Quantification of biomolecular binding reactions in their native environment is crucial for biology and medicine. However, reliable methods are rare. We have developed a new immobilization-free method in which thermophoresis, the movement of molecules in a thermal gradient, is used to determine binding curves; this method can be used to study binding in various buffers as well as in human blood serum. The assay does not rely on surface contact and requires only an unspecific fluorescence marker on one of the binding partners.

Aptamers are nucleic acid ligands selected in vitro for their ability to bind to specific molecular targets.[1–4] They are promising candidates for diagnostic applications because of their affinity and specificity—comparable to that of antibodies—and the ease with which novel aptamers can be designed.[5] Aptamers have been implemented in a variety of sensing technologies [6] including optical approaches like “aptamer beacons”,[7] electronic-sensing strategies,[8] and techniques based on changes in mass [9] or force.[10] In most aptamer-based binding assays, the signal transduction mechanism depends on the molecular recognition mechanism. As a result the aptamers must be designed not only to adopt an appropriate conformation to bind to a target (recognition) but also to undergo a binding-induced conformational change, which affects the fluorescence of a dye [8] or the electron transfer [9] of a redox tag to an electrode (signal transduction). This linkage between target recognition and signal transduction sets obstacles for the design of aptamers. Often aptamers must be modified with two labels, which results in reduced binding affinity or even complete suppression of binding.[11] These restrictions can be reduced by separating the molecular recognition from the signal transduction by using additional competitor oligonucleotides, complementary to the aptamer, as signal transduction elements.[12]