Plasmon Field-Enhanced Fluorescence Energy Transfer for Hairpin Aptamer Assay Readout

Abstract

Surface plasmon field-enhanced fluorescence energy transfer is employed for sensitive optical readout of a reversible hairpin aptamer assay that is suitable for continuous monitoring of low-molecular-weight chemical analytes. A hairpin aptamer specific to adenosine and adenosine triphosphate with Alexa Fluor 647 fluorophore attached to its 5′ end was anchored via 3′ end thiol to a gold thin film. Molecular spacers were used to control the distance of the fluorophore from the surface in the aptamer “off” and “on” states. The specific binding of the target analyte changes the aptamer conformation, which alters the distance of the fluorophore from the gold surface and translates to variations in the detected fluorescence intensity. The plasmonically mediated fluorescence signal increases the measured signal-to-noise ratio and allows for real-time observation of the analyte binding. Theoretical as well as experimental study of the optical signal dependence on fluorophore orientation, design of spacers, and angular distribution of collected light is presented for rational design of the assay. The detected sensor signal increased by a factor as large as 23 upon switching the aptamer from the “off” to “on” state due to the hairpin opening associated with the specific capture of target analyte.

Multiresonant plasmonic nanostructure for ultrasensitive fluorescence biosensing

“…The coupling of this fluorophore with the enhanced intensity of electric field | E → | 2 of resonantly excited PSPs at these wavelengths was studied using numerical simulations. The fluorophore was represented as an infinitesimally small electric dipole placed at a distance of 15 nm from the gold surface, which was chosen with respect to a typical immunoassay experiment considering the size of immunoglobulin G antibodies (13.7 × 8 × 4 nm [43]) and the fact that it is above the distance where strong quenching occurs [44]. The fluorescence emission has dipole characteristics [45,46], and it cannot be excited when the orientation of the electric field E → is perpendicular to the emitter absorption dipole μ → ab .…”

Surface Plasmon Resonance: Material and Interface Design for Universal Accessibility

“…214 Sergelen et al recently used this system for adenosine and ATP detection using conformation-switching hairpin aptamers, end-labeled with a fluorophore. 215 In the “off” state of the aptamer, the fluorophore end orients toward the gold surface, bringing the reporter within the attenuation distance of the metal. In response to analyte binding, the hairpin opens, tethering the fluorophore within the SPFS enhancement region.…”

Characteristics of Fluorescence Emission Excited by Grating-Coupled Surface Plasmons

“…Only a surprisingly small number of articles have been published since then on this subject. These cover calculations of the plasmonic fields and enhancement optimization [16], various excitation and emission geometries [17], studies of fluorescence lifetime as a function of the dye/metal separation [18], and applications in biosensing [19]. We examine here the emission geometry of the excited fluorescence and the influence of the polarization (cf.…”