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Amplite® Fluorimetric Hypochlorite (Hypochlorous Acid) Assay Kit

Hypochlorite was measured with Amplite® Fluorimetric Hypochlorite/Hypochlorous Acid Assay Kit in a 96-well solid black plate.
Hypochlorite was measured with Amplite® Fluorimetric Hypochlorite/Hypochlorous Acid Assay Kit in a 96-well solid black plate.
Hypochlorite was measured with Amplite® Fluorimetric Hypochlorite/Hypochlorous Acid Assay Kit in a 96-well solid black plate.
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Spectral properties
Excitation (nm)555
Emission (nm)576
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
555
Emission (nm)
576
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® Fluorimetric Hypochlorite (Hypochlorous Acid) Assay Kit offers a sensitive fluorescence-based assay for measuring hypochlorite (hypochlorous acid) with high specificity. Upon selective reaction with hypochlorite (hypochlorous) the weakly fluorescent Oxirite™ Hypochlorite Sensor generates a strongly fluorescent product that gives more than 100-fold fluorescence enhancement. The fluorescence signal can be measured by a fluorescence microplate reader. With this Fluorimetric Hypochlorite (Hypochlorous Acid) Assay Kit, as low as 0.4 µM hypochlorite was detected in a 100 µL reaction volume.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

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 10 - 30 min
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
To achieve the best results, it’s strongly recommended to use the black plates. 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 (200X):
Add 50 µL of DMSO (Component D) into the vial of Oxirite™ Hypochlorite Sensor (Component A) to make 200X stock solution. Protect from light.

PREPARATION OF STANDARD SOLUTION

Hypochlorite standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13846

Add 50 µL of Hypochlorite Standard (Component C) into 450 µL of Assay Buffer (Component B) to get 100 mM Hypochlorite standard solution (H7). Take 100 mM (H7) Hypochlorite standard solution and  perform 1:10 serial dilutions in Assay Buffer (Component B) to get 10, 1, 0.1, and 0.01 mM serially diluted Hypochlorite standards (H6 - H3). Then, 0.01 mM Hypochlorite standard (H3) and perform 1:3 in Assay Buffer (Component B) to get 0.003 and 0.001 mM diluted Hypoclorite standards (H2 - H1). 

PREPARATION OF WORKING SOLUTION

Add 25 µL of 200X 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.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Hypochlorite standards and test samples in a 96-well solid black microplate. H= Hypochlorite Standards (H1 - H7, 0.001 to 100 mM), BL=Blank Control, TS=Test Samples. 

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

Table 2. Reagent composition for each well.

WellVolumeReagent
H1 - H750 µLSerial Dilutions (0.001 to 100 mM)
BL50 µLAssay Buffer
TS50 µLtest sample
  1. Prepare Hypochlorite standards (H), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 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 standard, 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 Hypochlorite working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)555
Emission (nm)576

Images


Citations


View all 1 citations: Citation Explorer
Antioxidant microspheres as drug delivery vehicles for the prevention of post-traumatic osteoarthritis
Authors: Kavanaugh, Taylor Elizabeth
Journal: (2017)

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