Tali Dadosh

Rapid electronic detection of probe-specific microRNAs using thin nanopore sensors

Small RNA molecules have an important role in gene regulation and RNA silencing therapy, but it is challenging to detect these molecules without the use of time-consuming radioactive labelling assays or error-prone amplification methods. Here, we present a platform for the rapid electronic detection of probe-hybridized microRNAs from cellular RNA. In this platform, a target microRNA is first hybridized to a probe. This probe:microRNA duplex is then enriched through binding to the viral protein p19. Finally, the abundance of the duplex is quantified using a nanopore. Reducing the thickness of the membrane containing the nanopore to 6 nm leads to increased signal amplitudes from biomolecules, and reducing the diameter of the nanopore to 3 nm allows the detection and discrimination of small nucleic acids based on differences in their physical dimensions. We demonstrate the potential of this approach by detecting picogram levels of a liver-specific miRNA from rat liver RNA.

Measurement of the conductance of single conjugated molecules

Electrical conduction through molecules depends critically on the delocalization of the molecular electronic orbitals and their connection to the metallic contacts. Thiolated (- SH) conjugated organic molecules are therefore considered good candidates for molecular conductors: in such molecules, the orbitals are delocalized throughout the molecular backbone, with substantial weight on the sulphur–metal bonds. However, their relatively small size, typically ∼1 nm, calls for innovative approaches to realize a functioning single-molecule device. Here we report an approach for contacting a single molecule, and use it to study the effect of localizing groups within a conjugated molecule on the electrical conduction. Our method is based on synthesizing a dimer structure, consisting of two colloidal gold particles connected by a dithiolated short organic molecule, and electrostatically trapping it between two metal electrodes. We study the electrical conduction through three short organic molecules: 4,4′-biphenyldithiol (BPD), a fully conjugated molecule; bis-(4-mercaptophenyl)-ether (BPE), in which the conjugation is broken at the centre by an oxygen atom; and 1,4-benzenedimethanethiol (BDMT), in which the conjugation is broken near the contacts by a methylene group. We find that the oxygen in BPE and the methylene groups in BDMT both suppress the electrical conduction relative to that in BPD.

Managing light polarization via plasmon–molecule interactions within an asymmetric metal nanoparticle trimer

The interaction of light with metal nanoparticles leads to novel phenomena mediated by surface plasmon excitations. In this article we use single molecules to characterize the interaction of surface plasmons with light, and show that such interaction can strongly modulate the polarization of the emitted light. The simplest nanostructures that enable such polarization modulation are asymmetric silver nanocrystal trimers, where individual Raman scattering molecules are located in the gap between two of the nanoparticles. The third particle breaks the dipolar symmetry of the two-particle junction, generating a wavelength-dependent polarization pattern. Indeed, the scattered light becomes elliptically polarized and its intensity pattern is rotated in the presence of the third particle. We use a combination of spectroscopic observations on single molecules, scanning electron microscope imaging, and generalized Mie theory calculations to provide a full picture of the effect of particles on the polarization of the emitted light. Furthermore, our theoretical analysis allows us to show that the observed phenomenon is very sensitive to the size of the trimer particles and their relative position, suggesting future means for precise control of light polarization on the nanoscale.

Plasmonic Control of the Shape of the Raman Spectrum of a Single Molecule in a Silver Nanoparticle Dimer

We study surface-enhanced Raman scattering (SERS) of individual organic molecules embedded in dimers of two metal nanoparticles. The good control of the dimer preparation process, based on the usage of bifunctional molecules, enables us to study quantitatively the effect of the nanoparticle size on the SERS intensity and spectrum at the single molecule level. We find that as the nanoparticle size increases the total Raman intensity increases and the lower energy Raman modes become dominant. We perform an electromagnetic calculation of the Raman enhancement and show that this behavior can be understood in terms of the overlap between the plasmonic modes of the dimer structure and the Raman spectrum. As the nanoparticle size increases, the plasmonic dipolar mode shifts to longer wavelength and thereby its overlap with the Raman spectrum changes. This suggests that the dimer structure can provide an external control of the emission properties of a single molecule. Indeed, clear and systematic differences are observed between Raman spectra of individual molecules adsorbed on small versus large particles.

Fabrication of nanoscale gaps in integrated circuits

Nanosize objects such as metal clusters present an ideal system for the study of quantum phenomena and for the construction of practical quantum devices. Integrating these small objects in a macroscopic circuit is, however, a difficult task. So far, nanoparticles have been contacted and addressed by highly sophisticated techniques not suitable for large-scale integration in macroscopic circuits. We present an optical lithography method that allows for the fabrication of a network of electrodes separated by gaps of controlled nanometer size. The main idea is to control the gap size with subnanometer precision using a structure grown by molecular-beam epitaxy.

Nanoparticles and nanogaps: controlled positioning and fabrication

Abstract We present a novel method for fabrication of contacts to nanosize particles. The method is based on conventional optical lithography of GaAs/AlGaAs molecular beam epitaxy grown structures. Taking advantage of the difference in etch rate between GaAs and AlGaAs a narrow gap is formed between metal contacts deposited on the side of a mesa structure. We demonstrate electrostatic trapping of charged metal clusters into these structures using alternating electric fields.

Deposition of collagen type I onto skeletal endothelium reveals a new role for blood vessels in regulating bone morphology.

Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix. Highlighted article: Collagen I is deposited by osteoblasts onto endothelial cells within bone and serves as a template for mineralisation, with ossification thus spatially and temporally following vascular patterning.

Controlling Nanogap Quantum Dot Photoconductivity through Optoelectronic Trap Manipulation

Nanoscale devices are being extensively studied for their tunable electronic and optical properties, but the influence of impurities and defects is amplified at these length scales and can lead to poorly understood variations in characteristics of semiconducting materials. By performing a large ensemble of photoconductivity measurements in nanogaps bridged by core-shell CdSe/ZnS semiconductor nanocrystals, we discover optoelectronic methods for affecting solid-state charge trap populations. We introduce a model that unifies previous work and transforms the problem of irreproducibility in nanocrystal electronic properties into a reproducible and robust photocurrent response due to trap state manipulation. Because traps dominate many physical processes, these findings may lead to improved performance and device tunability for various nanoscale applications through the control and optimization of impurities and defects.

3D visualization of mitochondrial solid-phase calcium stores in whole cells

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.

SNARE priming is essential for maturation of autophagosomes but not for their formation

Significance The contribution of SNARE-mediated membrane fusion to the different stages along the autophagic process is not well understood. In this work we demonstrate that inhibition of SNARE priming leads to impairment of the autophagic flux manifested by the accumulation of mature autophagosomes that do not fuse with the lysosomes. Our results suggest that SNARE priming is essential for the fusion of the autophagosome with the lysosome but not for autophagosome formation. Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy. αSNAP, the adaptor molecule for the SNARE-priming enzyme N-ethylmaleimide-sensitive factor (NSF) is known to be crucial for intracellular membrane fusion processes, but its role in autophagy remains unclear. Here we demonstrated that knockdown of αSNAP leads to inhibition of autophagy, manifested by an accumulation of sealed autophagosomes located in close proximity to lysosomes but not fused with them. Under these conditions, moreover, association of both Atg9 and the autophagy-related SNARE protein syntaxin17 with the autophagosome remained unaffected. Finally, our results suggested that under starvation conditions, the levels of αSNAP, although low, are nevertheless sufficient to partially promote the SNARE priming required for autophagy. Taken together, these findings indicate that while autophagosomal–lysosomal membrane fusion is sensitive to inhibition of SNARE priming, the initial stages of autophagosome biogenesis and autophagosome expansion remain resistant to its loss.