This PDF file contains the front matter associated with SPIE Proceedings Volume 7909, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Semiconductor quantum dots (QDs) are highly interesting fluorophores for a large variety of spectroscopic applications. Although their fluorescence properties are well investigated, accurate size determination of QDs is still a problem. TEM techniques can image the inorganic core/shell system of QDs, but size determination of polymer coated QDs is difficult. SEC (size exclusion chromatography) compares the QD size only with standard polymers and their sizes, and is therefore not easy to use on nanoparticles. As QDs are fluorescent, single molecule spectroscopy methods such as fluorescence correlation spectroscopy (FCS) can be used to determine QDs diffusion coefficients and hence their hydrodynamic radii. Moreover, this method for size determination requires only very low QD concentrations, which is a mayor advantage compared to other techniques such as dynamic light scattering. Within our contribution we present the size determination of commercially available and self-modified QDs with FCS. The commercial QDs (QD525, QD565, QD605, QD655 and QD705 - purchased from Invitrogen Inc.) have a rather thick polymer shell and are functionalized with streptavidin, biotin or carboxylic groups. The self-modified QDs consist of the same commercial core/shell QDs and are modified with a polymer shell and several bio-functionalization groups. For all nanoparticles the diffusion coefficients were measured by FCS and the hydrodynamic radii were calculated according to the Stokes-Einstein equation. The obtained results are in good agreement with the size information provided by Invitrogen Inc., which demonstrates that FCS is an important technique for QD size determination at very low concentrations.

Gold nanoparticles may assist in the delivery of anti-cancer drugs, such as doxorubicin, deeper into cells and tumors in vitro and in vivo. However, the ideal shape, size, and surface chemistry of the particles have not been well determined. This is especially difficult in the case of doxorubicin, which has multiple modes of action, reacting differently in cancer cells vs. normal cells and in cytoplasm vs. nucleus. We begin to address these issues here by examining the cytotoxicity of two sizes of Au-doxorubicin particles, as well as examining the tracking of injected Au-doxorubicin in mice in vivo. Finally, we examine mechanisms of toxicity in different cell lines, finding that nanoparticles may assist in overcoming anti-apoptotic mechanisms in cancer cells.

Due to the ever increasing production of nanosized materials for a variety of novel applications (e.g. biomedicine), increased research has focused upon understanding the potential toxicity of these nanomaterials. In order to determine the potential toxicity of nanoparticles (NPs), it is essential that, in parallel to biochemical and toxicological testing, their specific route of uptake (if any), as well as their possible and/or subsequent intracellular localisation is investigated. By using a novel 3D in vitro triple cell co-culture model of the human epithelial airway barrier (containing epithelial cells and macrophages (apical-layer) and dendritic cells (basolateral-layer) which has been shown to mimic the architecture of the lung in vivo, in combination with a novel air-liquid interface cell exposure system, it is possible to mimic the exposure of NPs to the lung in vitro. Using both conventional and state-of-the art toxicological tests, in addition to light, laser scanning (including digital image restoration) and transmission electron microscopy methods, it has been possible to determine the interaction of both fluorescent (specifically designed core-shell nanoparticles with shell-embedded fluorophores for use in biomedicine) and electron dense NPs with the in vitro triple cell co-culture. It has been observed that the material of the engineered NPs (either gold or iron oxide) has no significant influence upon the toxicity and intracellular localisation over time. The results of these studies have shown that despite different compositions, specific NPs intended for use in biomedical applications, when exposed realistically (exposure method/concentration/primary contact cells) cause minimal effects to the lung in vitro.

Nano- and micro-particulates firmly attach with the surface of various biological systems. In some chronic pulmonary disease such as asbestosis and silicosis, causative particulates will induce chronic inflammatory disorder, followed by poor prognosis diseases. However, nano- and micro-scale specific toxicity of silica particulates is not well examined enough to recognize the risk of nano- and micro-particulates from the clinical aspect. To clarify the effect of the size and structure of silica particulates on the cellular damage and the biological response, we assessed the cytotoxicity of the various kinds of silica particles including amorphous and crystalline silica, in mouse alveolar macrophage culture, focusing on the fibrotic and inflammatory response. Our study showed that the cytotoxicity, which depends on the particle size and surface area, is correlated with their inflammatory response. By contrast, production of TGF-β, which is one of the fibrotic agents in lung, by addition of crystal silica was much higher than that of amorphous silica. We conclude that fibrosis and inflammation are induced at different phases and that the size- and structure-differences of silica particulates affect the both biological responses, caused by surface activity, radical species, and so on.

In the emerging area of nanotechnology a key issue is related to the potential impacts of the novel nanomaterials on the environment and human health so that this technology can be used with minimal risk. Specifically designed to combine on a single structure multipurpose tags and properties, nanomaterials need a comprehensive characterization of both chemicophysical properties and toxicological evaluation, which is a challenging endeavor: the in vitro toxicity assays that are employed for nanotoxicity assessments do not accurately predict in vivo response. To overcome these limitations and gain a deeper understanding of nanoparticle-cell interactions, we have employed cnidarian models, in particular the freshwater polyp Hydra vulgaris, not opposed to more complex and evoluted systems, but to add valuable information, at an intermediate level between prokaryotes and vertebrates, on both cytoxicity and on pollution affecting the environment. By testing CdSe/CdS core shell nanocrystals in vivo, at whole animal level, we investigated the impact of their properties on uptake, accumulation, biodistribution, elicitation of behavioural responses. Spanning from animal to cell biology, we provide an analysis on metal based and semiconductor NC, discussing the crucial role played by the synthesis route and chemical surface on the toxicity for living organisms.

The photoluminescence (PL) properties of quantum dots (QD) are of significant interest in the development of new methods for bioanalysis. Multiplexed solid-phase nucleic acid hybridization assays that use immobilized QDs as donors in fluorescence resonance energy transfer (FRET) are one such example, and offer several unique advantages over other methods. In this work, new interfacial chemistry is described for the immobilization of red-emitting CdSe/ZnS QDs on glass beads for use in hybridization assays. The beads were chemically modified with a dithiolate surface ligand and the QDs immobilized via self-assembly. Further derivatization of the QDs with dithiolate-terminated probe oligonucleotides enabled a hybridization assay that could detect unlabeled target down to nanomolar levels with discrimination of single base-pair mismatches. The use of beads as an immobilization platform afforded shorter analysis times and superior reusability compared to previous studies using optical fibers. Hybridization between probe, target, and Alexa Fluor 647 (A647) labeled reporter oligonucleotides in a sandwich format generated a spectroscopic signal by introducing the proximity needed for FRET between the QDs and A647. The results indicate clear directions for the optimization of solid-phase hybridization assays, and are important for the future development of true multiplexed biosensors based on QDs and FRET.

Semiconductor nanocrystals (quantum dots - QDs) possess unique photophysical properties that make them highly interesting for many biochemical applications. Besides their common use as fluorophores in conventional spectroscopy and microscopy, QDs are well-suited for studying Förster resonance energy transfer (FRET). Size-dependent broadband absorption and narrow emission bands offer several advantages for the use of QDs both as FRET donors and acceptors. QD-based FRET pairs can be efficiently used as biological and chemical sensors for highly sensitive multiplexed detection. In this contribution we present the use of several commercially available QDs (Qdot® Nanocrystals - Invitrogen) as FRET donors in combination with commercial organic dyes as FRET acceptors. In order to investigate the FRET process within our donor-acceptor pairs, we used biotinylated QDs and streptavidin-labeled dyes. The well-known biotinstreptavidin molecular recognition enables effective FRET from QDs to dye molecules and provides defined distances between donor and acceptor. Steady-state and time-resolved fluorescence measurements were performed in order to investigate QD-to-dye FRET. Despite a thick polymer shell around the QDs, our results demonstrate the potential of these QDs as efficient donors both for steady-state and time-resolved FRET applications in nano-biotechnology.

Plasmonic nanoparticles with silver cores and silica shells containing Eu fluorophores near the surface have been produced by wet chemistry method and their spontaneous emission properties characterized. Fluorescence amplification and decreased lifetime is interpreted within the Purcell framework which highlights the role of surface plasmon polariton modes of the nanoparticle. These behave as energy-storing resonators, with values of the Q factor between 50 and 170 at the fluorophore wavelength of 615 nm, and very small mode volumes, in the order of 10⁴ nm³, producing high Purcell factors of over 4000. Comparison of experiment with theoretical calculations by using the Mie theory shows that the values of cavity Q factors are moderated by the nonradiative rate of fluorophore molecules close to metal. The criteria for laser action in such composite nanoparticles are also presented, including lasing frequencies and threshold gain.

In this paper we report on the synthesis of robust water-soluble gold nanoparticles, suitable for biological applications. We show that specifically functionalized nanoparticles such as nanospheres, nanorods and hollow gold particles can migrate either on the cellular membrane or be endocytosed. A theoretical model is employed to discuss the potential use of these particles as tools for laser treatment of endothelial cells. Our studies open up new avenues towards manipulation of cells using gold nanoparticles and laser treatment.

Cancer is a multigenic complex disease where multiple gene loci contribute to the phenotype. The ability to simultaneously monitor differential expression originating from each locus results in a more accurate indicator of degree of cancerous activity than either locus alone. Metal nanoparticles have been thoroughly used as labels for in vitro identification and quantification of target sequences. We have synthesized nanoparticles with assorted noble metal compositions in an alloy format and functionalized them with thiol-modified ssDNA (nanoprobes). These nanoprobes were then used for the simultaneous specific identification of several mRNA targets involved in cancer development - one pot multicolor detection of cancer expression. The different metal composition in the alloy yield different "colors" that can be used as tags for identification of a given target. Following a non-cross-linking hybridization procedure previously developed in our group for gold nanoprobes, these multicolor nanoprobes were used for the molecular recognition of several different targets including differently spliced variants of relevant genes (e.g. gene products involved in chronic myeloid leukemia BCR, ABL, BCR-ABL fusion product). Based on the spectral signature of mixtures, before and after induced aggregation of metal nanoparticles, the correct identification could be made. Further application to differentially quantify expression of each locus in relation to another will be presented. The differences in nanoparticle stability and labeling efficiency for each metal combination composing the colloids, as well as detection capability for each nanoprobe will be discussed. Additional studies will be conducted towards allele specific expression studies.

Cerium-doped lanthanum fluoride colloidal nanocrystals (NCs) offer a way to improve radiation therapy through the enhanced absorption of high-energy photons. The use of Monte Carlo simulation allows the direct calculation of the macroscopic dose enhancement factor (MDEF), a figure of merit for NC-enhanced radiation therapy. Our simulations of brachytherapy using an Ir-192 source agree with previous work on the subject for gold NCs and show effectiveness of LaF₃:10%Ce NCs to be approximately 50% that of gold. Polyethylene-glycol-capped LaF₃:10%Ce NCs were synthesized, isolated, suspended in phosphate buffered saline (PBS), and characterized with transmission electron microscopy, dynamic light scattering, photoluminescence spectroscopy, and absorption spectroscopy. LaF3:10%Ce NCs were used in radiation dose enhancement experiments that involved an incoming 662 keV gamma flux from dual Cs-137 sources to test the mortality of Saccharomyces cerevisiae. At a small loading of 1.8 mg NC/g of PBS, the experiment did not produce a measurable increased mortality. To understand the results, additional Monte Carlo simulations revealed that the photon energy of 662 keV gamma rays is far from optimal, providing only a 4% increase in dose for a concentration of 18 mg of NCs / g of PBS. Further simulations showed that the optimal photon energy for this technique is 60 keV, tripling the absorbed dose for a concentration of 18 mg of NCs / g of PBS.

Quantum dots (QDs) are semiconductor nanocrystals with extensive imaging and diagnostic capabilities, including the potential for single molecule tracking. Commercially available QDs offer distinct advantages over organic fluorophores, such as increased photostability and tunable emission spectra, but their cadmium selenide (CdSe) core raises toxicity concerns. For this reason, replacements for CdSe-based QDs have been sought that can offer equivalent optical properties. The spectral range, brightness and stability of InP QDs may comprise such a solution. To this end, LANL/CINT personnel fabricated moderately thick-shell novel InP QDs that retain brightness and emission over time in an aqueous environment. We are interested in evaluating how the composition and surface properties of these novel QDs affect their entry and sequestration within the cell. Here we use epifluorescence and transmission electron microscopy (TEM) to evaluate the structural properties of cultured Xenopus kidney cells (A6; ATCC) that were exposed either to commercially available CdSe QDs (Qtracker® 565, Invitrogen) or to heterostructured InP QDs (LANL). Epifluorescence imaging permitted assessment of the general morphology of cells labeled with fluorescent molecular probes (Alexa Fluor® ® phalloidin; Hoechst 33342), and the prevalence of QD association with cells. In contrast, TEM offered unique advantages for viewing electron dense QDs at higher resolution with regard to subcellular sequestration and compartmentalization. Preliminary results show that in the absence of targeting moieties, InP QDs (200 nM) can passively enter cells and sequester nonspecifically in cytosolic regions whereas commercially available targeted QDs principally associate with membranous structures within the cell. Supported by: NIH 5R01GM084702.

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.

Semiconductor quantum dots (QD) are a class of NP with photophysical properties that are ideally suited for optical multiplexing and use as donors in fluorescence resonance energy transfer (FRET). A new strategy is presented for the development of multiplexed DNA hybridization assays using immobilized QDs in a microfluidic system. Green- or red-emitting QDs were immobilized via self-assembly with a multidentate-thiol-derivatized glass slide, and subsequently conjugated with amine-terminated probe oligonucleotides using carbodiimide activation. Immobilized QD-probe conjugates were then passivated with adsorbed non-complementary oligonucleotides to achieve selectivity in microfluidic assays. Target nucleic acid sequences hybridized with QD-probe conjugates and were labeled with Cy3 or Alexa Fluor 647 as acceptor dyes for the QD donors, where FRET-sensitized dye emission provided a signal for the detection of picomolar quantities of target. The simultaneous immobilization of green- and red-emitting QDs at different ratios within a microfluidic channel was demonstrated as a step toward multiplexed assays.

We report silver nanostructure-enhanced fluorescence of a europium (Eu) chelate, BHHCT-Eu-DPBT, which was covalently bound in Ag/SiO₂ nanocomposites. This design enhances the europium signal intensity by more than one order of magnitude, and accelerates the decay time from 0.3 ms down to 60 microseconds, at low excitation conditions. These nanocomposites were bright enough to be observed in time-gated fluorescence microscopy under 365 nm LED excitation. The increased brightness and reduced lifetime of such fluorescent core-shell nanocomposites will enhance their applicability for ultrasensitive bioassays and bioimaging, especially with time-gating.

We report for the first time continuous flow synthesis of NaYF₄:Yb³⁺/Er³⁺ upconverting nanocrystals in a capillary-based microfluidic reaction system. Two sequential temperature steps were employed for heating with an initial high temperature (180°C) to burst nuclei and a subsequent low temperature (110°C) to promote growth of the nanocrystals in order to obtain high-performance nanocrystals. The prepared nanocrystals emit bright green and red emissions under excitation of 974 nm diode laser. Our research opens the door for the synthesis of upconverting nanocrystals in microfluidic systems.

A large number of different approaches for the aqueous phase transfer of quantum dots have been proposed. Surface ligand exchange with small hydrophilic thiols, such as L-cysteine, yields the lowest particle hydrodynamic diameter. However, cysteine is prone to dimer formation, which limits colloidal stability. We demonstrate that precise pH control during aqueous phase transfer dramatically increases the colloidal stability of InP/ZnS quantum dots. Various bifunctional thiols have been applied. The formation of disulfides, strongly diminishing the fluorescence QY has been prevented through addition of appropriate reducing agents. Bright InP/ZnS quantum dots with a hydrodynamic diameter <10 nm and long-term stability have been obtained. Finally we present in vitro studies of the quantum dots functionalized with the cell-penetrating peptide maurocalcine.

We investigate the use of energy delivery using microwave radiation for both synthesis of nanoparticles as well as a hybrid imaging technique known as thermoacoustic tomography (TAT). In each instance, the absorption of microwave radiation is converted into heat. In the case of nanoparticle synthesis, water is used as the solvent and heated to induce synthesis of the nanostructures. For this aqueous synthesis technique, we demonstrate the use of both pulsed and continuous wave (CW) microwave systems operating at 2.45 GHz. In this report, we concentrate on ZnO nanostructures including nanorods, nanowire arrays and nanobelts. These are compared with nanowire arrays and nanobelts grown by vapor transport through both electron microscopy and photo-excited luminescence. We also review the use of iron oxide (Fe3O4) nanoparticles as contrast agents in TAT as previously reported. Here, we measured the properties of the colloidal nanoparticles in the microwave regime and compared the absorption with the TAT signal produced by our thermoacoustic imaging system at 3 GHz. The nanoparticles directly absorb the microwave radiation and produce a thermo-acoustic signal. The results from nanoparticles are compared to the signal produced by deionized water. The results demonstrate that microwaves represent an efficient method for the delivery of energy for both synthesis and biomedical imaging.