at 37°C for 30 min, then add 0.50 mL of KI solution and 2.00 mL of sulfuric acid solution in S1, react at 37°C for 20 min, make up to volume, use distilled water as reference, and measure the absorbance value at the wavelength (360 nm) selected in step S2; wherein the optical path d in the process of measuring the absorbance value A is 1 cm, and the incident light wavelength λ is 360 nm; 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 absorbance coefficient, L / mmol·cm), K=11.54L / mmol·cm; V is the total volume of the reaction system; and v is the sample volume.

[0016] The assay principle of this embodiment is: glucose oxidase catalyzes the oxidation of glucose to produce H2O2, and H2O2 reacts with I in a sulfuric acid medium. - A redox reaction occurs to generate I2, and the reaction equation is as follows:

[0017] 2I - + H2O2 +2H + →I2 +2H2O

[0018] Example 1: Take 8 10 mL colorimetric tubes, add 0.00, 0.20, 0.40, 0.60, 0.80, 1.00, 1.50, 2.00 mL of 1 mmol / L glucose standard solution in sequence, complete the enzyme-catalyzed oxidation reaction and iodine generation reaction according to the above method, distilled water to make up the volume, and perform full wavelength scanning one by one. The scanning results are shown in the attached Figure 1 .

[0019] Read the absorbance A at 288 nm and 360 nm. Use the actual glucose concentration C (mmol / L) in the reaction solution as the horizontal axis and A as the vertical axis to draw a glucose working curve. See the attached Figure 2 .

[0020] By the attached Figure 1 And attached Figure 2 It can be seen that at wavelengths of 288nm and 360nm, the absorbance has a good linear relationship with the glucose concentration of the reaction solution, and the effective detection range of glucose is 0-8.0×10 -5 mol / L, when the glucose concentration in the reaction solution exceeds 8.0×10 -5 mol / L, due to the decrease in oxygen content in the solution, the absorbance increase slowed down and the linearity deteriorated; the glucose blank solution was measured 11 times in parallel, the standard deviation was calculated, and the glucose detection limit D was calculated by dividing 3 times the standard deviation by the slope of the curve. 288nm =1.57×10 -6 mol / L,D 360nm =2.72×10 -6mol / L, which meets the requirements of clinical blood glucose detection. To overcome the interference caused by the color of serum itself, the measurement wavelength is 360nm. The slope of the glucose working curve at 360nm is 11.54, from which the apparent millimolar absorption coefficient of glucose K=11.54L / mmol·cm is derived, which means the absorbance corresponding to the complete reaction of 1mmol / L glucose.

[0021] Example 2: Serum contains amino acids, vitamins, inorganic substances, lipids, nucleic acid derivatives, hormones, etc. The above amino acids and lipids are themselves yellowish and clear, similar to the color of iodine solution. In order to investigate their interference, the following experiment was performed: 0.05 ml of quality control serum (containing 6 mmol / L glucose) was taken, diluted to 10 ml with distilled water, and full wavelength scanning was performed in the wavelength range of 200-700 nm, see Appendix Figure 3 The original quality control serum has only one absorption peak at 280 nm, and the absorbance at 360 nm is 0. After adding glucose oxidase to the quality control serum, a new peak appears at 360 nm. See the attached Figure 4 The peak absorbance increases with the increase of the volume of quality control serum. It can be seen that when measuring the absorbance at 360nm, the coexisting substances in the serum do not affect the glucose determination.

[0022] Example 3: Take 0.80 ml of 1 mmol / L glucose standard solution and measure the absorbance A (optical path d is 1 cm, incident light wavelength λ is 360 nm) using a UV-visible spectrophotometer according to the method of Example 1. The measurement was repeated 6 times to calculate the average value and relative standard deviation RSD. At this time, the glucose concentration in the reaction solution was 0.08 mmol / L. The relative error RE was calculated based on the measured value. The results are shown in Table 1.

[0023] Table 1 Precision test of clear glucose determination

[0024] The average glucose reaction concentration is 0.9470 mmol / L. As can be seen from the above table, the relative standard deviation RSD=3.59%, indicating that the UV spectrophotometric method for determining glucose has good accuracy. Compared with the true value, the relative error RE is close to 5%, which meets the requirements of optical analysis.

[0025] Example 4: The serum samples collected in this 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. 100 μL of serum was collected in a colorimetric tube, and the absorbance A was determined using a UV-visible spectrophotometer according to a method for determining serum glucose concentration in the example (optical path d is 1 cm, and the incident light wavelength λ is 360 nm), and a spike recovery experiment was performed. The results are shown in Table 2.

[0026] Table 2 Human serum glucose analysis results (n=6)

[0027] It can be seen from the above table that the blood glucose measurement value of a healthy human body is between 4.59-5.32mmol / L, which is in line with the normal blood glucose concentration range (3.89-6.11mmol / L), and the highest recovery rate reaches 98.62%, indicating that the method of this embodiment has high accuracy.

[0028] Example 5: Take 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, containing 6 mmol / L of glucose) respectively, and measure the glucose concentration thereof by ultraviolet photometry in a method for measuring serum glucose concentration in an embodiment, perform 5 parallel measurements, and calculate the mean value (x), standard deviation (s) and coefficient of variation (RSD). The results are shown in Table 3.

[0029] Comparative Example 1 Quality control serum (Jiangxi Tekang Technology Co., Ltd., clinical biochemical quality control product, 5 ml / bottle, containing 6 mmol / L glucose) was taken at 0.05, 0.10, 0.15, 0.20, and 0.25 mL, 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.

[0030] Table 3 Comparison of the results of UV spectrophotometry and dual enzyme method (n=5)

[0031] 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 UV spectrophotometry method is smaller and the process is more convenient.

[0032] From the above results, it can be seen that in the method for determining the serum glucose concentration of this embodiment, glucose oxidase is used to catalyze the oxidation of glucose in the serum to produce H2O2, and H2O2 reacts with I in a sulfuric acid medium. - The redox reaction generates I2, and the absorbance is measured by a UV-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, economical and applicable, and is suitable for clinical blood sugar detection.

[0033] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for measuring serum glucose concentration, characterized in that: The specific steps include: S1: Prepare solutions: prepare 15KU / L glucose oxidase solution, 1mmol / L glucose standard solution, 0.1mol / L potassium iodide solution, and 0.5mol / L sulfuric acid solution respectively; S2: Establishing a glucose working standard curve: Using a spectrophotometer to measure the absorbance values ​​of glucose reaction solutions of different concentrations at a wavelength of 360 nm, establishing a glucose working curve based on the corresponding relationship between the absorbance value and the glucose concentration, and calculating the slope K of the glucose working curve equation; S3: Collect serum samples: serum samples include clinical serum and quality control serum; S4: Analysis and detection: add the serum sample and glucose oxidase solution into a 10 mL colorimetric tube, incubate at 37°C for 30 min, then add the KI solution and sulfuric acid solution in S1, react at 37°C for 20 min, make up to volume, use distilled water as a reference, and measure the absorbance value at the wavelength selected in step S2; 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 360 nm; K is the slope of the glucose working curve equation, K=11.54 L / mmol·cm; V is the total volume of the reaction system; and v is the sample volume.

2. A method for measuring serum glucose concentration according to claim 1, characterized in that: The specific steps of preparing 15KU / L glucose oxidase in step S1 are: weighing 30 mg of glucose oxidase standard, dissolving it with 10 ml of double distilled water, and storing it at 4°C.

3. A method for measuring serum glucose concentration according to claim 1, characterized in that: The specific steps of preparing the 1 mmol / L glucose standard solution in step S1 are as follows: weighing 19.8 mg of glucose monohydrate solid and dissolving it in 100 ml of double distilled water.

4. A method for measuring serum glucose concentration according to claim 1, characterized in that: The specific steps of preparing the 0.1 mol / L potassium iodide solution in step S1 are as follows: weigh 1 g of potassium iodide solid and put it into a small reagent bottle, add water to the 60 mL mark, shake to dissolve, and prepare as needed.

5. A method for measuring serum glucose concentration according to claim 1, characterized in that: The specific step of preparing the 0.5 mol / L sulfuric acid solution in step S1 is to absorb 2.7 mL of concentrated sulfuric acid into 100 mL of ultrapure water and mix well.

6. A method for measuring serum glucose concentration according to claim 1, characterized in that: In step S4, 100 μL of serum specimen and 50 μL of glucose oxidase solution are added to a 10 mL colorimetric tube, incubated at 37° C. for 30 min, and then 0.5 mL of KI solution and 2.00 mL of sulfuric acid solution in S1 are added. After reacting at 37° C. for 20 min, the volume is fixed, and distilled water is used as a reference. The absorbance value at the wavelength selected in step S2 is measured, and the enzyme blank is subtracted.

7. A 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 360 nm.

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