Gang Bao Georgia Institute of Technology
(and Emory University)
Jennifer West Rice University
Shuming Nie Emory University
(and Georgia Institute of Technology)
Andrew Tsourkas University of Pennsylvania
Y1: Nanoprobes and Nanocarriers for Biological Imaging
Thursday PM, April 20, 2006
Room 2005 (Moscone West)
9:30 AM - **Y1.1
Quantum Dots Complexed to Epidermal Growth Factor Reveal that Cellular Filopodia Function as Natural Biosensors.
Thomas Jovin 1 , Diane Lidke 1 , Donna Arndt-Jovin 1 Show Abstract
1 Dept. of Molecular Biology, Max-Plank-Institut for Biophysical Chemistry, Goettingen Germany
Biotinylated epidermal growth factor (EGF) associated with streptavidin-conjugated Quantum Dots (QDs) binds to its cognate cell surface receptor (EGFR, ErbB1, HER1). Widefield and laser scanning microscopy have revealed free diffusion of the ligand-receptor complexes on the cell surface, including filopodia, cellular extensions with an F-actin bundle core. Interaction of at least two complexes leads to activation of the cytoplasmic receptor tyrosine domain and initiation of a systematic mechanism of retrograde transport from the filopodia to the cell body. A FRET assay exploiting the QDs as donors indicates that endocytosis of the complexes takes place at the base of the filopodia. We have measured the rates of free diffusion and retrograde transport as a function of cell type, temperature, presence of kinase and cytoskeletal inhibitors, and mutations or manipulation of the expression levels of putative protein components of the transport mechanism. Cytochalasin D, a disruptor of the actin cytoskeleton, abolishes transport. Diffusion constants and transport rates were determined with single molecule sensitivity by tracking individual QD-receptor complexes, exploiting the high temporal and intensity sensitivity provided by an electron multiplying CCD camera. The QDs, the associated receptors (visualized as a fusion protein with eGFP), and F-actin are transported with the same rate on filopodia, implying a coupling of receptor movement to the treadmilling of the F-actin bundles. We conclude that filopodia act as non-linear biosensors for growth factors located far from the cell body, mediating cellular responses via the directed transport of activated receptors.Ref.: Lidke et al, J. Cell Biol. (2005) 170: 619-626.
10:00 AM - **Y1.2
Engineering of Nanocarriers for Enhanced Tumor Visualization.
Vladimir Torchilin 1 Show Abstract
1 , Northeastern University, Boston, Massachusetts, United States
Enhanced tumor imaging is required for better diagnosis and therapy control. With this in mind, novel nanoparticular tumor-targeted contrast agents have been prepared capable of enhanced visualization of varioustumors. These agents were engineered in several steps. First, liposomes and micelles have been prepared loaded with various contrast metals (such as 111-In for gamma-scintigraphy and with Gd for magnetic resonance imaging)via the liposome membrane- or micelle core-incorporated amphiphilic polychelating polymers capable of carrying multiple heavy metal moieties. Then, the liposome surface was modified with sterically-protecting poly(ethylene glycol) for prolonged liposome circulation and better accumulation in tumors via the enhanced permeability and retention effect. Lastly, for still better tumor targeting, long-circulating contrast metal-loaded liposomes and micelles were additionally modified with amonoclonal antibody specific towards intact nucleosomes and recognizing various tumors via cancer cell surface-bound nucleosomes. These nucleosomes originate from the apoptotically dying tumor cells and attach to thesurface of neighboring live tumor cells making them good targets for antinucleosomal antibodies. New engineered long-circulating, contrast-loaded, and tumor-targeted nanocarriers have been successfullyused for the fast and efficient tumor visualization in various tumor models in mice using gamma-scintigraphy and MRI imaging modalities.
10:30 AM - **Y1.3
Deciphering the Mechanism of mRNA Transport in the Nucleus by Tracking Individual mRNA Molecules.
Sanjay Tyagi 1 Show Abstract
1 , Public Health Research Institute, Newark, New Jersey, United States
Cell biologists have wondered for many years how mRNAs, associated with an entourage of proteins, travel from the sites of transcription to the nuclear pores through dense nucleoplasm. Early theorists postulated active transport mechanisms, or proposed that transcriptionally active genes need to lie close to the periphery of the nucleus to allow the export of mRNP complexes through nearby pores. More recent studies indicate that mRNPs can diffuse within the nucleus, but their motility is dependent upon ATP. To resolve this apparent contradiction, we have developed a system of fluorogenic probes and mRNA constructs that allows us to track individual mRNA molecules as they are transcribed, move within the nucleus, exit from the nuclear pores, and spread throughout the cytoplasm. Our probes are small hairpin-shaped oligonucleotides called molecular beacons with an internally quenched fluorophore whose fluorescence is restored upon hybridization to a specific nucleic acid sequence. To obtain single-molecule sensitivity, we engineered a host cell line to express an mRNA possessing multiple molecular beacon binding sites. The binding of multiple molecular beacons to each mRNA molecule rendered them so intensely fluorescent that individual mRNA molecules could be detected and their motion could be tracked. We found that the mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. Their diffusion is restricted to extra-nucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the particles to resume their motion after they become stalled. This provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process.
11:15 AM - **Y1.4
Specificity of RNA Imaging in Live Cells using Molecular Beacons.
Phi Santangelo 1 Show Abstract
1 Biomedical Engineering , Georgia Institute of Technology, Atlanta, Georgia, United States
11:45 AM - Y1.5
Porous 3D Containers for Cellular Encapsulation and Drug Delivery.
Timothy Leong 1 , Zhiyong Gu 1 , Barjor Gimi 3 , David Gracias 1 2 Show Abstract
1 Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States, 3 The Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins Medicine, Johns Hopkins University, Baltimore, Maryland, United States, 2 Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Encapsulation devices are widely used in medicine to enclose and deliver a variety of cells and drugs for therapeutic applications. Most encapsulation systems consist of a polymeric or gel based matrix within which the therapeutic constituents are dispersed. For example, the concept of using “artificial cells”-transplanted therapeutic cells encapsulated in a nanoporous membrane was put forward in the mid 1970’s. Encapsulation was proposed as a means to protect the cells from the external immune response while at the same time allowing the bidirectional diffusion of nutrients to the encapsulated cells and therapeutic biochemicals from the encapsulated cells to the external environment. In encapsulation devices for drug release, a biodegradable polymer matrix dissolves slowly, and facilitates sustained release of drugs in-vivo. While polymeric encapsulation systems have some favorable attributes in biomedical engineering including biodegradability, biocompatibility, ease of fabrication and encapsulation some challenges do still exist. These include biochemical instability, mechanical rupture, polydispersivity, and broad pore size distribution. Additionally, it is challenging to detect and track polymeric devices in-vivo. In contrast to polymeric, hydrogel, and sol-gel based processes that have been used for encapsulation and delivery, conventional silicon (Si) based microfabrication has high reproducibility and stability. However, Si based microfabrication is principally a 2D process; 3D micropatterned devices remain largely unexplored.Recently, we developed a strategy to fabricate 3D metallic porous containers using a process that utilizes conventional microfabrication and self-assembly. The boxes represent the first 3D metallic microfabricated encapsulation devices that can be fabricated in a precise manner with controlled shape and surface porosity. The fabrication process is extremely versatile-boxes can be built in a large range of sizes and surface porosity in a highly parallel (cost-effective) manner. The fabrication process is fully compatible with CMOS and MEMS fabrication so it is easy to envision embedding “smart” electronic modules including imagers and remote identification (RFID) on the surfaces of the boxes. We have demonstrated that the boxes can be used for encapsulation and release of cells. We have also demonstrated that the encapsulation devices can be non-invasively detected and tracked using magnetic resonance imaging (MRI), which is an advantage over polymeric delivery systems that can be detected only when loaded with a contrast agent. This feature points to the possibility of using MR imaging to monitor transplanted encapsulated cells in-vivo. The faces of the boxes can be easily modified using bioinert metals and polymeric coatings to enhance biocompatibility.
12:00 PM - Y1.6
Gold Nanocages: Engineering the Structure for Biomedical Applications.
Jingyi Chen 1 , Benjamin Wiley 1 , Xingde Li 1 , Younan Xia 1 Show Abstract
1 , Univ. of Washington, Seattle, Washington, United States
Galvanic replacement reaction between Ag template and HAuCl4 in an aqueous solution transforms 30-200 nm Ag nanocubes into Au nanoboxes and nanocages (nanoboxes with porous walls). By controlling the molar ratio of Ag to HAuCl4, the extinction peak of resultant structures can be continuously tuned from the blue (400 nm) to the near infrared (1200 nm). These hollow Au nanostructures are characterized by extraordinarily large cross-sections for both absorption and scattering. Optical coherence tomography measurements indicate that the 36-nm nanocage has a scattering cross-section of ~0.8*10-15 m2 and an absorption cross-section of ~7.3*10-15 m2. The absorption cross-section is more than five orders of magnitude larger than those of conventional organic dyes. Exposure of Au nanocages to a camera flash resulted in the melting and conversion of Au nanocages into spherical particles due to photothermal heating. Discrete dipole approximation calculations suggest that the magnitudes of both scattering and absorption cross-sections of Au nanocages can be tailored by controlling their dimensions, as well as the thickness and porosity of their walls. Bioconjugated Au nanocages with specific antibody can serve as a molecular probe to target a specific cancer cell line. This novel class of hollow nanostructures is being developed as both a contrast agent for optical imaging in early-stage tumor detection, and as a therapeutic agent for photothermal cancer treatment.
12:15 PM - Y1.7
Nanoprobe Fabrication by Field Manipulation of Nanotubes
Joshua Freedman 1 , Gary Friedman 1 , Yury Gogotsi 2 , Adam Fontecchio 1 Show Abstract
1 Electrical and Computer Engineering, Drexel University, Philadelphia, Pennsylvania, United States, 2 Materials Engineering, Drexel University, Philadelphia, Pennsylvania, United States
Using electric and magnetic fields, carbon nanotubes (CNTs) have been placed at the tips of microelectrodes, and nanopipettes to create minimally invasive biological probes. Methods of CNT manipulation are explored as nanofabrication techniques using dielectrophoretic, electrophoretic, and magnetic forces. Dielectrophoretic assembly is accomplished by the placement of conductive microprobes in close vicinity to plane electrodes and the application of an alternating current through a solution. The nanotubes preferentially align themselves normal to the surface, and adhere via van der Waals forces. Electrophoretic assembly uses direct current through the CNT solution resulting in Columbic attraction of the inherently charged CNTs, which under these conditions align themselves parallel to the probe tip. Magnetic nanotubes, formed by the capillary filling of CNTs with iron oxide nanoparticles, are employed in magnetic assembly methods which utilized the presence of strong field gradients to align and position the tubes at the ends of submicron tipped pipettes.The goal of these efforts is the creation of a variety of nanoscale tools, including a subcellular probe which is minimally invasive to biological material. The CNT tip, which responds to magnetic manipulation, will offer additional degrees of freedom for in vitro studies of biological cell tissue. The potential applications of this device include nanofluidic delivery and abstraction, more precise electrochemical and electrophysiological measurements in biological environments, and externally guidable tips for force microscopy and Raman spectroscopy probes.
12:30 PM - Y1.8
Biologically Activated Carbon Nanotube Probe for Scanning Force Microscope.
Sang Ahn 1 , Byong Park 1 , Ki Jung 2 1 , Jinho Choi 1 , Dal Kim 1 Show Abstract
1 , Korea Research Institute of Standards and Science, Daejeon Korea (the Republic of), 2 , Nanofocus Inc., Seoul Korea (the Republic of)
12:45 PM - Y1.9
Development of a Biophotonic Imaging Agent for Intra-operative Imaging of Cancer Foci.
Mandana Veiseh 1 , S-Bahram Bahrami 1 , Patrik Gabikian 2 , Miqin Zhang 3 , Richard Ellenbogen 2 , James Olson 3 Show Abstract
1 , Fred Hutchinson Cancer Research Center, Seattle, Washington, United States, 2 Department of Neurosurgery, University of Washington, Seattle, Washington, United States, 3 Department of Materials Science & Engineering, University of Washington, Seattle, Washington, United States
Thursday, 4/20new abstract11:45 am Y1.9Development of a Biophotonic Imaging Agent for Intra-operative Imaging of Cancer Foci. Mandana Veiseh
Y2: Quantum Dot Bioconjugates for Optical Bioimaging
Thursday PM, April 20, 2006
Room 2005 (Moscone West)
2:30 PM - **Y2.1
Using Confocal Microscopy for the Identification of Putative Synaptic Contacts.
Robert Bailey 1 Show Abstract
1 Center for Neuroscience, University of California, Davis, California, United States
Thursday 4/20New Abstract1:30 pm *Y2.1Using Confocal Microscopy for the Identification of Putative Synaptic Contacts. Robert E. Bailey
3:00 PM - **Y2.2
Bioaffinity Quantum Dots for Single-Molecule Imaging and Tracking in Living Cells
Amit Agrawal 1 Show Abstract
1 Department of Biomedical Engineering, Emory University and George Institute of Technology, Atlanta, Georgia, United States
Quantum dots (QDs) are tiny light-emitting nanoparticles that are emerging as a new class of fluorescent probes for biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. These properties are most promising for improving the sensitivity of molecular imaging and quantitative cellular analysis by 1-2 orders of magnitude. However, the use of quantum dots for single-molecule imaging inside live cells has encountered significant problems such as QD aggregation in living cells, difficulties in transport and molecular binding in the crowded intracellular environment, and problems in tracking individual QDs in three dimensions. We have systematically examined the obstacles in intracellular QD imaging, and have overcome a number of the key problems. First, we have developed a cellular method that allows single QD probes to be efficiently delivered into living cells, without the problems associated with endocytosis and organelle trapping. Second, we have systematically studied the particle surface chemistry and have determined that certain chemical modifiers can be used to avoid the aggregation problem inside living cells. Third, we have successfully applied scanning-disk confocal microscopy to image single QDs in living cells at high resolution and sensitivity. The real-time images show active QD transport by molecular motors along both microtubule and actin-filament tracks. These advances have opened new possibilities in examining the inner workings of live cells at the single-molecule level.
3:30 PM - Y2.3
Imaging Quantum-Dot Motion and Binding at Biosurfaces.
Jack Rife 1 , James Long 1 , John Wilkinson 1 , Lloyd Whitman 1 Show Abstract
1 , Naval Research Laboratory, Washington, District of Columbia, United States
3:45 PM - Y2.4
Simultaneous AFM & NSOM Studies of Quantum Dot Labeled Breast Cancer Cells
Siyuan Lu 1 , Anubhuti Bansal 1 , Anupam Madhukar 1 , Henry Lin 1 , Ram Datar 1 Show Abstract
1 , University of Southern California, Los Angeles, California, United States
The functional elimination and down regulation of certain cell surface receptor proteins has shown to play a key role in the acquisition of invasion and metastasis functions by cancerous cells. Over expression of erbB2 or Her2/Neu receptor protein, a member of the EGFR (epidermal growth factor receptor) family, in ductal breast cancer cells has been associated with an increase in the potential for invasion. In this study, we have utilized the simultaneous nano-scale (<100nm) topographical and optical fluorescence imaging capability of an optical fiber cantilever based AFM (atomic force microscope) / NSOM (near-field scanning optical microscopy) system to examine and compare the density and distribution of the Her2/Neu receptor proteins on the surface of two breast cancer cell lines: the SK-BR-3 and the MDA-MB-231. The Her2/Neu receptors on the surface of these cells are labeled with CdSe nanocrystal quantum dots (QDs) via specific antigen – antibody interaction. The high photostability of the QDs enables increased observation time and thus enhanced signal to noise ratio. A systematic series of samples of both cell types with varying ratio of surface receptor density to the QD uptake density are prepared through controlled variation of the QD adsorption time. The density of Her2/Neu protein on the SK-BR-3 is known to be nearly fifty times higher than on the MDA-MB-231. Consequently, comparison of the distribution of the surface receptors for the two types of cell populations with varying density of QDs offers the opportunity of examining clustering with increasing density.
4:15 PM - **Y2.5
Luminescent Quantum Dots for Molecular Imaging and Drug Delivery
Xiaohu Gao 1 Show Abstract
1 Bioengineering, University of Washington, Seattle, Washington, United States
The development of high-sensitivity and high-specificity probes beyond the intrinsic limitations of organic dyes and fluorescent proteins is of considerable interest to many areas of research, ranging from in vitro ultrasensitive detection to in vivo medical imaging. Recent advances have shown that nanometer-sized semiconductor quantum dots (QDs) can be covalently linked with biorecognition molecules such as peptides, antibodies, nucleic acids, and small-molecule inhibitors for use as fluorescent probes. In comparison with organic fluorophores, QDs exhibit unique optical and electronic properties such as size- and composition-tunable fluorescence emission, large absorption coefficient, and significantly improved brightness and photostability. Due to their broad excitation profiles and narrow/symmetric emission spectra, high-quality QDs are also well suited for optical multiplexing, in which multiple colors and intensities are combined to encode thousands of genes, proteins, and small-molecule libraries. In this context, we present recent developments in bioconjugated QD probes and their applications in ultrasensitive molecular, cellular imaging and drug delivery. Despite their relatively large sizes (2-6 nm in diameter), bioconjugated QD probes behave like fluorescent proteins, and do not suffer from serious kinetics or steric-hindrance problems. In this “mesoscopic” size range, QDs and other types of nanoparticles also have more surface areas and functionalities that can be used for linking to multiple diagnostic (e.g., radioisotopic or magnetic) and therapeutic (e.g., anticancer) agents. These recent developments toward nanomedicine are expected to open new opportunities in molecular imaging, multiplexed profiling, disease diagnosis and treatment.
4:45 PM - Y2.6
Quantum Dot-Peptide Assemblies for Selective Intracellular Delivery and Labeling.
Thomas Pons 1 4 , James Delehanty 2 , Igor Medintz 2 , Florence Brunel 3 , Philip Dawson 3 , Hedi Mattoussi 1 Show Abstract
1 Optical Sciences Division - code 5611, Naval Research Laboratory, Washington, District of Columbia, United States, 4 Chemical and Biomolecular Dept, Johns Hopkins University, Baltimore, Maryland, United States, 2 Center for Bio/Molecular Science and Engineering - code 6900, Naval Research Laboratory, Washington, District of Columbia, United States, 3 Departments of Cell Biology & Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States
Several methods have been developed to deliver liposomes, nanoparticles and other “cargos” into the cell cytoplasm, among which peptide-mediated delivery is one of the most attractive, due to its efficiency and ease of implementation. We demonstrate the use of a cell-penetrating peptide conjugated to QDs for the selective intracellular labeling of several eukaryotic cell lines.A bifunctional oligoarginine cell penetrating peptide (based on the HIV-1 Tat protein motif) bearing a polyhistidine tract was synthesized and used to facilitate the trans-membrane delivery of QD-bioconjugates. The polyhistidine sequence allows the peptide to tightly self-assemble onto the QD surface via metal-affinity based coordination, while the oligoarginine sequence allows specific QD-conjugate delivery across the cell membrane and intracellular labeling. Our studies showed that this non-invasive peptide-facilitated delivery is concentration-dependent, and allows control over the amount of delivered conjugates by simply titrating the number of peptides immobilized on each QD. Upon internalization, QDs display punctate-like staining patterns within the cell cytoplasm that are partly co-localized within endocytic vesicles. The efficacy of selective cellular labeling using QD-peptide assemblies is highlighted by performing a multicolor QD staining, where the presence or absence of peptides on the QD surface controls cellular uptake. The toxicity effects of extended versus limited incubation with QD-peptide conjugates on the cell viability are evaluated using a metabolic specific assay. Clear differences in cell viability are noted, with a short exposure time required for efficient uptake of QD-peptide with minimal toxicity effects. Finally, we investigate the stability of the self-assembled QD-peptide conjugates inside the cells and the evolution of QD staining pattern over time, as cells process the endocytic compartments.
5:00 PM - Y2.7
Synthesis, Surface Functionalization, and Clinical Diagnostic Applications of Zinc Selenide Quantum Dots
Jun Wang 1 , Stelios Andreadis 1 , T Mountziaris 2 1 Show Abstract
1 Chemical and Biological Engineering, State University of New York, Buffalo, New York, United States, 2 Chemical Engineering, University of Massachusetts, Amherst, Massachusetts, United States
Fluorescent labeling of biological molecules is a technique that is used widely for analytical purposes in biotechnology and bioengineering. Semiconductor nanocrystals or quantum dots have emerged as a new class of fluorescent markers with distinct advantages over the traditional organic dyes. Their attractive properties include narrow, symmetric, and very bright emission, continuous excitation by any wavelength smaller than the emission wavelength, resistance to photobleaching, as well as excellent stability that allows their use in lengthy experiments. The ability to synthesize different populations of quantum dots with narrow emission spectra permits multiplexing, a property that is very important for simultaneous detection of several analytes, that would be very tedious and expensive if done sequentially.The focus of our work is the development of new strategies for functionalizing the surface of II-VI nanocrystals (e.g., ZnSe, CdSe, etc.) and their use in new applications in biological sensing and DNA analysis. Highly luminescent ZnSe quantum dots have been synthesized using a liquid-phase technique utilizing a hot coordinating solvent in which the nanocrystals are grown by injection of suitable precursors. The synthesis of ZnSe quantum dots is carried out in a stirred batch reactor containing liquid hexadecylamine at 260 degrees C. The precursors are diethylzinc diluted in heptane and selenium powder dispersed in trioctylphosphine. The mixture of reactants is injected into the batch reactor and the time of reaction is used to determine the size and luminescence emission wavelength of the quantum dots. Capping of the ZnSe quantum dots with a ZnS layer to obtain a core-shell structure was found to increase the fluorescence intensity and stability of the quantum dots. Surface ligand exchange reactions with mercapto-cabroxylic acids was used for obtaining water-soluble ZnSe quantum dots that are stable in storage for several days, fluorescent, and suitable for biological labeling applications. Conjugating water-soluble ZnSe and (ZnSe)ZnS core-shell quantum dots with oligonucleotides was found to increase their fluorescence emission intensity by up to an order of magnitude in a dose-dependent way. Quantum dots conjugated with longer DNA strands exhibited stronger fluorescence emission intensity that reached a plateau after the surface of the dots was saturated with a 50-mer. At the same time, the stability of quantum dots in water was substantially improved. Hybridization of q-dot-conjugated oligos increased the fluorescence emission intensity even further. Ongoing experiments are focusing on the use of quantum dots in high-throughput clinical diagnostic applications, such as real time PCR, DNA microarrays, and immunodiagnostics.
5:15 PM - Y2.8
Quantum Dot-Protein/Peptide Bioconjugates as a Platform for Monitoring Proteolytic Enzymatic Activity
Igor Medintz 1 , Aaton Clapp 2 , Florence Brunel 4 , Ellen Goldman 1 , Thomas Pons 2 3 , Philip Dawson 4 , Hedi Mattoussi 1 Show Abstract
1 Center for Bio/Molecular Science and Engineering Code 6900, U.S. Naval Research Laboratory, Washington, District of Columbia, United States, 2 Division of Optical Sciences Code 5611, U.S. Naval Research Laboratory, Washington, District of Columbia, United States, 4 Dept. Cell Biology & Chem & the Skaggs Inst. for Chem. Bio. , The Scripps Research Insititute, San Diego, California, United States, 3 Chemical & Biomolecular Engineering Dept., Johns Hopkins University, Baltimore, Maryland, United States
Luminescent semiconductor nanocrystals or quantum dots (QDs) offer several unique benefits for fluorescence resonance energy transfer-based biological assays that are not available to conventional organic fluorophores. These include: 1-the ability to size-tune fluorescent donor emission as a function of QD core size to better match overlap with a particular acceptor 2-the option of arraying multiple acceptors around a QD which increases the overall FRET efficiency and 3-the possibility of exciting a QD population at any wavelength to the blue of its absorption band edge, which can substantially reduce direct excitation of the acceptor. In this investigation, we apply QD-protein and QD-peptide bioconjugates to detect the activity of a variety of enzymatic proteases in vitro. We first use generic dye-labeled protein substrates immobilized on QDs and engaged in efficient FRET for ‘proof of concept’ demonstration of sensing enzymatic digestion. These QD-protein bioconjugates were exposed to increasing concentrations of the endopeptidases proteinase K or papain. Digestion of the dye-labeled protein substrate displaces the dye away from the QD surface and alters FRET efficiency. Measurement of changes in the FRET efficiency allowed monitoring of the specific protease activity. A calibration curve that correlates the number of dye-labeled proteins/peptides immobilized on the QD in each solution (free of enzymes) to the measured FRET efficiency was used for subsequent conversion of the FRET changes into units of enzymatic activity. The second set of QD-bioconjugates employed dye-labeled peptides as specific substrates for a variety of peptidases of clinical or therapeutic interest. For both types of bioconjugates, changes in FRET efficiency were also monitored when inhibitors were added to the assay solutions. Assays were carried out in both excess enzyme and excess substrate formats, and analysis of the data was carried using the Michaelis-Menten model of enzymatic activity. Quantitative monitoring of enzymatic activity was demonstrated, and Michaelis-Menten descriptors such as Vmax (maximum reaction rate or enzymatic velocity), KM (Michaelis constant) and Ki (inhibitor dissociation constant) were derived from the analysis. The data, subsequent analyses and some practical applications for this technology will also be discussed.
5:30 PM - Y2.9
Glutathione-Capped CdTe Quantum Dots: Synthesis and Applications in Cell Imaging.
Yuangang Zheng 1 , Shujun Gao 1 , Jackie Ying 1 , Emril Mohamed Ali 1 Show Abstract
1 , Institute of Bioengineering and Nanotechnology, Singapore Singapore
5:45 PM - Y2.10
Control of the Optical Properties and Specific Delivery of Quantum Dots with Small Molecules for Imaging in Living Cells.
Elizabeth Jares-Erijman 1 , Carla Spagnuolo 1 , Sandra Miskoski 1 , Luciana Giordano 1 , Maria Etchehon 1 , Ekaterina Papoucheva 2 , Thomas Jovin 3 , Gertrude Bunt 2 Show Abstract
1 Dept. of Organic Chemistry, FCEN - UBA, Buenos Aires Argentina, 2 Dept. of Molecular Biology of Neuronal Signals, MPI f. Experimental Medicine, Goettingen Germany, 3 Dept. of Molecular Biology, MPI f. Biophysical Chemistry, Goettingen Germany
We have modified Qdot surfaces with small molecules designed to control the emission properties and to provide specific delivery to genetically engineered peptides and proteins. Modulation by irreversible or reversible FRET of the emission properties of Qdots by association with small molecules such as photochromic dithienylethenes enables the identification of nanoparticles selected in time and space. A specific probe delivery strategy has been developed involving the synthesis of specially designed compounds that combine chemoselective chemistry with native chemical ligation, resulting in the specific labeling of proteins in living eukaryotic cells. The method requires a transthioesterification step between a thioester in a small molecule bearing a fluorophore and the thiol group in a protein modified so as to express an N-terminal cysteine. The ligated thioester intermediate undergoes spontaneous S → N acyl rearrangement (see diagram below), thereby covalently linking the protein to the fluorophore of interest. In order to optimize the methodology for eukaryotic cells, a GFP construct that carries a modified tobacco etch virus (TEV) protease recognition site at its N-terminus was expressed in mammalian cells together with a TEV protease. The modification involves the introduction of a cysteine immediately downstream of the cleavage position (P') in the recognition sequence, resulting in the exposure of a N-terminal cysteine upon TEV protease cleavage. The N terminal cysteine thus produced can react chemoselectively with the thioester derivatives of cell permeable luminescent labels.Cells transfected with the GFP fusion protein were selectively labeled in vivo with different dyes-containing benzyl-based thioesters. Among these are TMR, fluorescein, and biotin; the latter can be visualized with fluorescent or Qdot-conjugated (strept)avidin.
Y3: Poster Session
Friday AM, April 21, 2006
Salons 8-15 (Marriott)
9:00 PM - Y3.1
Labeling of Individual Carbon Nanotube by Non-covalent Bonding with Quantum Dots
Ning-Yu Wu 1 , Tri-Rung Yew 1 Show Abstract
1 Material Science and Engineering, National Tsing Hua University, Hsinchu Taiwan
This paper presents an approach to label individual carbon nanotube (CNT), which enables its observation under simple fluorescent microscope, via non-covalently bonding quantum dots (QDs) to its sidewall by π-stacking. This makes it much easier to observe CNTs, which have been developed for electronic, optical, chemical, and biological applications. This work will emphasize the synthesis of the bi-functional pyrenyl-based molecule with mercapto and pyrenyl groups. The pyrenyl group can be attached to the sidewall of CNTs strongly via non-covalent π-stacking. The mercapto group can be bonded to the shell surface of luminescent core/shell QD, like ZnS-capped CdSe QDs. Linked by this bi-functional molecule, the whole sidewall of the CNT can be covered by a large number of QDs. This CNT-QDs linkage makes it possible to observe individual CNT directly under fluorescent microscope, rather than using sophisticated instrument like atomic force microscope (AFM), transmission and scanning electron microscope. Furthermore, this CNT-QDs system can be used as a platform for bio-labeling applications by further modifying the pyrenyl-based bi-functional linker so as to observe their movement under simple fluorescent microscope.
9:00 PM - Y3.2
Biomimetic Synthesis of Titania Nanoparticles.
Melanie Tomczak 1 , Lawrence Drummy 1 , Morley Stone 1 , Rajesh Naik 1 Show Abstract
1 Materials and Manufacturing Directorate, Air Force Research Laboratory, WPAFB, Ohio, United States
Peptide-templated synthesis of inorganic nanomaterials has been a vigourous area of research in the past several years. We and others have shown that peptides based on diatom silaffin proteins precipitate silica nanoparticles at room temperature and neutral pH. These experiments have been extended by using peptide phage display, which has identified peptides that bind and/ or nucleate a wide variety of inorganic particles from magnetic to semiconducting to photoexcitable. Here we demonstrate synthesis of spherical titanium dioxide nanoparticles using a peptide-template. We found that the ratio of the water-soluble titanium precursor (Ti-BALDH) to peptide, as well as other reaction components, play a key role in the structure of the resulting nanoparticles. We will show the structural characterization of the nanoparticles and discuss how the peptide template may be affected during synthesis.
9:00 PM - Y3.3
Design and Synthesis of Multifunctional Probes for Imaging and Therapeutics.
Yu-San Liu 1 , Chi Hui Liang 1 , Ching-An Peng 1 Show Abstract
1 Mock family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California, United States
Gang Bao Georgia Institute of Technology
(and Emory University)
Jennifer West Rice University
Shuming Nie Emory University
(and Georgia Institute of Technology)
Andrew Tsourkas University of Pennsylvania
Y4: Nanoparticle Probes for Biological Imaging
Friday AM, April 21, 2006
Room 2005 (Moscone West)
9:45 AM - Y4.1
Magnetic Nanoparticle Probes as a Contrast Agent for Molecular Imaging Using MRI.
Gang Bao 1 , Nitin Nitin 1 , Leslie LaConte 1 , Charles Glaus 1 Show Abstract
1 Biomedical Engineering Dept, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States
Friday, April 21new abstract8:45 am Y4.1Magnetic Nanoparticle Probes as a Contrast Agent for Molecular Imaging Using MRI. Gang Bao
10:00 AM - **Y4.2
Studying Single-Molecule Molecular Motors
Paul Selvin 1 Show Abstract
1 , University of Illinois, UC, Urbana, Illinois, United States
We have achieved 1.5 nm resolution using fluorescenceimaging of single molecules, approximately 300 times better than the diffraction limit of conventional light. We call the technique Fluorescence Imaging with One Nanometer Accuracy (FIONA). We have looked at a variety of kinesins, and find some surprising results when a particular kind of kinesin (Kar3), is coupled to a microtubule binder(Cik1 or Vik1). Also, we have been able to increase the time resolution to 1 msec, from a previous value of 500 msec. Using this increased time-resolution, we have looked at molecular motors -- kinesin and dynein-- inside living cells. We find that both kinesin and dynein move cargo8 nm per ATP (the universal food of the cell), in opposite directions in a cell. Amazingly, these two molecular motors do not engage in a tug-of-war, but appear to be cooperative, giving the cargo extra speed.Lastly, we have invented a knock off of FIONA which is capable of looking at non-fluorescent objects. With this technique, we see cargoes moving in frog eggs, and find that heterotrimeric kinesin also takes 8 nm steps.
10:30 AM - **Y4.3
Targeting and Imaging with Polymersomes.
Dan Hammer 1 Show Abstract
1 Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
We describe recent advances in the construction, functionalization, and packaging of polymersomes. We have successfully constructed polymersomes solely from a single biodegradable polyethylene oxide-polycaprolactone diblock copolymer. This technology offers an advantage over existing methods in which biodegradable polymers are blended with non-biodegradable polymers. Efforts to extend the palate of polymers to make polymersomes will be discussed. Further, we will describe wide ranging methods to functionalize polymersomes with peptides and antibodies for a spectrum of targeting applications, and how systematic construction of the polymersome interface allows us to design polymersomes for adhesion. Finally, we have now been able to encapsulate a spectrum of different agents within and on polymersomes, including porphyrins for near –IR optical imaging; tat-peptides for cell internalization; other imaging agents, such as ones for magnetic resonance; and chemotherapeutic drugs. Thus, we are approaching the full potential of polymersomes as multi-functional carriers that can be targeted, imaged, and used to release therapeutics. Since polymersomes can be made in sizes down to a hundred nanometers, they can be considered a multi-functional nanotechnological material. Additional authors: I.-W. Chen, J. Lin, and D. Levine (Penn).
11:15 AM - **Y4.4
‘Activatable’ Nanostructured Probes for Molecular Imaging Applications
Antony Chen 1 , Julie Czupryna 1 , Daniel Thorek 1 , Andrew Tsourkas 1 Show Abstract
1 Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States
The study of human health and disease at the molecular level has led to tremendous advancements in the development of therapeutics and clinical diagnostics. It is now possible to distinguish between healthy and diseased states by patterns of gene expression, levels of enzymatic activity, and the structural makeup of tissue. This knowledge is now being extended to aid in the design of novel probes that are capable of sensitively and specifically detecting diseased states in vivo. Of particular interest are activatable (sensing) probes, which posses a quenched/unique signal until activated enzymatically or upon binding a specific target. Our lab is particular interested in the development of activatable probes that can be monitored via magnetic resonance (MR) or optical imaging platforms. For example, we are utilizing ‘magnetic relaxation switching’ (MRS) to detect biomolecules both in vitro and in vivo via MR. MRS refers to the process whereby clusters of superparamagnetic iron-oxide nanoparticles become dramatically more efficient at dephasing the spin-spin relaxation times (T2 relaxation times) of the surrounding protons than the monodisperse nanoparticles. We have designed and synthesized various magnetic relaxation switch bioconjugates such that nanoparticle clustering is induced in the presence of the target protein, nucleic acid, or enzyme. Similarly, we are developing various optically-based nanostructured probes that specifically elicit a detectable signal in the presence of the target biomolecule. We hope that these approaches to identifying molecular signatures of disease will allow clinicians to make more accurate diagnoses and will lead to more personalized patient care.
11:45 AM - Y4.5
Synthesis and Biomedical Applications of AuFe-C Core-shell Nanoparticles.
He Li 1 , Hongjie Dai 2 , Lei Xing 3 , Robert Sinclair 1 Show Abstract
1 Department of Materials Science and Engineering, Stanford University, Stanford, California, United States, 2 Department of Chemistry, Stanford University, Stanford, California, United States, 3 Department of Radiation Oncology, Stanford University, Stanford, California, United States
12:00 PM - Y4.6
Nanoscale Size Effect of Magnetic Nanocrystals for MRI and Their in vivo Utilization for Breast Cancer Diagnosis
Jinwoo Cheon 1 Show Abstract
1 Chemistry, Yonsei Univ, Seoul Korea (the Republic of)
Although the use of nanocrystals as probes for biomedical system is quite attractive, their successful in vivo application for the diagnosis of cancer is still difficult. Here, we report a novel protocol for the development of high quality bio-compatible magnetic nanocrystals with narrow size distribution and single crystallinity. We also experimentally elucidate the nanoscale relationship between the nanoparticle size and magnetic resonance (MR) contrast enhancement effects. Upon conjugation to cancer targeting antibody, these nanocrystal conjugates are utilized as excellent MRI probes for in vivo diagnosis of breast cancer with taking advantage of their superior properties (i.e. strong MRI enhancement, high colloidal stability, and specific binding ability).
12:15 PM - Y4.7
Development of Phospholipid Coated Targeted Iron Oxide Nanoparticles as MR Contrast Agents.
Kristi Hultman 1 , Stephen O'Brien 1 , Truman Brown 2 , Paul Harris 3 , Adrienne Grzenda 4 , Amanda Willis 5 Show Abstract
1 Applied Physics, Columbia University, New York, New York, United States, 2 Radiology and Biomedical Engineering, Columbia University, New York, New York, United States, 3 Medicine-Oncology, Columbia University, New York, New York, United States, 4 Surgery, Columbia University, New York, New York, United States, 5 Chemistry, Columbia University, New York, New York, United States
12:30 PM - Y4.8
Optically Tagged Calcium Phosphate-based Nanoparticles for Probing Gene Delivery Processes
Hyunbin Kim 1 , Renato Camata 2 , Rakesh Kapoor 2 , Selvarangan Ponnazhagan 3 Show Abstract
1 Dept of Materials Science and Engineering, University of Alabama at Birmingham, Birmingham, Alabama, United States, 2 Dept of Physics, University of Alabama at Birmingham, Birmingham, Alabama, United States, 3 Dept of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States
Trans-membrane cellular uptake of calcium phosphate nanoparticles has been widely used for several decades as the basis of a variety of simple gene transfection protocols. Using easily available ingredients, a particulate dispersion formed during co-precipitation of plasmid DNA and calcium phosphate is given to cells, which incorporate the DNA-calcium phosphate conjugates through endocytosis. A fraction of the incorporated DNA, which contains coding sequences and control regions, eventually finds its way to the cell nucleus leading to the expression of specific proteins. Despite its routine use in transient and stable transfection, these methods exhibit variable and often irreproducible efficiency. Although such problems can be circumvented in select cases by additional chemical treatments, the additives involved are often toxic to cells and require careful optimization. Basic investigations that could elucidate the mechanisms of calcium phosphate gene transfer and enable improvements of this versatile non-viral gene transfer methodology have been limited, however, because of the heterogeneous nature of calcium phosphate nanoparticles commonly employed in these studies. Moreover, there are currently no efficient probes capable of tracking the intracellular kinetics of gene transfer, making detailed studies of gene delivery processes virtually impossible. Rare-earth doped particles are well known for efficient infrared-to-visible upconversion. In this work, we focus on the development of custom-designed calcium phosphate nanoparticles of well-controlled size, structure and chemical composition that are doped with Er3+ or Yb3+. The upconverted fluorescence of these doped calcium phosphate nanoparticles may allow tracking of different intracellular processes involved in trasfection. The nanoparticles are produced using nanoparticle beam pulsed laser deposition, in which a hydroxyapatite target containing ~0.1-5 at. % of the rare-earth species is ablated in inert gas. Nanoparticles formed are size selected, and delivered to flat silicon surfaces or dispersed in balance solutions for subsequent mixing with plasmid DNA and exposure to cells for gene transfer experiments. Nanoparticle sizes can be tuned in the 3-20 nm range while nanoparticle concentrations may be varied between 108 and 1012 cm-2 on flat surfaces and between 105 and 109 cm-3 in solution. We have targeted primarily the control over crystal phase make-up of the calcium phosphate nanoparticles, which determines their dissolution behavior, and the rare-earth dopant concentration. If the dissolution kinetics of these DNA-carrying nanoparticles can be matched to the temporal sequence of specific cell cycles, there is a clear opportunity to exert control over the gene transfer process. In-vitro measurements of gene transfer efficiencies as a function of calcium phosphate nanoparticle size, composition, and chemical environment (silicon surface or balance solution) will also be discussed.
12:45 PM - Y4.9
Nanomaterial Molecular Rulers: Probing Distance and Orientation in dye-labeled Biomolecules
Mani Prabha Singh 1 , Geoffrey Strouse 1 , Travis Jennings 1 Show Abstract
1 Deptt. of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, United States
Optical molecular rulers are an important technology for measuring conformational changes in biomolecular systems following specific binding interactions. Typically this is limited to distances < 10 nm due to the limitations of two interacting dipoles between a molecular donor and a molecular acceptor, which obey a 1/R6 distance dependence. Energy transfer between an organic dye (FAM) to a 1.5-2 nm Au nanoparticle (Au NP) was studied. To understand the mechanism involved and the factors affecting energy transfer from a local donor dipole to a small metal nanoparticle in the absence of surface plasmon resonance band, the distance dependence, donor type, and donor orientation were varied. The studies involved two sets of experiments where first the length of double stranded DNA (ds-DNA), a rigid spacer, separating the dye and the Au NP was varied to test the distance dependence on the energy transfer efficiency. In the second set of experiments the length of the spacer was kept constant (60bp ds-DNA) but the position of the dye was varied on the strand. This study not only imposed distance dependence but also an orientation dependence of the dye relative to the Au NP. Photoluminescence and fluorescence lifetime experiments were performed on both. The results of these experiments showed that the quenching efficiency of the Au NP decreased with increasing distance (R) between the donor and the acceptor and also followed a 1/R4 dependence.