Glucose detection kit (GOD-POD microplate method)

Cat. No.: A1371397

GRADE & PURITY BioReagent

Overview

Glucose (Glucose, Dextrose, Glu), also known as corn sugar, is the most widely distributed and most important monosaccharide in nature. It is a polyhydroxy aldehyde with the chemical formula C₆H₁₂O₆ and a molecular weight of 180.16. Enzymatic methods are commonly used for glucose determination in biochemical assays, with the glucose oxidase method and hexokinase method being the most frequently used. These methods are characterized by high sensitivity, accuracy, and precision; mild reaction conditions; specificity for glucose without interference from other sugars or reducing substances; simple operation; and suitability for automated analyzers.

The Glucose Assay Kit (GOD-POD Microplate Method), also known as the Glucose Oxidase Method or Glucose Oxidase-Peroxidase Coupling Method, operates on the following principle: Under the catalysis of glucose oxidase, glucose is oxidized to gluconic acid, consuming oxygen from the solution and producing hydrogen peroxide. The hydrogen peroxide then reacts with a chromogenic substrate to form a red quinone compound. In the initial reaction phase, the amount of hydrogen peroxide produced is proportional to the glucose concentration, which can be detected using a microplate reader at 505 nm.

This kit can be used for the quantitative determination of glucose content in samples such as human or animal serum, plasma, cerebrospinal fluid, cells, and tissues. However, it is not suitable for direct detection of glucose in urine. The concentration of the Glu Standard provided is 5 mmol/L (equivalent to 90 mg/dL). This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.

Before use, mix the Phenol Reagent and Enzyme Reagent at a 1:1 ratio to prepare the GOD-POD Working Solution. Store at 4°C.

Reagents, consumables and Equipments not provided

• Microplate reader

• Centrifuge tubes or 96-well plates, homogenizer, centrifuge, water bath or incubator

• Physiological saline or PBS

Procedure (For Reference Only)

1. Sample Preparation

1.1 Serum, Plasma, Cerebrospinal Fluid Samples

Separate serum or plasma from the test sample should be non-hemolyzed. Measure directly. If the concentration exceeds the linear range (30 mmol/L), dilute with physiological saline or PBS before measurement.

1.2 Cell Samples

(1) Take an appropriate number of cells (generally recommended > 10⁶ cells). Centrifuge at 1000 g for 10 minutes. Discard the supernatant, retaining the pellet.

(2) Wash 1-2 times with PBS or physiological saline. Centrifuge at 1000 g for 10 minutes. Discard the supernatant, retaining the pellet.

(3) Add 200–300 μL of PBS or physiological saline for homogenization. Disrupt cells by sonication in an ice bath (300 W power, 3-5 seconds each time with 30-second intervals, repeated 3-5 times). Manual homogenization can also be used. The prepared homogenate should not be centrifuged. Alternatively, add 1-2% Triton X-100 and incubate in an ice bath for 30–60 minutes. The prepared lysate should not be centrifuged.

1.3 Tissue Samples

Accurately weigh an appropriate amount of tissue sample. Add physiological saline or PBS at a ratio of sample weight (g): saline/PBS (mL) = 1:9. Homogenize manually or mechanically in an ice bath. Centrifuge at 2500–3000 g for 10 minutes and collect the supernatant.

2. Sample Addition

Take a 96-well plate and set up Blank, Standard, and Assay wells according to the table below. Add the various solutions sequentially, mix thoroughly, and incubate in a 37°C water bath or 45°C incubator for 15 minutes.

Addition (μL)	Blank Well	Standard Well	Assay Well
ddH₂O	2.5	—	—
Glu Standard (5mmol/L)	—	2.5	—
Sample to be tested	—	—	2.5
GOD-POD Working Solution	250	250	250

3. Measurement

After cooling, measure the absorbance at 505 nm. Zero the reader with the Blank well. Read the absorbance of the Standard and Assay wells, recorded as A_Standard and A_Assay, respectively.

4. Calculation

Glu (mmol/L) = Glu (mmol/L) = A_Assay / A_Standard × 5 

Glu (mg/L) = A_Assay / A_Standard × 900

Reference Interval:

Healthy adults, fasting blood glucose: 3.9–6.1 mmol/L (70–110 mg/dL)

Note: Glu Standard (5mmol/L) = 90 mg/dL = 900 mg/L

Performance Characteristics

Appearance	Colorless to light yellow clear liquid
Linear Range	0–30 mmol/L, r > 0.990
Coefficient of Variation	Intra-assay < 2-5%, Inter-assay < 5%
Blank Absorbance	< 0.2 (1 cm light path)
Stability	6 months (sealed)

Notes

1. The prepared GOD-POD Working Solution should be stored at 4°C protected from light and is stable for 1 week. Avoid repeated freeze-thaw cycles of refrigerated reagents to prevent inactivation or reduced efficiency.

2. Use serum or plasma anticoagulated with potassium oxalate-sodium fluoride (which inhibits glucose breakdown) for measurement. This method can be directly used to detect glucose content in cerebrospinal fluid. If the test samples cannot be measured promptly, store them at 2-8°C; they are stable for up to 3 days.

3. Although this method is often used for quantitative determination of urine glucose, it cannot be measured directly. First, perform a semi-quantitative test on the urine sample using the Benedict's method. Based on the approximate content obtained, dilute the urine sample with distilled water to bring the glucose concentration below 3 mg/mL. Then perform the assay, multiplying the result by the dilution factor. Untreated urine contains high concentrations of reducing substances like uric acid that can interfere with the peroxidase reaction, potentially causing falsely low results.

4. Low-concentration samples may also turn red over extended periods. Therefore, measure promptly after the 15-minute incubation; avoid prolonged delays.

5. If the microplate reader is not zeroed, the blank reference range is typically between 0.04 and 0.09, and the 5 mmol/L standard reference range is typically between 0.25 and 0.45. Reference ranges may vary depending on factors such as instrument and operating method.

6. The detection limit of this kit is 0.1 mmol/L, and the upper limit is 30 mmol/L. Visually, concentrations ≤ 0.6 mmol/L appear almost colorless, concentrations around 0.7 mmol/L show a faint red color, and concentrations ≥ 2.5 mmol/L show a distinct red color. Generally, results near the upper limit are more accurate than those near the lower limit.

7. The linear range of this method reaches up to 30 mmol/L. If the sample glucose concentration is too high, results may be falsely low. Dilute the sample with physiological saline or PBS and re-assay, multiplying the result by the dilution factor.

8. Use the reagents as soon as possible after opening to avoid affecting subsequent experimental results.

9. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature,Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.

Contents & Storage

A1371397	Component	200 T	500 T	Storage
A1371397A	Phenol Reagent	25 mL	65 mL	RT. Store in the dark.
A1371397B	Enzyme Reagent	25 mL	65 mL	-20℃. Avoid freeze/thaw cycle. Store in the dark.
A1371397C	Glu Standard (5mmol/L)	1 mL	1 mL	2-8℃.
A1371397D	ddH₂O	1 mL	1 mL	RT.