Microscale thermophoresis

Microscale thermophoresis (MST) is a technology for the interaction analysis of biomolecules. Microscale thermophoresis is the directed movement of particles in a microscopic temperature gradient. Any change of the hydration shell of biomolecules due to changes in their structure/conformation results in a relative change of the movement along the temperature gradient and is used to determine binding affinities. MST allows measurement of interactions directly in solution without the need of immobilization to a surface (immobilization-free technology).

Affinity

Stoichiometry

Thermodynamic parameters

Additional information

MST is based on the directed movement of molecules along temperature gradients, an effect termed thermophoresis. A spatial temperature difference ΔT leads to a depletion of molecule concentration in the region of elevated temperature, quantified by the Soret coefficient S_T: c_hot/c_cold = exp(-S_T ΔT)

Thermophoresis depends on the interface between molecule and solvent. Under constant buffer conditions, thermophoresis probes the size, charge and solvation entropy of the molecules. The thermophoresis of a fluorescently labeled molecule A typically differs significantly from the thermophoresis of a molecule-target complex AT due to size, charge and solvation entropy differences. This difference in the molecule's thermophoresis is used to quantify the binding in titration experiments under constant buffer conditions.

The thermophoretic movement of the fluorescently labelled molecule is measured by monitoring the fluorescence distribution F inside a capillary. The microscopic temperature gradient is generated by an IR-Laser, which is focused into the capillary and is strongly absorbed by water. The temperature of the aqueous solution in the laser spot is raised by up to ΔT=5 K. Before the IR-Laser is switched on a homogeneous fluorescence distribution F_cold is observed inside the capillary. When the IR-Laser is switched on, two effects, separated by their time-scales, contribute to the new fluorescence distribution F_hot. The thermal relaxation time is fast and induces a binding-dependent drop in the fluorescence of the dye due to its local environmental-dependent response to the temperature jump. On the slower diffusive time scale (10 s), the molecules move from the locally heated region to the outer cold regions. The local concentration of molecules decreases in the heated region until it reaches a steady-state distribution.

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