Comparing Transient Oligonucleotide Hybridization Kinetics Using DNA-PAINT and Optoplasmonic Single-Molecule Sensing on Gold Nanorods

Abstract

We report a comparison of two photonic techniques for single-molecule sensing: fluorescence nanoscopy and optoplasmonic sensing. As the test system, oligonucleotides with and without fluorescent labels are transiently hybridized to complementary 'docking' strands attached to gold nanorods. The measured single-molecule kinetics is compared to discern the influence of fluorescent labels as well as factors arising from different sensing geometries. Our results demonstrate that DNA dissociation is not significantly altered by the fluorescent label, while DNA association is affected by geometric factors in the two techniques. These findings open the door to exploiting plasmonic sensing and fluorescence nanoscopy in a complementary fashion, which will aid in building more powerful sensors and uncovering the intricate effects that influence the behavior of single molecules.

Anomalous DNA hybridisation kinetics on gold nanorods revealed via a dual single-molecule imaging and optoplasmonic sensing platform

“…Different concentrations are used for optoplasmonic and DNA-PAINT measurements due to the different distributions of detectable docking sites. 2 In the case of DNA-PAINT (Fig. 3(b)), 5 min of idle time in the measurement is delineated by break lines.…”

“…A detailed discussion of the discrepancy in measuring association rate of DNA hybridisation on both techniques is provided in our previous work. 2…”

“…Examples for this are the plasmonic enhancement of fluorescence signals adjacent to nanoparticles, 16–18 and the enhancement of the label-free signals from whispering-gallery mode (WGM) sensors. 1,2,19–23…”

From Whispering Gallery Mode Resonators to Biochemical Sensors

“…We can easily question the way in which a single-molecule detection would be possible in common practice and how to reach enough specificity to ensure targeted detection. 192 In most cases, "single-molecule" sensing is only obtained statistically or using larger nanoparticles embedding hundreds or even thousands of molecules to detect in order to achieve a sufficient signal shift and be experimentally viable. It is therefore important to contextualize the manipulation not to mislead the reader with too optimistic theoretical LOD values.…”

“Grafting-To” Covalent Binding of Plasmonic Nanoparticles onto Silica WGM Microresonators: Mechanically Robust Single-Molecule Sensors and Determination of Activation Energies from Single-Particle Events

“…Detection down to the single-molecule level has been made possible through enhancing the WGM signal by attaching plasmonic nanoparticles (PNPs) to a ~100 μm diameter glass microsphere WGM resonator [ 10 , 11 , 12 , 13 , 14 ]. The integration of PNPs with WGMs, resulting in so-called optoplasmonic WGM sensors, led to major breakthroughs in biosensing; for example, the real-time detection of the single-molecule hybridization events of DNA oligonucleotides and the detection of single-molecule WGM signals corresponding to disulfide bond formation [ 3 , 15 , 16 , 17 , 18 ]. PNPs with different geometries, such as nanorods, nanostars, and nanoshells, can be employed for assembling a single-molecule optoplasmonic WGM sensor [ 4 ].…”