TLR5-Derived, TIR-Interacting Decoy Peptides to Inhibit TLR Signaling.

TLR5, which is activated by flagellin, plays an important role in initiating immune response to a broad spectrum of motile bacterial pathogens. TLRs induce intracellular signaling via dimerization of their TIR domains followed by adapter recruitment through multiple interactions of receptor and adapter TIRs. Here, a library of cell-permeable decoy peptides derived from the TLR5 TIR was screened for TLR5 signaling inhibition in the HEK-Blue-mTLR5 reporter cell line. The peptide demonstrating the strongest inhibition, 5R667, corresponded to the second helix of the region between the third and fourth ß-strands (helix C″). In addition to the TLR5-induced cytokine expression, 5R667 inhibited cytokine expression elicited by TLR4, TLR2, and TLR9. 5R667 also suppressed the systemic cytokine induction elicited by LPS administration in mice. 5R667 binding specificity was studied by time-resolved fluorescence spectroscopy in a cell-based assay. 5R667 demonstrated a multispecific binding pattern with respect to TIR domains: It bound TIRs of TLR adapters of the MyD88-dependent pathway, Toll/interleukin-1 receptor domain-containing adapter protein/MyD88 adapter-like (TIRAP) and MyD88, and also the TIR of TLR5. TR667, the peptide derived from the TIRAP region, which is structurally homologous to 5R667, demonstrated binding and inhibitory properties similar to that of 5R667. The surface-exposed residues within TIR regions represented by 5R667 and TR667 form motifs, which are nearly 90% conserved in vertebrate evolution and are distinctive of TLR5 and TIRAP TIR domains. Thus, we have identified an evolutionary conserved adapter recruitment motif within TLR5 TIR, the function of which can be inhibited by selective cell-permeable decoy peptides, which can serve as pan-specific TLR inhibitors.

Fluorophore Interactions with the Surface Modes and Internal Modes of a Photonic Crystal.

The metal-ligand complex tris(2,2'-bipyridine)ruthenium(II) chloride (Ru probe) displays a broad emission spectrum ranging from 540 to 730 nm. The emission spectra of Ru probe were measured when placed on top of a one-dimensional photonic crystal (1DPC), which supports both Bloch surface wave (BSW) and internal modes for wavelengths below 640 nm and only internal modes above 640 nm. The S-polarized emission spectra, with the electric vector parallel to the 1DPC surface, were found to be strongly dependent on the observation angle through the coupling prism. Also, the usual single broad-emission spectrum of Ru probe on glass was converted into two or more narrow-band-spectrum on the 1DPC, with emission band maxima dependent on the observation angle. The two S-polarized emission band peaks for Ru probe were found to be consistent with coupling to the BSW and first internal mode (IM1) of the 1DPC. The same spectral shifts and changes in emission maxima were observed by using Kretschmann and reverse Kretschmann illuminations. As the coupling requires the emitter to be in proximity with the photonic structure, we calculated near- and far-field distributions of a dipole directly located on the 1DPC surface. Finite-Difference Time-Domain (FDTD) simulations were performed to confirm fluorophore coupling to the BSW and internal modes (IMs). Both the measured and simulated results showed that IM coupled emission is significant. Coupling to the IM mode occurred at longer wavelengths where the 1DPC did not support a BSW. These results demonstrate that a simple Bragg grating, without a BSW mode, can be used for detection of surface-bound fluorophores.

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity.

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking. GTP-binding/GTPase activity is required for TG2 maintenance of the aggressive cancer phenotype. Thus, identifying agents that convert TG2 from the closed to the open GTP-binding/GTPase inactive conformation is an important cancer prevention/treatment strategy. Sulforaphane (SFN) is an important diet-derived cancer prevention agent that is known to possess a reactive isothiocyanate group and has potent anticancer activity. Using a biotin-tagged SFN analog (Biotin-ITC) and kinetic analysis we show that SFN covalently and irreversibly binds to recombinant TG2 to inhibit transamidase activity and shift TG2 to an open/extended conformation, leading to a partial inhibition of GTP binding. We also show that incubation of cancer cells or cancer cell extract with Biotin-ITC results in formation of a TG2/Biotin-ITC complex and that SFN treatment of cancer cells inhibits TG2 transamidase activity and shifts TG2 to an open/extended conformation. These findings identify TG2 as a direct SFN anticancer target in epidermal squamous cell carcinoma.

Converting the guided-modes of Bloch surface waves with surface pattern.

The guided-modes of Bloch surface waves, such as the transverse electric modes (TE00 and TE01 modes), can simultaneously exist in a low-refractive-index ridge waveguide with subwavelength thickness that are deposited on an all dielectric one-dimension photonic crystal. By using the finite difference frequency domain method, coupled mode theory and finite-difference time-domain method, the conversion between the guided-modes has been investigated. This conversion can be realized in a broadband wavelength with surface pattern of this low-index ridge. This conversion is useful for developing lab-on-a-chip photonic devices, such as a mode converter that can maintain the output mode purity over 90% with working wavelength ranging from 590 to 680 nm, and a power splitter that can maintain the splitting ratio over 8:2 with wavelength ranging from 530 to 710 nm.

Sodium-Sensitive Contact Lens for Diagnostics of Ocular Pathologies.

The ability to measure all the electrolyte concentrations in tears would be valuable in ophthalmology for research and diagnosis of dry eye disease (DED) and other ocular pathologies. However, tear samples are difficult to collect and analyze because the total volume is small and the chemical composition changes rapidly. Measurements of electrolytes in tears is challenging because typical clinical assays for proteins and other biomarkers cannot be used to detect ion concentrations tears. Here, we report the contact lens which is sensitive to sodium ion (Na+), one of the dominant electrolytes in tears. The Na ions in tears is diagnostic for DED. Three sodium-sensitive fluorophores (SG-C16, SG-LPE and SG-PL) were synthesized by derivatizing the sodium green with 1-hexadecyl amine, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine or poly-L-lysine, respectively. These probes were bound to modern silicone hydrogel (SiHG) contact lens, Biofinity from Cooper Vision. Doped lenses were tested for sodium ion dependent spectral properties of probes within the contact lens. The probes displayed changes in intensity and lifetime in response to Na+ concentration, were completely reversible, no significant probe wash-out from the lenses, were not affected by proteins in tears and were not removed after repeated washing. These results are the first step to our long-term goal, which is a lens sensitive to all the electrolytes in tears. We presented design, synthesis and implementation of three new sodium sensitive probes within a silicon hydrogel lens. Contact lenses to measure the other electrolytes in tears can be developed using the same approach by synthesis and testing of new ion-sensitive fluorophores.

Fluorescent contact lens for continuous non-invasive measurements of sodium and chloride ion concentrations in tears.

Rapid and non-invasive measurement of hydration status is medically important because even mild levels of dehydration can have a significant impact on physical and cognitive performance. Despite the potential value of determining whole-body hydration based on the electrolytes found in tears, very few tests are available. An area of intense interest is the development of a contact lens which could measure ion concentrations in tears, specifically that of sodium (Na+) and chloride (Cl-) ions, the dominant electrolytes in blood plasma and tears. Here, we describe a method to make fluorescent contact lenses which allow determination of Na+ and Cl- ion concentrations in tears. Fluorophores known to be sensitive to Na+ and Cl- were derivatized to bind non-covalently to two commercially-available silicone hydrogel (SiHG) contact lenses-the Biofinity (Comfilcon A) or MyDay (Stenfilcon A) lenses. The sodium- and chloride-sensitive fluorophores displayed spectral changes in the physiological range for Na+ and Cl- ions in tears. The lenses for both Na+ and Cl- ions were completely reversible. The sodium responses were not sensitive to protein interference including human lysozyme, human serum albumin and mucin type 2. The chloride sensitivity was similar with both lenses, but the sodium-sensitive range was different in the Biofinity and MyDay lenses. We also fabricated a lens with both the Na+ and Cl- probes in a single MyDay lens resulting in a contact lens that independently measured Na+ and Cl- concentrations without physical separation of the fluorophores. Our findings indicated that a sodium and chloride-sensitive contact lens (NaCl-lens) could be used for rapid non-invasive detection of whole-body hydration, as well as associated diseases or other infections.

Blocking TIR Domain Interactions in TLR9 Signaling.

Interaction of TLR9 with ligands activates NF-κB, leading to proinflammatory cytokine production. Excessive TLR activation is a pathogenic factor for inflammatory diseases. This study has examined cell-permeating decoy peptides (CPDPs) derived from the TLR9 Toll/IL-1R resistance (TIR) domain. CPDP 9R34, which included AB loop, ß-strand B, and N-terminal BB loop residues, inhibited TLR9 signaling most potently. CPDPs derived from α-helices C, D, and E (i.e., 9R6, 9R9, and 9R11) also inhibited TLR9-induced cytokines but were less potent than 9R34. 9R34 did not inhibit TLR2/1, TLR4, or TLR7 signaling. The N-terminal deletion modification of 9R34, 9R34-ΔN, inhibited TLR9 as potently as the full length 9R34. Binding of 9R34-ΔN to TIR domains was studied using cell-based Förster resonance energy transfer/fluorescence lifetime imaging approach. Cy3-labeled 9R34-ΔN dose-dependently decreased fluorescence lifetime of TLR9 TIR-Cerulean (Cer) fusion protein. Cy3-9R34-ΔN also bound TIRAP TIR, albeit with a lesser affinity, but not MyD88 TIR, whereas CPDP from the opposite TIR surface, 9R11, bound both adapters and TLR9. i.p. administration of 9R34-ΔN suppressed oligonucleotide-induced systemic cytokines and lethality in mice. This study identifies a potent, TLR9-specific CPDP that targets both receptor dimerization and adapter recruitment. Location of TIR segments that represent inhibitory CPDPs suggests that TIR domains of TLRs and TLR adapters interact through structurally homologous surfaces within primary receptor complex, leading to formation of a double-stranded, filamentous structure. In the presence of TIRAP and MyD88, primary complex can elongate bidirectionally, from two opposite ends, whereas in TIRAP-deficient cells, elongation is unidirectional, only through the αE side.

Extending the Propagation Distance of a Silver Nanowire Plasmonic Waveguide with a Dielectric Multilayer Substrate.

Chemical-synthesized silver nanowires have been proven as an efficient architecture for plasmonic waveguides, but the high propagation loss prevents their widely applications. Here, we demonstrate that the propagation distance of the plasmons along a silver nanowire can be extended if this nanowire was placed on a dielectric multilayer substrate containing a photonic band gap but not placed on a commonly used glass substrate. The propagation distance at 630 nm wavelength can reach 16 µm, even when the silver nanowire is as thin as 90 nm in diameter. Experimental and simulation results further show that the polarization of this propagating plasmon mode was nearly parallel to the surface of the dielectric multilayer, so it can be excited by a transverse-electric polarized Bloch surface wave propagating along a polymer nanowire with diameter at only about 170 nm on the same dielectric multilayer. Numerical simulations were also carried out and are consistent with the experiment results. Our work provides a platform with which to extend the propagation distance of the plasmonic waveguide and also for the integration between photonic and plasmonic waveguides on the nanometer scale.

Contact lens to measure individual ion concentrations in tears and applications to dry eye disease.

Dry eye disease (DED) affects millions of individuals in the United States and worldwide, and the incidence is increasing with an aging population. There is widespread agreement that the measurement of total tear osmolarity is the most reliable test, but this procedure provides only the total ionic strength and does not provide the concentration of each ionic species in tears. Here, we describe an approach to determine the individual ion concentrations in tears using modern silicone hydrogel (SiHG) contact lenses. We made pH (or H3O+, hydronium cation,/OH-, hydroxyl ion) and chloride ion (two of the important electrolytes in tear fluid) sensitive SiHG contact lenses. We attached hydrophobic C18 chains to water-soluble fluorescent probes for pH and chloride. The resulting hydrophobic ion sensitive fluorophores (H-ISF) bind strongly to SiHG lenses and could not be washed out with aqueous solutions. Both H-ISFs provide measurements which are independent of total intensity by use of wavelength-ratiometric measurements for pH or lifetime-based sensing for chloride. Our approach can be extended to fabricate a contact lens which provides measurements of the six dominant ionic species in tears. This capability will be valuable for research into the biochemical processes causing DED, which may improve the ability to diagnose the various types of DED.

Radiative decay engineering 8: Coupled emission microscopy for lens-free high-throughput fluorescence detection.

Fluorescence spectroscopy and imaging are now used throughout the biosciences. Fluorescence microscopes, spectrofluorometers, microwell plate readers and microarray imagers all use multiple optical components to collect, redirect and focus the emission onto single point or array imaging detectors. For almost all biological samples, except those with regular nanoscale features, emission occurs in all directions. With the exception of complex microscope objectives with large collection angles (NA ≤ 0.5), all these instruments collect only a small fraction of the total emission. Because of the increasing knowledge base on fluorophores within near-field (<200 nm) distances from plasmonic and photonic structures we can anticipate the development of compact devices in which the sample to be detected is located directly on solid state detectors such as CCDs or CMOS cameras. Near-field interactions of fluorophores with metallic or dielectric multi-layer structures (MLSs) can capture a large fraction of the total emission. Depending on the composition and dimensions of the MLSs, the spatial distribution of the sample emission results in distinct optical patterns on the detector surface. With either plain glass slides or MLSs the most commonly used front focal plane (FFP) images reveal the x-y spatial distribution of emission from the sample. Another approach, which is often used with two or three-dimensional nanostructures, is back focal plane (BFP) imaging. The BFP images reveal the angular distribution of the emission. The FFP and BFP images occur at certain distances from the sample which is determined by the details of the optical components. Obtaining these images requires multiple optical components and distances which are too large for the compact devices. For devices described in this paper, the images will be detected at a fixed distance between the sample and some arbitrary distance below the MLS which is determined by the geometry and thicknesses of the components. We refer to measurements at these locations as out-of-focal plane (OFP) imaging. Herein we describe a method to measure the optical fields at micron and multi-micron distances below the MLS, which will represent the images seen by an optically coupled array detector. The possibility of sub-surface optical images is illustrated using five different multi-layer structures. This is accomplished using an optical configuration which allows measurement at a front focal plane (FFP), back focal plane (BFP) or any OFP locations. Our OFP imaging method provides a link between the FFP images which reveals the surface distribution of fluorophores with the BFP images that reveal the angular distribution of emission. This linkage can be useful when examining structures which have nanoscale features due to fluorescence or leakage radiation from nanostructures.

Directing fluorescence with plasmonic and photonic structures.

Fluorescence technology pervades all areas of chemical and biological sciences. In recent years, it is being realized that traditional fluorescence can be enriched in many ways by harnessing the power of plasmonic or photonic structures that have remarkable abilities to mold the flow of optical energy. Conventional fluorescence is omnidirectional in nature, which makes it difficult to capture the entire emission. Suitably designed emission directivity can improve collection efficiency and is desirable for many fluorescence-based applications like sensing, imaging, single molecule spectroscopy, and optical communication. By incorporating fluorophores in plasmonic or photonic substrates, it is possible to tailor the optical environment surrounding the fluorophores and to modify the spatial distribution of emission. This promising approach works on the principle of near-field interaction of fluorescence with spectrally overlapping optical modes present in the substrates. In this Account, we present our studies on directional emission with different kinds of planar metallic, dielectric, and hybrid structures. In metal-dielectric substrates, the coupling of fluorescence with surface plasmons leads to directional surface-plasmon-coupled emission with characteristic dispersion and polarization properties. In one-dimensional photonic crystals (1DPC), fluorophores can interact with Bloch surface waves, giving rise to sharply directional Bloch surface wave-coupled emission. The interaction of fluorescence with Fabry-Pérot-like modes in metal-dielectric-metal substrates and with Tamm states in plasmonic-photonic hybrid substrates provides beaming emission normal to the substrate surface. These interesting features are explained in the context of reflectivity dispersion diagrams, which provide a complete picture of the mode profiles and the corresponding coupled emission patterns. Other than planar substrates, specially fabricated plasmonic nanoantennas also have tremendous potential in controlling and steering fluorescence beams. Some representative studies by other research groups with various nanoantenna structures are described. While there are complexities to near-field interactions of fluorescence with plasmonic and photonic structures, there are also many exciting possibilities. The routing of each emission wavelength along a specific direction with a given angular width and polarization will allow spatial and spectral multiplexing. Directional emission close to surface normal will be particularly useful for microscopy and array-based studies. Application-specific angular emission patterns can be obtained by varying the design parameters of the plasmonic/photonic substrates in a flexible manner. We anticipate that the ability to control the flow of emitted light in the nanoscale will lead to the development of a new generation of fluorescence-based assays, instrumentation, portable diagnostics, and emissive devices.

Antigenic properties of the HIV envelope on virions in solution.

The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro.

Extracting surface wave-coupled emission with subsurface dielectric gratings.

Conventional surface plasmons (SPs) or Bloch surface waves (BSWs) have a wave vector exceeding that of light in vacuum, and, therefore, the surface plasmon-coupled emission (SPCE) or Bloch surface wave-coupled emission (BSWCE) cannot escape from the corresponding structures. With the aid of a high-refractive-index prism or an oil-immersion objective, the SPCE or BSWCE can be coupled into free space. But the large volumes of the prism and objective are certainly unfavorable for miniaturization of the optical systems or inconvenient for applications such as the optical displays. Here we experimentally demonstrate a new method to extract the SPCE or BSWCE with a subsurface dielectric grating. The experimental results verify that the chip-like substrate with two decorated sides can bring out the directional fluorescence emission in free space. The emitting direction and emitting patterns can be tuned by the period size and dimensionality of the gratings. Our work provides a new strategy to realize free-space directional fluorescence emission at a very low cost and compact configuration, which has potential applications in fluorescence-based sensing, imaging, light-emitting diodes, optical displays, and other near-field optical devices.

Radiative decay engineering 7: Tamm state-coupled emission using a hybrid plasmonic-photonic structure.

There is a continuing need to increase the brightness and photostability of fluorophores for use in biotechnology, medical diagnostics, and cell imaging. One approach developed during the past decade is to use metallic surfaces and nanostructures. It is now known that excited state fluorophores display interactions with surface plasmons, which can increase the radiative decay rates, modify the spatial distribution of emission, and result in directional emission. One important example is surface plasmon-coupled emission (SPCE). In this phenomenon, the fluorophores at close distances from a thin metal film, typically silver, display emission over a small range of angles into the substrate. A disadvantage of SPCE is that the emission occurs at large angles relative to the surface normal and at angles that are larger than the critical angle for the glass substrate. The large angles make it difficult to collect all of the coupled emission and have prevented the use of SPCE with high-throughput and/or array applications. In the current article, we describe a simple multilayer metal-dielectric structure that allows excitation with light that is perpendicular (normal) to the plane and provides emission within a narrow angular distribution that is normal to the plane. This structure consists of a thin silver film on top of a multilayer dielectric Bragg grating, with no nanoscale features except for the metal or dielectric layer thicknesses. Our structure is designed to support optical Tamm states, which are trapped electromagnetic modes between the metal film and the underlying Bragg grating. We used simulations with the transfer matrix method to understand the optical properties of Tamm states and localization of the modes or electric fields in the structure. Tamm states can exist with zero in-plane wavevector components and can be created without the use of a coupling prism. We show that fluorophores on top of the metal film can interact with the Tamm state under the metal film and display Tamm state-coupled emission (TSCE). In contrast to SPCE, the Tamm states can display either S or P polarization. The TSCE angle is highly sensitive to wavelength, which suggests the use of Tamm structures to provide both directional emission and wavelength dispersion. Metallic structures can modify fluorophore decay rates but also have high losses. Photonic crystals have low losses but may lack the enhanced light-induced fields near metals. The combination of plasmonic and photonic structures offers the opportunity for radiative decay engineering to design new formats for clinical testing and other fluorescence-based applications.

Back focal plane imaging of directional emission from dye molecules coupled to one-dimensional photonic crystals.

Bloch surface waves (BSWs) on one-dimensional photonic crystals (1DPCs) have been used to beam the fluorescence emission from the dye molecules. All dielectric 1DPC displays its low propagating loss, narrow resonance and the absence of absorption or quenching. In this paper, back focal plane imaging reveals that in addition to the BSW mode, a guided mode and a cavity mode also exist in the 1DPC which all couple with the excited dye molecules. The appearance of these modes is sensitive to the wavelength of the fluorescence and alters the beaming effect by the 1DPC. Numerical simulations verify the existence of these modes which are consistent with the experimental results. Comparisons between the Bloch surface wave coupled emission and surface plasmon coupled emission are also presented for a clearer understanding of the multilayered film enabled directional emission.

Effect of metal film thickness on Tamm plasmon-coupled emission.

Tamm plasmons (TPs) are the result of trapping optical energy at the interface between a metal film and a one-dimensional photonic crystal. In contrast to surface plasmons, TPs display unique properties such as the ability to undergo direct optical excitation without the aid of prisms or gratings, being populated using both S- and P-polarized light, and importantly, they can be created with incident light normal to the surface. This latter property has recently been used to obtain Tamm plasmon-coupled emission (TPCE), which beams along a path directly perpendicular to the surface. In this paper the effects of metal film thickness on the TPCE are investigated using back focal plane (BFP) imaging and spectral resolutions. The observed experimental results are in agreement with the numerical simulations. The present work provides the basic understanding needed to design structures for TPCE, which in turn has potential applications in the fabrication of active materials for light emitting devices, fluorescence-based sensing, using microarrays, and imaging.

Radiative decay engineering 6: fluorescence on one-dimensional photonic crystals.

During the past decade the interactions of fluorophores with metallic particles and surfaces has become an active area of research. These near-field interactions of fluorophores with surface plasmons have resulted in increased brightness and directional emission. However, using metals has some disadvantages such as quenching at short fluorophore-metal distances and increased rates of energy dissipation due to lossy metals. These unfavorable effects are not expected in dielectrics. In this article, we describe the interactions of fluorophores with one-dimensional (1D) photonic crystals (PCs), which have alternating layers of dielectrics with dimensions that create a photonic band gap (PBG). Freely propagating light at the PBG wavelength will be reflected. However, similar to metals, we show that fluorophores within near-field distances of the 1DPC interacts with the structure. Our results demonstrate that these fluorophores can interact with both internal modes and Bloch surface waves (BSWs) of the 1DPC. For fluorophores on the surface of the 1DPC, the emission dominantly occurs through the 1DPC and into the substrate. We refer to these two phenomena together as Bragg grating-coupled emission (BGCE). Here we describe our preliminary results on BGCE. 1DPCs are simple to fabricate and can be handled and reused without damage. We believe that BGCE provides opportunities for new formats for fluorescence detection and sensing.

Largely enhanced single-molecule fluorescence in plasmonic nanogaps formed by hybrid silver nanostructures.

It has been suggested that narrow gaps between metallic nanostructures can be practical for producing large field enhancement. We design a hybrid silver nanostructure geometry in which fluorescent emitters are sandwiched between silver nanoparticles and silver island film (SIF). A desired number of polyelectrolyte layers are deposited on the SIF surface before the self-assembly of a second silver nanoparticle layer. Layer-by-layer configuration provides a well-defined dye position. It allows us to study the photophyical behaviors of fluorophores in the resulting gap at the single molecule level. The enhancement factor of a fluorophore located in the gap is much higher than those on silver surfaces alone and on glass. These effects may be used for increased detectability of single molecules bound to surfaces which contain metallic structures for either biophysical studies or high sensitivity assays.

Targeting TLR4 signaling by TLR4 Toll/IL-1 receptor domain-derived decoy peptides: identification of the TLR4 Toll/IL-1 receptor domain dimerization interface.

Agonist-induced dimerization of TLR4 Toll/IL-1R (TIR) domains initiates intracellular signaling. Therefore, identification of the TLR4-TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating decoy peptides, each of which represents a nonfragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides--4R1, 4R3, 4BB, 4R9, and 4αE--potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by Förster resonance energy transfer using time-resolved fluorescence spectroscopy, Bodipy-TMR-X-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean expressed in HeLa or HEK293T cells, whereas 4R3 was partially active, and 4R9 was least active. These findings suggest that the area between the BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the decoy peptide approach, in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function, and then their specific targets are identified by Förster resonance energy transfer to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions.

Plasmon- and Waveguide-Coupled Fluorescence at the Ultraviolet Region.

Surface plasmon-coupled emission (SPCE) has been well studied for its coupled, directional, and enhanced P-polarized radiation due to the interactions of fluorophores with surface plasmon polaritons (SPPs) on thin metal films. Such surface plasmon polariton-assisted directional fluorescence has various applications in biosensing. Herein, we demonstrate 2-aminopurine (2AP, a UV-absorbing and -emitting fluorophore) emission coupling to modes in aluminum-based plasmon-coupled waveguides (Al-PCWs). Directional emission from 2-aminopurine on plasmon-coupled waveguides was observed at specific angles as P-polarized SPCE and/or as P- or S-polarized waveguide-coupled emission (WGCE). All S-polarized waveguide modes showed clear angularly resolved emission as compared to that of P-polarized surface plasmon-coupled emission or P-polarized waveguide-coupled emission. The coupling angles, efficiencies, and polarizations of the modes were sensitive to the optical properties and overall dimensions of the top dielectric layer in PCWs. The effective plasmon-coupled waveguide can consist of either a thin probe-containing layer on top of the undoped silica film, or a single dielectric PVA layer with probes distributed throughout the film on the Al layer. The former structures with probes confined to the top of the undoped silica layer showed much higher angular resolutions and coupling efficiencies, as well as mode-dependent changes in lifetimes. These results demonstrate that the plasmon and waveguide modes can be used for selective detection of surface-bound and bulk fluorophores, simultaneously.