Voltage Sensor Probes (VSPs) are a Fluorescence Resonance Energy Transfer (FRET)-based voltage-sensing assay technology, used to measure changes in cells’ membrane potential. This FRET-based measurement is restricted to the plasma membrane and is therefore more relevant than non-FRET (single dye) based approaches that measure signals throughout the cell.


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.
  • Universal methodology—applicable to any target that changes the membrane potential
  • Highly accurate, real-time optical readout—results correlate with patch clamp data
  • Robust HT assays have been described for voltage and ligand-gated targets (Table 1)
Target Reference Chemical classes
GABAA a3 ß3 γ2, a4 ß3 γ2, a4 ß3 δ Adkins, C.E. et al. (2001), J. Biol. Chem. 276 : 38934-38939 Agonists, antagonists, inverse agonist, allosteric modulators
NaV1.5, NaV1.7 Felix, J.P. et al. (2004), ASSAY & Drug Devel. Technol. 2 : 260-268 Antagonists, allosteric modulators
NaV1.8 Liu, C.J. et al. (2006), ASSAY & Drug Devel. Technol. 4 : 37-48 Antagonists, allosteric modulators
NaV1.5, NaV1.7, CaV 2.2, CaV 1.2 Bugianesi, R.M. et al. (2006), ASSAY & Drug Devel. Technol. 4 : 21-35 Antagonists

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 coumarin-phospholipid FRET donor (CC2-DMPE). At rest, the cell interior has a relatively negative potential, and the oxonol dye is in close proximity to the coumarin donor, resulting in efficient FRET. When the cell is depolarized, the oxonol dye will associate to the interior of the cell membrane, thus separating the FRET pair and disrupting FRET (Figure 1).

VSP FRET Pair

Figure 1. The VSP FRET pair consists of a mobile, voltage-sensitive acceptor and an outer membrane-bound donor. In resting cells (with a relatively negative internal potential), both members of the FRET pair bind to the outer surface of the cell membrane, resulting in efficient FRET. When the cells are depolarized, however, the donor remains on the outer surface, but the mobile acceptor rapidly translocates to the inner surface of the cell membrane, resulting in diminished FRET. The emission ratio (the ratio of the donor emission to acceptor emission) reports changes in potential and is low in polarized cells and increases in depolarized cells.

Discover more about the Mechanism of Voltage Sensor Probe Technology and Applications of Voltage Sensor Probes:

The Oxonol Substrates and Loading Concentrations

Invitrogen offers two oxonols; voltage sensitivity and temporal responses depend upon the particular dye combination used in assays. The two oxonols [DiSBAC2(3) or DiSBAC4(3)] differ in their loading characteristics, response times to voltage changes, and the types of applications they are most suited for. Most ion channel screening applications have been conducted with DiSBAC2(3) because it is more water-soluble, sensitive, stable, and easier to load than DiSBAC4(3), and can remain in the extracellular media. The time response of this oxonol is ~500 ms. In contrast, the more hydrophobic DiSBAC4(3) responds to changes to the membrane potential in ~20 ms. Because of its hydrophobicity, DiSBAC4(3) requires Pluronic®-127 surfactant (Sigma-Aldrich, St. Louis, MO) for cellular loading and a washing step to remove excess dye. DiSBAC4(3) is useful for applications that require faster response time or no added dye in the extracellular solution.

For most cell types, optimal CC2-DMPE and DiSBAC2(3) loading concentrations fall between 0.5–20 µM. However, higher CC2-DMPE concentrations (up to 40 µM) may be necessary for some cell types. DiSBAC2(3) loading concentrations should be optimized below 20 µM, due to solubility issues. Optimal DiSBAC4(3) loading concentrations range between 2–3 µM.

Background Signal Suppression
VABSC-1 Dye (Voltage Assay Background Suppression Compound) is a unique dye that optionally can be added to suppress background signals.

Contents and Storage
The Voltage Sensor Probes Sets (Cat. nos. K1016 and K1046) provide sufficient reagent volumes for performing approximately 20 sets (each set is a 96-well assay plate done in 100-µl volume) of assays, each using 5 µM CC2-DMPE and 10 µM of either DiSBAC2(3) or DiSBAC4(3). VABSC-1 (Cat. no. K1019) is supplied as 1 g of powder which is sufficient for performing approximately 100 sets (each set is a 96-well assay plate done in 100-µl volume) assays, each using 250 µM VABSC-1. CC2-DMPE, DiSBAC2(3) and DiSBAC4(3) are also available in larger sizes for high-throughput screening applications.

For Research Use Only. Not for use in diagnostic procedures.

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