Introduction: Biosensors based on plasmonic gold nanorods have recently reached single-molecule sensitivity owing to the sensitivity of the nanorod plasmon to local dielectric changes. Previous studies have utilized gold nanorods to track protein-binding to the nanorod surface, but a system with tunable protein affinities, ie – aptamer-based systems, are attractive in that they are tunable to function at a range of biologically-relevant timescales and concentrations. Additionally, inhibition of nonspecific nanorod surface interactions is essential, as biological fluids contain a concentrated mixture of many proteins. In SPR studies, PEG molecule coatings have been demonstrated to effectively reduce surface charge and subsequently inhibit electrostatic attractions between large biomolecules and gold surfaces. Here, protein-targeting DNA aptamers were mixed with short, PEG molecules and appended to a gold nanorod surface. The plasmon wavelength was monitored to track single protein-aptamer binding events and the effect of PEG in suppressing non-specific interactions was investigated.
Method: Following immobilization of gold nanorods on a coverslip, varying ratios of thiolated DNA aptamers and thiol-PEG were added to the coverslip. Next, protein solutions were introduced to the sample in a flow chamber setup. We employed dark-field, single-particle scattering spectroscopy to track the nanorod plasmon shifts due to single protein-aptamer binding events in real-time.
Results: In samples with mixed PEG:aptamer coverage, step-wise plasmon scattering intensity changes were observed upon protein addition, indicative of single protein binding events. In control samples without aptamer or PEG, non-specific interactions induced similar step-wise intensity changes. However, on nanorods coated with PEG alone, no plasmon scattering intensity change was observed, even at high protein concentrations.
Discussion: Single protein-aptamer binding events were successfully measured on samples containing a mixed PEG:aptamer surface, as indicated by step-wise changes in the plasmon scattering intensity. Additionally, surface coating with PEG successfully suppressed non-specific interactions with the nanorod surface. Future studies with this system include tuning the binding affinity of the aptamer to match biologically relevant concentrations and protein detection in biological fluids.
