Description
Glucose (Dextrose, Glu), chemical formula C₆H₁₂O₆, molecular weight 180.16, is the most widely distributed and important monosaccharide in nature, belonging to polyhydroxy aldehydes. Enzymatic methods for determining glucose are commonly used in biochemical detection, with the most frequently used being the glucose oxidase method and the hexokinase method. The characteristics of these enzymatic methods are:High sensitivity, accuracy, and precision;Use mild reaction conditions;Specific for glucose, not interfered with by other sugars and reducing substances;Simple operation;Suitable for automatic analyzers.Detection Principle: Under the catalysis of glucose oxidase, glucose is oxidized to gluconic acid, simultaneously consuming oxygen in the solution. The generated hydrogen peroxide reacts with an oxidative chromogen to form a red quinone compound. The amount of hydrogen peroxide produced in the initial reaction is proportional to the glucose concentration. Colorimetric determination is performed using a spectrophotometer at 505 nm. This kit is specifically designed for the quantitative determination of glucose content in human or animal serum, plasma, cerebrospinal fluid, cells, tissues, and other samples. It is not suitable for direct detection of glucose in urine.*Note: Glu Standard (5 mmol/L) = 90 mg/dL.*G1501761Component200TStorageG1501761APhenol Reagent80 mLRT. Store in the dark.G1501761BEnzyme Reagent80 mL-20℃. Store in the dark.G1501761CGlu Standard (5 mmol/L)1.5 mL2-8℃G1501761DddH₂O1.5 mLRTUser-Prepared Instruments and ReagentsNormal saline or PBSCentrifuge tubes, Homogenizer, Centrifuge, Water bath or incubator, Spectrophotometer, 1.0 mL CuvetteExperimental Procedure1. Reagent PreparationShortly before use, mix the Phenol Reagent and Enzyme Reagent in equal volumes to prepare the GOD-POD Working Solution. Store at 4°C.2. Sample Preparation2.1 Serum, Plasma, Cerebrospinal Fluid SamplesSerum or plasma separated from the test sample should not be hemolyzed. Detect directly. If the concentration exceeds the linear range (30 mmol/L), dilute with normal saline or PBS before assay.2.2 Cell Samples(1) Take an appropriate amount of cells (generally recommended >10⁶), centrifuge at 1000 g for 10 min, discard the supernatant, keep the pellet.(2) Wash 1-2 times with PBS or normal saline, centrifuge at 1000 g for 10 min, discard the supernatant, keep the pellet.(3) Add 200-300 µL of PBS or normal saline and homogenize. Ultrasonicate on ice (power 300 W, 3-5 s each time, 30 s interval, repeat 3-5 times). The prepared homogenate should not be centrifuged.*Alternatively, manually homogenize (prepared homogenate should not be centrifuged). Or lyse with 1-2% Triton X-100 on ice for 30-60 min (prepared lysate should not be centrifuged).*2.3 Tissue SamplesAccurately weigh an appropriate amount of tissue. Add normal saline or PBS at a ratio of 1:9 (mass (g) : volume (mL)). Homogenize manually or mechanically on ice. Centrifuge at 2500-3000 g for 10 min. Collect the supernatant.3. Assay SetupRefer to the table below to set up Blank, Standard, and Test tubes. Add solutions sequentially, mix well, and incubate at 37°C in a water bath or 45°C in an incubator for 15 minutes.Reagent (mL)Blank TubeStandard TubeTest TubeddH₂O0.008//Glu Standard (5 mmol/L)/0.008/Test Sample//0.008GOD-POD Working Solution0.80.80.8 4. Measurement After cooling, transfer to a 1.0 mL cuvette. Measure the absorbance at 505 nm. Zero the instrument with the Blank tube. Read the absorbances of the Standard tube and Test tube, recorded as A standard and A test, respectively. 5. Result Calculation Glu (mmol/L) = A test / A standard × 5 Glu (mg/L) = A test / A standard × 900 Reference Interval Healthy adults fasting glucose: 3.9 - 6.1 mmol/L (70 - 110 mg/dL) *Note: Glu Standard (5 mmol/L) = 90 mg/dL = 900 mg/L*Precautions1. The prepared GOD-POD Working Solution should be stored at 4°C protected from light and is valid for 1 week. Avoid repeated freeze-thaw cycles for low-temperature reagents to prevent inactivation or decreased efficiency.2. Use serum or plasma anticoagulated with potassium oxalate-sodium fluoride (inhibits glucose decomposition) for testing. Cerebrospinal fluid can be detected directly. If test samples cannot be assayed immediately, store at 2-8°C; stable for 3 days.3. Urine glucose is often quantified using this method, but cannot be detected directly. First, perform a semi-quantitative test on the urine sample using Benedict's method. Based on the approximate content, dilute the urine with distilled water so that the glucose content is below 3 mg/mL before detection. Multiply the result by the dilution factor. This is because untreated urine contains high concentrations of reducing substances like uric acid, which affect the peroxidase reaction and may cause falsely low results.4. Low-concentration samples will also turn red over time. Therefore, detection should be performed promptly after 15 minutes; the time should not be too long.5. Without zeroing the microplate reader, the typical reference range for the blank is 0.04-0.09, and for the 5 mmol/L standard is 0.25-0.45. Reference ranges may vary due to differences in instruments and operating methods.6. The lower detection limit of this kit is 0.1 mmol/L, and the upper limit is 30 mmol/L. Visual observation: concentration ≤ 0.6 mmol/L is almost colorless; concentration ≥ 0.7 mmol/L shows light red; concentration ≥ 2.5 mmol/L shows red. Generally, results are more accurate near the upper limit than near the lower limit.7. The linear range of this method can reach 30 mmol/L. If the sample glucose concentration is too high, the result may be falsely low. Dilute with normal saline or PBS and re-assay, multiplying the result by the dilution factor.8. Use reagents promptly after opening to avoid affecting subsequent experimental results.9. For your safety and health, please wear lab coats and disposable gloves during operation.10. This kit is for scientific research use only and is not intended for clinical diagnosis or other purposes