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

Abstract

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.

Keywords: biosensor; immunoassays; rolling circle amplification; single molecule; surface plasmon resonance; surface plasmon-enhanced fluorescence.

Figure 1

Optical setup employed (a) in the combined SPR and surface PEF spectroscopy and (b) for scanning confocal microscopy measurements with the use of a laser band pass filter (LBP), polarizer (POL), dichroic mirror (DM), neutral density filter (NDF), laser notch filter (LNF), fluorescence band pass filter (FBP), and a photomultiplier (PMT). (c) Schematics of the optically probed biointerface with tethered DNA chains generated by RCA.

Figure 2

Schematics of the (a) biointerface for the affinity capture of circular padlock probe (PL) via the immobilized capture strands (CS*), growth of ssDNA chain (with repeating sequences LS*, GS*, and CS* specified in Table 1), and its reaction with short labeling strands (LS) conjugated with the Cy5 emitter. (b) Visualization of the guiding of RCA-generated long ssDNA chains with the use of mixed ssDNA brush carrying strands with CS* and GS. As a control, a scrambled guiding sequence (rGS) was used.

Figure 3

Example of (a) SPR and PEF signal kinetics upon the affinity capture of padlock molecules PL followed by RCA and labeling with Cy5-LS measured at an angle of incidence of θ = 56.7° and (b) measured angular reflectivity and fluorescence scans between the reaction steps.

Figure 4

(a) Dependence of the measured fluorescence signal for excitation via the resonantly excited SPs on the sensor surface for varied average distance between ssDNA chains D (dashed lines between measured data are guides for eye) and schematics of ssDNA chains taking (b) dense brush and (c) sparse random coil conformation.

Figure 5

Comparison of the calibration curves for the PEF biosensor for an RCA assay with direct labeling of padlock molecules, RCA on the biointerface carrying randomized rGS strands, and RCA on the biointerface with short complementary GS strands. The measurements were performed after exposure of ssDNA to high ionic strength solution and rinsing the surface with a running buffer. The black line represents a fit with the Langmuir model function, the red curve represents the linear fit, and the blue dashed line is a guide for eyes. Error bars were determined as the standard deviation of readout noise.

Figure 6

(a) Fluorescence microscopy observation of the fluorescence signal from the sensor chip surface carrying RCA chains labeled with LS-Cy5 that were generated with the use of padlock affinity-captured from a solution with the padlock concentration of c = 40 fM to 40 pM and c = 0 as the control experiment. (b) Comparison of the average distance between the affinity-captured padlock molecules on the surface depending on its concentration in the solution contacted with the sensor surface.

Figure 7

(a) Schematics of the RCA implementation to sandwich immunoassay on a metallic surface with mixed thiol SAM. (b) SPR and PEF readout of the formation of the biointerface and sandwich assay with the concentration of target IL6 analyte of c = 47.6 nM (specific) and c = 0 (control) followed by RCA and reaction with complementary fluorophore-tagged BA labeling strands.