Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology


ROSFluor™ Series

Fluorescent Probes for the Detection of Reactive Oxygen Species


Reactive Oxygen Species

  • Reactive oxygen species (ROS) play a key role in many pathogenic processes, such as:
  • • carcinogenesis
  • • inflammation
  • • ischemia-reperfusion injury
  • • signal transduction

  • It is believed that each reactive oxygen species has a certain role in living cells.
  • For example:
  • • H2O2 is an endothelium-derived hyperpolarizing factor in human and mice
  • • Induced by singlet oxygen (1O2) but not by H2O2, p38 mitogen-activated protein kinase mediates caspase-3 activation
       during apoptosis
  • • Hydroxyl radical (.OH) plays an important role as second messenger in T cell activation

  • MoBiTec and its partner Goryo offer fluorescent probes for highly seletive detection of each ROS to get more insights in their biological functions:
  • HPF & APF – For the detection of hydroxyl radical (.OH), peroxynitrite (ONOO-), and hypochlorite (ClO-)
  • NiSPY-3 – For the detection of peroxynitrite (ONOO-)
  • HySOx – For the detection of hypochlorous acid (HClO)
  • HYDROP - For the detection of hydrogen peroxide (H2O2)
  • OxiORANGE™ - For the detection of hydroxyl radical (.OH) or hypochlorous acid (HClO)

Hydroxyphenyl Fluorescein (HPF) & Aminophenyl Fluorescein (APF)

Measuring Principle


  • HFP and AFP selectively detect highly reactive oxygen species like the hydroxyl radical (.OH) and peroxynitrite (ONOO-)
  • They do not react with other reactive oxygen species (.O-
    , H2O2, 1O2, .NO, etc.)
  • Quantitative analysis of H2O2 is possible by HRP
  • Specific detection of hypochlorite (OCl-) is possible by combination of HPF and APF
  • HPF and APF are not subject to autoxidaton, which is a prerequisite for reliable data
  • For live-cell imaging
  • References

Measuring Principle

  • HPF and APF are almost non-fluorescent in neutral solution. When they react with highly reactive oxygen species (hROS), they generate fluorescein that displays a high fluorescence intensity (absorption: 490 nm, emission: 515 nm).

Reaction of HPF, APF, and DCFH with Different Reactive Oxygen Species

.OH 730 1200 7400
ONOO- 120 560 6600
OCl- 6 3600 86
1O2 5 9 26
8 6 67
H2O2 2 <1 190
.NO 6 <1 150
ROO. 17 2 710
Autoxidation <1 <1 2000

Tab. 1

  • Each fluorescent probe (final concentration: 10 μM, 0.1 % DMF as co-solvent) reacted with various reactive oxygen species in phosphate buffer (0.1 M, pH 7.4).
  • Fluorescent intensity of HPF, APF, and DCFH was measured at 515, 515, 520 nm by excitation at 490, 490, 500 nm, respectively.

NiSPY-3 – Nitrative Stress Sensing Pyrromethene Dye

Fluorescence spectra of NiSPY

Left: Fluorescence spectra of NiSPY-3 solution (10 µM NiSPY-3 in 0.1 M phosphate buffer pH 7.4 containing 0.1% DMF as a co-solvent) upon addition of peroxynitrite (final 0, 1, 2, 5,10, 20 µM).
Right: Fluorescence response of NiSPY-3 in various ROS generation systems


  • NiSPY-3 specifically detects peroxynitrite (ONOO-)
  • It does not react with other reactive oxygen species (.OH, .O-
    , H2O2, 1O2, .NO, etc.)
  • Fluorescent intensity is not increased by the presence of hydroxyl radical, singlet oxygen, hydrogen peroxide, hypochlorite, nitric oxide, or superoxide
  • For live-cell imaging


  • NiSPY-3 (Nitrative Stress Sensing Pyrromethene Dye) is a fluorescent probe for the detection of nitrosative stress. NiSPY-3 reacts specifically with peroxynitrite (ONOO-).
  • NiSPY-3 does not show any fluorescence in neutral solution. When NiSPY-3 reacts with peroxynitrite, it shows strong fluorescence (excitation: 490 nm, emission: 515 nm).

HySOx – Fluorophore to Detect Hypochlorous Acid in Living Cells


Reactivity of HySOx with various ROS
Fluorescence intensity of HySOx increases up to ~170-fold when it reacts with hypochlorous acid. Other ROS do not increase fluorescent intensity of HySOx under identical conditions.

Hypochlorous Acid Production

Hypochlorous acid production during phagocytosis of Zymosan A by U937 cells


Alteration in the fluorescent spectrum of 5 µM HySOx solution in various hypochlorous acid concentrations. The reaction of HySOx with hypochlorous acid saturates at the molar ratio of 1:2.
Inset: fluorescence intensities of HySOx against hypochlorous acid concentrations.


  • Highly specific detection of hypochlorous acid (HOCl)
  • Low reactivity with other reactive oxygen species
  • Fast reactivity and slow photobleaching
  • Suitable for time-lapse imaging
  • λex: 553 nm; λem: 574 nm


  • HySOx is a fluorescent probe that shows excellent selectivity to hypochlorous acid (HOCl). HySOx is almost non-fluorescent in neutral solutions. When HySOx reacts with hypochlorous acid, it generates a highly fluorescent product.

HySOx Selectivity

Dose-Dependent Changes in Spectrum and Intensity of HySOx Fluorescence

Application Example

HYDROP – For the Detection of Hydrogen Peroxide


Reaction of HYDROP with various ROS
After addition of hydrogen peroxide the fluorescence intensity of hydrolyzed HYDROP increases.


Visualization of hydrogen peroxide production by RAW 264.7 cells using HYDROP. Green-fluorescent signal is overlayed to DIC image. Hydrogen peroxide production was induced by the addition of 0.001 µg/ml phorbol myristate acetate (PMA). Cells were stained with 1 µM HYDROP for 20 min. Bar: 25 µm.


  • Highly specific for hydrogen peroxide (H2O2) detection
  • Suitable for time-lapse imaging because of slow photobleaching
  • HYDROP is a fluorescent probe which is non-fluorescent under physiological conditions, but shows strong fluorescence upon reaction with hydrogen peroxide
  • λex: 492 nm; λem: 518 nm


  • The HYDRPO probe is cell permeable. Within the cell HYDROP is hydrolyzed by intracellular esterases. The hydrolyzed product cannot cross the cell membrane and thus stays within the cell. The hydrolysis also increases the reactivity of the probe with hydrogen peroxide. HYDROP and its hydrolyzed product are almost non-fluorescent in neutral solutions. After reaction with hydrogen peroxide, a highly fluorescent product is generated.

ROS Selectivity

Application Example

OxiORANGE™ - For Detection of Hydroxyl Radical (.OH) or Hypochlorous Acid (HClO)

OxiORANGE Spectra

Fluorescence and Reactivity
Absorbance/fluorescence spectra (left) and reactivity with various ROS (right).

OxiORANGE: ROS-Detection

Bright and Stable Fluorescence
OxiORANGE™ (1 µM, top, orange) or competitor product (5 µM, bottom, deep red) were added to the medium and incubated for 30 minutes. After the medium was exchanged to HBSS, 1 mM H2O2 was added to stimulate ROS production. Bright signal from OxiORANGE™ was detected. DIC image (gray), Hoechst 33342 (blue), and OxiORANGE™ (orange) or competitor product (red) were overlaid.
(right: before stimulus; left: 15 minutes after addition of 1 mM H2O2)

OxiORANGE: Localisation within Cells

OxiORANGE™ Tends to Localize within Mitochondria.
HeLa cells were stained with 0.5 µM of OxiORANGE™, 0.25 µM of MitoTrackerGREEN, and 0.2 µg/mL of Hoechst 33342 for 30 minutes. Stimulated with 100 µM of hydrogen peroxide, for 30 min. Observed by fluorescence microscopy.


  • For live-cell imaging
  • High photostability, suitable for time-lapse imaging of intracellular ROS generation
  • For multicolor imaging with green and blue fluorophores
  • λex: 553 nm; λem: 577 nm
  • Localizes within mitochondria because of its positive charge
  • Fluorescence is stable after mild fixation (3-4 % PFA, 20 min)


  • OxiORANGE™ is an orange fluorescent probe for the detection of hydroxyl radical (.OH) or hypochlorous acid (HClO) in living cells. Its nearly red fluorescence spectrum allows multicolor imaging with green (e.g., GFP, FITC) and blue (e.g., Hoechst 33342) fluorophores. Because of its positive charge, OxiORANGE™ tends to localize within mitochondria. It shows a high photostability and is suitable for time-lapse imaging of intracellular hROS generation.


  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
SK3001-01 Hydroxyphenyl Fluorescein (HPF) Liquid type 1mg 589,00 PDF
SK3002-01 Aminophenyl Fluorescein (APF) Liquid type 1mg 589,00 PDF
SK3003-01 NiSPY-3 1mg 716,00 PDF
GC3006-01 HySOx 20μg X 5 627,00 PDF
GC3007-01 HYDROP 30nmolx3 527,00 PDF
GC3004-01 OxiORANGE 1mg 627,00 PDF
  • All prices are in EURO excl. VAT and shipping. Only available in Europe.


  • GORYO: OxiORANGE™ Flyer (PDF)