Plasmon-enhanced Fluorescence Research Articles

More Symmetrical "Hot Spots" Ensure Stronger Plasmon-Enhanced Fluorescence: From Au Nanorods to Nanostars.

Plasmon-enhanced fluorescence (PEF) is considered to be a powerful signal amplification technology to overcome intrinsic shortcomings of photobleaching and brightness of the traditional fluorescent dyes. Nevertheless, exploitation of PEF-based probes for bioimaging application is still at a very early stage. In this work, a simple but powerful gold nanostar (Au NST)@SiO2-based PEF probe with 20 symmetric "hot spots" was developed for highly sensitive "lighting up" in situ imaging of intracellular microRNAs (miRNAs). By regulating the thickness of the silica shell, the distance between Au NSTs and fluorescent dyes was controlled, and the optimum fluorescence enhancement (21-fold) was obtained with the silica shell thickness of approximately 22 nm. Thanks to the 20 more powerful "hot spots" that can produce stronger localized electric fields, the Au NST-based PEF probe exhibits stronger PEF effects than the traditional plasmonic nanostructures such as gold nanorods (Au NRs), gold nanobipyramids (Au NBPs), and triangular gold nanoprisms (Au NPRs), resulting in high sensitivity and improved detection limit (LOD) of 0.21 pM for miRNA-21 analysis. Moreover, not only cancer cells (MCF-7 and Hela) and normal cells (L02) with distinct miRNA-21 expression levels can be discriminated but also tumor cells in co-cultured mixtures can be recognized, indicating its promising potential in clinical diagnosis.

Ultrasensitive antibody-aptamer plasmonic biosensor for malaria biomarker detection in whole blood

Development of plasmonic biosensors combining reliability and ease of use is still a challenge. Gold nanoparticle arrays made by block copolymer micelle nanolithography (BCMN) stand out for their scalability, cost-effectiveness and tunable plasmonic properties, making them ideal substrates for fluorescence enhancement. Here, we describe a plasmon-enhanced fluorescence immunosensor for the specific and ultrasensitive detection of Plasmodium falciparum lactate dehydrogenase (PfLDH)—a malaria marker—in whole blood. Analyte recognition is realized by oriented antibodies immobilized in a close-packed configuration via the photochemical immobilization technique (PIT), with a top bioreceptor of nucleic acid aptamers recognizing a different surface of PfLDH in a sandwich conformation. The combination of BCMN and PIT enabled maximum control over the nanoparticle size and lattice constant as well as the distance of the fluorophore from the sensing surface. The device achieved a limit of detection smaller than 1 pg/mL (<30 fM) with very high specificity without any sample pretreatment. This limit of detection is several orders of magnitude lower than that found in malaria rapid diagnostic tests or even commercial ELISA kits. Thanks to its overall dimensions, ease of use and high-throughput analysis, the device can be used as a substrate in automated multi-well plate readers and improve the efficiency of conventional fluorescence immunoassays.

Highly Sensitive Immunofluorescence Assay of Prostate-Specific Antigen Using Silver Nanoparticles

We consider the possibilities of applying the plasmon-enhanced fluorescence of monoclonal antibodies labelled with fluorescein isothiocyanate (FITC) to the prostate-specific antigen (PSA) for increasing the immunofluorescence analysis sensitivity. An immunochemical test system featuring two center binding using a pair of noncompeting monoclonal antibodies in combination with silver plasmon nanoparticles was used for the first time for determining the PSA concentration. The advantages over the standard PSA immunofluorescence assay are as follows. The intensity of the recorded fluorescent signal is enhanced by 2.3–3.2 times in the presence of silver nanoparticles in comparison with the signal of the test system on the intact surface of a polystyrene plate. The signal-to-noise ratio is increased by up to two times. In addition to the plasmon-enhanced fluorescence, the effect of an intermediate polyelectrolyte layer for enhancement of the adsorption capacity of the primary PSA antibodies is shown, which, in turn, affects the fluorescence signal intensity. Growth of the plasmon fluorescence enhancement factor with increasing concentration of the labelled antibodies indicates suppression of the self-quenching of the fluorescent labels by metal nanoparticles.

Probing the Mechanisms of Strong Fluorescence Enhancement in Plasmonic Nanogaps with Sub-nanometer Precision

Plasmon-enhanced fluorescence is demonstrated in the vicinity of metal surfaces due to strong local field enhancement. Meanwhile, fluorescence quenching is observed as the spacing between fluorophore molecules and the adjacent metal is reduced below a threshold of a few nanometers. Here, we introduce a technology, placing the fluorophore molecules in plasmonic hotspots between pairs of collapsible nanofingers with tunable gap sizes at sub-nanometer precision. Optimal gap sizes with maximum plasmon enhanced fluorescence are experimentally identified for different dielectric spacer materials. The ultrastrong local field enhancement enables simultaneous detection and characterization of sharp Raman fingerprints in the fluorescence spectra. This platform thus enables in situ monitoring of competing excitation enhancement and emission quenching processes. We systematically investigate the mechanisms behind fluorescence quenching. A quantum mechanical model is developed which explains the experimental data and will guide the future design of plasmon enhanced spectroscopy applications.

Plasmon-Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs)-nanoscale phospholipid vesicles secreted by cells-present new opportunities for molecular diagnosis from non-invasive liquid biopsies. Single EV protein analysis can be extremely valuable in studying EVs as circulating cancer biomarkers, but it is technically challenging due to weak detection signals associated with limited amounts of epitopes and small surface areas for antibody labeling. Here, a new, simple method that enables multiplexed analyses of EV markers with improved sensitivities is reported. Specifically, plasmon-enhanced fluorescence detection is implemented that amplifies fluorescence signals using surface plasmon resonances excited by periodic gold nanohole structures. It is shown that fluorescence signals in multiple channels are amplified by one order of magnitude, and both transmembrane and intravesicular markers can be detected at the single EV level. This approach can offer additional insight into understanding subtypes, heterogeneity, and production dynamics of EVs during disease development and progression.

Enhancing luminescent down-shifting of Eu-doped phosphors by incorporating plasmonic silver nanoparticles for silicon solar cells

This study experimentally demonstrated enhanced fluorescence emissions from luminescent down-shifting (LDS) Eu-doped phosphors under the effects of localized surface plasmon resonance (LPSR) from silver nanoparticles (Ag NPs). The plasmonic effects of the Ag NPs was examined in terms of plasmon-enhanced Raman scattering and plasmonic absorption. The fluorescence emission of the Eu-doped phosphors was examined in terms of photoluminescence (PL). Overlap between the plasmonic absorption band of Ag NPs and the PL emission band of Eu-doped phosphors increased fluorescence emissions by enhancing excitation. This study also developed an optical process model describing LSPR (Ag NPs), LDS (Eu-doped phosphors), and plasmon-enhanced fluorescence (PEF; Eu-doped phosphors with Ag NPs) on silicon solar cells. PEF was experimentally confirmed in terms of PL, external quantum efficiency (EQE), and photovoltaic current–voltage measurements. Compared to the cell with only an anti-reflection layer of SiO2/SOG, the inclusion of Ag NPs with the Eu-doped phosphors enhanced the PL emission intensity by 1.1243 times at a wavelength of 514 nm and also improved the EQE response by 8.3% (from 57.28% to 62.05%) and conversion efficiency by 7.1% (from 12.04% to 12.89%).

Multiresonant plasmonic nanostructure for ultrasensitive fluorescence biosensing

Abstract A novel metallic nanostructure for efficient plasmon-enhanced fluorescence readout of biomolecular binding events on the surface of a solid sensor chip is reported. It is based on gold multiperiod plasmonic grating (MPG) that supports spectrally narrow plasmonic resonances centered at multiple distinct wavelengths. They originate from diffraction coupling to propagating surface plasmons (SPs) forming a delocalized plasmonic hotspot associated with enhanced electromagnetic field intensity and local density of optical states at its surface. The supported SP resonances are tailored to couple with the excitation and emission transitions of fluorophores that are conjugated with the biomolecules and serve as labels. By the simultaneous coupling at both excitation and emission wavelengths, detected fluorescence intensity is enhanced by the factor of 300 at the MPG surface, which when applied for the readout of fluorescence immunoassays translates to a limit of detection of 6 fM within detection time of 20 min. The proposed approach is attractive for parallel monitoring of kinetics of surface reactions in microarray format arranged on a macroscopic footprint. The readout by epi-fluorescence geometry (that inherently relies on low numerical aperture optics for the imaging of the arrays) can particularly take advantage of the reported MPG. In addition, the proposed MPG nanostructure can be prepared in scaled up means by UV-nanoimprint lithography for future practical applications.

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

Enhancement of optical emission on plasmonic nanostructures is intrinsically limited by the distance between the emitter and nanostructure surface, owing to a tightly-confined and exponentially-decaying electromagnetic field. This fundamental limitation prevents efficient application of plasmonic fluorescence enhancement for diversely-sized molecular assemblies. We demonstrate a three-dimensionally-tapered gap plasmon nanocavity that overcomes this fundamental limitation through near-homogeneous yet powerful volumetric confinement of electromagnetic field inside an open-access nanotip. The 3D-tapered device provides fluorescence enhancement factors close to 2200 uniformly for various molecular assemblies ranging from few angstroms to 20 nanometers in size. Furthermore, our nanostructure allows detection of low concentration (10 pM) biomarkers as well as specific capture of single antibody molecules at the nanocavity tip for high resolution molecular binding analysis. Overcoming molecule position-derived large variations in plasmonic enhancement can propel widespread application of this technique for sensitive detection and analysis of complex molecular assemblies at or near single molecule resolution.

Target-Triggered Assembly of Nanogap Antennas to Enhance the Fluorescence of Single Molecules and Their Application in MicroRNA Detection.

Nanogap antennas are plasmonic nanostructures with a strong electromagnetic field generated at the gap region of two neighboring particles owing to the coupling of the collective surface plasmon resonance. They have great potential for improving the optical properties of fluorophores. Herein, nanogap antennas are constructed using an aqueous solution-based method to overcome the defects of weak fluorescence and photobleaching associated with traditional organic dyes, and a highly sensitive nanogap antenna-based sensing strategy is presented for the detection of low-abundance nucleic acid biomarkers via a target-triggered strand displacement amplification (SDA) reaction between two DNA hairpins that are tagged to the tips of gold nanorods (Au NRs). In the presence of targets, end-to-end Au NR dimers gradually form, and the fluorophores quenched by the Au NRs exhibit a dramatic fluorescence enhancement due to the plasmon-enhanced fluorescence effect of nanogap antennas. Meanwhile, the SDA reaction results in secondary amplification of fluorescence signals. Combined with single-molecule counting, this method applied in miRNA-21 detection can achieve a low detection limit of 97.2 × 10-18 m. Moreover, accurate discrimination between different cells through miRNA-21 imaging demonstrates the potential of this method in monitoring the expression level of low-abundance nucleic acid biomarkers.

Plasmon-Induced Suppression of Exciton Self-Trapping in Polymer-Bound Pseudoisocyanine J-Aggregates

Features of plasmon-enhanced fluorescence in pseudoisocyanine (PIC) J-aggregates formed in polyelectrolyte films at liquid nitrogen temperature have been studied. Due to efficient exciton self-trap...

Surface Plasmon-Enhanced Optical Formaldehyde Sensor Based on CdSe@ZnS Quantum Dots.

For the first time, a reproducible surface plasmon-enhanced optical sensor for the detection of gaseous formaldehyde was proposed, which was fabricated by depositing a mixture of CdSe@ZnS quantum dots (QDs), fumed silica (FS), and gold nanoparticles (GNs) on the surface of a silica sphere array to meet the urgent requirement of a rapid, sensitive, and highly convenient formaldehyde detection method. Because of the spectral overlap between QDs and GNs, plasmon-enhanced fluorescence was observed in the film of QDs/FS/GNs. When exposed to formaldehyde molecules, the enhanced fluorescence was quenched linearly with the increase of formaldehyde concentration in the range of 0.5-2.0 ppm. The reason is attributed to the nonradiative electron transfer from QDs to the carbonyl of formaldehyde molecules with the assistance of amino groups. Our results demonstrate that the designed sensors are capable of detecting ultralow concentration gaseous formaldehyde at room temperature with a fast response-recovery time and excellent selectivity, stability, and reproducibility. This work provides a simple and low-cost approach for optical formaldehyde sensor fabrication and shows promising applications in environmental detection.

Parametric optimization of visible wavelength gold lattice geometries for improved plasmon-enhanced fluorescence spectroscopy.

We report the exploitation of spectroplasmonics for innovations in optical transducer development, specifically in the well-established application of labeled fluorescent analytes known as fluorescence spectroscopy. Presented herein are comprehensive analyses of nanoscale plasmonic lattice feature geometries using finite-difference time-domain software to determine the largest surface electric ($E$E) field enhancement resulting from localized surface plasmon resonance for reducing the limit of detection of plasmon-enhanced fluorescence. This parametric optimization of the critical dimensions of the plasmon resonance of noble metal nanostructures will enable improved excitation and emission enhancement of fluorophores used in visible wavelength fluorescence spectroscopy.

Immobilization and plasmon-enhanced fluorescence of EGFP on Al nanohole arrays

Enhanced Green Fluorescent Protein has been covalently immobilized on thin perforated Al films with short-order nanohole arrays using (3-Glycidyloxypropyl)trimethoxysilane and native aluminum oxide layer. The nanoholes’ diameter varied in range 60–120 nm, allowing to tune the surface plasmon resonance frequency. The fluorescence intensity enhancement up to factor 2.75 for the nanohole arrays with the largest overlap between EGFP fluorescence band and surface plasmon resonance.

Strong Plasmon Enhancement of the Saturation Photon Count Rate of Single Molecules.

Plasmon resonances have appeared as a promising method to boost the fluorescence intensity of single emitters. However, because research has focused on the enhancement at low excitation intensity, little is known about plasmon–fluorophore coupling near the point where the dye saturates. Here we study plasmon-enhanced fluorescence at a broad range of excitation intensities up to saturation. We adopt a novel DNA-mediated approach wherein dynamic single-molecule binding provides a controlled particle–fluorophore spacing, and dynamic rebinding circumvents artifacts due to photobleaching. We find that near saturation the maximum photon count rate is enhanced by more than 2 orders of magnitude at the optimal particle–fluorophore spacing, even for a dye with a high intrinsic quantum yield. We compare our results to a numerical model taking into account dye saturation. These experiments provide design rules to maximize the photon output of single emitters, which will open the door to studying fast dynamics in real time using single-molecule fluorescence.

Control of quantum dot emission by colloidal plasmonic pyramids in a liquid crystal.

We study the plasmon-enhanced fluorescence of a single semiconducting quantum dot near the apex of a colloidal gold pyramid spatially localized by the elastic forces of the liquid crystal host. The gold pyramid particles were manipulated within the liquid crystal medium by laser tweezers, enabling the self-assembly of a semiconducting quantum dot dispersed in the medium near the apex of the gold pyramid, allowing us to probe the plasmon-exciton interactions. We demonstrate the effect of plasmon coupling on the fluorescence lifetime and the blinking properties of the quantum dot. Our results demonstrate that topological defects around colloidal particles in liquid crystal combined with laser tweezers provide a platform for plasmon exciton interaction studies and potentially could be extended to the scale of composite materials for nanophotonic applications.

Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas

Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quencher.

UV-Laser Interference Lithography for Local Functionalization of Plasmonic Nanostructures with Responsive Hydrogel.

A novel approach to local functionalization of plasmonic hotspots at gold nanoparticles with biofunctional moieties is reported. It relies on photocrosslinking and attachment of a responsive hydrogel binding matrix by the use of a UV interference field. A thermoresponsive poly(N-isopropylacrylamide)-based (pNIPAAm) hydrogel with photocrosslinkable benzophenone groups and carboxylic groups for its postmodification was employed. UV-laser interference lithography with a phase mask configuration allowed for the generation of a high-contrast interference field that was used for the recording of periodic arrays of pNIPAAm-based hydrogel features with the size as small as 170 nm. These hydrogel arrays were overlaid and attached on the top of periodic arrays of gold nanoparticles, exhibiting a diameter of 130 nm and employed as a three-dimensional binding matrix in a plasmonic biosensor. Such a hybrid material was postmodified with ligand biomolecules and utilized for plasmon-enhanced fluorescence readout of an immunoassay. Additional enhancement of the fluorescence sensor signal by the collapse of the responsive hydrogel binding matrix that compacts the target analyte at the plasmonic hotspot is demonstrated.

Thinking outside the shell: novel sensors designed from plasmon-enhanced fluorescent concentric nanoparticles.

The alteration of photophysical properties of fluorophores in the vicinity of a metallic nanostructure, a phenomenon termed plasmon- or metal-enhanced fluorescence (MEF), has been investigated extensively and used in a variety of proof-of-concept demonstrations over the years. A particularly active area of development in this regard has been the design of nanostructures where fluorophore and metallic core are held in a stable geometry that imparts improved luminosity and photostability to a plethora of organic fluorophores. This minireview presents an overview of MEF-based concentric core-shell sensors developed in the past few years. These architectures expand the range of applications of nanoparticles (NPs) beyond the uses possible with fluorescent molecules. Design aspects that are being described include the influence of the nanocomposite structure on MEF, notably the dependence of fluorescence intensity and lifetime on the distance to the plasmonic core. The chemical composition of nanocomposites as a design feature is also discussed, taking as an example the use of non-noble plasmonic metals such as indium as core materials to enhance multiple fluorophores throughout the UV-Vis range and tune the sensitivity of halide-sensing fluorophores operating on the principle of collisional quenching. Finally, the paper describes how various solid substrates can be functionalized with MEF-based nanosensors to bestow them with intense and photostable pH-sensitive properties for use in fields such as medical therapy and diagnostics, dentistry, biochemistry and microfluidics.

Mixed metal zero-mode guides (ZMWs) for tunable fluorescence enhancement.

Zero-mode waveguides (ZMWs) are capable of modifying fluorescence emission through interactions with surface plasmon modes leading to either plasmon-enhanced fluorescence or quenching. Enhancement requires spectral overlap of the plasmon modes with the absorption or emission of the fluorophore. Thus, enhancement is limited to fluorophores in resonance with metals (e.g. Al, Au, Ag) used for ZMWs. The ability to tune interactions to match a wider range of fluorophores across the visible spectra would significantly extend the utility of ZMWs. We fabricated ZMWs composed of aluminum and gold individually and also in mixtures of three different ratios, (Al : Au; 75 : 25, 50 : 50, 25 : 75). We characterized the effect of mixed-metal ZMWs on single-molecule emission for a range fluorophores across the visible spectrum. Mixed metal ZMWs exhibited a shift in the spectral range where they exhibited the maximum fluorescence enhancement allowing us to match the emission of fluorophores that were nonresonant with single metal ZMWs. We also compared the effect of mixed-metal ZMWs on the photophysical properties of fluorescent molecules due to metal–molecule interactions. We quantified changes in fluorescence lifetimes and photostability that were dependent on the ratio of Au and Al. Tuning the enhancement properties of ZMWs by changing the ratio of Au and Al allowed us to match the fluorescence of fluorophores that emit in different regions of the visible spectrum.

A novel liquid biopsy-based approach for highly specific cancer diagnostics: mitigating false responses in assaying patient plasma-derived circulating microRNAs through combined SERS and plasmon-enhanced fluorescence analyses.

Studies have shown that microRNAs, which are small noncoding RNAs, hold tremendous promise as next-generation circulating biomarkers for early cancer detection via liquid biopsies. A novel, solid-state nanoplasmonic sensor capable of assaying circulating microRNAs through a combined surface-enhanced Raman scattering (SERS) and plasmon-enhanced fluorescence (PEF) approach has been developed. Here, the unique localized surface plasmon resonance properties of chemically-synthesized gold triangular nanoprisms (Au TNPs) are utilized to create large SERS and PEF enhancements. With careful modification to the surface of Au TNPs, this sensing approach is capable of quantifying circulating microRNAs at femtogram/microliter concentrations. Uniquely, the multimodal analytical methods mitigate both false positive and false negative responses and demonstrate the high stability of our sensors within bodily fluids. As a proof of concept, microRNA-10b and microRNA-96 were directly assayed from the plasma of six bladder cancer patients. Results show potential for a highly specific liquid biopsy method that could be used in point-of-care clinical diagnostics to increase early cancer detection or any other diseases including SARS-CoV-2 in which RNAs can be used as biomarkers.