A FLIM Microscopy Based on Acceptor-Detected Förster Resonance Energy Transfer.

Time-resolved donor-detected Förster resonance energy transfer (trDDFRET) allows the observation of molecular interactions of dye-labeled biomolecules in the ∼10-100 Å region. However, we can observe longer-range interactions when using time-resolved acceptor-detected FRET (trADFRET), since the signal/noise ratio can be improved when observing the acceptor emission. Therefore, we propose a new methodology based on trADFRET to construct a new fluorescence lifetime microscopy (FLIM-trADFRET) technique to observe biological machinery in the range of 100-300 Å in vivo, the last frontier in biomolecular medicine. The integrated trADFRET signal is extracted in such a way that noise is canceled, and more photons are collected, even though trADFRET and trDDFRET have the same rate of transfer. To assess our new methodology, proof of concept was demonstrated with a set of well-defined DNA scaffolds.

Dual-Channel Stopped-Flow Apparatus for Simultaneous Fluorescence, Anisotropy, and FRET Kinetic Data Acquisition for Binary and Ternary Biological Complexes.

The Stopped-Flow apparatus (SF) tracks molecular events by mixing the reactants in sub-millisecond regimes. The reaction of intrinsically or extrinsically labeled biomolecules can be monitored by recording the fluorescence, F(t), anisotropy, r(t), polarization, p(t), or FRET, F(t)FRET, traces at nanomolar concentrations. These kinetic measurements are critical to elucidate reaction mechanisms, structural information, and even thermodynamics. In a single detector SF, or L-configuration, the r(t), p(t), and F(t) traces are acquired by switching the orientation of the emission polarizer to collect the IVV and IVH signals however it requires two-shot experiments. In a two-detector SF, or T-configuration, these traces are collected in a single-shot experiment, but it increases the apparatus' complexity and price. Herein, we present a single-detector dual-channel SF to obtain the F(t) and r(t) traces simultaneously, in which a photo-elastic modulator oscillates by 90° the excitation light plane at a 50 kHz frequency, and the emission signal is processed by a set of electronic filters that split it into the r(t) and F(t) analog signals that are digitized and stored into separated spreadsheets by a custom-tailored instrument control software. We evaluated the association kinetics of binary and ternary biological complexes acquired with our dual-channel SF and the traditional methods; such as a single polarizer at the magic angle to acquire F(t), a set of polarizers to track F(t), and r(t), and by energy transfer quenching, F(t)FRET. Our dual-channel SF economized labeled material and yielded rate constants in excellent agreement with the traditional methods.

"Green" functionalization of pristine multi-walled carbon nanotubes with long-chain aliphatic amines.

Short pristine multi-walled carbon nanotubes (MWNTs) were functionalized with a series of long-chain (including polymeric) aliphatic amines, namely octadecylamine (ODA), 1,8-diaminooctane (DO), polyethylene glycol diamine (PEGDA) and polyethylenimine (PEI), via two "green" approaches: (1) gas-phase functionalization (for volatile ODA and DO) and (2) direct heating in the melt (for polymeric PEGDA and PEI). Both of them consist in one-step reaction between MWNTs and amine without the use of organic solvents. The nanostructures obtained were characterized by using infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. It was observed that both solvent-free methods were efficient in the nanotube functionalization, and the nanostructures of variable solubility and morphology were obtained depending on the amines attached. ODA, PEGDA and PEI-functionalized MWNTs were found to be soluble in propanol, meanwhile the MWNTs-PEGDA and MWNTs-PEI were soluble in water as well. The attachment of 1,8-diaminooctane onto MWNTs resulted in cross-linked stable nanostructure.

Ecotoxicological effects of carbon nanomaterials on algae, fungi and plants.

The ecotoxicological effects of carbon nanomateriales (CNMs), namely fullerenes and carbon nanotubes, on algae, fungi and plants are analyzed. In different toxicity tests, both direct and indirect effects were found. The direct effects are determined by nanomaterial chemical composition and surface reactivity, which might catalyze redox reactions in contact with organic molecules and affect respiratory processes. Some indirect effects of carbon nanoparticles (CNPs) are physical restraints or release of toxic ions. Accumulation of CNPs in photosynthetic organs provokes obstruction in stomata, foliar heating and alteration in physiological processes. The phytotoxicity studies of CNMs should be focused on determining phytotoxicity mechanisms, size distribution of CNPs in solution, uptake and translocation of nanoparticles by plants, on characterization of their physical and chemical properties in rhizosphere and on root surfaces. More studies on plants and algae, as a part of food chain, are needed to understand profoundly the toxicity and health risks of CNMs as ecotoxicological stressors. Correct and detailed physical and chemical characterization of CNMs is very important to establish the exposure conditions matching the realistic ones. Ecotoxicity experiments should include examinations of both short and long-term effects. One must take into account that real carbon nanomaterials are complex mixtures of carbon forms and metal residues of variable chemistry and particle size, and the toxicity reported may reflect these byproducts/residues/impurities rather than the primary material structure. One more recommendation is not only to focus on the inherent toxicity of nanoparticles, but also consider their possible interactions with existing environmental contaminants.

Genotoxic properties of nylon-6/MWNTs nanohybrid.

We performed the micronucleus test to determine the level of biocompatibility of pristine multi-walled carbon nanotubes (MWNTs) and MWNTs functionalized with nylon-6, (referred to as the nylon-6/ MWNTs nanohybrid), when they interact with human lymphocytes in a cell culture medium. A comparative genotoxic analysis demonstrated a better degree of biocompatibility of nylon-6/MWNT with human lymphocytes, as compared to pristine MWNTs, for the concentration range of 10-60 microg/ml. An evidence was found that pristine MWNTs act as clastogenic agents and possibly as aneuploidogenic agents, increasing the frequency of genotoxic bioindicators. On the other hand, nylon-6/ MWNTs nanohybrid were observed to induce cell death via apoptosis, which could be attributed to residual impurities of epsilon-caprolactam.