Alan L. Huston

Cellular Uptake and Fate of PEGylated Gold Nanoparticles Is Dependent on Both Cell-Penetration Peptides and Particle Size

Numerous studies have examined how the cellular delivery of gold nanoparticles (AuNPs) is influenced by different physical and chemical characteristics; however, the complex relationship between AuNP size, uptake efficiency and intracellular localization remains only partially understood. Here we examine the cellular uptake of a series of AuNPs ranging in diameter from 2.4 to 89 nm that are synthesized and made soluble with poly(ethylene glycol)-functionalized dithiolane ligands terminating in either carboxyl or methoxy groups and covalently conjugated to cell penetrating peptides. Following synthesis, extensive physical characterization of the AuNPs was performed with UV-vis absorption, gel electrophoresis, zeta potential, dynamic light scattering, and high resolution transmission electron microscopy. Uptake efficiency and intracellular localization of the AuNP-peptide conjugates in a model COS-1 cell line were probed with a combination of silver staining, fluorescent counterstaining, and dual mode fluorescence coupled to nonfluorescent scattering. Our findings show that AuNP cellular uptake is directly dependent on the surface display of the cell-penetrating peptide and that the ultimate intracellular destination is further determined by AuNP diameter. The smallest 2.4 nm AuNPs were found to localize in the nucleus, while intermediate 5.5 and 8.2 nm particles were partially delivered into the cytoplasm, showing a primarily perinuclear fate along with a portion of the nanoparticles appearing to remain at the membrane. The 16 nm and larger AuNPs did not enter the cells and were located at the cellular periphery. A preliminary assessment of cytotoxicity demonstrated minimal effects on cellular viability following peptide-mediated uptake.

Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.

Multiple UV wavelength excitation and fluorescence of bioaerosols

A two-wavelength excitation bioaerosol sensor has been developed and characterized for classifying various types of aerosols, including biological organisms and non-biological interferents. Single aerosols, smaller than 10 μm, are interrogated with 266 nm and 355 nm laser pulses separated in time by 400 ns. Fluorescence signals excited by these pulses are detected in three broad spectral bands centered at 350 nm, 450 nm and 550 nm. The results indicate that bacterial spores, vegetative bacterial cells and proteins can be differentiated based on the two wavelength excitation approach.

Some characteristics of a droplet whispering-gallery-mode laser

We report lasing characteristics of 40-60-microm-diameter Rhodamine 590/water solution droplets pumped by a 20-nsec-duration Q-switched laser. The Rhodamine/water solution provides a useful model system for studying the properties of oscillators based on whispering-gallery-wave spherical cavities. The low threshold for lasing, 10(4) W/cm(2) for 10(-4) M solutions, is consistent with particle size and a cavity Q factor of 10(4). Portions of the droplet lase purely in transverse electric (TE) modes, while other portions contain both TE and lower-Q transverse magnetic modes. In the far field, the lasing droplet approximates a coherent point source emitting in all directions.

Self-Assembled Quantum Dot-Sensitized Multivalent DNA Photonic Wires

Combining the inherent scaffolding provided by DNA structure with spatial control over fluorophore positioning allows the creation of DNA-based photonic wires with the capacity to transfer excitation energy over distances greater than 150 Å. We demonstrate hybrid multifluorophore DNA-photonic wires that both self-assemble around semiconductor quantum dots (QDs) and exploit their unique photophysical properties. In this architecture, the QDs function as both central nanoscaffolds and ultraviolet energy harvesting donors that drive Förster resonance energy transfer (FRET) cascades through the DNA wires with emissions that approach the near-infrared. To assemble the wires, DNA fragments labeled with a series of increasingly red-shifted acceptor-dyes were hybridized in a predetermined linear arrangement to a complementary DNA template that was chemoselectively modified with a hexahistidine-appended peptide. The peptide portion facilitated metal-affinity coordination of multiple hybridized DNA-dye structures to a central QD completing the final nanocrystal-DNA photonic wire structure. We assembled several such hybrid structures where labeled-acceptor dyes were excited by the QDs and arranged to interact with each other via consecutive FRET processes. The inherently facile reconfiguration properties of this design allowed testing of alternate formats including the addition of an intercalating dye located in the template DNA or placement of multiple identical dye acceptors that engaged in homoFRET. Lastly, a photonic structure linking the central QD with multiple copies of DNA hybridized with 4-sequentially arranged acceptor dyes and demonstrating 4-consecutive energy transfer steps was examined. Step-by-step monitoring of energy transfer with both steady-state and time-resolved spectroscopy allowed efficiencies to be tracked through the structures and suggested that acceptor dye quantum yields are the predominant limiting factor. Integrating such DNA-based photonic structures with QDs can help create a new generation of biophotonic wire assemblies with widespread potential in nanotechnology.

Remote optical fiber dosimetry

Abstract Optical fibers offer a unique capability for remote monitoring of radiation in difficult-to-access and/or hazardous locations. Optical fiber sensors can be located in radiation hazardous areas and optically interrogated from a safe distance. A variety of remote optical fiber radiation dosimetry methods have been developed. All of the methods take advantage of some form of radiation-induced change in the optical properties of materials such as: radiation-induced darkening due to defect formation in glasses, luminescence from native defects or radiation-induced defects, or population of metastable charge trapping centers. Optical attenuation techniques are used to measure radiation-induced darkening in fibers. Luminescence techniques include the direct measurement of scintillation or optical excitation of radiation-induced luminescent defects. Optical fiber radiation dosimeters have also been constructed using charge trapping materials that exhibit thermoluminescence or optically stimulated luminescence (OSL).

Selecting Improved Peptidyl Motifs for Cytosolic Delivery of Disparate Protein and Nanoparticle Materials

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptides ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

Broadband thermal optical limiter

The limiting behavior of nigrosin dye dissolved in carbon disulfide was investigated in an f/5 defocusing geometry using 6 ns duration 532 nm laser excitation. Nigrosin dye is a broadband visible light absorber that is used here in conjunction with the large thermal nonlinearity of carbon disulfide solvent to defocus intense incident visible light. A limiting threshold energy of 40 nJ, corresponding to a fluence of only 100 mJ/cm2 in the solution, was observed with device absorption adjusted to 53%.

Excited-state absorption-enhanced thermal optical limiting in C 60

We report on optical limiting of Q-switched Nd:YAG radiation at 532 nm in an f/5 defocusing geometry using liquid solutions of C60 in 1-chloronaphthalene. The nonlinear optical-limiting mechanism is C60 excited-state absorption and enhanced thermal lensing in the solvent. A limiting threshold energy of 65 nJ, corresponding to an incident fluence of ∼0.3 J/cm2 at the focus in the solution, was observed with a device absorption of 25%.

Laser‐heated radiation dosimetry using transparent thermoluminescent glass

Laser‐stimulated thermoluminescence emission is reported from a novel transparent glass phosphor exposed to γ‐ray or ultraviolet radiation. Laser‐heated radiation dosimetry measurements, using this effect, are reported. A unique laser heating method permits the stimulation of the thermoluminescence without significantly raising the bulk temperature of the glass.