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Amplite® Colorimetric Hypochlorite (Hypochlorous Acid) Assay Kit

Hypochlorite was measured with Amplite® Colorimetric Hypochlorite/Hypochlorous Acid Assay Kit in a 96-well clear bottom plate. As low as 0.5uM sodium hypochlorite (NaClO) was detected with 3-5 minutes incubation.
Hypochlorite was measured with Amplite® Colorimetric Hypochlorite/Hypochlorous Acid Assay Kit in a 96-well clear bottom plate. As low as 0.5uM sodium hypochlorite (NaClO) was detected with 3-5 minutes incubation.
Hypochlorite was measured with Amplite® Colorimetric Hypochlorite/Hypochlorous Acid Assay Kit in a 96-well clear bottom plate. As low as 0.5uM sodium hypochlorite (NaClO) was detected with 3-5 minutes incubation.
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Hypochlorite anion (ClO-) and its protonated form, hypochlorous acid (HClO) are critical reactive oxygen species (ROS) in biological systems. Uncontrolled production of hypochlorite (hypochlorous acid) can lead to tissue damage and diseases including arthritis, renal failure and cancers. In addition, sodium hypochlorite (NaClO) has been widely used as a bleaching agent for surface cleaning, odor removal and water disinfection in our daily lives. Exposure to large amount of sodium hypochlorite can lead to poisoning with the symptoms of serious breathing problems, stomach irritation, redness and pain on skin and eye. Therefore, highly selective and sensitive detection of hypochlorite (hypochlorous acid) is of toxicological and environmental importance. Amplite® Colorimetric Hypochlorite (Hypochlorous Acid) Assay Kit offers a sensitive absorption-based assay for measuring hypochlorite (hypochlorous acid) with high specificity. Upon selective reaction with hypochlorite (hypochlorous) the colorless Oxirite™ Hypochlorite Sensor generates a strong color product. The color signal can be measured by a absorption microplate reader at ~580 nm. With this Colorimetric Hypochlorite (Hypochlorous Acid) Assay Kit, trace amount of hypochlorite can be detected.

Platform


Absorbance microplate reader

Absorbance555 ± 5 nm
Recommended plateClear bottom

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare Hypochlorite working solution (50 µL)
  2. Add Hypochlorite standards or test samples (50 µL)
  3. Incubate at room temperature for 3 - 5 min
  4. Monitor absorbance increase at OD of 555 ± 5 nm

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Oxirite™ Hypochlorite Sensor stock solution (20X):
Add 500 µL of DMSO (Component D) into the vial of Oxirite™ Hypochlorite Sensor (Component A) to make 20X Oxirite™ Hypochlorite Sensor stock solution.

 

PREPARATION OF STANDARD SOLUTION

Hypochlorite standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13845

Add 100 μL of Hypochlorite Standard (Component C) into 400 μL of Assay Buffer (Component B) to get 1% Hypochlorite standard solution (H7). Take 1% Hypochlorite standard solution (H7) and perform 1:3 serial dilutions in Assay Buffer (Component B) get serially diluted Hypochlorite standards (H6 - H1).

PREPARATION OF WORKING SOLUTION

Add 250 µL of 20X Oxirite™ Hypochlorite Sensor stock solution into 5 mL of Assay Buffer (Component B), and mix well to make Hypochlorite working solution. Note: This Hypochlorite working solution is enough for one 96-well plate. It is not stable, use it promptly. Protect from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Hypochlorite standards and test samples in a 96-well clear bottom microplate. H = hypochlorite standard (H1 - H7, 0.001% to 1%), BL = blank control, TS = test sample.

BLBLTSTS
H1H1......
H2H2......
H3H3  
H4H4  
H5H5  
H6H6  
H7H7  

Table 2. Reagent composition for each well.

WellVolumeReagent
H1-H750 µLSerial Dilution (0.001% to 1%)
BL50 µLAssay Buffer (Component B)
TS50 µLTest Sample
  1. Prepare Hypochlorite standards (H), blank controls (BL), and test samples (TS) in a 96-well clear bottom microplate as provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of Hypochlorite working solution to each well of Hypochlorite standards, blank control, and test samples to make the total Hypochlorite assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 3 - 5 minutes, protected from light.

  4. Monitor the absorbance increase with an absorbance plate reader at OD = 555 ± 5 nm.

Product Family


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Citations


View all 2 citations: Citation Explorer
A MXene-Based Bionic Cascaded-Enzyme Nanoreactor for Tumor Phototherapy/Enzyme Dynamic Therapy and Hypoxia-Activated Chemotherapy
Authors: Zhang, Xiaoge and Cheng, Lili and Lu, Yao and Tang, Junjie and Lv, Qijun and Chen, Xiaomei and Chen, You and Liu, Jie
Journal: Nano-Micro Letters (2022): 1--21
Non-invasive Control of Bacterial Biofilms by Wireless Electrostimulation
Authors: Wang, Hao and Tampio, Alex JF and Xu, Yikang and Nicholas, Brian D and Ren, Dacheng
Journal: ACS Biomaterials Science & Engineering (2019)

References


View all 60 references: Citation Explorer
Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation
Authors: Martin DE, De Almeida JF, Henry MA, Khaing ZZ, Schmidt CE, Teixeira FB, Diogenes A.
Journal: J Endod (2014): 51
Effect of hypochlorite oxidation on cholinesterase-inhibition assay of acetonitrile extracts from fruits and vegetables for monitoring traces of organophosphate pesticides
Authors: Kitamura K, Maruyama K, Hamano S, Kishi T, Kawakami T, Takahashi Y, Onodera S.
Journal: J Toxicol Sci (2014): 71
Green synthesis of carbon dots with down- and up-conversion fluorescent properties for sensitive detection of hypochlorite with a dual-readout assay
Authors: Yin B, Deng J, Peng X, Long Q, Zhao J, Lu Q, Chen Q, Li H, Tang H, Zhang Y, Yao S.
Journal: Analyst (2013): 6551
Comparative antimicrobial activities of aerosolized sodium hypochlorite, chlorine dioxide, and electrochemically activated solutions evaluated using a novel standardized assay
Authors: Thorn RM, Robinson GM, Reynolds DM.
Journal: Antimicrob Agents Chemother (2013): 2216
Analysis of the germination kinetics of individual Bacillus subtilis spores treated with hydrogen peroxide or sodium hypochlorite
Authors: Setlow B, Yu J, Li YQ, Setlow P.
Journal: Lett Appl Microbiol (2013): 259
A simple yet effective chromogenic reagent for the rapid estimation of bromate and hypochlorite in drinking water
Authors: Zhang J, Yang X.
Journal: Analyst (2013): 434
Effect of hypochlorite-based disinfectants on inactivation of murine norovirus and attempt to eliminate or prevent infection in mice by addition to drinking water
Authors: Takimoto K, Taharaguchi M, Sakai K, Takagi H, Tohya Y, Yamada YK.
Journal: Exp Anim (2013): 237
Use of pyrogallol red and pyranine as probes to evaluate antioxidant capacities towards hypochlorite
Authors: Perez-Cruz F, Cortes C, Atala E, Bohle P, Valenzuela F, Olea-Azar C, Speisky H, Aspee A, Lissi E, Lopez-Alarcon C, Bridi R.
Journal: Molecules (2013): 1638
Enhancement of anti-cholinesterase activity of aqueous samples by hypochlorite oxidation for monitoring traces of organophosphorus pesticides in water
Authors: Kanno A, Kawakami T, Takahashi Y, Onodera S.
Journal: J Toxicol Sci (2012): 389
Colorimetric determination of hypochlorite with unmodified gold nanoparticles through the oxidation of a stabilizer thiol compound
Authors: Zhang J, Wang X, Yang X.
Journal: Analyst (2012): 2806