In Situ Monitoring of Rolling Circle Amplification on a Solid Support by Surface Plasmon Resonance and Optical Waveguide Spectroscopy

Abstract

The growth of surface-attached single-stranded deoxyribonucleic acid (ssDNA) chains is monitored in situ using an evanescent wave optical biosensor that combines surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The "grafting-from" growth of ssDNA chains is facilitated by rolling circle amplification (RCA), and the gradual prolongation of ssDNA chains anchored to a gold sensor surface is optically tracked in time. At a sufficient density of the polymer chains, the ssDNA takes on a brush architecture with a thickness exceeding 10 μm, supporting a spectrum of guided optical waves traveling along the metallic sensor surface. The simultaneous probing of this interface with the confined optical field of surface plasmons and additional more delocalized dielectric optical waveguide modes enables accurate in situ measurement of the ssDNA brush thickness, polymer volume content, and density gradients. We report for the first time on the utilization of the SPR/OWS technique for the measurement of the RCA speed on a solid surface that can be compared to that in bulk solutions. In addition, the control of ssDNA brush properties by changing the grafting density and ionic strength and post-modification via affinity reaction with complementary short ssDNA staples is discussed. These observations may provide important leads for tailoring RCA toward sensitive and rapid assays in affinity-based biosensors.

Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection

“…The generated DNA strands carry repeating motifs CS*, GS*, and LS* that are complementary to PL (see Figure a). For the used RCA reaction time of 60 min, the previous study shows that the PL was, on average, rolled over 159 times yielding chains with the contour length of 12.9 kb. The LS* segments carried by long RCA chains were used for the fluorescence detection by affinity reacting with labeled Cy5-LS ssDNA strands.…”

“…The RCA was performed after the coupling of CS* and PL molecules, leading to the increase in the SPR signal on the specific surface by ΔR = 1.52 mRIU (and not measurable on the control surface). This change is associated with the prolongation of CS* sequences anchored to dAb as is proven by the strong increase of the fluorescence intensity after labeling with BA-Cy5 (let us note that this labeling sequence was chosen based on the previous work due to its ability to partially cross-link the RCA chains 45 ). The specific plasmonically enhanced fluorescence response of ΔF = 1.88 × 10 5 cps is comparable to that measured in the experiments with model PL experiments (see Figure 3).…”

“…Then, the fluorescence response Δ F was weaker and led to an only 13-fold improved LOD with respect to directly labeled PL. Based on the previous study, it can be estimated that RCA strands are formed by rolling the PL by about 159 times for the used conditions. This value corresponds to the maximum increase in the number of anchored fluorophores per affinity-captured PL molecules with respect to the direct labeling, and the achieved maximum enhancement in LOD approaches this factor.…”

“…Our previous work investigated the growth of several tens of micrometer long ssDNA chains generated by RCA on a gold surface with high grafting density by using the combined SPR and PEF method. 45 Here, we follow up on this work by studying the impact of conformation of RCA-generated ssDNA chains on the dual enhancement efficiency (optical and enzymatic) when it is applied for detecting a low concentration of the target analyte. When decreasing the analyte concentration, the transition from densely packed to sparsely attached individual DNA strands occurs on the surface.…”

Can classical surface plasmon resonance advance via the coupling to other analytical approaches?

“…Stengel and Knoll used this method for studying elongation of double stranded DNA tethered to a solid gold surface by the DNA polymerase enzyme and distinguishing of the binding kinetics of enzyme and assembly of the DNA duplex was possible (Stengel and Knoll, 2005). Similar instrument extended with optical waveguide spectroscopy was lately used for the observation of the elongation speed and conformation changes of the single stranded DNA generated on a solid sensor surface by the rolling circle amplification (Figure 4A) (Lechner et al, 2021). As illustrated in Figure 4B, these strands formed a dense polyelectrolyte brush architecture that can be collapsed by incorporating calcium ions.…”