Recent developments in aptamer chemistry open up opportunities for new tools for protein biosensing. In this work, we present an approach to use immobilized slow off-rate modified aptamers (SOMAmers) site-specifically labeled with a nitroxide radical via azide-alkyne click chemistry as a means for detecting protein binding. Protein binding induces a change in rotational mobility of the spin label, which is detected via solution-state electron paramagnetic resonance (EPR) spectroscopy. We demonstrate the workflow and test the protocol using the SOMAmer SL5 and its protein target, platelet-derived growth factor B (PDGF-BB). In a complete site scan of the nitroxide over the SOMAmer, we determine the rotational mobility of the spin label in the absence and presence of target protein. Several sites with sufficiently tight affinity and large rotational mobility change upon protein binding are identified. We then model a system where the spin-labeled SOMAmer assay is combined with fluorescence detection via diamond nitrogen-vacancy (NV) center relaxometry. The NV center spin-lattice relaxation time is modulated by the rotational mobility of a proximal spin label and thus responsive to SOMAmer-protein binding. The spin label-mediated assay provides a general approach for transducing protein binding events into magnetically detectable signals.
Keywords: EPR spectroscopy; aptamer; protein assay; quantum sensing; spin labeling.