An infrared-laser is used to generate precise microscopic temperature gradients within thin glass capillaries that are filled with a sample in a buffer or bioliquid of choice. The fluorescence of molecules is used to monitor the motion of molecules along these temperature gradients. The fluorescence can be either intrinsic (e.g. tryptophane) or of an attached dye or fluorescent protein (e.g. GFP).
Extensive research conducted at the Biophysics Department of the Ludwig-Maximilians-University Munich (LMU) identified the solvation entropy and the hydration shell of molecules as the driving force. Any change of the hydration shell of biomolecules due to changes in their primary, secondary, tertiary and/or quaternary structure affects the thermophoretic movement and is used to determine binding affinities with high accuracy. The dependency on the energetic of the hydration shell allows for sensitivity unmatched by any other technology.
The experimental procedure is straightforward and eliminates expensive and tedious sample preparation.
The Monolith instruments use a capillary format that reduces the overall costs and the setup costs which are typically associated with standard molecular interaction technologies. It also allows to avoid regular maintenance and time consuming assay preparation steps since no valves or pumps are needed and experiments are performed in free solution without surface coupling.
The concentration of a fluorescent molecule is kept constant and the concentration of a binding partner is increased. 4 ul of the respective samples are filled in MST capillaries by simple capillary forces. The IR-Laser is used to create a localized microscopic temperature gradient in the capillary. Simultaneously, local changes of fluorescence intensity due to the motion of labeled molecules in the glass capillaries are observed. For fluorescence read out either a label/fluorescent protein (NT.115/NT.115Pico) or a source for intrinsic fluorescence like tryptophane is used (NT.LabelFree).
Fluorescent molecules or particles are initially distributed evenly and diffuse freely in solution. By switching on the IR-Laser, the molecules experience a thermophoretic force in the temperature gradient and typically move out of the heated spot. In the steady state, this molecule flow is counterbalanced by ordinary mass diffusion. After turning off the laser, the molecules diffuse back to re-establish a homogeneous distribution. The following stages are recorded for each sample: fluorescence signal before turning the IR laser on, thermophoresis of molecules and back diffusion after switching the laser off.
The signal is recorded in all capillaries with varying concentration of the not labeled/fluorescent ligand. Any change of thermophoretic properties is observed as a change in fluorescence intensity.
NanoTemper's unique MST technology has the following features and benefits:
Broad application range
MicroScale Thermophoresis Technology (MST) is based on a physical principle used for the first time in biomolecule analytics
What is MicroScale Thermophoresis?
MicroScale Thermophoresis (MST) is a powerful new technology.
MicroScale Thermophoresis is based on a physical principle used for the first time in bioanalytics, it detects changes in the hydration shell, charge or size of molecules. It allows measuring a wide range of biomolecular interactions under close-to-native conditions: Label-Free, immobilizationFree, in any buffer or complex bioliquid. NanoTemper's unique technology is ideal for basic research applications requiring flexibility in the experimental scale, as well as for pharmaceutical research applications including small molecules profiling.
A technology by NanoTemper
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