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

We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

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

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.

Dual Monitoring of Surface Reactions in Real Time by Combined Surface-Plasmon Resonance and Field-Effect Transistor Interrogation.

By combining surface plasmon resonance (SPR) and electrolyte gated field-effect transistor (EG-FET) methods in a single analytical device we introduce a novel tool for surface investigations, enabling simultaneous measurements of the surface mass and charge density changes in real time. This is realized using a gold sensor surface that simultaneously serves as a gate electrode of the EG-FET and as the SPR active interface. This novel platform has the potential to provide new insights into (bio)adsorption processes on planar solid surfaces by directly relating complementary measurement principles based on (i) detuning of SPR as a result of the modification of the interfacial refractive index profile by surface adsorption processes and (ii) change of output current as a result of the emanating effective gate voltage modulations. Furthermore, combination of the two complementary sensing concepts allows for the comparison and respective validation of both analytical techniques. A theoretical model is derived describing the mass uptake and evolution of surface charge density during polyelectrolyte multilayer formation. We demonstrate the potential of this combined platform through the observation of layer-by-layer assembly of PDADMAC and PSS. These simultaneous label-free and real-time measurements allow new insights into complex processes at the solid-liquid interface (like non-Fickian ion diffusion), which are beyond the scope of each individual tool.