Amplex™ Red and Amplex™ UltraRed Reagents

In the presence of horseradish peroxidase (HRP), the Amplex Red and UltraRed reagents react in a 1:1 stoichiometry with H₂O₂ to produce highly fluorescent, strongly absorbing resorufin (ex/em ∼570/585 nm) or Amplex UltraRed (ex/em ∼568/581 nm), respectively. These oxidized reaction products have a broad red emission with little interference from blue and green autofluorescence found in most biological samples. Because H₂O₂ is produced in many different enzymatic reactions, the Amplex Red and UltraRed reagents coupled with HRP can be used to detect the activity of many different enzymes. These reagents are also used to detect H₂O₂ released from biological samples, including cells and isolated mitochondria.

Features of the Amplex Red reagent:
• Sensitive and fast detection of HRP activity or H₂O₂ for solution-based assays and ELISAs
• Detection of H₂O₂ produced in many different enzymatic reactions
• Measurement with either fluorescence or absorbance, permitting either fluorogenic or chromogenic detection

The Amplex Red reagent has been used to develop a variety of novel fluorogenic and chromogenic assays for enzymes that produce hydrogen peroxide. When coupled with added horseradish peroxidase, these assays that generate H₂O₂ permit the ultrasensitive quantitation of a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline, and acetylcholine.

The Amplex UltraRed reagent offers brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays compared to Amplex Red. The Amplex UltraRed reagent is also less sensitive to acidic pH and exhibits improved stability in the presence of H₂O₂ or thiols such as dithiothreitol (DTT). For HRP-based ELISA applications, this results in at least two-fold greater sensitivity compared to Amplex Red.

Benefits of the Amplex UltraRed reagent over Amplex Red:
• Brighter signals and enhanced sensitivity for HRP activity and H₂O₂
• Less sensitive to acidic pH
• Improved stability in the presence of H₂O₂ or thiols such as DTT

The Amplex Red and Amplex UltraRed reaction products resorufin and Amplex UltroxRed have high extinction coefficients so they can be measured either fluorometrically or spectrophotometrically with absorbance at ∼560 nm, providing flexibility in detection of H₂O₂, various enzyme assays, and HRP-based ELISAs.