Principle, Process and Advantages of Microscale Thermophoresis Technology

The study of biomolecular interactions can help to explore the signal transduction pathways and regulatory mechanisms, physiological and biochemical metabolic pathways of organisms. 2010, Wienken et al. reported for the first time the use of MST technique to study the interactions of proteins and small molecules in biological solutions. Based on the advantages of fluorescence detection technology, microscale thermophoresis (MST) technique combines fluorescence detection with thermophoretic phenomena to provide an efficient, accurate and sensitive detection method.

MST has been successfully applied to study interactions between biomolecules, mainly including interactions between oligonucleotides, interactions between proteins and DNA, interactions between proteins and proteins, interactions between proteins and small molecules, and interactions between proteins and liposomes.

Application of microscale thermophoresis (Jerabek et al., 2014)

Principle of MST

The temperature change (ΔT) in the reaction system can lead to a decrease in the number of particles in the region of elevated temperature. The thermoswimming motion is quantified by the Soret (Soret) coefficient ST: Chot/Ccold = exp(-STΔT). The weakening of the thermoswimming motion is due to the molecule-solvent cross-interface interaction. With constant buffer, thermoswimming motions can be analyzed for molecular size, charge and solvent entropy values. If the thermoswimming motions of protein and protein-ligand complexes are significantly different, the formation of the complexes causes changes in molecular size, charge and solvent energy. Even if the binding does not significantly change the protein size and charge, MST can still detect whether binding has occurred by detecting the change in solvent entropy value.

Process of MST

A 6ul sample volume is filled in the MST capillary and then used to create a local temperature gradient. Changes in regional fluorescence intensity due to the movement of labeled molecules in the glass capillary are then observed. Both labeled fluorescent dyes/fusion expressed fluorescent proteins can be used to emit light (NT.115 system) and tryptophan autofluorescence can be detected (NT.LabelFree system). Various parameters of the interaction between these two molecules are obtained by detecting the effect of different concentrations of the binding molecules on the distribution of fluorescent molecules in the thermophoretic dynamic equilibrium state.

The fluorescent molecules are initially freely and uniformly distributed. Under IR laser irradiation, the molecules are subjected to the force of thermophoretic motion and move from the heated region to the low temperature region. Meanwhile, the molecules are subjected to concentration gradients and mass diffusion forces. Finally the molecules reach equilibrium under the thermophoretic action force and mass diffusion force and form a steady state. After turning off the IR laser, the molecular diffusion reconstructs the uniform distribution state. The following figure shows the process.

MicroScale Thermophoresis (Schubert et al., 2015).

(A) Technical setup of the MST. (B) MST time trace—a profile depicting the movement of molecules in a temperature gradient. (C) Results of a typical MST experiment

Advantages of MST

• Sample consumption is low, minimum only 4ul is required.
• The detection speed is fast, and the detection can be completed in 10min.
• Wide range of affinity assays, from pM to mM
• A wide range of different sample types, such as animal, fungal, bacterial, plant extracts, biological fluids
• Affinity tests for a variety of different samples, from proteins and ions to proteins and cells. No fear of molecular weight and interaction objects
• Assay in the most natural liquid environment, no fixation required
• No buffer limitations
• No time-consuming maintenance