Abstract
Biochemical affinity assays inherently involve interactions of heterogeneous nature. We report a methodology to discriminate between and accurately characterize specific and nonspecific interactions in force-induced dissociation assays. Ligand-coupled superparamagnetic particles are incubated on surfaces coated with a mixture of specific receptors and nonspecifically interacting proteins. Consequently, a mixed population of surface bound particles is formed with different binding natures. Magnetic field gradients are used to apply translational forces on the bound particles. Using a multicomponent dissociation analysis, we are able to make a distinction between weak nonspecific interactions, strong nonspecific interactions, and specific interactions. We validate the model by comprehensive experiments in which the biochemical components and applied forces are varied. The low-force data yield reliable values for the spontaneous dissociation rates of single-molecule specific bonds, and at high forces, the bond barriers are modified by the applied force. The results generate a new perspective for applications of magnetic force affinity assays in studies of heterogeneous molecular biorecognition.
Original language | English |
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Pages (from-to) | 9287-9294 |
Number of pages | 8 |
Journal | Analytical Chemistry |
Volume | 84 |
Issue number | 21 |
DOIs | |
Publication status | Published - 2012 |