Hydrogen peroxide (H₂O₂) content detection kit (micro method)

Cat. No.: H1492752

GRADE & PURITY BioReagent

Descripción general

Hydrogen peroxide (H₂O₂) is the most common reactive oxygen species (ROS) in living organisms. It is a by-product of active oxygen metabolism, primarily produced by enzymes like SOD and XOD, and degraded by enzymes such as CAT and POD. H₂O₂ is not only a significant ROS but also a hub for the interconversion of reactive oxygen species. On one hand, H₂O₂ can directly or indirectly oxidize biological macromolecules like nucleic acids and proteins within cells, damaging cell membranes and thereby accelerating cellular aging and disintegration. On the other hand, H₂O₂ is also a key regulatory factor in many oxidative stress responses. It can activate factors like NF-κB, and these H₂O₂-related signaling pathways are associated with many diseases such as asthma, inflammatory arthritis, arteriosclerosis, and neurodegenerative diseases. H₂O₂ is also closely related to processes like cell apoptosis and proliferation.

Detection Principle: H₂O₂ oxidizes ferrous ions (Fe²⁺) to ferric ions (Fe³⁺). The Fe³⁺ then forms a purple complex with xylenol orange in a specific solution. The absorbance at 580 nm is directly proportional to the H₂O₂ concentration, allowing for the quantification of H₂O₂ levels.

Detection Range: 1-100 µM

Sensitivity: 1 µM

Applicable Samples: Animal/plant tissues, cells, bacteria, serum (plasma), urine.

H1492752	Component	96T	480T	Storage
H1492752A	Reaction Buffer	5 mL	25 mL	-20℃. Store in the dark.
H1492752B	H₂O₂ Standard (1M)	0.1 mL	0.1 mL	-20℃. Store in the dark.
H1492752C	Assay Buffer (10×)	13 mL	65 mL	2-8℃

Please check the quantity of each component before the experiment.

An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.

User-Provided Instruments and Reagents

Type	Name	Notes
Instrument	Microplate Reader	Capable of measuring absorbance at 580 nm.
Consumables	96-well Microplate / Ultrafiltration tubes	Standard transparent plate / 10 kDa MWCO
Reagents	PBS (pH 7.4) / Deionized Water / 30% ZnSO₄ solution	For washing cells/bacteria / Reagent preparation / Protein removal
Others	Homogenizer (for tissue samples), incubator, ice bucket, low-temperature centrifuge, adjustable pipettes and tips	Using a multichannel pipette for large-scale detection can improve efficiency.

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Precautions
Reaction Buffer	Ready-to-use; equilibrate to room temperature before use.	Protect from light during the experiment; aliquot and store at -20°C in the dark.
H₂O₂ Standard (1M)	Ready-to-use; equilibrate to room temperature before use.	Protect from light during the experiment; aliquot and store at -20°C in the dark.
Assay Buffer (1×)	Dilute the 10× Assay Buffer 1:10 with deionized water before use; equilibrate to room temperature.	The diluted buffer can be stored at 4°C for at least 2 months. Used for diluting H₂O₂ standard and samples.

2. Standard Preparation

Standard Curve Setup:

First, prepare a 2 mM H₂O₂ Standard: Dilute 2 µL of the 1M H₂O₂ Standard with 998 µL of Assay Buffer (1×).

Then, prepare a 100 µM H₂O₂ Standard: Dilute 50 µL of the 2 mM H₂O₂ Standard with 950 µL of Assay Buffer (1×).

Using the 100 µM H₂O₂ Standard, prepare further dilutions as shown in the table below.

• Prepare fresh standard solutions for each experiment.

• Prepared standards must be used within 4 hours.

• If the sample is a cell suspension, it is recommended to prepare the H₂O₂ standards using the culture medium.

Standard Working Solution	100µM Standard (μL)	Assay Buffer (1×) (μL)	Concentration (µM)
1	200	0	100
2	100	100	50
3	40	160	20
4	20	180	10
5	10	190	5
6	4	196	2
7	2	198	1
Blank	0	200	0

3. Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. When ready for the experiment, thaw samples on ice. Note that this may affect sample stability, and results might be lower than expected. The following substances interfere with detection and should be avoided in samples: Ferric salts, iron salts, sucrose, glucose, ascorbic acid, SDS (>0.2%), sodium azide.

3.1 Animal Tissues:

Wash the tissue with cold PBS to remove as much blood as possible. Blot dry, weigh 0.1 g, and add 1 mL of pre-cooled Assay Buffer (1×). Homogenize the sample on ice. Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.

3.2 Plant Tissues:

Weigh approximately 0.1 g of sample, add 1 mL of pre-cooled Assay Buffer (1×), and grind. Disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.

3.3 Cells/Bacteria:

Collect 5×10⁶ cells or bacteria. Wash with cold PBS, then add 1 mL of pre-cooled Assay Buffer (1×). Homogenize on ice or disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.

3.4 Plasma, Serum, and Urine (and other biological fluids):

Remove proteins and use the supernatant. Protein removal methods:

• Use a 10 kDa ultrafiltration tube: filter and collect the filtrate.

• Mix sample : 30% ZnSO₄ solution = 20 : 1, vortex, then centrifuge at 10000 g, room temperature for 5 min, and collect the supernatant.

4. Assay Steps

4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 580 nm.

4.2 Assay System Setup:

Reagent	Standard Well (μL)	Test Well (μL)
Standard (various conc.)	60	0
Sample	0	60
Reaction Buffer	40	40

4.3 Mix the reaction system thoroughly and incubate at 37°C for 10 minutes.

4.4 Absorbance Measurement: Read the absorbance at 580 nm, recorded as A _blank , A _standard , and A _test .

5. Result Calculation

The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.

5.1 Data Processing

Calculate ΔA _standard = A _standard - A _blank , ΔA _test = A _test - A _blank .

5.2 Standard Curve Plotting

Plot the standard curve with standard concentration as the y-axis and ΔA _standard as the x-axis. Substitute ΔA _test into the equation to obtain the y value (µM).

5.3 Sample H₂O₂ Concentration Calculation

(1) Based on sample mass:

H₂O₂ Content (nmol/g fresh weight) = y × V _sample ÷ (W × V _sample ÷ V _total ) × n = y ÷ W × n

(2) Based on cell or bacterial count:

H₂O₂ Content (nmol/10⁴ cells) = y × V _sample ÷ (500 × V _sample ÷ V _total ) × n = y ÷ 500 × n

(3) Based on liquid volume:

H₂O₂ Content (nmol/mL) = y × V _sample ÷ V _sample × n = y × n

Parameter Description:

• 1 µM = 1 nmol/mL;

• V _sample : Volume of sample added;

• V _total : Volume of Assay Buffer (1×) added, 1 mL;

• n: Sample dilution factor;

• W: Sample mass, g;

• 500: Cell or bacterial count, in units of 10⁴.

6. Result Presentation

Typical Standard Curve: y = 207.21x + 1.4921, R² = 0.9988

Example-1: 0.1 g of corn tissue was processed and assayed according to the procedure using a 96-well plate.

Measured: ΔA _test = A _test - A _blank = 0.278 - 0.048 = 0.230

Substituting into the standard curve gives y = 49.15 µM.

Calculated based on sample mass:

H₂O₂ Content (nmol/g) = y ÷ W × n = 491.5 nmol/g.

Precautions

1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

2. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.

3. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.

4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.

5. This product is for scientific research use only. Not intended for clinical diagnosis.

Frequently Asked Questions

Q: What should I do if the sample ΔA _test is too high or too low?

A: If the sample ΔA _test is greater than the ΔA _standard of the 100 µM standard, the H₂O₂ content in the sample is too high. Dilute the sample appropriately with Assay Buffer (1×) (multiply by the dilution factor in the calculation). If the sample ΔA _test is less than 0.005, increase the sample amount.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light,Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

Each component has a shelf life of 1 year under corresponding storage conditions. H1492752A, H1492752B: Store at -20℃ long term (12 months). Store in the dark. H1492752C: Store at 2-8℃ long term (12 months).