Voltage Sensor Probes (VSPs) are a Fluorescence Resonance Energy Transfer (FRET)-based voltage-sensing assay technology used to measure changes in cellular membrane electrical potential. VSPs are ideal for screening compounds that modulate ion channels because they provide:
• High-throughput capabilities-screen 32,000 samples in one day
• Highly sensitive detection-accurately and precisely report a 1% change in the Emission Ratio per mV
• Rapid detection response-sub-second time response
• Ratiometric readout (the Emission Ratio)-reduces experimental errors arising from well-to-well variations in
• cell number, addition artifacts, dye loading, plate inconsistencies, temperature fluctuations, and signal intensities. Allows for highly accurate, real-time optical readout of changes in membrane potential
• Universal methodology-applicable to any target that changes the membrane potential
• Highly accurate, real-time optical readout-results correlate with patch clamp data
How VSPs Work
VSPs use a FRET pair composed of one of two highly fluorescent, mobile, voltage-sensitive acceptor oxonols [DiSBAC2(3) or DiSBAC4(3)] and a fluorescent, membrane-bound coumarinphospholipid FRET donor (CC2-DMPE). When the cell interior has a relatively negative potential, the oxonol dye will bind to the exterior of the cell membrane, resulting in efficient FRET. When the cell interior has a relatively positive potential, however,
the oxonol dye will bind to the interior of the cell membrane, thus separating the FRET pair and disrupting FRET.
DiSBAC2(3) is used for most ion channel screening applications because it is more sensitive, easier to load, more stable than DiSBAC4(3), and can remain in the extracellular media. The time response of this oxonol is ∼500 ms. DiSBAC4(3) requires a surfactant for cellular loading and a washing step to remove excess dye, but responds to changes to the membrane potential in ∼20 ms. This dye is useful for applications that require faster response time or no added dye in the extracellular solution.
VABSC-1 Dye (Voltage Assay Background Suppression Compound) performs in conjunction with CC2-DMPE to suppress background signal, providing optimal performance and minimal toxicity.