Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water.

Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes.

Photoluminescence enhancement of graphene oxide emission by infiltration in an aperiodic porous silicon multilayer.

Graphene oxide (GO) is a photoluminescent material whose application in integrated optoelectronics has been strongly limited due to poor emission intensity and handling procedures not compatible with standard microelectronic ones. In this work, a hybrid GO-porous silicon (GO-PSi) structure is realized in order to investigate the emission properties of GO infiltrated into an aperiodic porous multilayered matrix. A photoluminescence enhancement by a factor 32, compared to the same amount of GO deposited on a flat silicon surface, is demonstrated. Photoluminescence measurements also show wavelength modulation of the emitted signal.

One-pot synthesis of a gold nanoparticle-Vmh2 hydrophobin nanobiocomplex for glucose monitoring.

HydrophobinVmh2 is a small amphiphilic protein, which self-assembles on different surfaces and naturally interacts with glucose. Here, we report on the synthesis of a nanobiocomplex made of polyethylene glycol, Vmh2 and gold nanoparticles by a one-step process and on its ability to recognise glucose in an aqueous solution at 0.3-0.6-1.2 mg ml(-1) concentrations. Even though the Vmh2 proteins are intrinsically bonded to the gold core, effective glucose interaction monitoring was demonstrated by using dynamic light scattering, ultraviolet-visible, polarization-modulated infrared reflection-absorption and x-ray photoelectron spectroscopies. Experimental results highlighted an affinity constant of 7.3 ± 0.3 mg ml(-1) between the nanobiosystem and the sugar, and a detection sensitivity of 0.13 ± 0.06 a.u./mg ml(-1).

Oligopeptide-heavy metal interaction monitoring by hybrid gold nanoparticle based assay.

Phytochelatins are small peptides that can be found in several organisms, which use these oligopeptides to handle heavy metal elements. Here, we report a method for monitoring interactions between lead(ii) ions in aqueous solutions and phytochelatin 6 oligopeptide bioconjugated onto pegylated gold nanorods (PEG-AuNrs). This study is the first step towards a high sensitive label free optical biosensor to quantify heavy metal pollution in water.

Interaction of Thermus thermophilus ArsC enzyme and gold nanoparticles naked-eye assays speciation between As(III) and As(V).

The thermophilic bacterium Thermus thermophilus HB27 encodes chromosomal arsenate reductase (TtArsC), the enzyme responsible for resistance to the harmful effects of arsenic. We report on adsorption of TtArsC onto gold nanoparticles for naked-eye monitoring of biomolecular interaction between the enzyme and arsenic species. Synthesis of hybrid biological-metallic nanoparticles has been characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV-vis), dynamic light scattering (DLS) and phase modulated infrared reflection absorption (PM-IRRAS) spectroscopies. Molecular interactions have been monitored by UV-vis and Fourier transform-surface plasmon resonance (FT-SPR). Due to the nanoparticles' aggregation on exposure to metal salts, pentavalent and trivalent arsenic solutions can be clearly distinguished by naked-eye assay, even at 85 µM concentration. Moreover, the assay shows partial selectivity against other heavy metals.

Characterization by LC-MS/MS of oxidized products identified in synthetic peptide somatostatin and cetrorelix submitted to forced oxidative stress by hydrogen peroxide: Two case studies.

In a broader scenario, the forced degradation studies provided by the ICH guidelines for Q1A, Q1B, and Q2B degradation studies allow to know the CQA of the molecule used as a drug product, to determine the appropriate analytical methods, excipients, and storage conditions ensuring the quality of the drug, its efficacy, and patient safety. In this study, we focused our attention on understanding how oxidative stress is performed by H2 O2 -impacted small synthetic peptides that do not contain residues susceptible to oxidation such as methionine. Among the amino acids susceptible to oxidation, methionine is the most reactive and depending on the structure of the protein where it is exposed, it tends to oxidize by converting into methionine sulfone or methionine sulfoxide by oxidation of its sulfur atom. Scouting experiments obtained by forced oxidative stress conditions are presented on two small synthetic peptides that do not contain any methionine residues spiked with different amounts of H2 O2 , and they are analyzed by LC-MS/MS. Less frequent oxidation products than those commonly observed on proteins/peptides-containing methionine have been characterized on both peptides. The study demonstrated that somatostatin, by means of one residue of tryptophan on the molecule, can generate traces of several oxidized products detected by UPLC-MS. Furthermore, even at a negligible level, oxidation on tyrosine and proline in cetrorelix that does not contain methionine nor tryptophan has been detected by UHPLC-MS/MS. Identification and quantification of oxidized species were achieved by high-resolution MS and MS/MS experiments. Thus, FDSs undoubtedly aid the evaluation of the CQAs as an important component of the characterization package as recommended by HAs and ICH, facilitating the understanding of unforeseen features of the studied molecule used as drugs.

Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein.

A fusion protein designed in order to combine the fluorescence emission of the Green Fluorescent Protein (GFP) with the adhesion ability of the class I hydrophobin Vmh2 was heterologously produced in the yeast Pichia pastoris. The Vmh2-GFP fusion protein has proven to be a smart and effective tool for the study of Vmh2 self-assembling. Since the two proteins were linked by the specific cutting site of the thrombin, the fusion protein was used as the active biological element in the realization of a thrombin biosensor. When the thrombin present in the target solution specifically hydrolyzed its cleavage sequence, a consequent decrease in the fluorescence intensity of the sample could be observed. The Vmh2-GFP based assay allowed quantification of thrombin in solution with a detection limit of 2.27aM. The specificity of the assay with respect to other proteases and proteins granted the measurement of thrombin added to healthy human plasma with same high sensitivity and a limit of detection of 2.3aM. Further advantages of the developed biosensor are the simplicity of its design and preparation, and the low requirements in terms of samples, reagents and time.

Arsenate reductase from Thermus thermophilus conjugated to polyethylene glycol-stabilized gold nanospheres allow trace sensing and speciation of arsenic ions.

Water sources pollution by arsenic ions is a serious environmental problem all around the world. Arsenate reductase enzyme (TtArsC) from Thermus thermophilus extremophile bacterium, naturally binds arsenic ions, As(V) and As (III), in aqueous solutions. In this research, TtArsC enzyme adsorption onto hybrid polyethylene glycol-stabilized gold nanoparticles (AuNPs) was studied at different pH values as an innovative nanobiosystem for metal concentration monitoring. Characterizations were performed by UV/Vis and circular dichroism spectroscopies, TEM images and in terms of surface charge changes. The molecular interaction between arsenic ions and the TtArsC-AuNPs nanobiosystem was also monitored at all pH values considered by UV/Vis spectroscopy. Tests performed revealed high sensitivities and limits of detection equal to 10 ± 3 M-12 and 7.7 ± 0.3 M-12 for As(III) and As(V), respectively.

Optically monitored drug delivery patch based on porous silicon and polymer microneedles.

Fabrication and characterization of an optically monitored hybrid patch for local administration of drugs, based on polymeric micro-needles and a porous silicon free-standing membrane, are reported. The micro-needles are realized by an innovative photolithographic approach that allows fine tuning of geometrical parameters, using polyethylene glycol and a commercial photo-catalyzer. The porous silicon multilayer not only increases the storage of a relevant amount of the drug, but also offers a continuous, naked-eye monitoring of the drug delivery process. As a proof-of-concept experiment, we report our results on the release of a dye molecule (fluorescein, 332 Da) in a phosphate saline buffer.

The amphiphilic hydrophobin Vmh2 plays a key role in one step synthesis of hybrid protein-gold nanoparticles.

We report a simple and original method to synthesize gold nanoparticles in which a fungal protein, the hydrophobin Vmh2 from Pleurotus ostreatus and dicarboxylic acid-terminated polyethylene-glycol (PEG) has been used as additional components in a one step process, leading to hybrid protein-metal nanoparticles (NPs). The nanoparticles have been characterized by ultra-violet/visible, infrared and X-ray photoelectron spectroscopies, dynamic light scattering and also by electron microscopy imaging. The results of these analytical techniques highlight nanometric sized, stable, hybrid complexes of about 12 nm, with outer surface rich in functional chemical groups. Interaction with protein and antibodies has also been exploited.