Monitoring Single DNA Docking Site Activity With Sequential Modes of an Optoplasmonic Whispering-Gallery Mode Biosensor

TL;DR

This study tackles the challenge of resolving activity at individual DNA docking sites within multiplexed optoplasmonic whispering-gallery-mode sensors. It introduces ratio-based tracing of sequential polar WGM mode shifts, defining ratio identifiers (RRAs) as $\left(\frac{A_2}{A_1},\frac{A_3}{A_2}\right)$ to map docking-site activity at single-molecule resolution. The authors demonstrate that RRAs cluster by docking site across multiple experiments, enabling discrimination of transient interactions from permanent hybridization and revealing site blocking as docking sites become inaccessible. The work suggests broad applicability of RRAs to other multiplexed sensing platforms and outlines future directions using engineered docking-site architectures for angstrom-level resolution, potentially impacting single-molecule biosensing and molecular dynamics studies.

Abstract

In recent years, there has been rapid advancement in single-molecule techniques, driven by their unparalleled precision in studying molecules whose sizes are beyond the diffraction limit. Among these techniques, optoplasmonic whispering gallery mode sensing has demonstrated great potential in label-free single-molecule characterization. It combines the principles of localized surface plasmon resonance (LSPR) and whispering gallery mode (WGM) sensing, offering exceptional sensing capabilities, even at the level of single ions. However, current optoplasmonic WGM sensing operates in a multiplexed channel, making it challenging to focus on individual binding sites of analyte molecules. In this article, we characterize different binding sites of DNA analyte molecules hybridizing to docking strands on the optoplasmonic WGM sensor, using the ratio of the resonance shift between sequential polar WGM modes. We identify specific docking sites that undergo transient interactions and eventually hybridize with the complementary analyte strands permanently.

Figures (5)

• Figure 1: (a)The schematic graph of the whispering gallery polar modes. Gold nanorods are attached to the WGM sensor and overlap with three adjacent modes corresponding to the mode order. (b) Transmission spectrum showing the 3 modes used for the detection of DNA molecules in experiments. (c) Schematic graph of experimental design. An incident laser beam is focused onto the coverslip surface, establishing the total internal reflection. The generated evanescent wave is subsequently coupled into the WGM. The LSPR is excited by the gold nanorods deposited on the surface of the WGM microsphere. (d) Example data-trace of optoplasmonic sensing signals showing the resonance shift, $\Delta \lambda$, experienced by each biosensing mode.
• Figure 2: Spike amplitudes of Mode 1 and 2 (black dots) collected from first 45mins of 1 $\mu$M ImT22 hybridization experiment. Linear fitting is carried out for the event number greater than 1 (With bin width=0.02). Corresponding slopes ($a$) are shown and denoted as different colors. Inset figure is the zoomed-in view of Mode 1 and 2 amplitudes within 20fm. Among all collected data, only 12 data points appeared only once, the remaining 115 data points are shown in repeated slopes.
• Figure 3: Part of spike amplitude ratios (Ratios between 0 and 1) histogram of Mode3 and Mode2 collected from four continuous optoplasmonic sensing experiments (Bin width=0.02). The ratios among different experiments are labelled with specific colors.
• Figure 4: Overall RRAs, RRAs with similar values form clusters. The red boxes (Group 1) in the inset indicate points likely from the same docking sites, while the blue box (Group 2) shows a cluster that may represent one or more docking sites.
• Figure 5: (a)Example of a transient interaction (Highlighted event) captured by sequential polar modes and their amplitude. The ratio identifier (RRA) denotes the corresponding amplitude ratios between (Mode2/Mode1 & Mode3/Mode2). (b) The step signal with the same RRA is detected after the spike event. (c) Spike (Green star) and step (Red stop sign) signals with the same RRAs captured within four continuous experiments. No spike signals are observed after a step signal, indicating the site is no longer accessible. (d) Event rates per minute for the four continuous experiments. Events captured by both Mode 1 and 2 are denoted as blue dots, by both Mode 2 and 3 are denoted as red dots.