Europium Tetracycline (EuTc)
sensitive fluorescent dye to assay peroxide
The EuTc-Hydrogen Peroxide Assay is based on a novel, Europium-based fluorescent reagent that is used to detect free or enzymatically produced H2O2. Europium tetracycline (EuTc) detects hydrogen peroxide in a fast and easy method, and without the need for additional enhancer solutions. This makes EuTc a sensitive reagent to monitor catalases, peroxidases or glucose-oxidase activities through in vitro enzymatic assays, or to study substances that inhibit or activate these enzymes.
Hydrogen peroxide (H2O2) is a reactive metabolic by-product that is a key regulator in a number of oxidative stress-related states. Functioning through NFκB and other factors, hydroperoxide-mediated pathways have been linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down’s syndrome. Due to its implication in these many disease states, there is much interest in sensitive assays to monitor hydrogen peroxide in biochemical, clinical, as well as in environmental samples. In addition, there is much research on the enzymes that produce and eliminate hydrogen peroxide, and on their inhibitors.
Active Motif’s EuTc-Hydrogen Peroxide Assay is based on a novel fluorescent reagent that is used to detect free or enzymatically produced H2O2. Europium Tetracycline (EuTc) detects hydrogen peroxide in a fast and easy method, and without the need for additional enhancer solutions. This makes EuTc a sensitive reagent to monitor catalases, peroxidases or glucose-oxidase activities through in vitro enzymatic assays, or to study substances that inhibit or activate these enzymes.
The main advantages of using EuTc for luminescent peroxide detection are:
- Fast, 10-minute incubation time
- Applicable to turbid samples
- Functions at neutral pH
- No need for enzymatic amplification
- Large Stoke’s shift decreases interference of background fluorescence
- Long decay time enables time-resolved, or time-gated, measurement
EuTc alone is weakly fluorescent. But, EuTc combines rapidly with free H2O2 to form a EuTc-H2O2 complex that is 15-fold brighter than EuTc alone (Figure 1). Measuring fluorescent intensity makes it possible to quantify the amount of H2O2 present in a sample (Figure 2). Thus, the basic principle of the assays is based upon the following equilibrium:
EuTc (weakly fluorescent) + H2O2 ↔ EuTc-H2O2 (strongly fluorescent)
Figure 1: Spectral properties of the EuTc dye and the EuTc-H2O2 complex.
Figure 2: Calibration graph for H2O2 concentration.
Lifetime measurement for reduced background
In addition to measuring H2O2 levels via increases in fluorescent intensity, EuTc can also be used to measure changes in the decay time (lifetime) of the EuTc-H2O2 complex (Figure 3). The use of lifetime-based measurement has significant advantages when measuring hydrogen peroxide levels in biological fluids as such samples typically have high levels of auto-fluorescence that can interfere with intensity-based measurements. Because the EuTc-H2O2 signal is relatively long lived (Table 1), lifetime measurement can completely eliminate any background contributed by the light source, the biological sample (typically ~0.1-10 ns) or by free EuTc.
Figure 3: Kinetic determination of peroxidase activity.
|EuTc||406 nm||617 nm||30 µs|
|EuTc-H2O2||406 nm||617 nm||60 µs|
|Table 1: Lifetime measurements of free and peroxide-bound EuTc.|
The EuTc-Hydrogen Peroxide Assay has been described and used in the following publications:
- “A Europium Ion-based luminescent sensing probe for hydrogen peroxide” by Wolfbeis et al (2002) Angewandte Chemie International Edition 41(23):4495-4498.
- “Determination of the activity of catalase using a europium(III)-tetracycline-derived fluorescent substrate” by Wu et al (2003) Analytical Biochemistry 320:129-135.
- “Detection of Hydrogen Peroxide in River Water via a Microplate Luminescence Assay with Time-Resolved (“Gated”) Detection” by Lei et al (2003) Microchim. Acta 143:269-274.
- “Time-Resolved Luminescence Imaging of Hydrogen Peroxide Using Sensor Membranes in a Microwell Format” by Schaeferling et al (2003) Applied Spectroscopy 57(11):1386-1392.
- “Time-resolved enzymatic determination of glucose using a fluorescent europium probe peroxide” by Wu et al (2004) Anal. Bioanal. Chem. 380:619-626.
- “Nonenzymatic direct assay of Hydrogen Peroxide at neutral pH using the Eu3Tc Fluorescent Probe” by Duerkop et al (2005) Journal of Fluorescence 15(5):755-761.
- “Enhancement of Europium Luminescence in Tetracycline-europium Complexes in the presence of urea Hydrogen peroxide” by da Silva et al (2005) Journal of Fluorescence 15(5):667-671.
- “A Novel method for Time-Resolved Fluorimetric Determination and Imaging of the Activity of peroxidase, and Its Application to an Enzyme-Linked Immunosorbent Assay” by Lin et al (2006) Chemistry 12 (10):2730-2738.
- “Detecting hydrogen peroxide in leaves in vivo - a comparison of methods” by Snyrychova et al (209) Pysiologica Plantarum 135:1-18.
EuTc is supplied as a dry powder that can be stored at room temperature. After resuspension, it should be stored at -4°C in the dark. Enough reagent is supplied for 1000 rxns in 96-well plates, or 100 rxns in cuvettes.