Single-molecule Characterisation of DNA Hybridization via Fluorescence Microscopy and Optoplasmonic Sensing Approaches

Abstract

The single-molecule study provides an unprecedented deep view into the biological process, unveiling the hidden heterogeneity that is hard to observe in ensemble methods. Recently, various techniques have shown the detection of biomolecules with single-molecule level sensitivity. However, each technique has its unique advantages and drawbacks. Single-molecule techniques can influence the molecular systems that they intend to detect in different ways. For example, fluorescence labels may affect the kinetics and dynamics of biomolecular interactions, while plasmonic-based approaches use local field enhancements that are not uniform across the involved plasmonic nanostructures, both of which can play a significant role in the observed statistics. Before one can combine the information obtained from fluorescence and optoplasmonic techniques, single-molecule experiments must be compared and cross-validated.

Here we first compare the detection of DNA hybridization on fluorescence-based imaging technique and optoplasmionic sensors. We investigate the impact of (i) the presence of labels, and (ii) the potential influence of the plasmonic nanoparticle surface. Our measurements reveal that the dissociation rates of hybridized DNA strands are approximately the same for both techniques. Our study establishes the equivalence of both techniques for this DNA molecular test system and can serve as the basis for combining these techniques in other single-molecule studies. With optoplasmonic sensing, our results indicate that one may benefit from the larger binding efficiency of fluorescence imaging while the impact of the label is checked with the optoplasmonic sensing platform.

To further combine the two single-molecule characterisation systems, we developed the first optical setup that integrates optoplasmonic and fluorescence-based detection. These sub-platforms provide complementary insights into single-molecule processes. We record the fluorescence and optoplasmonic sensor signals for individual, transient DNA hybridisation events. The hybridisation events are observed in the same sample cell and over a prolonged time (i.e. towards high binding site occupancies). A decrease in the association rate over the measurement duration is reported. Our dual optoplasmonic sensing and imaging platform offers insight into the observed phenomenon, revealing that irreversible hybridisation events accumulate over detected step signals in optoplasmonic sensing. Our results point to novel physicochemical mechanisms that result in the stabilisation of DNA hybridisation on optically-excited plasmonic nanoparticles.