Regensburg 2010 – wissenschaftliches Programm
BP 23.10: Vortrag
Donnerstag, 25. März 2010, 12:45–13:00, H43
Biomolecule Binding quantified with Thermophoresis — •Christoph J Wienken, Philipp Baaske, Dieter Braun, and Stefan Duhr — Systems Biophysics, Center for Nanoscience, LMU München, Germany
Methods to measure biomolecule interactions are essential for medicine, biology and pharmaceutical industry. We use thermophoresis, the directed movement of molecules in a temperature gradient, to quantify a wide range of interactions like protein protein, protein DNA and protein small molecule.
By combining highly defined microfluidics with all-optical heating and detection, the thermophoretically induced concentration change is measured with high precision. We fluorescently label one binder and track the changes of thermophoretic depletion while titrating the binding partner. The results are quantitative binding curves allowing to measure dissociation coefficients in the picomolar to milimolar range.
Advantages of the method are low volume consumption, fast response time and surface-free detection. However the measurement in various physiological buffers is the hallmark of the approach. Affinities can be measured in highly complex biological fluids as blood serum.
Thermophoresis is performed in bulk fluid, and significant background signals from surface binding are avoided. Competing surface-based methods such as ELISA or surface plasmon resonance have to measure on non-physiological surfaces that are prone to unspecific biomolecule adsorption and do not allow measurements in undiluted biological liquids.