AlphaScreen® relies on hydrogel-coated donor and acceptor beads providing functional groups for conjugation to biomolecules. The beads come in proximity when the biomolecules interact by molecular binding, e.g. antibody-antigen or receptor-ligand. Laser excitation at 680 nm of a photosensitizer present on the donor bead results in the production of singlet oxygen. The singlet oxygen migrates to react with a chemiluminescent coating on the acceptor bead. The chemiluminescent coating then activates fluorophores emitting light at 520 - 620 nm. This reaction cascade results in a huge amplification of the signal. The short lifetime of singlet oxygen in aqueous solution (~4 μs) allows diffusion over a distance up to ~200 nm. This means that if donor and acceptor beads are not in proximity no signal is produced.
AlphaScreen® is a homogeneous method that is well suited for miniaturization and is used in drug discovery screening. It has proven to be a reliable and sensitive method in many detection assays, including:
- GPCR secondary messengers like cyclic AMP or IP3.
- Enzyme assays ( , helicases, proteases, phosphatases...)
A laser emitting at 680 nm is the excitation source required and high-sensitivity luminescence using filters is recommended for AlphaScreen® measurements. Allbelow are suitable for AlphaScreen®.
AlphaScreen® is a registered trademark of PerkinElmer.