Anisotropic Fluorescence Emission and Photobleaching at the Surface of One-Dimensional Photonic Crystals Sustaining Bloch Surface Waves. II. Experiments

Abstract

Photonic crystal (PC) enhanced fluorescence has been proposed as a novel tool for early disease detection in liquid biopsy. Photobleaching of the emitters has never been deeply investigated, despite its cross section is expected to increase due to the large field intensity enhancement in PC. Herein, we report on the experimental investigation of the anisotropic effects arising when fluorescence excitation and emission are coupled to differently polarized modes of the same PC structure. In particular, we experimentally characterized the anisotropic photobleaching taking place during the operation of one dimensional PC (1DPC) biosensors sustaining Bloch surface waves (BSW) at their truncation edge. The experimental results are compared to the description provided by our theoretical model reported in a twin article. We demonstrate experimentally that photobleaching affects the response of a cancer biomarkers' detection assay and propose a procedure to compensate for it. Moreover, we describe the experimental use of BSW coupled fluorescence and of the strong localization of the BSW at the 1DPC surface to selectively probe the rotational diffusion kinetics of proteins bound at the 1DPC surface with high spatial resolution and for different binding strengths.

Anisotropic Fluorescence Emission and Photobleaching at the Surface of One-Dimensional Photonic Crystals Sustaining Bloch Surface Waves. I. Theory

“…The geometry and the choice of materials result from an optimization aimed at developing highly resolved BSW label-free and fluorescence biosensors [16]. The design was used to fabricate the 1DPC used in experiments on anisotropic fluorescence emission and PB [15]. The specific 1DPC design does not affect the general validity of the present theoretical results, which could be recalculated for different cases, if needed.…”

“…Here we make use of the emission spectrum of the dye Dylight650, with MAX = 667 nm, which was used in our experiments [15].…”

“…Figure 1d shows a simplified sketch of the fluorescence excitation and detection apparatus used in the experiments. A more detailed description can be found elsewhere [15].…”

“…( 6) were used to calculate, from the curves in Figure From Figure 4a we found that, for isotropically angularly distributed emitters, 𝑅 0 = 6.1. Such a value depends on the specific 1DPC design and dye spectrum and on the distance d. It is of particular relevance the dependency on d, since it can vary much in the real experimental case, due to the presence of any capture or blocking biomolecular spacer, as sketched in Figure 1 [15]. Figure 4b In the calculations, we took into account that the spacer affects also the local intensity of the excitation field, which however changes by less than 1%.…”

Multiresonant plasmonic nanostructure for ultrasensitive fluorescence biosensing

“…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 . In the reported experiments, fluorophores were conjugated to proteins by flexible molecular linkers with high degree of rotational freedom, which leads to randomizing its orientation in the fluorescence lifetime and consequently to an isotropic emission profile.…”