Joining Caffeic Acid and Hydrothermal Treatment to Produce Environmentally Benign Highly Reduced Graphene Oxide.

Reduced graphene oxide (rGO) is a promising graphene-based material, with transversal applicability to a wide range of technological fields. Nevertheless, the common use of efficient-but hazardous to environment and toxic-reducing agents prevents its application in biological and other fields. Consequently, the development of green reducing strategies is a requirement to overcome this issue. Herein, a green, simple, and cost-effective one-step reduction methodology is presented. Graphene oxide (GO) was hydrothermally reduced in the presence of caffeic acid (CA), a natural occurring phenolic compound. The improvement of the hydrothermal reduction through the presence of CA is confirmed by XRD, Raman, XPS and TGA analysis. Moreover, CA polymerizes under hydrothermal conditions with the formation of spherical and non-spherical carbon particles, which can be useful for further rGO functionalization. FTIR and XPS confirm the oxygen removal in the reduced samples. The high-resolution scanning transmission electron microscopy (HRSTEM) images also support the reduction, showing rGO samples with an ordered graphitic layered structure. The promising rGO synthesized by this eco-friendly methodology can be explored for many applications.

Functionalized-ferroelectric-coating-driven enhanced biomineralization and protein-conformation on metallic implants.

In the context of bone regeneration, it is important to have platforms that with appropriate stimuli can support the attachment and direct the growth, proliferation and differentiation of cells. In the orthopedic field, metals and alloys are still the dominant materials used as implants, though their bioinert character leads to failure or to the need for multiple revision procedures. To respond to this situation here we exploit an alternative strategy for bone implants or repairs, based on charge mediating signals for bone regeneration, envisaged as a type of biological micro-electromechanical system (BioMEM). This strategy includes coating metallic 316L-type stainless steel substrates with ferroelectric LiTaO3 layers functionalized via electrical charging or UV-light irradiation. We show that the formation of surface calcium phosphates and protein adsorption are considerably enhanced for 316L-type stainless steel functionalized ferroelectric coatings. Our findings go beyond the current knowledge and demonstrate that the protein conformation is sensitive to the type of charge functionalization of the ferroelectric coatings. Our approach can be viewed as a set of guidelines for the development of electrically functionalized platforms that can stimulate tissue regeneration, promoting direct integration of the implant in the host tissue and hence contributing ultimately to reducing implant failure.

MC3T3-E1 pre-osteoblast response and differentiation after graphene oxide nanosheet uptake.

Nano-graphene oxide (GO) and its functionalized derivatives have aroused a great interest for drug delivery, tissue engineering and photothermal cancer therapy, but their biocompatibility has not yet been fully assessed. The aim of the present study was to evaluate the proliferation and differentiation of MC3T3-E1 pre-osteoblasts after the uptake of GO nanosheets (c.a. 400nm), functionalized with poly(ethylene glycol-amine) (PEG) and labelled with fluorescein isothiocyanate (FITC). Significant proliferation decrease and apoptosis increase were observed 3days after incorporation of FITC-PEG-GO by MC3T3-E1 cells. However, alterations on healthy pre-osteoblast differentiation into cells exhibiting osteoblast phenotype were not observed, as they showed normal alkaline phosphatase levels and matrix mineralization 12days after nanosheet uptake. The results suggest that 40µg/mL concentrations of these nanosheets would not affect the differentiation of healthy pre-osteoblasts, thus these PEG-GO nanosheets have potential to be used for biomedical applications after their internalization, as the induction of local hyperthermia on bone cancer.

Improved detection of domoic acid using covalently immobilised antibody fragments.

Antibody molecules, and antibody fragments in particular, have enormous potential in the development of biosensors for marine monitoring. Conventional immobilisation approaches used in immunoassays typically yield unstable and mostly incorrectly oriented antibodies, however, resulting in reduced detection sensitivities for already low concentration analytes. The 2H12 anti-domoic acid scFv antibody fragment was engineered with cysteine-containing linkers of two different lengths, distal to the antigen binding pocket, for covalent and correctly oriented immobilisation of the scFvs on functionalised solid supports. The Escherichia coli-produced, cysteine-engineered scFvs dimerised in solution and demonstrated similar efficiencies of covalent immobilisation on maleimide-activated plates and minimal non-covalent attachment. The covalently attached scFvs exhibited negligible leaching from the support under acidic conditions that removed almost 50% of the adsorbed wildtype fragment, and IC50s for domoic acid of 270 and 297 ng/mL compared with 1126 and 1482 ng/mL, respectively, for their non-covalently adsorbed counterparts. The expression and immobilisation approach will facilitate the development of stable, reusable biosensors with increased stability and detection sensitivity for marine neurotoxins.

Covalent and oriented immobilization of scFv antibody fragments via an engineered glycan moiety.

Antibody-derived fragments have enormous potential application in solid-phase assays such as biomarker detection and protein purification. Controlled orientation of the immobilized antibody molecules is a critical requirement for the sensitivity and efficacy of such assays. We present an approach for covalent, correctly oriented attachment of scFv antibody fragments on solid supports. Glycosylated scFvs were expressed in Escherichia coli and the C-terminal, binding pocket-distal glycan tag was oxidized for covalent attachment to amine-functionalized beads. The glycosylated scFvs could be immobilized at salt concentrations that precluded nonspecific adsorption of unglycosylated molecules and the covalently attached antibody fragments exhibited 4-fold higher functional activity than ionically adsorbed scFvs. The glyco-tethered scFvs were stable in NaCl concentrations that removed greater than 90% of adsorbed scFvs and they exhibited improved stability of antigen binding over both adsorbed scFvs and soluble, nonimmobilized scFvs in accelerated degradation tests. The simple expression and immobilization approach reported is likely to find broad application in in vitro antibody tests.

Chitosan gelation induced by the in situ formation of gold nanoparticles and its processing into macroporous scaffolds.

This work describes a simple synthetic route to induce chitosan (CHI) gelation by the in situ formation of gold nanoparticles (AuNPs). AuNPs were obtained by thermal treatment (e.g., 40 and 80 °C) of CHI aqueous solutions containing HAuCl(4) and in the absence of further reducing agents. The CHI hydrogels resulting after AuNP formation were submitted to unidirectional freezing and subsequent freeze-drying via ISISA (ice-segregation-induced self-assembly) process for the preparation of CHI scaffolds. The study of AuNP-CHI scaffolds by SEM and confocal fluorescence microscopy revealed a morphological structure characteristic of the hydrogel nature of the samples subjected to the ISISA process. Interestingly, not only the morphology but also the dissolution and swelling degree of the resulting CHI scaffolds were strongly influenced by the strength of the hydrogels obtained by the in situ formation of AuNP. We have also studied the catalytic activity AuNP-CHI scaffolds in the reduction of p-nitrophenol. The negligible dissolution and low swelling degree obtained in certain AuNP-CHI scaffolds allowed them to be used for more than four cycles with full preservation of the reaction kinetics.

Multiwall carbon nanotube scaffolds for tissue engineering purposes.

The use of scaffolds composed of a major fraction of multiwall carbon nanotubes (MWCNT, up to 89 wt.%) and a minor one of chitosan (CHI), and with a well-defined microchannel porous structure as biocompatible and biodegradable supports for culture growth is described. Cell adhesion, viability and proliferation onto the external surface of MWCNT/CHI scaffolds with C2C12 cell line (myoblastic mouse cell), which is a multipotent cell line able to differentiate towards different phenotypes under the action of some chemical or biological factors, has been evaluated in vitro and quantified by MTT assays. The evolution of the C2C12 cell line towards an osteoblastic lineage in presence of the recombinant human bone morphogenetic protein-2 (rhBMP-2) has also been studied both in vitro (e.g., following the appearance of alkaline phosphatase activity) and in vivo (e.g., by implantation of MWCNT/CHI scaffolds adsorbed with rhBMP-2 in muscle tissue and evaluation of the ectopic formation of bone tissue).

Highly fluorescent rhodamine B nanoparticles entrapped in hybrid glasses.

Rhodamine B (RB) nanoparticles entrapped in hybrid glasses show enhanced fluorescent emission (approximately 220-fold larger than that of single RB molecules) thanks to the configuration control of the self-assembled aggregates that form the supramolecular architecture of the nanoparticles. The fluorescence performance reported in this work is around 1 order of magnitude larger than that recently reported for fluorescent Nile Red nanoparticles. The fluorescence enhancement results from the use of a highly efficient fluorescent dye such RB and the formation of larger nanoparticles. Note that the later implies the presence of a large number of emitting centers involved in the fluorescence emission.