Metal-enhanced Fluorescence Substrate Research Articles

Metal-enhanced fluorescence biosensor integrated in capillary flow-driven microfluidic cartridge for highly sensitive immunoassays

Point-of-care testing (POCT) for low-concentration protein biomarkers remains challenging due to limitations in biosensor sensitivity and platform integration. This study addresses this gap by presenting a novel approach that integrates a metal-enhanced fluorescence (MEF) biosensor within a capillary flow-driven microfluidic cartridge (CFMC) for the ultrasensitive detection of the Parkinson's disease biomarker, aminoacyl-tRNA synthetase complex interacting multi-functional protein 2 (AIMP-2). Crucial point to this approach is the orientation-controlled immobilization of capture antibody on a nanodimple-structured MEF substrate within the CFMC. This strategy significantly enhances fluorescence signals without quenching, enabling accurate quantification of low-concentration AIMP-2 using a simple digital fluorescence microscope with a light-emitting diode excitation source and a digital camera. The resulting platform exhibits exceptional sensitivity, achieving a limit of detection in the pg/mL range for AIMP-2 in human serum. Additionally, the CFMC design incorporates a capillary-driven passive sample transport mechanism, eliminating the need for external pumps and further simplifying the detection process. Overall, this work demonstrates the successful integration of MEF biosensing with capillary microfluidics for point-of-care applications.

Synthesis of SiO2@Ag Nanocomposite for Investigating Metal-Enhanced Fluorescence and Surface-Enhanced Raman Spectroscopy.

SiO2@Ag nanocomposite (NC) has been synthesized by the chemical reduction and Stӧber method for Metal-enhanced fluorescence (MEF) of Rhodmine 6G (R6G) and Surface-enhanced Raman spectroscopy (SERS) of Malachite green (MG). As-synthesized SiO2@Ag NC indicated SiO2 nanosphere (NS) and Ag nanoparticle (NP) morphologies. The SiO2@Ag NC was high quality with a well-defined crystallite phase with average sizes of 24nm and 132nm for Ag NP and SiO2 NC, respectively. By using SiO2@Ag NC, the photoluminescence (PL) intensity of the R6G (at 59.17ppm) was increased approximately 133 times. The SERS of the MG (at 1.0ppm) with SiO2@Ag NC as substrate clearly observed vibrational modes in MG dye at 798, 916, 1172, 1394, and 1616cm-1. As a result, the SERS enhancement factor (EFSERS) at 1172cm-1 obtained 6.3 × 106. This initial study points to the potential of SiO2@Ag NC as a promising material for MEF and SERS substrates to detect dyes at low concentrations.

Ag–ZnO Nanocomposites as a 3D Metal-Enhanced Fluorescence Substrate for the Fluorescence Detection of DNA

Fluorescence enhancement, including metal-enhanced fluorescence (MEF), has been widely explored in the field of biosensor technology. Two- and three-dimensional (2D and 3D) MEF substrate approaches have been developed for biosensors to detect target biomarkers such as genes and proteins. However, several issues remain, not least poor reproducibility caused by limited recognition and diffusion of biomolecules among target molecules, biological probes, and MEF substrates in aqueous environments. Here, an Ag–ZnO nanocomplex as a 3D MEF substrate was developed in two steps: hydrothermal synthesis of ZnO nanowire (NW) as a building block of Ag nanoparticles (NPs), and photochemical deposition of AgNPs on the ZnO NW. Fluorescence enhancement on Ag–ZnO nanocomposites (NCs) compared with the glass substrate was 14.8-, 16.9-, 11.4-, and 14.2-fold in the presence of 10, 20, 40, and 60 base pair (bp) double-stranded (ds) DNA, respectively. The MEF effects on glass, ZnO NW, and Ag–ZnO NCs were analyzed by measuring the fluorescence lifetime of fluorescein (FAM)-labeled dsDNA, and the average lifetimes were 9.76, 9.34, and 7.86 ns, respectively. Ag–ZnO NCs showed a 50-fold higher sensitivity for the detection of dsDNA than Ag nanoisland film (AgNIF) as a 2D MEF substrate. Therefore, Ag–ZnO NCs may have potential as a 3D MEF substrate. The MEF phenomenon might involve broad-range interactions between the surface of the metal nanostructure on Ag–ZnO NCs and fluorophores in various scenarios. Our Ag–ZnO NCs could facilitate the development of sensitive MEF-based biosensors for DNA detection.

Preparation of 2D Periodic Nanopatterned Arrays through Vertical Vibration-Assisted Convective Deposition for Application in Metal-Enhanced Fluorescence

The performance of a metal-enhanced fluorescence (MEF) substrate is fundamentally based on the orientation of the metal nanostructures on a solid substrate. In particular, two-dimensional (2D) periodic metallic nanostructures exhibit a strong confinement of the electric field between adjacent nanopatterns due to localized surface plasmon resonance (LSPR), leading to stronger fluorescence intensity enhancement. The use of vertical vibration-assisted convective deposition, a novel, simple, and highly cost-effective technique for preparing the 2D periodic nanostructure of colloidal particles with high uniformity, was therefore proposed in this work. The influences of vertical vibration amplitude and frequency on the structure of thin colloidal film, especially its uniformity, monolayer, and hexagonal close-packed (HCP) arrangement, were also investigated. It was found that the vibration amplitude affected film uniformity, whereas the vibration frequency promoted the colloidal particles to align themselves into defect-free HCP nanostructures. Furthermore, the results showed that the self-assembled 2D periodic arrays of monodisperse colloidal particles were employed as an excellent template for a Au thin-film coating in order to fabricate an efficient MEF substrate. The developed MEF substrate provided a strong plasmonic fluorescence enhancement, with a detection limit for rhodamine 6G as low as 10−9 M. This novel approach could be advantageous in further applications in the area of plasmonic sensing platforms.

Fabrication of a Plasmonic Nanoantenna Array Using Metal Deposition on Polymer Nanoimprinted Nanodots for an Enhanced Fluorescence Substrate.

A simple and cost-effective method is proposed herein for a plasmonic nanoantenna array (PNAA) for the fabrication of metal-enhanced fluorescence (MEF) substrates in which fluorophores interact with the enhanced electromagnetic field generated by a localized surface plasmon to provide a higher fluorescence signal. The PNAA is fabricated by the deposition of a silver (Ag) layer on an ultraviolet (UV) nanoimprinted nanodot array with a pitch of 400 nm, diameter of 200 nm, and height of 100 nm. During deposition, raised Ag nanodisks and a lower Ag layer are, respectively, formed on the top and bottom of the imprinted nanodot array, and the gap between these Ag layers acts as a plasmonic nanoantenna. Since the thickness of the gap within the PNAA is influenced by the thickness of Ag deposition, the effects of the latter upon the geometrical properties of the fabricated PNAA are examined, and the electromagnetic field intensity distributions of PNAAs with various Ag thicknesses are simulated. Finally, the fluorescence enhancement factor (FEF) of the fabricated PNAA MEF substrate is measured using spotted Cy5-conjugated streptavidin to indicate a maximum enhancement factor of ~22× for the PNAA with an Ag layer thickness of 75 nm. The experimental results are shown to match the simulated results.

Fabrication of Ag nanorods on micropost array for a metal-enhanced fluorescence substrate with a high signal-to-background ratio

Selective fabrication of metallic nanostructures at the spotting area is required to increase the signal-to-background noise ratio (SBR) of the metal-enhanced fluorescence (MEF) substrate. As a simple and cost-effective fabrication method for MEF substrate with high SBR, a glancing angle deposition (GLAD) process of Ag material on the UV-imprinted micropost array (50 μm in height, 300 μm in diameter, and 600 μm in pitch) was proposed to selectively fabricate Ag nanorods on the top of micropost structure (spotting area). Ag nanorod formation at the bottom of the micropost decreased as the deposition angle in Ag GLAD increased. A deposition angle of 89° and deposition thickness of 500 nm were selected as the optimum GLAD conditions to maximize the SBR. The optimum Ag nanorods on micropost array (AgNMPA) MEF substrate provided 71-fold fluorescence signal enhancement and 25-times higher SBR than the bare glass substrate. It also provided 7-times higher SBR than the Ag nanorod MEF substrate, which has a similar Ag nanorod structure but is not selectively formed. The detection limit of AgNMPA was 16- and 4-times lower than that of the amine-functionalized glass substrate and commercial epoxy slide, respectively. Although the fluorescence signal of AgNMPA was similar to that of Ag nanorod substrate, the detection limit was 2-times lower because of the low signal standard deviation caused by the low background noise and clear spot shape.

Design and fabrication of a new fluorescence enhancement system of silver nanoparticles-decorated aligned silver nanowires

A new substrate, aligned Ag nanowires decorated with silver nanoparticle composite structure (AgNWs@AgNPs), was fabricated to investigate metal-enhanced fluorescence (MEF) and its mechanism. The new composite structure was fabricated via a three-phase interface assembly method followed by SnCl2 sensitization and AgNO3 reduction process. The size and distribution of the nanoparticles on silver nanowires increased with the sensitization and reduction cycles. The formation of AgNPs on the surfaces of AgNWs was confirmed by multiple characterization methods including scanning electron microscopy (SEM), transmission electron microscope (TEM), atomic force microscopy (AFM) and X-ray diffraction (XRD). The results show that the fluorescence intensity of the poly(3-hexylthiophene) (P3HT) on the composite structure was greatly enhanced compared with that on bare glass substrate, and the intensity increased with the increase in particle sizes and density. The mechanism was based on the increase in excitation rate and the radiation decay rate. The new type of substrate could serve as a good and efficient MEF substrate for high-performance fluorescence-based devices.

Development of a Protein Microarray Chip with Enhanced Fluorescence for Identification of Semen and Vaginal Fluid.

The detection of body fluids has been used to identify a suspect and build a criminal case. As the amount of evidence collected at a crime site is limited, a multiplex identification system for body fluids using a small amount of sample is required. In this study, we proposed a multiplex detection platform using an Ag vertical nanorod metal enhanced fluorescence (MEF) substrate for semen and vaginal fluid (VF), which are important evidence in cases of sexual crime. The Ag nanorod MEF substrate with a length of 500 nm was fabricated by glancing angle deposition, and amino functionalization was conducted to improve binding ability. The effect of incubation time was analyzed, and an incubation time of 60 min was selected, at which the fluorescence signal was saturated. To assess the performance of the developed identification chip, the identification of semen and VF was carried out. The developed sensor could selectively identify semen and VF without any cross-reactivity. The limit of detection of the fabricated microarray chip was 10 times better than the commercially available rapid stain identification (RSID) Semen kit.

Room-temperature excitonic emission with a phonon replica from graphene nanosheets deposited on Ni-nanocrystallites/Si-nanoporous pillar array.

Graphene nanosheets (GNSs) were grown on a Si nanoporous pillar array (Si-NPA) via chemical vapour deposition, using a thin layer of pre-deposited Ni nanocrystallites as catalyst. GNSs were determined to be of high quality and good dispersivity, with a typical diameter size of 15 × 8 nm. Light absorption measurements showed that GNSs had an absorption band edge at 3.3 eV. They also showed sharp and regular excitonic emitting peaks in the ultraviolet and visible region (2.06–3.6 eV). Moreover, phonon replicas with long-term stability appeared with the excitonic peaks at room temperature. Temperature-dependent photoluminescence from the GNSs revealed that the excitonic emission derived from free and bound excitonic recombination. A physical model based on band energy theory was constructed to analyse the carrier transport of GNSs. The Ni nanocrystallites on Si-NPA, which acted as a metal-enhanced fluorescence substrate, were supposed to accelerate the excitonic recombination of GNSs and enhanced the measured emission intensity. Results of this study would be valuable in determining the luminescence mechanism of GNSs and could be applied in real-world optoelectronic devices.

Gold Nanostar Substrates for Metal-Enhanced Fluorescence through the First and Second Near-Infrared Windows

Gold nanostars (AuNSs) are receiving increasing attention for their potential applications in bionanotechnology because of their unique optical properties related to their complex branched morphology. Their sharp features allow strong localized surface plasmon resonances, tunable in the near-infrared (NIR) region, and large enhancements of local electromagnetic fields. Here, the application of AuNSs in metal-enhanced fluorescence (MEF) in the NIR and second NIR (NIR-II) biological windows is explored for the first time. NIR/NIR-II fluorophores are incorporated onto monolayers of AuNSs with tunable plasmonic responses. Over 320-fold fluorescence enhancement is achieved in the NIR, confirming that AuNS substrates are promising NIR-MEF platforms for the development of ultrasensitive biosensing applications. Using fluorescence lifetime measurements to semiquantitatively deconvolute the excitation enhancement from emission enhancement, as well as modeling to simulate the electric field enhancement, we show tha...

Fluorescence enhancement of the conjugated polymer films based on well-ordered Au nanoparticle arrays

In this paper, well-ordered Au nanoparticle arrays on silicon substrates were employed as efficient metal-enhanced fluorescence (MEF) substrates for investigating the fluorescence properties of the conjugated polymer poly(3-hexylthiophene) (P3HT). The ordered Au nanoparticle arrays were fabricated by block copolymer self-assembly technology, and the particle sizes were controlled by adjusting the molar ratios of HAuCl4 precursor to vinyl pyridine units. The approach is economical and suitable to fabricate large-area MEF substrates. The results about fluorescence properties of P3HT showed that the fluorescence intensities of the P3HT films were improved on ordered Au nanoparticle arrays compared to those on bare silicon substrate and were significantly enhanced with the Au nanoparticle sizes increasing. The mechanism is based on localized surface plasmon resonances, coupling and propagating surface plasmons, and the emission enhancement mainly resulted from the increase of the excitation rate. This work provides a new way to prepare efficient MEF substrates for high-performance fluorescence-based devices.

Metal-Enhanced Fluorescence Lifetime Imaging and Spectroscopy on a Modified SERS Substrate

In this paper, we developed a metal-enhanced fluorescence (MEF) substrate by modification of the commercially available surface enhanced Raman spectroscopy (SERS) substrate that may meet the reproducibility and sensitivity challenge of MEF. In spite of many studies and interest on MEF from a number of research groups, application to real-world situations and its commercial use remain challenging mainly due to the difficulties in fabricating reproducible MEF substrates. Specifically, one of the challenges is achieving a standardized MEF substrate for reproducible fluorescence intensity enhancement and/or changes in lifetime. The gold standard klarite substrates for SERS were coated with a thin layer of silver nanoparticles for MEF studies. To test the newly developed MEF substrates, a monolayer of streptavidin conjugated Alexa-647 was assembled on biotinylated-glass or MEF substrates. We observed over 50-fold increase in the fluorescence intensity from a monolayer of streptavidin conjugated Alexa-647 on the biotinylated MEF substrate compared to the same on glass substrate. A significant reduction in the lifetime and increased photostability of Alexa-647 on MEF substrate was observed. Fluorescence lifetime imaging was performed on the monolayer of dye assembled on the modified SERS substrates. We expect this study will serve as a platform to encourage the future use of a standardized MEF substrate for a plethora of sensing applications.

Fabrication of a substrate for Ag-nanorod metal-enhanced fluorescence using the oblique angle deposition process

Metal-enhanced fluorescence (MEF) is a powerful technology to improve the sensitivity of fluorescence analysis by allowing fluorophores to interact with enhanced electromagnetic fields generated by the localised surface plasmon resonance effects of metallic nanostructures. To apply MEF technology to disposable DNA or protein microarray analysis, metallic nanostructures need to be fabricated on the full area of a glass slide at low cost. In this reported work an oblique angle deposition process was used to fabricate Ag nanorods on the whole area of a 25 × 75 mm2 glass slide to serve as an inexpensive and large-area MEF substrate. To examine the feasibility of the proposed substrate and maximise signal enhancement, Ag nanorods with different lengths were deposited on glass slides. Different concentrations of streptavidin-conjugated Cy5 in phosphate buffered saline were spotted onto the Ag nanorods and bare glass substrates, and the fluorescence signals were measured and compared. Longer Ag nanorods improved the fluorescence enhancement factor because of a higher surface plasmon resonance effect and the large surface area of the nanostructure with a high aspect ratio. A maximum enhancement factor of −23 was obtained from Ag nanorods that were 1000 nm long with comparable uniformity with the glass substrate.

Use of metal-enhanced fluorescence spectroscopy for detection of polycyclic aromatic hydrocarbons in diesel oil emulsions in artificial seawater

In this study, we employed the metal-enhanced fluorescence (MEF) method for the detection of polycyclic aromatic hydrocarbons (PAHs) in diesel oil emulsions in artificial seawater. Silver nanoparticles with an average diameter of 80 nm were immobilized onto silanized quartz as MEF substrates. Excited by illumination at 355 nm, a 4.6-fold increase in the fluorescence signals of PAHs was recorded when MEF substrates were used. The finite-difference time-domain (FDTD) method was used to provide mechanistic insights into the fluorescence enhancement. Monitoring water pollution has environmental significance and MEF spectroscopy is finding direct applications in this field.

Silver-Gold Nanocomposite Substrates for Metal-Enhanced Fluorescence: Ensemble and Single-Molecule Spectroscopic Studies.

In recent years, there has been a growing interest in the studies involving the interactions of fluorophores with plasmonic nanostructures or nanoparticles. These interactions lead to several favorable effects such as increase in the fluorescence intensities, increased photostabilities, and reduced excited-state lifetimes that can be exploited to improve the capabilities of present fluorescence methodologies. In this regard, we report the use of newly developed silver-gold nanocomposite (Ag-Au-NC) structures as substrates for metal-enhanced fluorescence (MEF). The Ag-Au-NC substrates have been prepared by a one-step galvanic replacement reaction from thin silver films coated on glass slides. This approach is simple and suitable for the fabrication of MEF substrates with large area. We have observed about 15-fold enhancement in the fluorescence intensity of ATTO655 from ensemble fluorescence measurements using these substrates. The fluorescence enhancement on the Ag-Au-NC substrates is also accompanied by a reduction in the fluorescence lifetime of ATTO655, which is consistent with the fluorophore-plasmon coupling mechanism. Single-molecule fluorescence measurements have been performed to gain more insight into the metal-fluorophore interactions and to unravel the heterogeneity in the interaction of individual fluorophores with the fabricated substrates. The single-molecule studies are in good agreement with the ensemble measurements and show maximum enhancements of ~50-fold for molecules located in proximity to the "hotspots" on the substrates. In essence, the Ag-Au-NC substrates have a very good potential for various MEF applications.

Synthesis of silver particles decorated anodic aluminium oxide substrates for metal-enhanced fluorescence

Silver nanoparticles for metal-enhanced fluorescence (MEF) have been successfully synthesised by pulsed electrodeposition of silver in the anodic aluminium oxide (AAO) templates. X-ray diffraction and scanning electron microscopy indicate that the as-synthesised samples were composed of face-centred cubic Ag nanoparticles and the average nanoparticle size increased with the increasing electrodeposition time. The UV–visible reflection spectra were measured to investigate the surface plasma resonance absorbance. Taking rhodamine B as the probing molecule, high-quality MEF spectra were observed by excitation with a 480 nm laser line. The enhancement ability could be well controlled by simply adjusting the deposition times. The high enhancement factor suggests that this substrate could be used to fabricate ‘hot spots’ for MEF.

Ensemble and Single Molecule Studies on the Use of Metallic Nanostructures to Enhance the Intrinsic Emission of Enzyme Cofactors

We present a strategy for enhancing the intrinsic emission of the enzyme cofactors flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH). Ensemble studies show that silver island films (SIFs) are the optimal metal enhanced fluorescence (MEF) substrates for flavins and gave emission enhancements of over 10-fold for both FAD and FMN. A reduction in the lifetime of FAD and FMN on SIFs was also observed. Thermally evaporated aluminum films on quartz slides were found to be the optimal MEF substrate for NADH and gave a 5-fold increase in the emission intensity of NADH. We present finite-difference time-domain (FDTD) calculations that compute the enhancement in the radiated power emitting from an excited state dipole emitting in the wavelength range of NADH in close proximity to an aluminum nanoparticle, and a dipole emitting in the emission wavelength of flavins next to a silver nanoparticle. These calculations confirm that aluminum serves as the optimal MEF substrate for NADH and silver was the optimal MEF substrate for flavins. This is because the plasmon resonance properties of aluminum lie in the UV-blue regime and that of silver lie in the visible region. We also present the results of single molecule studies on FMN which show SIFs can both significantly enhance the intrinsic emission from single FMN molecules, significantly reduce their lifetimes and also significantly reduce FMN blinking. This is the first report of the observation of MEF from cofactors both at the ensemble and single molecule level. We hope this study will serve as a platform to encourage the future use of metallic nanostructures to study cofactors using their intrinsic fluorescence to directly monitor enzyme binding reactions without the need of extrinsic labeling of the molecules.

Tuning the Intensity of Metal‐Enhanced Fluorescence by Engineering Silver Nanoparticle Arrays

It is demonstrated that silver nanoparticle (SNP) arrays fabricated by combining nanoimprint lithography and electrochemical deposition methods can be used as substrates for metal-enhanced fluorescence, which is widely used in optics, sensitive detection, and bioimaging. The method presented here is simple and efficient at controlling the nanoparticle density and interparticle distance within one array. Furthermore, it is found that the fluorescence intensity can be tuned by engineering the feature size of the SNP arrays. This is due to the different coupling efficiency between the emission of the fluorophores and surface plasmon resonance band of the metallic nanostructures.

Silver half-shell arrays with controlled plasmonic response for fluorescence enhancement optimization

Regular arrays of interconnected silver half-shells (HSs) deposited on self-organized polystyrene microspheres are proposed as plasmonic substrates for metal-enhanced fluorescence. An emission enhancement of 28 times was demonstrated for Rose Bengal fluorophore placed at about 1 nm above HSs. The enhancement correlates with the spectral overlap between the fluorophore emission and the plasmonic resonance of the HSs, indicating a surface plasmon-coupled emission mechanism for the amplification. As the overlap can be easily tuned by controlling the diameter of underlying microspheres, such plasmonic structures could be relevant for building fluorescence-based sensing devices with optimized efficiency for any given fluorophore.

Aluminum Nanoparticles as Substrates for Metal-Enhanced Fluorescence in the Ultraviolet for the Label-Free Detection of Biomolecules

We use finite-difference time-domain calculations to show that aluminum nanoparticles are efficient substrates for metal-enhanced fluorescence (MEF) in the ultraviolet (UV) for the label-free detection of biomolecules. The radiated power enhancement of the fluorophores in proximity to aluminum nanoparticles is strongly dependent on the nanoparticle size, fluorophore-nanoparticle spacing, and fluorophore orientation. Additionally, the enhancement is dramatically increased when the fluorophore is between two aluminum nanoparticles of a dimer. Finally, we present experimental evidence that functionalized forms of amino acids tryptophan and tyrosine exhibit MEF when spin-coated onto aluminum nanostructures.