Many CBRNE threat materials are optimally implemented as aerosols. However, aerosol threats present unique challenges for sensor development, test and evaluation since particles may disburse in a turbulent atmosphere differently from tracer gases. This presentation addresses the need for tracer aerosol particles with known size distributions to be released with agent target simulant aerosols to provide ground truth for sensor test and evaluation. A novel approach for achieving uniquely identifiable individual aerosol particles is described based on utilization of quantum dots (QDs) and/or other luminescent nanocrystals (NCs), to create a multiplexed spectral barcode in tracer aerosol particles. QDs are small, typically nanometer scale, compared to micron-sized polymer beads as host aerosol particles. They also possess desirable optical properties of narrow, efficient emission bands, and are typically long-lived compared to organic dye molecules that photodegrade in sunlight. Multiple QD subpopulations, each with a narrow emission band at a distinct peak wavelength, can be encapsulated in a polymer microbead, conferring a superposition emission profile having multiple narrow peaks. The relative intensities of the emission peaks can be controlled by adjusting the number of QDs in each subpopulation. This spectral emission profile effectively becomes an individual particle barcode. Multiple polymer bead samples can be prepared each with different emission pattern (barcode). These samples can be mixed with target materials to be simultaneously released as aerosols to provide test ground truth for the simulant. Proof-of-principle experiments assessing the feasibility for using combinations of embedded NC populations in micron-sized droplets, as well as potential challenges to practical implementation will be discussed.
Nanoparticle (NP) bioconjugates have an important role in the development of photothermal (PT) therapy, a promising noninvasive approach wherein the NP acts as a light harvesting antenna to convert light into thermal energy to control cellular function. NP-mediated PT control of cellular membrane potential has gained significant interest in recent years as membrane potential regulates proliferation, migration, action potentials (in neurons), and contraction (in muscle cells). Recently gold nanoparticles (AuNPs) and Au nanorods have been demonstrated to induce action potentials via light-induced thermal activation of membrane tethered NPs. Spherical AuNPs have an efficient plasmonic output and are easily modified to interface with the cell surface. We demonstrate here that 20 nm diameter spherical AuNPs (tethered to the plasma membrane by a cholesterol moiety) transduce incident 532 nm light into proximal membrane heating that induces depolarization of membrane potential. Using these NP bioconjugates, we show the ability to controllably induce action potentials in dorsal root ganglion neurons and to control the membrane potential of rat pheochromocytoma cells. The ability to use light-actuated NP conjugates to control cellular behavior is an emerging research field with implications for neuronal and muscle cell modulation as well as in cancer therapeutics.
Nanoparticle (NP)-mediated drug delivery offers the potential to overcome limitations of systemic delivery, including the ability to specifically target cargo and control release of NP-associated drug cargo. Doxorubicin (DOX) is a widely used FDA-approved cancer therapeutic; however, multiple side effects limit its utility. Thus, there is wide interest in modulating toxicity after cell delivery. Our goal here was to realize a NP-based DOX-delivery system that can modulate drug toxicity by controlling the release kinetics of DOX from the surface of a hard NP carrier. To achieve this, we employed a quantum dot (QD) as a central scaffold which DOX was appended via three different peptidyl linkages (ester, disulfide, hydrazone) that are cleavable in response to various intracellular conditions. Attachment of a cell penetrating peptide (CPP) containing a positively charged polyarginine sequence facilitates endocytosis of the ensemble. Polyhistidine-driven metal affinity coordination was used to self-assemble both peptides to the QD surface, allowing for fine control over both the ratio of peptides attached to the QD as well as DOX dose delivered to cells. Microplate-based Förster resonance energy transfer assays confirmed the successful ratiometric assembly of the conjugates and functionality of the linkages. Cell delivery experiments and cytotoxicity assays were performed to compare the various cleavable linkages to a control peptide where DOX is attached through an amide bond. The role played by various attachment chemistries used in QD-peptide-drug assemblies and their implications for the rationale in design of NPbased constructs for drug delivery is described here.
KEYWORDS: Energy transfer, Gold, Data modeling, Energy efficiency, Fluorescence resonance energy transfer, Nanocrystals, Metals, Semiconductors, Quantum dots, Nanolithography
We characterize energy transfer between luminescent 1.5 nm diameter gold nanocrystal (AuNC) acceptors and three structurally/functionally-distinct classes of emissive donors including organic dyes, metal chelates and semiconductor quantum dots (QDs). Energy transfer efficiencies within the donor-AuNC assemblies were evaluated with steady-state and time-resolved measurements. Donor quenching was observed for every donor-acceptor pair although AuNC sensitization was only observed from metal-chelates and QDs. Results were analyzed with Förster’s dipole-dipole coupling model (FRET) and dipole-metal damping models including nanosurface energy transfer (NSET) and nanovolume energy transfer (NVET). FRET dramatically underestimated energy transfer efficiencies while the damping models provided qualitatively better fits to the data although neither fully reproduces the experimental data. Analysis suggests that organic dye donor quenching without corresponding AuNC sensitization results from enhanced intersystem crossing between dye singlet and triplet states driven by AuNC magnetic dipoles. We further consider factors that account for the unique electronic properties of the ultra-small luminescent AuNCs including the high excited state densities, rapid dephasing time and strong electron confinement as well as paramagnetic properties. Overall, the results provide insight into requirements necessary for realizing applications based on AuNC acceptor sensitization.
Recent interest in quantum dots (QDs) stems from the plethora of potential applications that arises from their tunable absorption and emission profiles, high absorption cross sections, resistance to photobleaching, functionalizable surfaces, and physical robustness. The emergent use of QDs in biological imaging exploits these and other intrinsic properties. For example, quantum confined Stark effect (QCSE), which describes changes in the photoluminescence (PL) of QDs driven by the application of an electric field, provides an inherent means of detecting changes in electric fields by monitoring QD emission and thus points to a ready mean of imaging membrane potential (and action potentials) in electrically active cells. Here we examine the changing PL of various QDs subjected to electric fields comparable to those found across a cellular membrane. By pairing static and timeresolved PL measurements, we attempt to understand the mechanism driving electric-field-induced PL quenching and ultimately conclude that ionization plays a substantial role in initiating PL changes in systems where QCSE has traditionally been credited. Expanding on these findings, we explore the rapidity of response of the QD PL to applied electric fields and demonstrate changes amply able to capture the millisecond timescale of cellular action potentials.
The controlled delivery of nanomaterials to the plasma membrane is critical for the development of nanoscale probes that can eventually enable cellular imaging and analysis of membrane processes. Chief among the requisite criteria are delivery/targeting modalities that result in the long-term residence (e.g., days) of the nanoparticles on the plasma membrane while simultaneously not interfering with regular cellular physiology and homeostasis. Our laboratory has developed a suite of peptidyl motifs that target semiconductor nanocrystals (quantum dots (QDs) to the plasma membrane where they remain resident for up to three days. Notably, only small a percentage of the QDs are endocytosed over this time course and cellular viability is maintained. This talk will highlight the utility of these peptide-QD constructs for cellular imaging and analysis.
The simultaneous visualization, identification and targeting of neurons during patch clamp-mediated
electrophysiological recordings is a basic technique in neuroscience, yet it is often complicated by the
inability to visualize the pipette tip, particularly in deep brain tissue. Here we demonstrate a novel
approach in which fluorescent quantum dot probes are used to coat pipettes prior to their use. The strong
two-photon absorption cross sections of the quantum dots afford robust contrast at significantly deeper
penetration depths than current methods allow. We demonstrate the utility of this technique in multiple
recording formats both in vitro and in vivo where imaging of the pipettes is achieved at remarkable depths
(up to 800 microns). Notably, minimal perturbation of cellular physiology is observed over the hours-long
time course of neuronal recordings. We discuss our results within the context of the role that quantum dot
nanoprobes may play in understanding neuronal cell physiology.
There is considerable research in the area of manipulating light below the diffraction limit, with potential applications ranging from information processing to light-harvesting. In such work, a common problem is a lack of efficiency associated with non-radiative losses, e.g., ohmic loss in plasmonic structures. From this point of view, one attractive method for sub-wavelength light manipulation is to use Förster resonance energy transfer (FRET) between chromophores. Although most current work does not show high efficiency, biology suggests that this approach could achieve very high efficiency. In order to achieve this goal, the geometry and spacing of the chromophores must be optimized. For this, DNA provides an easy means for the self-assembly of these complex structures. With well established ligation chemistries, it is possible to create facile hierarchical assemblies of quantum dots (QDs) and organic dyes using DNA as the platform. These nanostructures range from simple linear wires to complex 3-dimensional structures all of which can be self-assembled around a central QD. The efficiency of the system can then be tuned by changing the spacing between chromophores, changing the DNA geometry such that the donor to acceptor ratio changes, or changing the number of DNA structures that are self-assembled around the central QD. By exploring these variables we have developed a flexible optical system for which the efficiency can be both controlled and optimized.
CdSe/ZnS semiconductor quantum dots (QDs) are ideal materials for biological sensing and cellular imaging applications due to their superior photophysical properties in comparison to fluorescent proteins or dyes and their ease of conjugation to biological materials. We have previously developed a number of in vitro FRET based biosensors in the laboratory for detection of proteases and biological and chemical agents. We would like to expand these biosensing capabilities into cellular systems, requiring development of QD cellular delivery techniques. Peptide mediated cellular delivery of QDs is ideal as peptides are small, easily conjugated to QDs, easily manipulated and synthesized, and can be designed with “handles” for further chemical conjugation with other cargo. Here we discuss four cell delivery peptides that facilitate QD uptake in live cells. Understanding these peptides will help us design better nanoparticle cellular delivery systems and advance our capabilities for in vivo biosensing.
Biocompatible nanoparticles have recently attracted significant attention due to increasing interest in their use for
biological sensing, cellular labeling and in vivo imaging. Semiconductor quantum dots (QDs) with good colloidal
stability as well as small hydrodynamic sizes are particularly useful within these applications. We have developed a
series of dihydrolipoic acid (DHLA) based surface ligands to enhance the colloidal stability and biocompatibility of
water soluble QDs. Modification of DHLA with poly(ethylene glycol) derivatives provided the QDs with extended
colloidal stability over a wide pH range and under high salt concentrations, which contrasts with the limited colloidal
stability provided by DHLA alone. Functionalization of the PEG termini enabled one to have easy access to the QD
surface and construct a variety of stable QD-biomolecules conjugates. A series of DHLA-based compact ligands with
zwitterionic character has also been explored to develop compact sized QDs without sacrificing the colloidal stability.
Despite their smaller sizes than the PEG analogs, the QDs coated with the zwitterionic ligands still have excellent
colloidal stability and minimize nonspecific interactions in biological environments. Recent studies of thiol-based
multidentate ligands and ligand exchange methods further improved the colloidal stability and fluorescence quantum
yields.
Currently there is considerable interest in using bioconjugated nanoparticles for in vivo imaging, biosensing and
theranostics. Luminescent CdSe/ZnS core shell semiconductor quantum dots (QDs) have unique optical properties
and bioconjugation capabilities that make them ideal prototypes for these purposes. We have previously described
the metal-affinity association between the imidazole groups of terminal hexahistidine residues of peptides and
proteins and the ZnS shell of quantum dots as a useful bioconjugation technique. We have also demonstrated that
QDs labeled with an oligohistidine-tagged cell penetrating peptide (CPP) derived from the HIV TAT-protein could
undergo specific endocytosis-mediated cellular uptake in both HEK293T/17 and COS-1 cells. However, the QDs
were predominantly sequestered in the endosomes. This remains a significant hindrance to future potential cellular
imaging applications which require the QDs to access other subcellular organelles. Here we describe the testing of
several cytosolic QD delivery modalities including microinjection, the commercial cytosolic delivery agent PULSin,
and the cytosolic delivery peptide Palm-1. Palm-1, a palmitylated peptide that is capable of both cellular uptake and
rapid endosomal escape in multiple cell lines without concomitant toxicity, is shown to be the superior method for
cytosolic delivery of QDs. Potential intracellular applications for this peptide are discussed.
KEYWORDS: Fluorescence resonance energy transfer, Energy transfer, Quantum efficiency, DNA photonics, Quantum dots, Energy efficiency, Quenching (fluorescence), Composites, Chromophores, Absorption
We demonstrate Förster resonance energy transfer (FRET) through DNA photonic wires self-assembled around a
central CdSe/ZnS semiconductor quantum dot (QD). The central QD acts as a nanoscaffold and FRET donor to a
series of acceptor dyes along a DNA strand. By utilizing a DNA intercalating dye, altering the location of the dyes
and using a series of increasingly red-shifted dyes along the DNA, we are able to track the efficiency of energy
transfer through the DNA photonic structure via steady-state spectroscopy. Data suggests that limiting factors for
efficient energy transfer are the sub-obtimal photophysical properties of acceptor dyes, including low quantum yields.
These issues may be addressed with improved configurations of QDs, DNA and dyes. The development of biophotonic wire assemblies utilizing the superior photophysical properties of QDs will have widespread application
in nanotechnology.
Luminescent semiconductor quantum dots (QDs) possess several unique optical and spectroscopic properties including
high quantum yields, broad absorption spectra coupled to narrow symmetric, size-tunable emissions allowing large
achievable Stokes shifts, and exceptional resistance to photo- and chemical degradation. These properties make QDs
unique enabling materials for the development of the next generation of highly efficient biosensors for health security
applications, particularly within the context of living and fixed cells. Paramount in this developmental process is
addressing the biocompatibility of the QD materials. We are developing robust and facile delivery schemes for the
selective intracellular delivery of QD-based nanoassemblies. These schemes are based upon the self-assembly and
subsequent cellular uptake of QD-peptide and QD-polymer bioconjugates. Cellular delivery experiments utilizing both
delivery schemes will be presented. The advantages and disadvantages of each approach will be discussed, including
the intracellular fate and stability of the QD-nanoassemblies.
Sarnoff Corporation and the Naval Research Laboratory, through support of the U.S. Department of Homeland
Security, are developing an automated, high throughput bio-aerosol physical enrichment system designed for use as
part of a biological-threat protection system. The Biological Aerosol-Capture-Enrichment (BioACE) system is a
bio-aerosol collection system that combines three unique technologies to create physically enriched aerosol samples
that can be subsequently interrogated by any number of bio-threat detection systems for the presence of threat
agents. An air-to-air concentrator uses an inertial separation technique to highly concentrate an aerosol sample
presented to a dual wavelength ultra-violet laser induced fluorescence (UVLIF) optical trigger used to discriminate
potential threat particles from non-threat particles conveyed in a collimated particle stream. This particle
classification information is used to trigger an electrostatic deposition mechanism to deposit only those particles
determined to be potential bio-threats onto a stainless steel substrate. Non-threat particles are discarded with the
exiting airflow.
The goal for the most recent development effort has been the integration and optimization of these technologies into
a unit capable of producing highly enriched particulate samples from ambient air containing variable background
aerosol loading and type. Several key technical and engineering challenges were overcome during the course of this
development including a unique solution for compensating particle velocity dispersion within the airflow,
development of a real-time signal acquisition and detection algorithm for determining material type on a particle by
particle basis at rates greater than 2000 particles per second, and the introduction of a robust method for transferring
deposited particulate into a 50ul wet sample suitable for most advanced bio-detection techniques.
This paper will briefly describe the overall system architecture and then concentrate on the various component and
system design tradeoffs required to optimize sample enrichment performance. A system performance model will be
presented along with detailed analysis of the optical system components and electronic signal processing needed for
achieving high concentration sample enrichment. Experimental methods and data obtained in the laboratory setting
and from real world environments will be described and used to support the performance model of the system.
Finally, a number of air sampling scenarios will be analyzed using the system performance model to determine the
applicability of the BioACE system to the various concepts of operation perceived to be needed for achieving a high
performance bio-threat detect-to-protect system.
A bioaerosol sensor based on dual wavelength fluorescence excitation and multiple wavelength elastic
scattering has been developed and characterized for classifying micron-sized particles on the fly. The UVLIF
instrument successfully completed a field trial in which we detected and correctly identified over 90% of the simulant releases over the 2 week testing period.
Sarnoff Corporation and the Naval Research Laboratory, through support from HSARPA, are developing an automated,
high throughput bio-aerosol physical enrichment system designed for use as part of a biological-threat protection system.
The Biological Aerosol-Capture-Enrichment (BioACE) system is a bio-aerosol collection system that combines three
unique technologies to create physically enriched aerosol samples that can be subsequently interrogated by any number
of bio-threat detection systems for the presence of threat agents. An air-to-air concentrator uses an inertial separation
technique to highly concentrate an aerosol sample presented to a dual wavelength ultra-violet laser induced fluorescence
(UVLIF) optical trigger used to discriminate potential threat particles from non-threat particles conveyed in a collimated
particle stream. This particle classification information is used to trigger an electrostatic deposition mechanism to
deposit only those particles determined to be potential bio-threats onto a stainless steel substrate. Non-threat particles are
discarded with the exiting airflow. A prototype laboratory system in which particle size dependent elastic scatter rater
than fluorescence provides the triggering signal has been experimentally qualified. This paper will present a detailed
overview of the prototype system and discuss the physical enrichment results achieved.
A partnership that includes the Naval Research Laboratory (NRL), MIT Lincoln Laboratories and the Edgewood Chemical and Biological Command is engaged in an effort to develop optical techniques for the rapid detection and classification of biological aerosols. This paper will describe two efforts at NRL: development of an improved UV fluorescence front-end trigger and the use of infrared absorption spectroscopy to classify biological aerosol particles. UV Laser-induced fluorescence (UVLIF) has been demonstrated to provide very high sensitivity for differentiating between biological and inorganic aerosol particles. Unfortunately, current UVLIF systems have unacceptably high false alarm rates due to interferences from man made and naturally occurring organic and biological particulates. We have developed a two-wavelength, UVLIF technique that offers a higher level of discrimination than is possible using single wavelength UVLIF. Infrared absorption spectroscopy coupled with multivariate analysis demonstrates a high potential for differentiation among members of biological and chemical sample classes. Two-wavelength UVLIF in combination with the IR interrogation of collected bioaerosols could provide a rapid, reagentless approach to specific classification of biological particles according to an operational level of discrimination - the degree of particle characterization required in order to signal the presence of pathogenic material.
We are developing a novel method to fluorescently label specific biological aerosols on-the-fly using an in-line electrospray technique. Fluorescently labeled biomarkers such as molecular beacons, aptamer beacons, or those constructed from antibodies, will be used to coat aerosol particles in an air stream. Single biological particles with appropriate receptors will be tagged with biomarkers that fluoresce at a particular wavelength allowing the particle to be identified in near real time using a simple laser induced fluorescence technique. The fluorescent markers are normally quenched in the absence of their target analyte, permitting the use of mixtures of different biomarkers for simultaneously identifying multiple types of biological particles. The technique can also be applied to inorganic particulate with a molecular surface composition that lends itself to epitopic binding. Some of the issues that are currently being investigated include the kinetics of biomarker binding in an aerosol stream, optimal electrospray geometries and the nondestructive charging of biological particles on the fly.
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.
In this paper, we describe a fiber-optic-coupled radiation sensor that can be placed directly at the site of a tumor in a patient that is undergoing radiation therapy. The sensor provides real-time feedback during the therapy procedure allowing for precise control of the delivered dose. It is expected that better control of the radiotherapy parameters will lead to increased survivability statistics and a better overall quality of life for cancer patients.
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