Plasmonic antennas have a profound impact on nanophotonics as they provide efficient means to manipulate light and enhance light-matter interactions at the nanoscale. However, the large absorption losses found in metals can severely limit the plasmonic applications in the visible spectral range. Here, we demonstrate the effectiveness of an alternative approach using all-dielectric nanoantennas based on silicon dimers to enhance the fluorescence detection of single molecules. The silicon antenna design is optimized to confine the near-field intensity in the 20 nm nanogap and reach a 270-fold fluorescence enhancement in a nanoscale volume of λ(3)/1800 with dielectric materials only. Our conclusions are assessed by combining polarization resolved optical spectroscopy of individual antennas, scanning electron microscopy, numerical simulations, fluorescence lifetime measurements, fluorescence burst analysis, and fluorescence correlation spectroscopy. This work demonstrates that all-silicon nanoantennas are a valid alternative to plasmonic devices for enhanced single molecule fluorescence sensing, with the additional key advantages of reduced nonradiative quenching, negligible heat generation, cost-efficiency, and complementary metal-oxide-semiconductor (CMOS) compatibility.
Optical nanoantennas have a great potential for enhancing light-matter interactions at the nanometer scale, yet fabrication accuracy and lack of scalability currently limit ultimate antenna performance and applications. In most designs, the region of maximum field localization and enhancement (i.e., hotspot) is not readily accessible to the sample because it is buried into the nanostructure. Moreover, current large-scale fabrication techniques lack reproducible geometrical control below 20 nm. Here, we describe a new nanofabrication technique that applies planarization, etch back, and template stripping to expose the excitation hotspot at the surface, providing a major improvement over conventional electron beam lithography methods. We present large flat surface arrays of in-plane nanoantennas, featuring gaps as small as 10 nm with sharp edges, excellent reproducibility and full surface accessibility of the hotspot confined region. The novel fabrication approach drastically improves the optical performance of plasmonic nanoantennas to yield giant fluorescence enhancement factors up to 10 4 −10 5 times, together with nanoscale detection volumes in the 20 zL range. The method is fully scalable and adaptable to a wide range of antenna designs. We foresee broad applications by the use of these in-plane antenna geometries ranging from large-scale ultrasensitive sensor chips to microfluidics and live cell membrane investigations. KEYWORDS: Optical nanoantennas, template stripping, electron beam lithography, fluorescence enhancement, plasmonics O ptical nanoantennas take advantage of the plasmonic response of noble metals to strongly confine light energy into nanoscale dimensions and breach the classical diffraction limit. 1−3 This confinement leads to a drastic enhancement of the interactions between a single quantum emitter and the light field, 4−7 enabling large fluorescence gains above a thousand fold, 8−13 ultrafast picosecond emission, 14−16 and photobleaching reduction. 17,18 As such, optical antennas hold great interest for ultrasensitive biosensing, especially for the detection of single molecules at biologically relevant micromolar concentrations. 19−21 Biosensing applications of nanoantennas require the largescale availability of narrow accessible gaps. Not only should nanogaps with sub-20 nm dimensions be reproducibly fabricated but also the gap region (plasmonic hotspot) must remain accessible to probe the target molecules. Despite impressive recent progress using electron beam, 22 focused ion beam, 23 or stencil lithographies 24−26 or alternatively with bottom-up self-assembly techniques, 6,7,9,13,16,27−30 the challenges of reliable narrow gap fabrication and hotspot accessibility remain major hurdles limiting the impact and performance of optical nanoantennas. For instance, when aiming for the fabrication of aperture antennas, electron beam lithography (EBL) using a positive-tone resist requires metal dry etching, which produces high line-edge roughness that are not suited for the definition ...
Nanoscale membrane assemblies of sphingolipids, cholesterol, and certain proteins, also known as lipid rafts, play a crucial role in facilitating a broad range of important cell functions. Whereas on living cell membranes lipid rafts have been postulated to have nanoscopic dimensions and to be highly transient, the existence of a similar type of dynamic nanodomains in multicomponent lipid bilayers has been questioned. Here, we perform fluorescence correlation spectroscopy on planar plasmonic antenna arrays with different nanogap sizes to assess the dynamic nanoscale organization of mimetic biological membranes. Our approach takes advantage of the highly enhanced and confined excitation light provided by the nanoantennas together with their outstanding planarity to investigate membrane regions as small as 10 nm in size with microsecond time resolution. Our diffusion data are consistent with the coexistence of transient nanoscopic domains in both the liquid-ordered and the liquid-disordered microscopic phases of multicomponent lipid bilayers. These nanodomains have characteristic residence times between 30 and 150 μs and sizes around 10 nm, as inferred from the diffusion data. Thus, although microscale phase separation occurs on mimetic membranes, nanoscopic domains also coexist, suggesting that these transient assemblies might be similar to those occurring in living cells, which in the absence of raft-stabilizing proteins are poised to be short-lived. Importantly, our work underscores the high potential of photonic nanoantennas to interrogate the nanoscale heterogeneity of native biological membranes with ultrahigh spatiotemporal resolution. KEYWORDS: optical nanoantennas, fluorescence correlation spectroscopy, FCS diffusion laws, biological membranes, lipid rafts T he spatiotemporal lateral organization and the biological function of the eukaryotic plasma membrane are intricately interlaced at the nanoscale. It is well accepted that the landscape of the cell membrane is highly heterogeneous and shaped by a variety of lipids and proteins that differ in their physicochemical properties. In the plane of the membrane, lateral heterogeneities resulting from the formation of specialized regions enriched in sphingolipids, cholesterol, and specific proteins are commonly known as lipid rafts.1−4 These lipid assemblies are thought to constitute a tightly packed, short-range, liquid-ordered (Lo) phase coexisting with a more liquid-disordered (Ld) phase within the surrounding fluid membrane.5−7 While the existence of phase separation in the plasma membrane has been debated for many years, a large number of recent experimental data convincingly demonstrates that lipid rafts in living cell membranes have nanoscopic dimensions and are highly dynamic.8−13 Importantly, lipid rafts play a crucial role in many cellular processes that include signal transduction, protein and lipid sorting, and immune response among others. 2,5,10,14,15 Understanding the formation mechanism and properties (e.g., size, composition) of lipid...
Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 μs. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.
The plasma membrane of living cells is compartmentalized at multiple spatial scales ranging from the nano- to the mesoscale. This nonrandom organization is crucial for a large number of cellular functions. At the nanoscale, cell membranes organize into dynamic nanoassemblies enriched by cholesterol, sphingolipids, and certain types of proteins. Investigating these nanoassemblies known as lipid rafts is of paramount interest in fundamental cell biology. However, this goal requires simultaneous nanometer spatial precision and microsecond temporal resolution, which is beyond the reach of common microscopes. Optical antennas based on metallic nanostructures efficiently enhance and confine light into nanometer dimensions, breaching the diffraction limit of light. In this Perspective, we discuss recent progress combining optical antennas with fluorescence correlation spectroscopy (FCS) to monitor microsecond dynamics at nanoscale spatial dimensions. These new developments offer numerous opportunities to investigate lipid and protein dynamics in both mimetic and native biological membranes.
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