The authors regret that some funding information was omitted from the Acknowledgements section of the manuscript. The complete Acknowledgements section should read as follows: Acknowledgements The authors thank Jing Jiang and Te-Wei Chang for help with the SEM. We thank Hoang Nguyen, Cindy Larson for the preparation of master nanopillar mold. Transmission experiments were carried out in the Frederick Seitz
A Photonic Crystal (PC) surface fabricated upon a quartz substrate using nanoimprint lithography has been demonstrated to enhance light emission from fluorescent molecules in close proximity to the PC surface. Quartz was selected for its low autofluorescence characteristics compared to polymer-based PCs, improving the detection sensitivity and signal-to-noise ratio (SNR) of PC Enhanced Fluorescence (PCEF). Nanoimprint lithography enables economical fabrication of the subwavelength PCEF surface structure over entire 1x3 in2 quartz slides. The demonstrated PCEF surface supports a transverse magnetic (TM) resonant mode at a wavelength of λ = 632.8 nm and an incident angle of θ = 11°, which amplifies the electric field magnitude experienced by surface-bound fluorophores. Meanwhile, another TM mode at a wavelength of λ = 690 nm and incident angle of θ = 0° efficiently directs the fluorescent emission toward the detection optics. An enhancement factor as high as 7500 × was achieved for the detection of LD-700 dye spin-coated upon the PC, compared to detecting the same material on an unpatterned glass surface. The detection of spotted Alexa-647 labeled polypeptide on the PC exhibits a 330 × SNR improvement. Using dose-response characterization of deposited fluorophore-tagged protein spots, the PCEF surface demonstrated a 140 × lower limit of detection compared to a conventional glass substrate.
Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by >8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels (“enhanced excitation”) and the spatially biased funneling of fluorophore emissions through coupling to PC resonances (“enhanced extraction”), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couples fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region <100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in <1 minute. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM.
A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ∼3×5 in.(2) surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ=632.8 nm laser (cyanine-5) and a dye excited by a λ=532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32× increase in fluorescent signal intensity for cyanine-5 conjugated streptavidin labeling, while a 25× increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm<λ<633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays.
All fluorescent assays would benefit from greater signal-to-noise ratios (SNRs), which enable detection of disease biomarkers at lower concentrations for earlier disease diagnosis and detection of genes that are expressed at the lowest levels. Here, we report an approach to enhance fluorescence in which surface adsorbed fluorophore-tagged biomolecules are excited on a photonic crystal surface that is coupled to an underlying Fabry-Perot type cavity through a gold mirror reflector beneath the photonic crystal. This approach leads to 6Â increase in signal-to-noise ratio of a dye labeled polypeptide compared to ordinary photonic crystal enhanced fluorescence. Fluorescence imaging is currently among the most widely used techniques for disease diagnosis, genomic/proteomic research, and monitoring processes in biological systems. 1 A variety of nano-patterned structures such as plasmonic gratings, nanoantennas, and photonic crystals are being studied for the purpose of enhancing fluorescence output. 2 These approaches seek to use a nanostructure to enhance the electric field intensity experienced by surface-bound fluorophores, so as to provide a gain mechanism that is not present upon an ordinary surface. Such surfaces have also been shown to incorporate additional signal enhancement mechanisms that include increased particle extinction coefficients, reduced fluorescence lifetimes, and directional emission. 3 Photonic crystals (PCs) exhibit remarkable optical properties due to excitation of resonant guided modes by the incident light, which results in a significant enhancement of the electromagnetic fields at the surface of this nanostructure. 4 This enhanced near field can be used to design highly sensitive chemical and biological sensors with specific PC resonances tailored by PC's geometry. 5 The fluorescence enhancement in PCs is attributable to a combination of processes including enhanced excitation of the molecule and enhanced coupling efficiency of the fluorescent emission to the far field. 6 Coupling of multiple resonators can lead to interesting optical properties like increase in Q, changes in electric fields, or modification of the far-field reflection properties, which can improve detection in sensing applications. 7 In this paper, we demonstrate such a coupled-cavity photonic crystal structure. The structure operates by coupling onedimensional (1D) PC modes to the modes of an underlying Fabry-Perot type optical cavity. This coupling of the two modes results in even higher evanescent fields on the surface of the PC when compared to the fields when the light is resonantly coupled to a PC without an underlying cavity coupled to it. Our experimental results are supported by a quantitative theoretical investigation of the cavity-coupled PC structure using rigorous coupled wave analysis (RCWA) electromagnetic modeling.Figures 1(a) and 1(b) compare the structure of the cavity-coupled PC biosensor to the solitary PC. Adding a layer of gold under the PC at a specific distance forms the cavity-coupled PC structure...
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