SLAS

Luminescence Detection Technologies

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Authored by: Paul Taylor, Boehringer Ingleheim Inc.

Contents

Glow Luminescence

Detailed article: Going with the glow

Glow Luminescence provides a long-lived (half life >2 hrs) luminescence signal deriving from the conversion of D-luciferin to oxyluciferin by luciferase in the presence of ATP and molecular oxygen.

GLOW.jpg
Image Source: PerkinElmer Life and Analytical Sciences

Flash Luminescence

Flash luminescence utilizes aequorin which is a photoprotein isolated from the jellyfish Aequoria Victoria. Upon binding to calcium, aequorin oxidizes coelenterazine to coelentramide and as a by product produces CO2 and light (half life of seconds). The primary application is monitoring GPCR stimulation with the production of flash luminescence.

FLASH.jpg
Image Source: PerkinElmer Life and Analytical Sciences

Amplified Luminescent Proximity Homogenous Assay (AlphaScreen)

AlphaScreen (Amplified Luminescent Proximity Homogenous Assay) is a bead-based format that uses donor and acceptor beads. When the beads are in close proximity due to a binding event, laser excitation at 680 nm initiates a cascade when a photosensitizer (phthalocyanine) in the donor bead excites ambient oxygen to the singlet state which then diffuses across to the acceptor bead and reacts with thioxene derivatives. The reaction generates chemiluminescence which transfers to a fluorophore in the acceptor bead, subsequently producing fluorescence in the 520-620 nm range. One of the unique aspects of this format is that due to the lifetime of unquenched singlet oxygen (4 μs), intermolecular distances of up to 200 nm can be probed which include larger biomolecules.

AlphaScreen.gif
Image Source: PerkinElmer Life and Analytical Sciences

Electrochemiluminescence (ECL)

Electrochemiluminescence (ECL) detection uses labels that emit light when electrochemically stimulated. Background signals are minimized because the stimulation mechanism (electrical current) is decoupled from the signal (light). Labels are generally stable, non-radioactive and typically offer a choice of coupling chemistries. Light emission is in the area of 620 nm and this minimizes compound interference. ECL has been developed as a process in which reactive species are generated from stable precursors at the surface of an electrode and the technology has the advantage of high sensitivity with a dynamic range of over six orders of magnitude. In earlier versions of this technology, assays used a ruthenium chelate label and a magnetic bead. Biological activity was directly related to the amount of ruthenium label that bound to the magnetic bead deriving from one assay component labeled with ruthenium and the other bound to the bead. On completion of the assay, assay components were aspirated through a detection cell containing an electrode, washed, exposed to a tripropylamine (TPA) containing buffer and then subjected to a series of redox reactions that generated light at 620 nm. A newer methodology has now been developed in which the plate surface itself contains the electrode, obviating the need for fluidic transfers.

ECL.jpg
Image Source: Global BioSystems

Bioluminescence Resonance Energy Transfer (BRET)

Bioluminescence Resonance Energy Transfer (BRET) is similar to FRET in that it utilizes a non-radiative energy transfer between a donor and an acceptor. The difference is that in BRET the donor molecule is not a fluorescent molecule, but rather a luminescent molecule (coelenterazine which is excited by the enzyme Renilla Luciferase (Rluc)). The acceptor molecule is a fluorescent protein such as green fluorescent protein (GFP). Living cells have the capability of expressing Rluc-GFP recombinantly fused proteins and this has provided useful insights into interactions in cell-based assays. The use of an enzyme to generate the donor luminescence provides the benefit of not needing an excitation source and this contributes to minimizing compound autofluorescence and inner filter effects. One limitation of BRET is been the degree of overlap of the donor and acceptor's emission spectra (spectral resolution = 50 nm). To address this in the newer BRET2 format, a coelenterazine analog (Deep Blue C) has been developed which provides a spectral resolution of 110 nm. However the quantum yield of the new analog is significantly lower, requiring more sensitive detection capability. Additional to BRET2 is the development of a GFP mutant with an emission maximum at 510 nm.

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