Observing the confinement potential of bacterial pore-forming toxin receptors inside rafts with nonblinking Eu(3+)-doped oxide nanoparticles.

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and ε-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 ± 0.05 µm(2). Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 µm(2)/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 ± 0.03 pN/µm at 37°C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family.

Synthetically encoded ultrashort-channel nanowire transistors for fast, pointlike cellular signal detection.

Nanostructures, which have sizes comparable to biological functional units involved in cellular communication, offer the potential for enhanced sensitivity and spatial resolution compared to planar metal and semiconductor structures. Silicon nanowire (SiNW) field-effect transistors (FETs) have been used as a platform for biomolecular sensors, which maintain excellent signal-to-noise ratios while operating on lengths scales that enable efficient extra- and intracellular integration with living cells. Although the NWs are tens of nanometers in diameter, the active region of the NW FET devices typically spans micrometers, limiting both the length and time scales of detection achievable with these nanodevices. Here, we report a new synthetic method that combines gold-nanocluster-catalyzed vapor-liquid-solid (VLS) and vapor-solid-solid (VSS) NW growth modes to produce synthetically encoded NW devices with ultrasharp (<5 nm) n-type highly doped (n(++)) to lightly doped (n) transitions along the NW growth direction, where n(++) regions serve as source/drain (S/D) electrodes and the n-region functions as an active FET channel. Using this method, we synthesized short-channel n(++)/n/n(++) SiNW FET devices with independently controllable diameters and channel lengths. SiNW devices with channel lengths of 50, 80, and 150 nm interfaced with spontaneously beating cardiomyocytes exhibited well-defined extracellular field potential signals with signal-to-noise values of ca. 4 independent of device size. Significantly, these "pointlike" devices yield peak widths of ∼500 µs, which is comparable to the reported time constant for individual sodium ion channels. Multiple FET devices with device separations smaller than 2 µm were also encoded on single SiNWs, thus enabling multiplexed recording from single cells and cell networks with device-to-device time resolution on the order of a few microseconds. These short-channel SiNW FET devices provide a new opportunity to create nanoscale biomolecular sensors that operate on the length and time scales previously inaccessible by other techniques but necessary to investigate fundamental, subcellular biological processes.

Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells.

Low concentrations of reactive oxygen species, notably hydrogen peroxide (H(2)O(2)), mediate various signalling processes in the cell. Production of these signals is highly regulated and a suitable probe is needed to measure these events. Here, we show that a probe based on a single nanoparticle can quantitatively measure transient H(2)O(2) generation in living cells. The Y(0.6)Eu(0.4)VO(4) nanoparticles undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants, leading to a recovery in luminescence. Our probe can be regenerated and reliably detects intracellular H(2)O(2) with a 30-s temporal resolution and a dynamic range of 1-45 microM. The differences in the timing of intracellular H(2)O(2) production triggered by different signals were also measured using these nanoparticles. Although the probe is not selective towards H(2)O(2), in many signalling processes H(2)O(2) is, however, the dominant oxidant. In conjunction with appropriate controls, this probe is a powerful tool for unravelling pathways that involve reactive oxygen species.

Organic functionalization of luminescent oxide nanoparticles toward their application as biological probes.

Luminescent inorganic nanoparticles are now widely studied for their applications as biological probes for in vitro or in vivo experiments. The functionalization of the particles is a key step toward these applications, since it determines the control of the coupling between the particles and the biological species of interest. This paper is devoted to the case of rare earth doped oxide nanoparticles and their functionalization through their surface encapsulation with a functional polysiloxane shell. The first step of the process is the adsorption of silicate ions that will act as a primary layer for the further surface polymerization of the silane, either aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GPTMS). The amino- or epoxy- functions born by the silane allow the versatile coupling of the particles with bio-organic species following the chemistry that is commonly used in biochips. Special attention is paid to the careful characterization of each step of the functionalization process, especially concerning the average number of organic functions that are available for the final coupling of the particles with proteins. The surface density of amino or epoxy functions was found to be 0.4 and 1.9 functions per square nanometer for GPTMS and APTES silanized particles, respectively. An example of application of the amino-functionalized particles is given for the coupling with alpha-bungarotoxins. The average number (up to 8) and the distribution of the number of proteins per particle are given, showing the potentialities of the functionalization process for the labeling of biological species.

Single lanthanide-doped oxide nanoparticles as donors in fluorescence resonance energy transfer experiments.

We used lanthanide-ion doped oxide nanoparticles, Y(0.6)Eu(0.4)VO(4), as donors in fluorescent resonance energy transfer (FRET) experiments. The choice of these nanoparticles allows us to combine the advantages of the lanthanide-ion emission, in particular the long lifetime and the large Stokes shift between absorption and emission, with the detectability of the nanoparticles at the single-particle level. Using cyanine 5 (Cy5) organic molecules as acceptors, we demonstrated FRET down to the single-nanoparticle level. We showed that, due to the long donor lifetime, unambiguous and precise FRET measurements can be performed in solution even in the presence of large free acceptor concentrations. Highly efficient energy transfer was obtained for a large number of acceptor molecules per donor nanoparticle. We determined FRET efficiencies as a function of Cy5 concentration which are in good agreement with a multiple acceptor-multiple donor calculation. On the basis of the donor emission recovery due to acceptor photobleaching, we demonstrated energy transfer from single-nanoparticle donors in fluorescence microscopy experiments.