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

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Anomalous DNA hybridisation kinetics on

gold nanorods revealed via a dual

single-molecule imaging and

optoplasmonic sensing platform

Narima Eerqing1,2,+,*, Hsin-Yu Wu1,2,+, Sivaraman Subramanian1,2, Serge

Vincent1,2, and Frank Vollmer1,2

1Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, United

Kingdom

2Department of Physics and Astronomy, University of Exeter, Stocker Road, Exeter EX4

4QL, United Kingdom

+T hese authors have contributed equally to this work and share f irst authorship

*Email :ne276@exeter.ac.uk /Tel : 0044 07840355637

ABSTRACT

Observing the hybridisation kinetics of DNA probes immobilised on plasmonic nanoparticles is key in

plamon-enhanced ﬂuorescence detection of weak emitting species, and refractive index based single-

molecule detection on optoplasmonic sensors. The role of the local ﬁeld in providing plasmonic signal

enhancements for single-molecule detection has been studied in great detail. Nevertheless, few stud-

ies have compared the experimental results in both techniques for single-molecule studies. Here

we developed the ﬁrst optical setup that integrates optoplasmonic and DNA-PAINT based detection

of oligonucleotides to compare these sub-platforms and provide complementary insights into single-

molecule processes. We record the ﬂuorescence and optoplasmonic sensor signals for individual,

transient 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.

Keywords: single-molecule, plasmonics, whispering-gallery modes, optoplasmonic, DNA-PAINT,

total internal reﬂection ﬂuorescence, localisation microscopy

INTRODUCTION

The interaction between DNA strands is key to many fundamental processes in the cell. The hybridisation

between DNA oligonucleotides is essential for our most sensitive methods of DNA detection, including

state-of-the-art single-molecule techniques

1–3

. Single-molecule techniques have enriched biomolecular

studies by providing details about the kinetics of biological reactions and physiological processes that is not

apparent in their corresponding bulk measurements. Powerful new approaches to single-molecule sensing

arXiv:2210.14686v2 [physics.optics] 27 Oct 2022

and imaging have emerged in the last few decades. One example is ﬂuorescence based single-molecule

imaging, which overcomes the diffraction limit by reconstructing images from high-precision temporal

modulation and the accumulation of single-molecule detection events

4–7

. Among these, photo-activated

localisation microscopy (PALM)

8,9

, stochastic optical reconstruction microscopy (STORM)

10,11

, and

DNA based point accumulation for imaging in nanoscale topography (DNA-PAINT)

12–15

have robustly

demonstrated single-molecule localisation microscopy at the nanoscale. On the other hand, noble metal

nanoparticles of various morphologies have drawn attention for their use in single-molecule sensing due

to their extraordinary optical properties derived from localised surface plasmon resonances (LSPRs). In

a large range of applications, plasmonic nanoparticles have been employed to amplify single-molecule

detection signals. 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)

sensors1,2,19–23.

Along with the development of various single-molecule techniques, it is becoming increasingly

important to compare and cross-validate their results

. Detecting a single-molecule process on two

different optical instruments enables one to gain a deeper understanding of the biomolecular system under

investigation. Recently, we reported a study in which we compare DNA hybridisation events observed

on plasmonic nanorods using an optoplasmonic sensor, with the results obtained on a single-molecule

imaging technique based on DNA-PAINT

. The optoplasmonic sensor measures single-molecule events

within the enhanced near ﬁeld of plasmonic gold nanorods (GNRs) that are attached to an optical WGM

surface. The signals are obtained indirectly via the shift in the resonance of the WGM. On the other

hand, DNA-PAINT provides signals via ﬂuorescence localization microscopy. Although DNA-PAINT

does not require plasmonic enhancement, we performed all DNA-PAINT experiments with molecular

interactions on the surface of GNRs to replicate the conditions of the optoplasmonic sensor system. We

found that both techniques deliver comparable results. Speciﬁcally, we found DNA dissociation kinetics

(i.e. off-rates) for both schemes lay within experimental error.

In this article, we demonstrate the ﬁrst use of a total internal reﬂection ﬂuorescence (TIRF) objective

to perform label-free optoplasmonic sensing and ﬂuorescence imaging of single molecules in one optical

platform. The TIRF objective is employed as an evanescent coupler similar to common coupling methods

such as prism, grating, end-ﬁbre and wave guide couplers to evanescently excite the WGMs on glass

microspheres while enabling single-molecule imaging capability. We use this platform to study the

hybridisation kinetics of DNA oligomers attached to gold nanorods (GNRs). We chose to study the

interaction between DNA oligonucleotides because recent reports show that DNA hybridisation kinetics

on GNRs are seemingly affected by the experimental single-molecule technique. In 2018, Weichun et

al.

studied the single-molecule ﬂuorescence enhancement from GNRs, reporting a disappearance of

DNA hybridisation events over time. This phenomenon was, however, not observed for ’docking’ strands

(immobilized single stranded DNA) bound to glass. Previously, Taylor et al.

conducted DNA-PAINT

experiments on gold nanorods to reconstruct single GNR geometry. They also reported a similar reduction

and disappearance of DNA hybridisation events for ’docking’ strands attached to GNRs. Weichun et

al.24 attributed this effect to the cleaving of Au −Sbonds and thus removal of the docking DNA strands

bound to the GNRs by hot electrons generated in the GNRs. Studying the decrease in DNA hybridisation

event frequency on our dual single-molecule ﬂuorescence imaging and optoplasmonic sensing platform

would provide more detailed insight into the mechanisms behind the reported phenomenon. By probing

single-molecule kinetics with ﬂuorescence and optoplasmonic refractive index based methods in parallel,

we obtain results which show that the anomalous disappearance of DNA hybridisation events over time

2/19

Figure 1. Design of the dual optoplasmonic sensing and imaging platform. Incident laser beam is

focused onto the back focal plane of a TIRF objective, establishing total internal reﬂection on the glass

coverslip surface. Generated evanescent wave is then coupled into the WGM. (a) Schematic of

DNA-PAINT imaging. The zoomed-in view shows the transient interaction between freely defusing

imager DNA strands and docking DNA strands immobilised on the GNRs. The imager denoted by a red

star is the one that contributes ﬂuorescence signals. (b) Typical intensity time trace extracted from

colocalised events in the 5×5 pixels (with red ROI box in the inset) around the GNR. The scale bar =

500 nm. (c) Schematic describing the optoplasmonic sensing principle. The zoomed-in view shows

imager strands interact with docking strands, wherein the imager denoted by a red star is the molecule

that contributes to the sensing signal. The inset depicts the chamber and microsphere positioning. (d)

Typical WGM resonance wavelength trace obtained by tracking the resonance peak position (with

Lorentzian lineshape shown in the inset). FC: ﬁber-coupled collimator; LF: laser-line ﬁlter; L: lens; HWP:

half-wave plate; PBS: polarizing beam splitter; M: Mirror; BS: non-polarizing beam splitter; BFP: back

focal plane; EF: emission ﬁlter; SM: switchable mirror; PD: photodetector.

on GNR arises from the probabilistic permanent binding of complementary strands and that this is not a

result of hot electron cleaving of Au −Sbonds.

DUAL SENSING AND IMAGING SETUP

Figure 1shows the schematic of the experimental setup that we have developed for dual single-molecule

imaging microscopy and optoplasmonic sensing (SIMOPS). Light from a tunable external cavity diode

laser (Toptica DL pro 780) is collimated, expanded, and focused onto the back focal plane of a TIRF

objective (CFI Apo TIRF 100X, 1.49 NA, Nikon). The total internal reﬂection (TIR) off a glass coverslip

is used to evanescently couple to optical WGMs of a fused silica microsphere similar to evanescent

coupling via prisms

. The reﬂected light is then collimated and collected by a lens L4, and a switchable

3/19

mirror (SM) onto a photodetector (PD). Alternatively, the evanescent ﬁeld originating from TIR off the

glass coverslip is used to excite plasmonic GNRs and dye molecules directly near the surface of the glass

coverslip. (Please refer to Supplementary Figure S1 for details of ﬁeld enhancements.) In this case, a

ﬂuorescence image is obtained by replacing the lens L4 with a switchable emission ﬁlter and collecting

the emitted photons on an EMCCD camera (Andor iXon 888).

Figure 1a shows a schematic of the DNA-PAINT measurements. Firstly, plasmonic GNRs are

immobilised on the surface of a glass coverslip that is placed in the sample chamber. The GNR attachment

can be monitored in real-time via the photoluminescence of the GNRs as shown in Figure 1b inset

(highlighted by the red box). Secondly, DNA docking strands are then anchored onto the GNRs via

mercaptohexyl linkers. The sample chamber is then rinsed to remove excess, unbound DNA strands.

Finally, complementary DNA strands with a ﬂuorescent label (DY782) are added to the sample chamber

(See Materials and Methods for more details). The transient hybridisation of freely-diffusing imager

strands and ﬁxed docking strands then produces an increased intensity at the GNR location. The intensity

integrated within a region-of-interest (ROI) of 5

5 pixels around the GNR position then provides the

intensity time traces as shown in Figure 1b. The captured ROIs are stacked and processed in Fiji (ImageJ)

via a single-molecule localisation microscopy package named ThunderSTORM26.

Figure 1c shows the schematic of the experiments performed using the optoplasmonic technique. In

this case, light is coupled to WGMs in an

80 µm

glass microsphere

placed near the coverslip surface.

GNRs are then attached to the microsphere surface. Subsequently, docking DNA strands are immobilised

on the GNRs with a protocol similar to that for the DNA-PAINT. Once the ﬂuorophore-labeled imager

strands are added to the sample chamber (Detailed protocol can be found in the Materials and Methods

section), hybridisation of the imager strands with the docked DNA strands is observed in the form of a

shift in WGM resonance frequency (See inset of Figure 1d ). These frequency shifts are recorded over

time to obtain the single-molecule time traces as shown in Figure 1d (See Materials and Methods for

further context).

MATERIALS AND METHODS

Materials

GNRs with an average diameter of 10 nm and length of 35 nm (i.e. longitudinal plasmon resonance at

λ≈

750 nm) were purchased from Nanopartz Inc.(A12-10-CTAB-750). All DNA oligos were purchased

from Euroﬁns Genomics and their sequences are listed in Table 1. Glass coverslips with a refractive

index of n

≈

1.52, with an aspect ratio of 22 mm

22 mm and a thickness of 170

±5 µm

, were

purchased from Thorlabs (Precision Glass Cover Slips, CG15CH). High-

glass microspheres (n

≈

1.45)

were fabricated with a 30 W CO

continuous wave laser (

λ≈10.6 µm

) purchased from Synrad 48-2,

Novanta Inc., WA, USA. PLL-g-PEG was purchased from SuSOS Surface Technology, Switzerland.

Tris(carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Sigma-Aldrich (Catalog Number

646547). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Sigma

Aldrich, and a 20 mM HEPES buffer with pH

≈

7 was prepared for use as the interaction buffer. All

chemicals for the buffers were purchased from Sigma Aldrich.

SIMOPS Setup Optics

Figure 1 provides the schematic of the SIMOPS setup. In order to couple light into WGMs with an

objective lens, a ﬁber-coupled tunable laser diode with nominal wavelength of 780 nm (DL pro, Toptica)

was collimated with a ﬁber collimator and then expanded by a beam expander (L1 and L2), resulting in a

4/19

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AnomalousDNAhybridisationkineticsongoldnanorodsrevealedviaadualsingle-moleculeimagingandoptoplasmonicsensingplatformNarimaEerqing1,2,+,*,Hsin-YuWu1,2,+,SivaramanSubramanian1,2,SergeVincent1,2,andFrankVollmer1,21LivingSystemsInstitute,UniversityofExeter,StockerRoad,ExeterEX44QD,UnitedKingdom2Departme...

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