Microscale thermophoresis

Microscale thermophoresis is based on the detection of a temperature-induced change in fluorescence of a target as a function of the concentration of a non-fluorescent ligand.

On the one hand it is based on a temperature related intensity change (TRIC) of the fluorescent probe, which can be affected by binding events.

The fluorescence of a target molecule can be extrinsic or intrinsic (aromatic amino acids) and is altered in temperature gradients due to two distinct effects.

[11][12] On the other hand, MST is also based on the directed movement of molecules along temperature gradients, an effect termed thermophoresis.

The temperature of the aqueous solution in the laser spot is raised by ΔT=1-10 K. Before the IR-Laser is switched on a homogeneous fluorescence distribution Fcold is observed inside the capillary.

The thermal relaxation induces a binding-dependent drop in the fluorescence of the dye due to its local environmental-dependent response to the temperature jump (TRIC).

This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant KD as result.

Principle of the MST technology: MST is performed in thin capillaries in free solution thus providing close-to-native conditions (immobilization free in any buffer, even in complex bioliquids) and a maintenance free instrument. When performing an MST experiment, a microscopic temperature gradient is induced by an infrared laser, and TRIC as well as thermophoresis are detected. TRIC depends on the fluorophore's microenvironment, which is typically changed in binding events. Thermophoresis, the movement of the molecule in the temperature gradient, depends on three parameters that typically change upon interaction. Thus, the overall MST signal is plotted against the ligand concentration to obtain a dose-response curve, from which the binding affinity can be deduced.