This PDF file contains the front matter associated with SPIE Proceedings Volume 6866, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

We have developed a new synthetic method for producing high-quality quantum dots (QDs) in aqueous solution for biological imaging applications. The glutathione-capped CdTe, ZnSe and Zn_1-xCd_xSe alloyed QDs derived are tunable in fluorescence emissions between 360 nm and 700 nm. They show high quantum yields (QYs) of up to 50%, with narrow bandwidths of 19-55 nm. The synthesis of glutathione-capped QDs is simple and cost-effective compared to the conventional organometallic approaches. It can be easily scaled up for the commercial production of alloyed nanocrystals of various compositions. We have also demonstrated the fabrication of magnetic quantum dots (MQDs) through a seed-mediated approach. The formation and assembly of these bifunctional nanocomposites have been elucidated by high-resolution transmission electron microscopy (HRTEM). The MQDs exhibit superparamagnetism and tunable emissions characteristic of the components in this hybrid system. We have created biocompatible silica-coated MQDs that effectively target the cell membranes.

We have studied phosphor doping of core / shell nanocrystals (NCs) where the impurity emitter resides in the shell. We have found that a two step synthesis can be used to create these non-toxic materials that efficiently transfer energy from the core to the doped shell. These core / shell NCs retain ample brightness when solubilized in water. We explored the functionalization of these materials in water as well to create ratiometric chemical sensing agents. First, we used a method of controlled polymerization to synthesize amphiphilic polymers to solubilize the intrinsically hydrophobic NCs into water. The polymer has a build in "chemical handle" which we use to functionalize the polymers closely bound to the NC with a fluorescent dye in aqueous solution. We have found that there exists efficient Förster resonant energy transfer from the shell doped phosphors to the surface bound dyes. Conjugation of the NC to an environmentally sensitive dye such as fluorescein has also demonstrated that non-toxic doped NCs can be used to develop ratiometric sensing / biological imaging agents. Last, we have found that the same technique can be applied to functionalize non-emissive magnetic nanocrystals as well.

Lead-iodide-based nanocrystals were synthesized by dissolution of commercial lead iodide powder in a coordinating solvent, tetrahydrofuran, and subsequent re-crystallization after an optimum addition of methanol. The nanocrystals were characterized by transmission electron microscopy, scanning electron microscopy, energy dispersive spectroscopy (EDS), steady state UV-visible optical absorption and photoluminescence spectroscopy, and by photoluminescence lifetime and quantum efficiency measurements. The radiation hardness of the synthesized material was tested using a ¹³⁷Cs gamma source. Scintillation was observed from the lead-iodide based material exposed to low-level gamma irradiation.

We report the design and synthesis of new bidentate ligands to promote biocompatibility of luminescent quantum dots (QDs) and gold nanoparticle (Au-NPs) alike. The ligands use commercially available methoxy-terminated hydroxypoly( ethylene glycol) (HO-PEG-OCH3) and thioctic acid (TA) coupled via a stable amide bond to make TA-PEG-OCH3 ligands. Following a simple transformation of the hydroxyl group into an amine group on the methoxy-PEG, the ligand was attached to thioctic acid via DCC coupling. The use of mono-hydroxy-terminated PEG simplifies the reaction and purification steps. Following ring opening of the TA to form DHLA-PEG-OCH3, cap exchange on CdSe-ZnS core-shell QDs provided homogeneous stable dispersions in buffer solutions. Furthermore, using either the newly designed ligand pure, or mixed with amine-functionalized ligands (DHLA-PEG-NH2) allowed tuning of the surface functionalities of the nanoparticles. Gel electrophoresis, absorption and fluorescence experiments confirmed cap exchange, while microinjection in live cells provided additional verification of the potential utility of these hydrophilic QDs in biology. These ligands were also applied to functionalize Au-NPs, with a substantial improvement of their stability under high salt conditions compared to citrate-functionalized dispersions.

Recent progress in the field of semiconductor nanocrystals or Quantum Dots (QDs) has seen them find wider acceptance as a tool in biomedical research labs. As produced, high quality QDs, synthesized by high temperature organometallic synthesis, are coated with a hydrophobic ligand. Therefore, they must be further processed to be soluble in water and to be made biocompatible. To accomplish this, the QDs are generally coated with a synthetic polymer (eg. block copolymers) or the hydrophobic surface ligands exchanged with hydrophilic material (eg. thiols). Advances in this area have enabled the QDs to experience a smooth transition from being simple inorganic fluorophores to being smart sensors, which can identify specific cell marker proteins and help in diagnosis of diseases such as cancer. In order to improve the biocompatibility and utility of the QDs, we report the development of a procedure to coat QDs with silk fibroin, a fibrous crystalline protein extracted from Bombyx Mori silkworm. Following the coating process, we characterize the size, quantum yield and two-photon absorption cross section of the silk coated QDs. Additionally, the results of biocompatibility studies carried out to compare the properties of these QD-silks with conventional QDs are presented. These natural polymer coatings on QDs could enhance the intracellular delivery and enable the use of these nanocrystals as an imaging tool for studying subcellular machinery at the molecular level.

We have previously assembled semiconductor quantum dot (QD)-based fluorescence resonance energy transfer (FRET) sensors that can specifically detect nutrients, explosives or enzymatic activity. These sensors utilized the inherent benefits of QDs as FRET donors to optimize signal transduction. In this report we functionalize QDs with the multi-subunit multi-chromophore b-phycoerythrin (b-PE) light harvesting complex using biotin-Streptavidin binding. FRET and gel electrophoretic analyses were used to characterize and confirm the QD-b-PE self-assembly. We found that immobilizing additional cell-penetrating peptides on the nanocrystal surface along with the b-PE was the key factor allowing the mixed surface QD-cargos to undergo endocytosis and intracellular delivery. Our findings on the intracellular uptake promoted by CPP were compared to those collected using microinjection technique, where QD-assemblies were delivered directly into the cytoplasm; this strategy allows bypassing of the endocytic uptake pathway. Intracellular delivery of multifunctional QD-fluorescent protein assemblies has potential applications for use in protein tracking, sensing and diagnostics.

Efficient Fluorescence (or Förster) Resonance Energy Transfer (FRET) pairs between fluorescent proteins and quantum dots (QDs) have a significant potential for ultrasensitive biochemical assays in disease detection and diagnosis. We have developed such FRET pairs using commercially available QDs as donors and fluorescent protein as acceptor, with polyhistidine-chelation as the means of bioconjugation. In this study we compared two brands of QDs with different surface coatings and found that the FRET pair containing EviTags from Evident Technology produced a higher FRET efficiency due to the shorter donor-acceptor distance. The polyhistidine binds directly to the ZnS capping layer of the EviTags, whereas the carboxyl QDots from Invitrogen, although having a higher quantum yield, require the addition of Ni²⁺ to the solution in order to facilitate chelation-mediated binding to outer surface of the polymer coating. These findings have significant implications to QD-based FRET assay design.

Quantum dots (Qdots) are nanoparticles exhibiting fluorescent properties that are widely applied for cell staining. We present here the development of quantum dots for specific targeting of apoptotic cells, for both apoptosis detection and staining of apoptotic "living" cells. These Qdots are functionalized with Annexin V, a 35-kDa protein that specifically interacts with the membrane of apoptotic cells: Annexin V recognizes and binds to phosphatidylserine (PS) moieties which are present on the outer membrane of apoptotic cells and not on this of healthy or necrotic cells. By using Annexin V, our Qdots probes are made specific for apoptotic cells. For that purpose, Qdots Streptavidin Conjugates are coupled to biotinylated Annexin V. Staining of apoptotic cells was checked using fluorescence and confocal microscopy techniques on nonfixed cells. It is shown here that Qdots are insensitive to bleaching after prolonged and frequent exposure as opposed to organic dyes and this makes them excellent candidates for time-lapse imaging purposes. We illustrate the application of our Qdots-based probes to continuously follow fast changes occurring on the membrane of apoptotic cells.

In this work, we demonstrate the application of quantum dots (QDs) to several biologically relevant applications. QDs are synthesized by biological and organometallic routes and the relative merits of these methods are identified. Our results indicate that QDs can be functionalized and specifically targeted to both mammalian and bacterial cells. In the case of mammalian cells, they can be targeted to an engineered sodium channel for the purpose of sensing. In both mammalian and bacterial cells, the interaction with bioconjugated QDs can lead to phototoxicity due to the generation of reactive oxygen species (ROS).

Luminescent semiconductor quantum dots (QDs) possess several unique optical and spectroscopic properties that are of great interest and promise in biology. These properties suggest that QDs will be integral to the development of the next generation of biosensors capable of detecting molecular processes in both living and fixed cells. 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. The QD-peptide structures are generated by the self-assembly of the peptide onto CdSe-ZnS core-shell QDs via metal ion coordination between the peptide's polyhistidine motif and the Zn-rich QD shell. The polymer-based QD assemblies are formed via the electrostatic interaction of aqueous cationic liposomes with available carboxylate moieties on the QD surface ligands. 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.

This paper describes the preparation of bioactive water-soluble fluorescent CdSe/ZnS semi-conductor quantum dots with a small hydrodynamic diameter of 10 nm. These quantum dots are functionalized with a biotinylated peptide that can be introduced at different ratios onto the surface of the quantum dots. Their ability to bind to streptavidin in solution is tested by using gel electrophoresis and fluorescence resonance energy transfer with a fluorescent labeled-streptavidin. The binding of these quantum dots to Agarose micrometric beads coated with streptavidin is also analyzed by fluorescent optical microscopy. A synthetic pegylated peptide is successfully used to prevent the non specific adsorption of streptavidin onto the quantum dots. A specific binding to the streptavidin results in the formation of a very stable streptavidin-quantum dot complex without any significant aggregation. The average number of streptavidin per quantum dot is found to be to 4 at the most. Such bioactivate quantum dots can be further conjugated to any biotinylated biomolecule and used in biological medium.

We have undertaken a study of the dynamics of CdSe/ZnS quantum dots in the blood vessels of the chicken embryo chorioallantoic membrane (CAM). We show proof of principle that fluorescence correlation spectroscopy can be used in this system to determine the concentrations and hydrodynamic radii of quantum dot solutions micro-injected into the CAM.

Noninvasive imaging of epidermal growth factor (EGF) receptor (EGFR) expression can provide valuable molecular information that could aid diagnostic and therapeutic decisions, particularly with targeted cancer therapies utilizing anti-EGFR antibodies. In this study we report on the development and validation of a nanoprobe for in-vivo imaging and discrimination of EGFR-overexpressing tumors from surrounding normal tissues that also expresses EGFR. Near-infrared quantum dots (QDs) were coupled to EGF using thiol-maleimide conjugation to create EGF-QD nanoprobes. These nanoprobes demonstrated excellent in-vitro and in-vivo binding affinity. In-vivo imaging demonstrated three distinct phases of tumor influx (~3min), clearance (~60min) and accumulation (1-6hrs) of EGF-QD nanoprobes. Both QD and EGF-QD demonstrated non-specific rapid tumor influx and clearance followed by an apparent dynamic equilibrium at ~60min. Subsequently (1-6hrs), while QD concentration gradually decreased in tumors, EGF-QDs progressively accumulated in tumors. At 24hrs, tumor fluorescence decreased to near baseline levels for both QD and EGF-QD. Ex vivo whole-organ, tissue-homogenate fluorescence, confocal microscopy and immunofluorescence staining confirmed tumor-specific accumulation of EGF-QD nanoprobes at an early time-point (4hrs). The favorable pharmacokinetics, the ability to discriminate EGFR-overexpressing tumors from surrounding normal tissues using low concentration (10-pmol) of EGF-QD nanoprobe underscores the clinical relevance of this probe to evaluate therapeutic intervention.

Nanomedical approaches to diseases such as cancer provide great promise with respect to diagnostic and therapeutic applications. The impact of nanomedicine versus conventional therapies will be realized with regard to their specific cell targeting capabilities. Semiconductor nanoparticles have distinct advantages due to their chemical conjugation and detection characteristics. The attachment of a peptide sequence, LTVSPWY, was completed. These nanoparticles successfully targeted in vitro and in vivo systems. This technology can be utilized as a base mechanism for the construction of a multifunctional nanomedical system. Nanomedicine has great potential for impacting the treatment of specific diseases and healthcare delivery methods.

Quantum dots have unique properties for long-term immunofluorescence imaging of molecular activities inside living cells. The key is how to deliver the quantum dot-conjugated antibodies into cells and further allow the antibodies freely move inside cells to bind target molecules. This study investigated the feasibility of using Pep-1, a cell penetration protein, to facilitate the internalization of quantum dot-conjugated antibodies for the labeling of two intracellular cervical cancer biomarkers: p16 and Mcm5. Quantum dots were directly conjugated with the antibodies to p16 and Mcm5 and, they were able to stain fixed cells and to differentiate biomarker positive and negative cells. The non-covalent binding between the conjugates and Pep-1 peptides allows the quick internalization of the quantum dot-conjugated antibodies into living cells. The internalized conjugates were concentrated in the perinuclear regions of the biomarker-positive HeLa cells. In the biomarker negative Um-Uc-3 cells, however, the conjugates concentrated in juxtaneclear region. Cells bearing with quantum dots still go through the mitosis process. Although the study indicates many questions need to be answered and many problems need to be solved, the use of cell penetration peptide is a promising method for the intracellular labeling of living cell molecules using quantum dots.

Chemically functionalized semiconductor quantum dot protocols were optimized for the specific labeling and imaging of neural cells, both neurons and macroglial cells. Beta-tubulin III was used to image primary cortical neurons and PC12 cells while glial fibrillary acidic protein (GFAP) was used to image primary spinal cord and cortical astrocytes and the rMC-1 retinal glial Muller cell line. Both proteins are the main components of intermediate filaments and are specific to the two classes of neural cells. We also specifically labeled and imaged at high resolutions using anti-GFAP conjugated quantum dots glial scars in situ in intact neural sensory retina in a rodent model of macular degeneration.

In this paper we report our work on the development of a human serotonin transporter (hSERT) antagonist that can be conjugated to quantum dots. This approach has been used to target and visualize the human serotonin transporter protein (hSERT). We demonstrate that labeling is blocked by the addition of high affinity hSERT antagonists such as paroxetine. This approach may be useful for the development of fluorescent assays to study the location and temporal dynamics of biogenic amine transporters and also holds promise for the development of plate-based high throughput assays used to identify novel transporter antagonists.

This letter demonstrates that the fluorescence intensity of CdSe-core CdS/ZnS-multishell quantum dots (QDs) in toluene is related via a power-law of ~1.9 to the input intensity of femtosecond laser at 5~130 GW/cm². This clearly indicates a broad range of optical intensity of two-photon excitation (TPE). The two-photon absorption (TPA) cross sections of QDs of core-size 2.9, 4.0 and 5.3 nm at 800 nm are 1980, 5680 and 14600 GM, respectively. Furthermore, the log-log plot of CdSe-core diameters versus the TPA cross sections shows the increase with a slope >3, indicating a nonlinear dependent relationship between TPA cross section and size of CdSe-core. The broad optical range of TPE and large TPA cross section make these QDs excellent candidates for two-photon fluorescent microscopy and bioimaging. Based on these two-photon properties of our QDs, we continue to investigate the bioimaging applications with two-photon microscopy. The results of the fluorescence images of living cells with the QDs demonstrate that QDs could be penetrate into cell membrane, then steadily and dispersedly distribute at the cytoplasm, which further indicates such QDs could be excellent candidate for two-photon microscopy applications.

Raman spectroscopy is now a well-established analytical tool for obtaining rapid and compound specific information for chemical analysis. However, Raman scattering - inelastic scattering of photons - cross sections are typically of the order of 10^-30 cm² per molecule and thus Raman signals are usually weak. In Surface Enhanced Raman Scattering (SERS) the signals can be greatly amplified by using specially structured metallic (usually Ag, Au, and Cu) substrates. SERS substrates can be fabricated by a variety of methods. Here, we report a method for fabricating SERS substrates from commercially available silver nanoparticle based printing inks. For dilute inks (~ 1-2% Ag by weight) the method involves the airbrushing of inks on heated (~100oC) quartz or polymer substrates followed by heating at 170oC for about 20 minutes. The heating treatment removes the polymer coating used to prevent aggregation of Ag particles in the colloidal suspension and allows partial sintering of particles. More concentrated inks (~ 20 - 30% Ag by weight) can be applied to various substrates at room temperature followed by the thermal treatment. SERS spectra of Rhodamine 6G, and β-carotene molecules are reported. SERS amplification factors of more than 106 can be easily obtained reproducibly.

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 clinical 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 of CdSe core should be considered. To overcome the problem, silicon nanocrystals, which have the potential of biocompatibility, could be a candidate of alternate probes. Silicon nanoparticles have been synthesized using several techniques. Recently, novel silicon nanoparticles were reported to be synthesized with the combination methods, radio frequency sputtering method and hydrofluoric-etching method In order to assess the biocompatibility of the Silicon nanoparticles, we performed two different cytotoxicity assays, cell iability/proliferation assay using the mitochondrial activity assay and cell membrane damage assay using the lactate dehydrogenase assay. At the 112 μg/mL of silicon nanoparticles (the maximum concentration in this study), we could detected the cell membrane damage of HeLa cells and the decrease of hepatocytes viability. We concluded that we could use the silicon nanoparticles as bioimaging marker but the attention should be given when Silicon nanoparticles were applied to cells in high concentration.