Amplex® Red Technology Overview
How does Amplex® Red reagent work?
Amplex® Red reagent is a colorless substrate that reacts with hydrogen peroxide (H2O2) with a 1:1 stoichiometry to produce highly fluorescent resorufin (excitation/emission maxima=570/585 nm) (Figure 1). Read more about Amplex® Red reagent chemistry.
Amplex® Red in Action
Amplex® Red reaction can be used to routinely detect as little as 10 picomoles of H2O2 in a 100 µL volume (50 nM, Figure 2), at least a 10-fold greater sensitivity than that attained with the commonly used scopoletin assay for hydrogen peroxide. In the scopoletin assay, HRP catalyzes conversion of the fluorescent scopoletin to a nonfluorescent product. Unlike scopoletin, the Amplex® Red reagent is a fluorogenic substrate with very low background fluorescence. Consequently, assays using Amplex® Red as the substrate result in an increase in fluorescence, not a decrease — an inherently superior method for enzymatic assays.
Other advantages of the Amplex® Red reaction over scopoletin-based H2O2 assays include high chemical stability of the Amplex® Red reagent and its fluorescent product, resorufin, and the long-wavelength spectra of resorufin. Because resorufin has excitation/emission maxima of ~570/585 nm (versus 360/460 nm for scopoletin), there is much less interference from autofluorescence in most biological samples.
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| Figure 1—Principle of coupled enzymatic assays using our Amplex® Red reagent. Oxidation of glucose by glucose oxidase results in generation of H2O2, which is coupled to conversion of the Amplex® Red reagent to fluorescent resorufin by HRP. | Figure 2—Detection of H2O2 using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188). The inset shows the sensitivity and linearity of the assay at low levels of H2O2. |
Enzyme-Coupled Assays
We have used our Amplex® Red reagent to develop a variety of novel fluorogenic and chromogenic assays for enzymes that produce hydrogen peroxide (see an example in Figure 3). Furthermore, these coupled assays permit the ultrasensitive quantitation of a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline and acetylcholine, as well as hydrogen peroxide. (See complete list of Amplex® Red enzyme-coupled assays available.)
Our patented PiPer Phosphate Assay Kit (P22061) and PiPer Pyrophosphate Assay Kit (P22062) also utilize our exclusive Amplex®Red technology for the continuous assays of enzymes that produce either inorganic phosphate or pyrophosphate.
View the enzyme cascades for all of the Amplex® Red Assays
Our patented PiPer Phosphate Assay Kit (P22061) and PiPer Pyrophosphate Assay Kit (P22062) also utilize our exclusive Amplex®Red technology for the continuous assays of enzymes that produce either inorganic phosphate or pyrophosphate.
Figure 3 — Example of enzyme-coupled Amplex® Red assays. The Amplex® Red Cholesterol Assay Kit (A12216) uses cholesterol oxidase to produce hydrogen peroxide, which is then detected by the Amplex® Red reagent in the presence of horseradish peroxidase (HRP). Similarly, the Amplex® Red Acetylcholine/ Acetylcholinesterase Assay Kit (A12217) uses choline oxidase to produce H2O2.
Amplex® UltraRed reagent - brighter and more sensitive
Our Amplex® UltraRed reagent (A36006) improves upon the performance of the Amplex® Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in horseradish peroxidase or horseradish peroxidase-coupled enzyme assays (Figure 4). Fluorescence of the oxidized Amplex® UltraRed reagent (Amplex® UltroxRed reagent) is also less sensitive to pH (Figure 5), and the substrate and its oxidation product exhibit greater stability that the Amplex® Red reagent in the presence of hydrogen peroxide (H2O2) or thiols such as dithiothreitol (DTT). We have also found that the Amplex® UltraRed reagent is the better choice when using cellular extracts (Figure 6).
 Figure 4 — Sensitivity of Amplex® UltraRed reagent. Detection of hydrogen peroxide was performed using Amplex® UltraRed reagent (red square) or Amplex® Red reagent (blue triangle). The inset shows the sensitivity and linearity of the Amplex® UltraRed assay at low levels of H2O2.  Figure 6 — Amplex® UltraRed reagent activity in cell extracts. Hydrogen peroxide production in Jurkat cell lysates was detected using Amplex® UltraRed reagent in HEPES (pH 7.0) (red squares) or Amplex® Red reagent in Tris-HCl (pH 7.5) (blue triangles). |
 Figure 5 — pH resistance of Amplex® UltraRed reagent. Comparison of pH-dependent fluorescence of Amplex® UltraRed reagent (solid blue circles) and Amplex® Red reagent (open blue squares). Fluorescence intensities were measured using excitation/emission of ~570/585 nm. |
Advantages of the Amplex® UltraRed reagent over chromogenic reagents
| Peroxidase Detection Reagent | Abs* | Em* | notes |
|---|---|---|---|
| Amplex® UltraRed reagent | 568 | 581 |
|
| Amplex® Red reagent | 570 | 585 |
|
| ABTS (2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) | 405 | n/a |
|
| TMB (3,3',5,5'-tetramethylbenzidine) | 450 (stopped reaction) 653 (kinetic assay) | n/a |
|
* Absorption (Abs) and fluorescence emission (Em) maxima, in nm. NA = Not applicable.
Customize your Amplex® Red and Amplex® UltraRed assays
We offer custom services with the Amplex® Red assays including:
For more information, please contact our Custom Assays Group
- Substitution of Amplex® Red reagent in an existing assay with Amplex® UltraRed reagent
- Special bulk packaging of our current assays
- Build your own enzyme-coupled Amplex® UltraRed or Amplex® Red assay
For more information, please contact our Custom Assays Group
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