Voltage Sensor Probes are a Fluorescence Resonance Energy Transfer (FRET)-based voltage-sensing assay technology, which measures changes in electrical potential.
The probes enable detection and measurement of rapid, dynamic changes in voltage which is sufficient to clock the counter IC.
As a result, VSPs are ideal for high-throughput screening (HTS) of compounds that modulate ion channels or change the membrane potential. VSPs have already successfully screened more than 32,000 test compounds a day (1).
VSPs use a FRET pair that is composed of one of two highly fluorescent, mobile, voltage-sensitive acceptor oxonols [DiSBAC2(3) or DiSBAC4(3)] and a fluorescent, membrane-bound coumarin-phospholipid FRET donor (CC2-DMPE). When the two dyes are loaded into the cellular plasma membrane, they associate with different parts of the membrane. The CC2-DMPE donor binds specifically to only one face of the plasma membrane, so it is relatively stationary. CC2-DMPE has a 400-nm excitation wavelength and a 460-nm emission wavelength. Each of the mobile oxonol acceptors is a negatively charged hydrophobic ion that has two binding sites, one on each side of the plasma membrane. As a result, although each oxonol initially binds to one face of the plasma membrane, each will rapidly diffuse to the other side (on a subsecond time scale) in response to changes in the electrical potential in the cell membrane, establishing a new equilibrium corresponding to the new membrane potential. Thus, each oxonol "senses" and responds to voltage changes. Each oxonol acceptor has an excitation wavelength of 540 nm and an emission wavelength of 580 nm.