Overcoming evanescent field decay using 3D-tapered nanocavities for on-chip targeted molecular analysis

Plasmonic nanostructures offer the potential for significant advancements in single-molecule fluorescence applications, such as DNA sequencing, disease detection, and observation of biological reactions. However, their widespread use in bioassays has been hindered by the significant variability in enhancement efficiency. Plasmonic fluorescence enhancement is critically dependent on both the electromagnetic (EM) field confinement at the plasmonic hotspot and the distance between the fluorophore and this hotspot. Since fluorophores are typically attached to biorecognition elements (antibodies, aptamers), their position relative to the hotspot is influenced by the size and number of molecules in the biomolecular complex. This distance-dependence drastically alters the plasmonic enhancement due to changes in radiative and non-radiative field components, leading to inconsistent and weak signal enhancement. While various nanoscale geometries have been explored to improve EM field confinement and fluorescence enhancement, creating a hotspot that overcomes the distance challenge has remained a significant hurdle. The development of a nanostructure capable of providing powerful and uniform fluorescence enhancement regardless of molecule size and position is crucial. This requires: (a) strong EM field confinement, (b) efficient coupling of the emitter to the field for emission enhancement, and (c) a hotspot geometry generating uniform EM field distribution, while also being large enough for assays involving protein-protein interactions (e.g., antibodies, ~15 nm). Metal-insulator-metal (MIM) structures using surface-plasmon-polariton (SPP) propagation are known for efficient EM energy confinement. Three-dimensionally (3D) tapered waveguides, utilizing adiabatic compression of the SPP mode, offer extreme volumetric nanoscale confinement. However, their potential for bioassays remained untapped due to their closed, monolithic structures which prevented molecular integration. The close packing of multiple metal-dielectric interfaces in thin MIM gaps can lead to the integration of multiple field profiles, creating a more homogeneous field distribution. Therefore, a 3D-tapered structure presents significant potential for achieving strong EM energy confinement within a tiny MIM gap, while offering open access to molecules.

Existing literature highlights the promise and challenges of plasmonic fluorescence enhancement. Studies demonstrate the potential for breakthroughs in single-molecule DNA sequencing (Eid et al., 2009; Flusberg et al., 2010), rapid disease detection (Ambrosi et al., 2009; Chen et al., 2011; De La Rica & Stevens, 2012), and observation of biological reactions (Acuna et al., 2012; Yokoe & Meyer, 1996). However, the distance-dependence of fluorescence enhancement (Lakowicz, 2005; Dragan et al., 2012; Feng et al., 2015) and the impact of molecule position (Anger et al., 2006; Bharadwaj et al., 2006; Kühn et al., 2008; Bardhan et al., 2008) have limited widespread application. Previous research has explored various nanoscale geometries to enhance EM field confinement (Kinkhabwala et al., 2009; Punj et al., 2013; Chikkaraddy et al., 2016; Flauraud et al., 2017), but engineering a hotspot that mitigates the distance-dependent decay has been elusive. The use of MIM structures and 3D-tapered waveguides (Volkov et al., 2009; Schnell et al., 2011; Gramotnev & Bozhevolnyi, 2014; Bermúdez-Ureña et al., 2015; Pile & Gramotnev, 2006; Gramotnev et al., 2007; Vedantam et al., 2009; Choo et al., 2012; Bao et al., 2012; Cai et al., 2010; Ko et al., 2010) for efficient EM energy confinement has shown promise, but their application to bioassays has been limited by design constraints.

The researchers designed and fabricated a fluidic channel-like 3D-tapered gap plasmon nanocavity to enable easy access of molecules in solution. The device was fabricated using gold and silica-coated silicon substrates. The fabrication process involved e-beam evaporation of gold, followed by focused ion beam (FIB) milling to create the 3D-tapered nanocavity. A hydrophilic SiO2 base attracts fluid into the nanocavity, facilitating molecular delivery and surface-specific binding. Arrays of nanocavities were also fabricated. Finite-difference time-domain (FDTD) simulations were used to optimize the device design for efficient excitation light coupling, optimal EM field confinement, and efficient longitudinal confinement at the tip. The simulations optimized parameters such as body width (*w*body), body length (*l*body), body height (*h*body), and taper angle (α). The simulations compared the 3D-tapered nanocavity with 2D-tapered nanocavities and tip-only structures. The 3D-taper resulted in efficient confinement of EM energy into a tiny cavity, significantly increasing the average transversal EM energy density at the tip. The optimized design achieved superior EM energy confinement and uniformity compared to other structures. The optimized device dimensions were then used for fabrication and subsequent fluorescence enhancement studies. Molecular layers and fluorescent labels were used to test the volumetric optical confinement and biosensing capabilities of the device. Biotin was assembled on the silica surface, and streptavidin-Alexa Fluor 750 (S-AF 750) was used as a fluorescent label. Both tail-end and full illumination modes were used for fluorescence imaging. The limit of detection for biomolecules was tested using two approaches: detection of low-concentration protein molecules in solution (10 pM-1 nM) and capture and detection of single or small arrays of molecules at the hotspot. Anti-biotin IgG antibodies tagged with DyLight 755 were used for antibody binding experiments with different tip lengths (500 nm and 20 nm). Atomic force microscopy (AFM) was used to analyze the antibody monolayer on flat silica surfaces. Fluorescence imaging was performed using a Leica DMI 6000 widefield fluorescence microscope, and scanning electron micrographs were taken using FEI Nova 600 and 200 dual beam systems. Image analysis was performed using Fiji (ImageJ) software. Different molecules, including dye molecules, aptamers, smaller proteins, and antibodies were used to assess the fluorescence enhancement with varying molecular sizes and heights from the silica surface. Enhancement factors were calculated by comparing fluorescence from the device with that from non-structured SiO2 control samples. An enhancement figure of merit, normalized with respect to dye quantum yield, was also used to compare device performance.

The 3D-tapered gap plasmon nanocavity demonstrated significantly improved fluorescence enhancement compared to other structures. FDTD simulations showed that the 3D taper provided superior |E|² uniformity within the gap, resulting in a nearly homogeneous EM field distribution at the tip. This improved uniformity is crucial for mitigating the distance-dependent decay of the evanescent field, a major limitation of conventional plasmonic structures. The simulations predicted a substantial increase in fluorescence enhancement, reaching values close to 2200 for a 20nm tip. The experimental results confirmed these predictions, showing uniform fluorescence enhancement for various molecular assemblies with heights ranging from <1 nm to ~20 nm. The experimental enhancement factor was close to 950 for 500 nm long tips and approximately 2200 for 20 nm tips, consistent with the simulation-based estimations. The device exhibited an enhancement figure of merit close to 260, significantly higher than values reported for other plasmonic nanostructures. The researchers achieved high sensitivity, detecting protein molecules at concentrations as low as 10 pM. Furthermore, they successfully captured and visualized single antibody molecules at the nanocavity tip, enabling single-molecule binding analysis. The 3D-tapered design enabled consistent and powerful fluorescence enhancement regardless of molecule size and position within the nanocavity, overcoming a key limitation of plasmonic fluorescence enhancement. The full-width half-maximum (FWHM) of the fluorescence enhancement profile covered 95.5% of the x-axis and 100% of the y-axis, demonstrating excellent uniformity. The near-uniform enhancement along the y-axis minimized the impact of fluorophore height variations, significantly improving the assay reliability. The enhancement level was dependent on the tip length, with shorter tips exhibiting greater enhancement.

The results demonstrate that the 3D-tapered gap plasmon nanocavity successfully overcomes the long-standing challenge of evanescent field decay in plasmonic fluorescence enhancement. The unique design, with its open-access nanotip and highly uniform EM field distribution, delivers a substantial improvement in the consistency and magnitude of fluorescence enhancement for diverse molecular assemblies. This achievement is particularly important for bioassays, where variations in molecule size and position can significantly impact signal reliability. The capability to detect single molecules and low concentrations of target biomolecules (10 pM) signifies the substantial potential of this technology for sensitive and specific biosensing applications. The high enhancement factor (EF ~2200) and figure of merit (260) are among the best reported for plasmonic nanostructures. The uniform enhancement across a wide range of molecule sizes is a key advantage, simplifying experimental procedures and improving the reliability of quantitative measurements. This technology holds significant promise for high-resolution analysis of protein function and behaviour. Future work could focus on increasing throughput and reducing device footprint by employing wafer-scale fabrication methods such as nanoimprinting, e-beam lithography, and template stripping.

This study presents a novel 3D-tapered gap plasmon nanocavity that significantly advances plasmonic fluorescence enhancement. The device achieves remarkably high and uniform enhancement factors, overcoming the limitations of evanescent field decay and molecule position-dependence. Its ability to detect single molecules and low-concentration biomarkers makes it a promising technology for various biosensing and molecular analysis applications. The high performance and versatility of this technology open up new avenues for research in single-molecule studies, high-throughput screening, and point-of-care diagnostics. Future research should focus on scaling up the fabrication process to enable high-throughput production of these devices.

The current fabrication process, utilizing focused ion beam lithography, is limited in throughput. The device footprint is relatively large compared to other nanoscale structures. The reliance on specific molecular binding may limit the applicability to certain biomolecules. Improved fabrication techniques and optimization of molecular binding strategies may enhance the overall performance and applicability of the device.