Marco Lazzarino

Nanoscale chemical mapping using three-dimensional adiabatic compression of surface plasmon polaritons

The fields of plasmonics, Raman spectroscopy and atomic force microscopy have recently undergone considerable development, but independently of one another. By combining these techniques, a range of complementary information could be simultaneously obtained at a single molecule level. Here, we report the design, fabrication and application of a photonic-plasmonic device that is fully compatible with atomic force microscopy and Raman spectroscopy. Our approach relies on the generation and localization of surface plasmon polaritons by means of adiabatic compression through a metallic tapered waveguide to create strongly enhanced Raman excitation in a region just a few nanometres across. The tapered waveguide can also be used as an atomic force microscope tip. Using the device, topographic, chemical and structural information about silicon nanocrystals may be obtained with a spatial resolution of 7 nm.

Acceleration of neuronal precursors differentiation induced by substrate nanotopography

Embryonic stem (ES) cell differentiation in specific cell lineages is a major issue in cell biology particularly in regenerative medicine. Differentiation is usually achieved by using biochemical factors and it is not clear whether mechanical properties of the substrate over which cells are grown can affect proliferation and differentiation. Therefore, we produced patterns in polydimethylsiloxane (PDMS) consisting of groove and pillar arrays of sub‐micrometric lateral resolution as substrates for cell cultures. We analyzed the effect of different nanostructures on differentiation of ES‐derived neuronal precursors into neuronal lineage without adding biochemical factors. Neuronal precursors adhered on PDMS more effectively than on glass coverslips. We demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. On higher pillar neuronal differentiation reaches ∼80% 96 h after plating and the largest differentiation enhancement of pillars over flat PDMS was observed during the first 6 h of culture. We conclude that PDMS nanopillars accelerate and increase neuronal differentiation. Biotechnol. Bioeng. 2011;108: 2736–2746.

Modulation of alpha-synuclein aggregation by dopamine analogs.

The action of dopamine on the aggregation of the unstructured alpha-synuclein (α-syn) protein may be linked to the pathogenesis of Parkinsons disease. Dopamine and its oxidation derivatives may inhibit α-syn aggregation by non-covalent binding. Exploiting this fact, we applied an integrated computational and experimental approach to find alternative ligands that might modulate the fibrillization of α-syn. Ligands structurally and electrostatically similar to dopamine were screened from an established library. Five analogs were selected for in vitro experimentation from the similarity ranked list of analogs. Molecular dynamics simulations showed they were, like dopamine, binding non-covalently to α-syn and, although much weaker than dopamine, they shared some of its binding properties. In vitro fibrillization assays were performed on these five dopamine analogs. Consistent with our predictions, analyses by atomic force and transmission electron microscopy revealed that all of the selected ligands affected the aggregation process, albeit to a varying and lesser extent than dopamine, used as the control ligand. The in silico/in vitro approach presented here emerges as a possible strategy for identifying ligands interfering with such a complex process as the fibrillization of an unstructured protein.

A Revertible, Autonomous, Self-Assembled DNA-Origami Nanoactuator

A DNA-origami actuator capable of autonomous internal motion in accord to an external chemical signal was designed, built, operated and imaged. The functional DNA nanostructure consists of a disk connected to an external ring in two, diametrically opposite points. A single stranded DNA, named probe, was connected to two edges of the disk perpendicularly to the axis of constrain. In the presence of a hybridizing target molecule, the probe coiled into a double helix that stretched the inner disk forcing the edges to move toward each other. The addition of a third single stranded molecule that displaced the target from the probe restored the initial state of the origami. Operation, dimension and shape were carefully characterized by combining microscopy and fluorescence techniques.

Atomic force microscope anodic oxidation studied by spectroscopic microscopy

Atomic force microscope (AFM) induced local oxidation is a versatile and promising nanofabrication process used successfully to produce quantum devices. Nevertheless, little information is available on the chemical and structural properties of the grown oxide. We address this open issue by a spectromicroscopic study of nanoscopic oxide patterns grown by AFM anodic oxidation on n-type silicon substrate. We show that AFM oxidation produces chemically uniform, stoichiometric SiO2, and that its chemical and structural properties do not depend on the applied voltage. The observed electrostatic shift of the oxide binding energies allows a simple estimation of the electrical properties of the AFM induced oxide.

Membrane stiffening by STOML3 facilitates mechanosensation in sensory neurons

Sensing force is crucial to maintain the viability of all living cells. Despite its fundamental importance, how force is sensed at the molecular level remains largely unknown. Here we show that stomatin-like protein-3 (STOML3) controls membrane mechanics by binding cholesterol and thus facilitates force transfer and tunes the sensitivity of mechano-gated channels, including Piezo channels. STOML3 is detected in cholesterol-rich lipid rafts. In mouse sensory neurons, depletion of cholesterol and deficiency of STOML3 similarly and interdependently attenuate mechanosensitivity while modulating membrane mechanics. In heterologous systems, intact STOML3 is required to maintain membrane mechanics to sensitize Piezo1 and Piezo2 channels. In C57BL/6N, but not STOML3−/− mice, tactile allodynia is attenuated by cholesterol depletion, suggesting that membrane stiffening by STOML3 is essential for mechanical sensitivity. Targeting the STOML3–cholesterol association might offer an alternative strategy for control of chronic pain.

Integration of confocal and atomic force microscopy images.

Atomic force microscopy (AFM) provides the possibility to map the 3D structure of viewed objects with a nanometric resolution, which cannot be achieved with other imaging methods such as conventional video imaging and confocal fluorescent microscopy. Video imaging with CCD cameras can provide an analysis of biological events with a temporal and spatial resolution not possible with AFM, while confocal imaging allows the simultaneous acquisition of immunofluorescence images. In this communication we present a simple method to combine AFM and confocal images to study differentiating embryonic stem (ES) cells-derived and dorsal root ganglia (DRG) neurons in culture. Neurons were grown on coverslips with micrometric markers that allow finding and imaging the same neuron with different microscopes. AFM and confocal images were registered using conventional methods used in Computer Science. The combination of these two techniques allows relating functional properties to morphological features of imaged neurons.

High-performance planar light-emitting diodes

High-speed planar light-emitting diodes fabricated within a single high-mobility quantum well are demonstrated. Devices were fabricated by photolithography and wet chemical etching starting from p-type modulation-doped Al0.5Ga0.5As/GaAs heterostructures grown by molecular beam epitaxy. Electrical and optical measurements from room temperature down to 1.8 K show high spectral purity, high external efficiency, and extremely short recombination times of the order of 50 ps. Time-resolved electroluminescence measurements demonstrate subnanosecond modulation time scale.

Asymmetrical twin cantilevers for single molecule detection

Recently, there has been growing interest in sensors based on frequency shift detection of mechanical resonators, that has led to extremely sensitive diagnostic methods for medicine and biology. Here the authors propose and demonstrate a strategy capable of detecting the presence of a few macromolecules, which uses an asymmetrical cantilever structure of several microns in size, operated at room temperature, and under ordinary vacuum conditions. The idea is to detect the presence of one or more molecules by detecting the mechanical cross-talk induced by the molecular link between a short cantilever and an array of longer cantilevers facing the short one.

A DNA Origami Nanorobot Controlled by Nucleic Acid Hybridization

A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.