This PDF file contains the front matter associated with Proceedings of SPIE Volume 6448, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

Fluorescent silicon nanomaterials have initiated great interest for their potential biological applications. In this paper, we report the synthesis of water-soluble, luminescent silicon nanoparticles with high quantum yield. The surfaces of the Si nanoparticles were capped by hydrophobic or hydrophilic organic molecules that passivate and protect the silicon particles from oxidation. The as-synthesized silicon nanoparticles displayed strong photoluminescence in the blue region of the visible spectrum. The attached organic molecules conveyed both water solubility and high stability, and coating had little adverse effect on the optical properties of nanoparticles. These results have major implications towards using colloidal silicon nanoparticles effectively in biological fluorescence imaging.

We report on colloidal synthesis of ZnO and novel ZnO/ZnS core/shell nanocrystals (NCs) from zinc alkoxy alkyl precursors in the MeIm/H₂O coordinating solvent. The results of NC structural and optical characterization are presented.

Nanomaterials, such as semiconductor Quantum Dots (QD) and Iron Oxide nanocrystals possess unique properties that are not available in their bulk phase. Some of these properties include the narrow emission spectra, superior brightness and higher photostability of QDs, and the superparamagnetic properties of Iron Oxide nanocrystals. In the past decade, these two nanomaterials have separately seen widespread use in a variety of biomedical applications ranging from multiplexed biomolecular detection to isolation and magnetic manipulation of disease cells and molecules respectively. Here, we describe a method for combining QDs and Iron Oxide nanocrystals into a micron-sized host material in a rapid fashion. The resulting beads are dual functional, i.e. they are optically encoded, and can be manipulated with a permanent magnet. The beads have great potential in biomedical applications because of the combined ability to enrich and detect multiple target molecules from heterogeneous and diluted biological samples. The development of multifunctional composite materials by combining novel nanomaterials is bound to open avenues for ultrasensitive and quantitative bioassays.

We report the phase transfer of hydrophobic CdSe/Zn_1-xMn_xS quantum dots (QDs) into water by encapsulation with octylamine-modified poly(acrylic acid) (PAA). CdSe/Zn_1-xMn_xS QDs are of interest as multimodality biological probes where the photoluminescence and magnetic relaxivity can be tuned independently. Different percentages of the carboxyl groups in PAA were grafted with octylamine molecules and the modified PAA was used for capping CdSe/Zn_1-xMn_xS QDs to make them water soluble. We investigated the optical properties and the solubility of the CdSe/Zn_1-xMn_xS QDs in water. It was found that the PAA with a modification percentage of ~ 45% resulted in soluble CdSe/Zn_1-xMn_xS QDs with the highest quantum yield (QY) in water of approximately 20%. The QY of CdSe/Zn_1-xMn_xS QDs in water was lower than the initial measurement in chloroform and dropped in the initial stages of phase transfer, stabilizing at 20%. Hydrodynamic size of the polymer encapsulated CdSe/Zn_1-xMn_xS QDs in water was evaluated by dynamic light scattering (DLS). The smallest average hydrodynamic diameter of ~ 30 nm was achieved when the molecular ratio of QDs to PAA for capping was 1 nanomole of QDs to 24 micromole of monomer unit.

Plasmon-resonant gold nanorods have outstanding potential as multifunctional agents for image-guided therapies. Nanorods have large absorption cross sections at near-infrared (NIR) frequencies, and produce two-photon luminescence (TPL) when excited by fs-pulsed laser irradiation. The TPL signals can be detected with single-particle sensitivity, enabling nanorods to be imaged in vivo while passing through blood vessels at subpicomolar concentrations. Furthermore, cells labeled with nanorods become highly susceptible to photothermal damage when irradiated at plasmon resonance, often resulting in a dramatic blebbing of the cell membrane. However, the straightforward application of gold nanorods for cell-specific labeling is obstructed by the presence of CTAB, a cationic surfactant carried over from nanorod synthesis which also promotes their nonspecific uptake into cells. Careful exchange and replacement of CTAB can be achieved by introducing oligoethyleneglycol (OEG) units capable of chemisorption onto nanorod surfaces by in situ dithiocarbamate formation, a novel method of surface functionalization. Nanorods with a dense coating of methyl-terminated OEG chains are shielded from nonspecific cell uptake, whereas nanorods functionalized with folate-terminated OEG chains accumulate on the surface of tumor cells overexpressing their cognate receptor, with subsequent delivery of photoinduced cell damage at low laser fluence.

We report on the single-particle properties of lanthanide-ion doped oxide nanoparticles. We have demonstrated that their size can be accurately determined from their luminosity. The optically determined size distribution is in very good agreement with the distribution obtained from transmission electron microscopy (TEM). We also showed that the photobleaching of these nanoparticles is related to a reduction process and that we can use it to sense in a concentration-dependent manner the presence of an oxidant like H₂O₂. Finally, we propose a way to perform nanoparticle-protein coupling and to determine the protein-nanoparticle ratio at the single-particle level.

Semiconductor quantum dots (QDs) have unique photophysical properties which make them excellent fluorescence resonance energy transfer donors. However, lack of facile methods for conjugating biomolecules such as DNA, proteins and peptides to QDs have limited their applications. In this report, we describe a general procedure for the preparation of a synthetic peptide that can be covalently attached to DNA segments and used to facilitate the self-assembly of the modified DNA onto water soluble QDs. To characterize this conjugation strategy, dye-labeled DNA is first reacted with the synthetic peptide and the resulting peptide-DNA then self-assembled onto QDs. QD attachment is verified by monitoring resonance energy transfer efficiency from the QD donor to the dye-labeled DNA acceptor. QD-DNA bioconjugates assembled using this method may find applications as molecular beacons and hybridization probes.

Colloidal quantum dots (QDs) are now commercially available in a bio-functionalized form and Förster resonance energy transfer (FRET) between bioconjugated dots and fluorophores within the visible range has been observed by several groups of researchers. We are particularly interested in the far-red region, as from a biological perspective, there are benefits in pushing to ~700 nm to minimize optical absorption (ABS) within tissue and avoiding cell autofluorescence. We report on FRET between streptavidin (STV) conjugated CdTe quantum dots, Qdot705-STV, with biotinylated Dy731-Bio fluorescent molecules in a donor-acceptor assay. We also highlight an unusual change in Dy731-Bio absorptivity during the streptavidin-biotin binding process that can be attributed to the structural reorientation. In moving to wavelengths beyond 700 nm, different alloy compositions are required for the quantum dot core and these introduce associated changes in the physical shape. These changes directly affect the fluorescence decay dynamics producing a marked biexponential decay with an extremely long lifetime component, a lifetime in excess of 100 ns. We compare and contrast the influence of the two QD relaxation processes upon the FRET dynamics in the presence of Dy731-Bio.

We present a single QD FRET study that allows us to probe the heterogeneity in QD-dye labeled protein conjugates. We first show that QDs are compatible with spFRET detection by demonstrating the equivalence of single particle and ensemble measurement modalities in terms of derived average FRET efficiencies and separation distances between a QD donor and dyes attached to specific sites on conjugated proteins. We then use spFRET data to demonstrate that the valence distribution of QD-protein conjugates follows Poisson statistics.

Homogeneous FRET (Forster Resonance Energy Transfer) fluoroimmunoassays (FIA) allow fast, inexpensive and highly sensitive monitoring of biochemical processes occurring on the nanometer scale. The technique is widely applied in high throughput screening (HTS) for in vitro diagnostics (IVD). Quantum dots (qdots) are usually applied as energy donors for FRET experiments in solution. In this contribution we show that commercially available biotinylated CdSe/ZnS core/shell qdots (Qdot 665 Biotin conjugate, Invitrogen Corp., USA) are excellent FRET acceptors in a time-resolved FIA based on interaction with lanthanide complex-labeled streptavidin as energy donor. The energy transfer experiments were performed on a modified commercial FIA reader system (KRYPTOR, Cezanne SA, France) using three di.erent lanthanide chelates (1 of terbium and 2 of europium). All three FRET donors showed efficient energy transfer to the qdots, evidenced both by nanocrystals emission sensitization and by a thousand fold increase of the qdot luminescence decay time, reaching some hundreds of microseconds. In a control experiment the unlabeled donors were used to rule out dynamic energy transfer between lanthanides and qdots. Due to the very high qdot absorption extremely large Forster radii of 104 A for terbium and 96 A for europium were achieved. FRET efficiency was up to 67 % and sub-picomolar detection limits were obtained for qdots in this type of homogeneous FIA. The use of qdots as energy acceptors potentially offers a broad scope of scientific and commercial applications such as ultra-sensitive FIA, the study of interactions within very large molecules, biomedical HTS and multiplexed analysis.

Energy transfer between semiconductor nanoparticles (quantum dots) and energy donors or acceptors modulates fluorescence, thus serving as a visual indicator of the interaction. Careful choice of conjugate or capping groups can thus make these particles serve as sensors for specific biological processes and as tools for targeted cell killing. Challenges include creation of stable conjugates, delivery to specific cell populations and intracellular regions, and characterization of fluorescence modulation by energy transfer.

The advance of nanotechnology has boosted the development of ultra-sensitive biosensors for biomedical applications. Most recently, optical detection based biosensors have been demonstrated in medical imaging and diagnosis employing nanocrystals such as fluorescent quantum dots (QDs) and plasmon resonant metal nanoparticles to achieve femto-molar detection. An intriguing but far less explored approach for biological diagnostics relies on an emerging ultrasensitive technology -- surface enhanced Raman scattering (SERS) spectroscopy. We have developed a stable SERS nano-tag by grafting hydrophilic polymer to gold nanoparticle-dye molecule complexes to preserve the spectral signature and fully control the aggregation states. The light-emitting power and scattered light of both QDs and SERS nano-tags have been recorded under the same experimental conditions using dark field microscope, fluorometer, and Raman instrument. A comparison in brightness, sensitivity level, and quantum efficiency between SERS nano-tags and near infrared (NIR) QDs has been assessed on both bulk colloidal solution and single particle measurements. Well-designed SERS nano-tags exhibit excellent advantages over NIR QDs.

Composition-tunable nanocrystals are fluorescent nanoparticles with a uniform particle size and with adjustable optical characteristics. When used for optical labeling of biomolecular targets these and other nanotechnology solutions have enabled new approaches which are possible because of the high optical output, narrow spectral signal, consistent quantum efficiency across a broad emission range and long lived fluorescent behavior of the nanocrystals. When coupled with spectral imaging the full potential of multiplexing multiple probes in a complex matrix can be realized. Spectral imaging can be used to improve sensitivity of narrowband fluorophores through application of chemometric image processing techniques used to reduce the influence of autofluorescence background. Composition-tunable nanocrystals can be complexed together to form nanoclusters which have the advantage of significantly stronger signal and therefore a higher sensitivity. These nanoclusters can be targeted in biomolecular systems using standard live-cell labeling and immunohistochemistry based techniques. Composition-tunable nanocrystals and nanoclusters have comparable mass and brightness across a wide emission range. This enables the production of nanocrystal-based probes that have comparable reactivity and sensitivity over a large color range. We present spectral imaging results of antibody targeted nanocrystal cluster labeling of target proteins in cultured cells and a Western blot experiment. The combination of spectral imaging with the use of clusters of nanocrystals further improves the sensitivity over either of the approaches independently.

Quantum dots are now recognised as valuable luminescent labels. Amongst their desirable characteristics is a relatively long luminescence decay time that can allow selective detection in the presence of shorter lived background fluorescence. Here we demonstrate that nanosecond time-resolved imaging of quantum dot samples is easily accomplished with a directly-gated cooled CCD system that does not require an image intensifier stage to achieve nanosecond time resolution. The approach is likely to find application both to imaging of quantum dots in the presence of autofluorescence from tissues and also for time-resolved measurements of resonance energy transfer from quantum dot donors to conventional acceptor labels.

We demonstrate the selective delivery of self-assembled luminescent semiconductor quantum dot (QD)-peptide bioconjugates into several eukaryotic cell lines. A 23-mer hetero-bifunctional peptide bearing a positively-charged oligoarginine domain and a terminal polyhistidine tract was synthesized and used to mediate the cellular internalization of the QD-bioconjugates. The polyhistidine tract allows the peptide to self-assemble onto the QD surface via metal-ion coordination while the oligoarginine domain mediates the specific uptake of the QD-bioconjugates via electrostatic interactions with cell surface receptors. In both HEK 293T/17 and COS-1 cells, this peptide-mediated delivery is concentration-dependent in terms of both the QD concentration and the peptide:QD ratio. Intracellularly, the QD signal is punctate in appearance and some, but not all, of the QDs are located within recycling endosomes as evidenced by their colocalization with transferrin. In both cell lines, the QD-bioconjugates elicit minimal cytotoxicity within the timeframe required for adequate cellular uptake. The specificity of this delivery strategy is demonstrated by performing a multicolor QD labeling, wherein the presence or absence of the peptide on the QD surface controls cellular uptake.

In this paper we report two different methodologies for labeling ligand-gated receptors. The first of these builds upon our earlier work with serotonin conjugated quantum dots and our studies with pegilated quantum dots to reduce non specific binding. In this approach a pegilated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphillic polymer derivative of poly acrylamide. These conjugates were used to image the GABA_C receptor in oocytes. An alternative approach was used to image tissue sections to study nicotinic acetylcholine receptors in the neuro muscular junction with biotinylated Bungerotoxin and streptavidin coated quantum dots.

The development of colloidal quantum dots (QDs) for biological imaging has brought a new level of sensitivity to live cell imaging. Single particle tracking (SPT) techniques in particular benefit from the superior photostability, high extinction coefficient and distinct emission spectra of QDs. Here we describe the use of QDs for SPT to study the dynamics of membrane proteins in living cells. We work with the RBL-2H3 mast cell model that signals through the high affinity IgE receptor, Fc&Vegr;RI. Using wide field or Total Internal Reflection Fluorescence (TIRF) microscopy we have achieved simultaneous imaging of two spectrally distinct QDs with frame rates of up to 750 frames/s and localization accuracy of ~10 nm. We also describe the imaging and analysis of QDs using a novel hyperspectral microscope and multivariate curve resolution analysis for multi-color QD tracking. The same QD-tag used for SPT is used to localize proteins at <10 nm resolution by electron microscopy (EM) on fixed membrane sheets.

Quantum dots (QDs) are light emitting semi-conductor nanocrystals with novel optical properties including superior photostability, narrow emission spectra with continuous excitation spectra. These properties make QDs especially suitable for multiplexed fluorescent labeling, live cell imaging, and in vivo animal imaging. The multiplexing potential has been recognized but real applications of biological/clinical significance are few. In this study, we used quantum dots to study epithelial mesenchymal transition (EMT), an important process involved in the bone metastasis of prostate cancer. Two prostate cancer cells lines with distinct molecular profiles, representing the two ends of the EMT process, were selected for this study. Four EMT-related biomarkers including E-cadherin, N-cadherin, Vimentin, and RANKL were stained with QD-antibody conjugates with elongation factor 1alpha as the internal control. Morphological information of the QD-stained cells was obtained by digital-color imaging and quantitative information obtained by spectra analysis using a spectrometer. Two types of analysis were performed: abundance of each biomarker in the same cell line relative to the internal control; and the relative abundance of these markers between the two cell lines. Our results demonstrate the feasibility of QDs for multiplexed profiling of FFPE cells/tissue of clinical significance; however, the standardization and quantification still awaits optimization.

Our group is investigating the use of ZnS-capped CdSe quantum dot (QD) bioconjugates combined with fluorescence endoscopy for improved early cancer detection in the esophagus, colon and lung. A major challenge in using fluorescent contrast agents in vivo is to extract the relevant signal from the tissue autofluorescence (AF). Our studies are aimed at maximizing the QD signal to AF background ratio (SBR) to facilitate detection. This work quantitatively evaluates the effect of the excitation wavelength on the SBR, using both experimental measurements and mathematical modeling. Experimental SBR measurements were done by imaging QD solutions placed onto (surface) or embedded in (sub-surface) ex vivo murine tissue samples (brain, kidney, liver, lung), using a polymethylmethacrylate (PMMA) microchannel phantom. The results suggest that the maximum contrast is reached when the excitation wavelength is set at 400±20 &mgr;m for the surface configuration. For the sub-surface configuration, the optimal excitation wavelength varies with the tissue type and QD emission wavelengths. Our mathematical model, based on an approximation to the diffusion equation, successfully predicts the optimal excitation wavelength for the surface configuration, but needs further modifications to be accurate in the sub-surface configuration.

Quantum dots (QDs) may serve as improved platforms for the complex modulation and ultra-sensitive imaging of molecular signaling in cells. The time course and spatial localization of activated ligand-receptor complexes and their trafficking within cells is becoming increasingly understood as vital for propagating cell signals. However, the movement and fate of ligand-receptor pairs inside cells is difficult to define with current technologies. We have studied the intracellular trafficking of TrkA receptors using QDs conjugated with nerve growth factor, a neuropeptide ligand critical for nervous system development and regulation. We find that NGF-QDs bind and activate TrkA surface receptors in PC12 neurons. Spatiotemporal maps of TrkA-NGF-QD endocytosis and translocation can be directly visualized with single QD resolution. Moreover, single molecule tracking experiments indicates that QDs complexes are actively shuttled over long distances within newly-sprouted neuronal processes. These results indicate that QDs can serve as effective high-resolution probe to track ligand-receptor function in the interior of cells.

We report the use of spatial arrays of single quantum dots (QD) as fluorescent probes to quantify deformations and displacements of bone tissue components (e.g. collagen and carbonated apatite) at the nanometer to micrometer level under mechanical load. Quantum dot bright emission and robustness allow nanometer localization and motion tracking by center of gravity (COG) analysis. Coupons of milled cortical bone are loaded in a purpose-built dynamic mechanical loading system that fits on a microscope stage. We used QD streptavidin conjugates to label the bone specimen prior to mechanical loading. COG of the laser-induced QD fluorescence diffraction spot is measured and tracked in real time (<0.1 sec) as the tissue is loaded quasi-statically. The technique has been validated by comparing the average values of tangent elastic moduli obtained by the QD/COG method to measurements made with an attached micro-strain gage and a calibrated load cell. Two or more colors of QD can be used to measure relative motions of different bone tissue components, as well as to measure small out-of-plane motions that cannot be detected otherwise.

Quantum dots (QDs) have brighter and longer fluorescence than organic dyes. Therefore, QDs can be applied to biotechnology, and have capability to be applied to medical technology. Currently, among the several types of QDs, CdSe with a ZnS shell is one of the most popular QDs to be used in biological experiments. However, when the CdSe QDs were applied to clinical technology, potential toxicological problems due to CdSe core should be considered. To eliminate the problem, silicon nanocrystals, which have the potential of biocompatibility, could be a candidate of alternate probes. Silicon nanocrystals have been synthesized using several techniques such as aerosol, electrochemical etching, laser pyrolysis, plasma deposition, and colloids. Recently, the silicon nanocrystals were reported to be synthesized in inverse micelles and also stabilized with 1-heptene or allylamine capping. Blue fluorescence of the nanocrystals was observed when excited with a UV light. The nanocrystals covered with 1-heptene are hydrophobic, whereas the ones covered with allylamine are hydrophilic. To test the stability in cytosol, the water-soluble nanocrystals covered with allylamine were examined with a Hela cell incorporation experiment. Bright blue fluorescence of the nanocrystals was detected in the cytosol when excited with a UV light, implying that the nanocrystals were able to be applied to biological imaging. In order to expand the application range, we synthesized and compared a series of silicon nanocrystals, which have variable surface modification, such as alkyl group, alcohol group, and odorant molecules. This study will provide a wider range of optoelectronic applications and bioimaging technology.

The application of quantum dots (QDs) as labels in immunoassay microarrays for the multiplex detection of 3- phenoxybenzoic acid (PBA) and atrazine-mercapturate (AM) has been demonstrated. PBA and AM are biomarkers of exposure to the pyrethroid insecticides and to the herbicide atrazine, respectively. Microarrays were fabricated by microcontact printing of the coating antigens in line patterns onto glass substrates. Competitive immunoassays were successfully performed using quantum dots (QD560 and QD620) as reporters. The multiplexed immunoassays were characterized by fluorescence microscopy and SEM. The application of QD fluorophores facilitates multiplex assays and therefore can contribute to enhanced throughput in biomonitoring.