Yuval Ebenstein

Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

Lasing from Semiconductor Quantum Rods in a Cylindrical Microcavity

For transient ellipsometry, the sample is placed between crossed polarizers at an angle of 54 between the sample normal and the laser beam. A laser diode at a wavelength k = 905 nm is employed and the residual birefringence of the sam- ple is compensated by a crystal compensator. The transmission through the el- lipsometry setup is measured in response to a step function of the electric field applied to the sample with a rise time of ~100 ls. The two-wave mixing is measured in a standard geometry (29) with the two beams having external angles of 30 and 60 to the sample normal and beam ra- tio 1:1. The laser source is a Kr ion laser at a wavelength of 647 nm. The time transients are measured by opening a shutter for both beams with a switching time of about 150 ls. After the measurements, the gratings are erased by a larg- er, non-Bragg matched erasing beam. In the beam fanning experiments, the same setup as in the two-wave mixing is used except only one beam is present. The angle of the beam to the sample normal is 60 and the applied electric field is inverted compared to two-wave mixing to enhance the fanning. Additionally, an aperture is placed in the beam path 50 cm behind the sample, which clips only 5 % of the unperturbed beam.

Fluorescence quantum yield of CdSe/ZnS nanocrystals investigated by correlated atomic-force and single-particle fluorescence microscopy

Correlated atomic-force and fluorescence microscopy are used to study single-particle versus ensemble fluorescence quantum yields (QY) of semiconductor nanocrystals by measuring a simultaneous map of the topography and the single-particle fluorescence. CdSe/ZnS nanocrystal quantum dots and quantum rods with high QY were investigated. A significant portion of dark particles is detected. Comparison with the ensemble solution QY shows that samples with higher QY have a larger fraction of bright particles accompanied by an increased single-particle QY. Saturated emission from single nanocrystals could not be detected because of particle darkening under high-power excitation.

Quantum Dots for In Vivo Small-Animal Imaging

Nanotechnology is poised to transform research, prevention, and treatment of cancer through the development of novel diagnostic imaging methods and targeted therapies. In particular, the use of nanoparticles for imaging has gained considerable momentum in recent years. This review focuses on the growing contribution of quantum dots (QDs) for in vivo imaging in small-animal models. Fluorescent QDs, which are small nanocrystals (1–10 nm) made of inorganic semiconductor materials, possess several unique optical properties best suited for in vivo imaging. Because of quantum confinement effects, the emission color of QDs can be precisely tuned by size from the ultraviolet to the near-infrared. QDs are extremely bright and photostable. They are also characterized by a wide absorption band and a narrow emission band, which makes them ideal for multiplexing. Finally, the large surface area of QDs permits the assembly of various contrast agents to design multimodality imaging probes. To date, biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Here we consider these novel breakthroughs in light of their potential clinical applications and discuss how QDs might offer a suitable platform to unite disparate imaging modalities and provide information along a continuum of length scales.

Beyond sequencing: optical mapping of DNA in the age of nanotechnology and nanoscopy.

Next generation sequencing (NGS) is revolutionizing all fields of biological research but it fails to extract the full range of information associated with genetic material. Optical mapping of DNA grants access to genetic and epigenetic information on individual DNA molecules up to ∼1 Mbp in length. Fluorescent labeling of specific sequence motifs, epigenetic marks and other genomic information on individual DNA molecules generates a high content optical barcode along the DNA. By stretching the DNA to a linear configuration this barcode may be directly visualized by fluorescence microscopy. We discuss the advances of these methods in light of recent developments in nano-fabrication and super-resolution optical imaging (nanoscopy) and review the latest achievements of optical mapping in the context of genomic analysis.

Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson–Crick base pairing

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters

The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods.

Aromatic Aldehyde and Hydrazine Activated Peptide Coated Quantum Dots for Easy Bioconjugation and Live Cell Imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.

Enzymatically Incorporated Genomic Tags for Optical Mapping of DNA-Binding Proteins

Affordable DNA sequencing is revolutionizing genetic research and is enabling multiple novel biomedical applications. Among the inherent properties of today’s high-throughput sequencing technologies is the fact that it compiles long-range sequences from the assembly of numerous short-read data.[1] This leads to two fundamental limitations: loss of long-range contextual information on the single-genome level and difficulties coping with repetitive or variable genomic regions. Optical mapping and its variants[2–10] rely on the visualization of individual, long (50 kb–1000 kb) DNA molecules and extraction of genomic information by fluorescent labeling of the DNA. These techniques lack the resolution of sequencing but offer genomic context and therefore are attractive both in combination with sequencing to aid in sequence assembly[11–13] and for investigation of genomic structural variations on the individual chromosome level.[14, 15] Such variations include deletions, duplications, copy-number variants (CNVs), insertions, inversions, and translocations, all of which have a major impact on the phenotypic variations within a population (or somatic mutations, important in cancer progression). In addition, the available information content of the genome extends beyond the sequence, and the long-range data offered by optical mapping may provide crucial information regarding the distribution of DNA-binding proteins such as transcription factors and histones along the genome.

Bacteriophage strain typing by rapid single molecule analysis.

Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.