Renewable nanocomposite layer-by-layer assembled catalytic interfaces for biosensing applications

A novel, easily renewable nanocomposite interface based on layer-by-layer (LbL) assembled cationic/anionic layers of carbon nanotubes customized with biopolymers is reported. A simple approach is proposed to fabricate a nanoscale structure composed of alternating layers of oxidized multiwalled carbon nanotubes upon which is immobilized either the cationic enzyme organophosphorus hydrolase (OPH; MWNT−OPH) or the anionic DNA (MWNT−DNA). The presence of carbon nanotubes with large surface area, high aspect ratio and excellent conductivity provides reliable immobilization of enzyme at the interface and promotes better electron transfer rates. The oxidized MWNTs were characterized by thermogravimetric analysis and Raman spectroscopy. Fourier transform infrared spectroscopy showed the surface functionalization of the MWNTs and successful immobilization of OPH on the MWNTs. Scanning electron microscopy images revealed that MWNTs were shortened during sonication and that LbL of the MWNT/biopolymer conjugates resulted in a continuous surface with a layered structure. The catalytic activity of the biopolymer layers was characterized using absorption spectroscopy and electrochemical analysis. Experimental results show that this approach yields an easily fabricated catalytic multilayer with well-defined structures and properties for biosensing applications whose interface can be reactivated via a simple procedure. In addition, this approach results in a biosensor with excellent sensitivity, a reliable calibration profile, and stable electrochemical response.

Sonochemical effects on ZnO particles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudos biológicos de nanopartículas de óxido de zinco: interação com proteínas e avaliação de “Burst” oxidativo de leucócitos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Imunohistoquímica em órgãos de tilápias-do-Nilo imunizadas com antígeno insolúvel de Streptococcus agalactiae

Pós-graduação em Medicina Veterinária - FCAV

Avaliação histológica e funcional do enxerto de neotraqueia de coelho desenvolvido por bioengenharia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação histológica e funcional do enxerto de neotraqueia de coelho desenvolvido por bioengenharia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic transformation of three sweet orange cultivars from explants of adult plants

The difficulty in adult tissue genetic transformation in woody species is still an obstacle to be overcome, including in most sweet orange cultivars of the Brazilian citrus industry. This work reports that, after in vitro culture adjustments, transgenic adventitious buds of 'Hamlin', 'Pra', and 'Valencia' sweet oranges (Citrus sinensis L. Osbeck) were recovered using adult material as explant source, in genetic transformation experiments via Agrobacterium tumefaciens. The transgenic buds were identified by the GUS histochemical analysis and confirmed by PCR analysis, which indicated the presence of an amplified fragment of 817 bp corresponding to the uidA gene sequence. The efficiencies of genetic transformation for 'Hamlin', 'Pra', and 'Valencia' sweet orange cultivars were 2.5, 1.4, and 3.7%, respectively. Media supplemented with auxins and cytokinins during co-culture, and media with high concentrations of cytokinins (3 mg L-1) during transgenic selection led to the transformation and, consequently, the regeneration of adequate number of adventitious buds for the three cultivars. The use of sonication during the explant disinfection was not effective to reduce endophytic contamination and reduced transformation efficiency.

Rapid sample preparation procedure for As speciation in food samples by LC-ICP-MS

This paper describes a rapid method for arsenic (As) speciation by LC-ICP-MS in several types of food samples. Prior to analysis, samples were milled and the As species extracted from biological tissues by sonication in only 2 min with a solution containing MeOH (10%, v/v) plus HNO3 (2%, v/v). As species were separated by LC using an anion exchange column. Method detection limits for AsB, As3+,DMA, MMA and As5+ were 1.3, 0.9, 0.6, 0.7 and 0.8 ng g(-1), respectively. Method accuracy and precision were traceable to Certified Reference Materials SRM1577 bovine liver from the National Institute of Standards and Technology, CE278 mussel tissue from the Institute of Reference Materials and Measurements and DOLT-3 dogfish liver tissue and DORM-3 fish protein from the National Research Council of Canada. Finally, the method was applied to speciate As in food samples (egg, fish muscle, beef and chicken) purchased in Brazilian markets.

Cellulose micro/nanofibres from Eucalyptus kraft pulp: Preparation and properties

There is growing interest in cellulose nanofibres from renewable sources for several industrial applications. However, there is a lack of information about one of the most abundant cellulose pulps: bleached Eucalyptus kraft pulp. The objective of the present work was to obtain Eucalyptus cellulose micro/nanofibres by three different processes, namely: refining, sonication and acid hydrolysis of the cellulose pulp. The refining was limited by the low efficiency of isolated nanofibrils, while sonication was more effective for this purpose. However, the latter process occurred at the expense of considerable damage to the cellulose structure. The whiskers obtained by acid hydrolysis resulted in nanostructures with lower diameter and length, and high crystallinity. Increasing hydrolysis reaction time led to narrower and shorter whiskers, but increased the crystallinity index. The present work contributes to the different widespread methods used for the production of micro/nanofibres for different applications. (C) 2012 Elsevier Ltd. All rights reserved.

Selective Permeation and Organic Extraction of Recombinant Green Fluorescent Protein (gfpuv) from Escherichia coli

Background Transformed cells of Escherichia coli DH5-α with pGFPuv, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. Material and Methods Cultures (37°C/100 rpm/ 24 h; μ = 0.99 h-1 - 1.10 h-1) of transformed (pGFP) Escherichia coli DH5-α, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75°C) cells were subjected to three freeze/thaw (-20°C/ 0.83°C/min) cycles interlaid by sonication (3 pulses/ 6 seconds/ 25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM β-mercaptoethanol β-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Results The sonication maximum released amount obtained from the cells was 327.67 μg gfpuv/mL (20.73 μg gfpuv/mg total proteins – BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 μg gfpuv/mL (29.74 μg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 μg/mg and 135.10 μg/mg. Conclusions The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.

Photoactive Carbon Nanostructures: From Multicomponent Arrays To Nanomaterials

Carbon has a unique ability to shape networks of differently hybridized atoms that can generate various allotropes and may also exist as nanoscale materials. The emergence of carbon nanostructures initially occured through the serendipitous discovery of fullerenes and then through experimental advances which led to carbon nanotubes, nanohorns and graphene. The structural diversity of carbon nanoscopic allotropes and their unique and unprecedentend properties, give rise to countless applications and have been intensively exploited in nanotechnology, since they may address the need to create smarter optoelectronic devices, smaller in size and with better performance. The versatile properties of carbon nanomaterials are reflected in the multidisciplinary character of my doctoral research where, in particular, I take advantage of the opportunities offered by fullerenes and carbon nanotubes in constructing novel functional materials. In this work, carbon nanostructures are incorporated in novel photoactive functional systems constructed through different types of interactions – covalent bonds, ion-pairing or self-assembly. The variety of properties exhibited by carbon nanostructures is successfully explored by assigning them a different role in a specific array: fullerenes are employed as electron or energy acceptors, whereas carbon nanotubes behave like optically inert scaffolds for luminescent materials or nanoscale substrates in sonication-induced self-assembly. All the presented systems serve as a testbed for exploring the properties of carbon nanostructures in multicomponent arrays, which may be advantageous for the production of new photovoltaic or optoelectronic devices, as well as in the design and control of self-assembly processes.

Photo-induced reversible assembly of colloidal polymer nanoparticles

Polymer nanoparticles functionalized on the surface with photo-responsive labels were synthesized. In a first synthetic step, polystyrene was copolymerized with the cross-linker divinylbenzene and poly(ethylene glycol) acrylate in a miniemulsion, to produce nano-sized spheres (~ 60 nm radius) with terminal hydroxyl groups, which were functionalized in a subsequent synthetic step with photo-responsive labels. For this purpose, two photo-active molecular structures were separately used: anthracene, which is well known to form covalently bonded dimers upon photo-excitation; and pyrene, which only forms short lived excited state dimers (excimers). Acid derivatives of these labels (9-anthracene carboxylic acid and 1-pyrene butyric acid) were bonded to the hydroxyl terminal groups of the nanoparticles through an esterification reaction, via the intermediate formation of the corresponding acid chloride.rnThe obtained labeled nanoparticles appeared to be highly hydrophobic structures. They formed lyophobic suspensions in water, which after analysis by dynamic light scattering (DLS) and ultramicroscopic particle tracking, appeared to equilibrate as a collection of singly dispersed nanoparticles, together with a few nanoparticle aggregates. The relative amount of aggregates decreased with increasing amounts of the surfactant sodium dodecyl sulfate (SDS), thus confirming that aggregation is an equilibrated state resulting from lyophobicity. The formation of such aggregates was corroborated using scanning electron microscopy (SEM). The photo-irradiation of the lyophobic aqueous suspensions of anthracene labeled nanoparticles (An-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. The obtained state of aggregation could be reverted by sonication. The possibility to re-aggregate the system in subsequent photo-excitation and sonication cycles was established. Likewise, the photo-irradiation of lyophobic aqueous suspensions of pyrene-labeled nanoparticles (Py-NP) resulted in the formation of higher aggregates, as evidenced by DLS and ultramicroscopy. These appeared to remain aggregated due to hydrophobic interactions. This system could also be re-dispersed by sonication and re-aggregated in subsequent cycles of photo-excitation and sonication.

Mikrobielle Synthese von Biopolymeren aus der nachwachsenden Rohstoffquelle Weizenstroh

Weizenstroh als erneuerbare Ressource zur Produktion von Biopolymeren und wichtigen Grundchemikalien stellt eine ökologisch sinnvolle Alternative dar. Durch die vom PFI durchgeführte Thermodruckhydrolyse konnte das Weizenstroh und die darin enthaltenen Zucker fast vollständig mobilisiert werden. Ein umfangreiches Screening nach Organismen, welche die Zucker des Weizenstrohs verwerten konnten, ergab, dass einige wenige Stämme zur PHB-Bildung aus Xylose befähigt waren (10 %). Zur PHB-Synthese aus Glucose waren indes ca. doppelt so viele Organismen in der Lage (20 %). Zwei der insgesamt 118 untersuchten Organismen zeigten besonders gute PHB-Bildung sowohl mit Xylose als auch mit Glucose als Substrat. Dabei handelte es sich um die hauseigenen Stämme Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2. Nach Enttoxifizierung der hemicellulosischen Fraktion konnte diese als C-Quelle im Mineral Medium eingesetzt werden. Burkholderia sacchari DSM 17165 und Hydrogenophaga pseudoflava DSM 1034, sowie die hauseigenen Isolate Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 wurden für die Synthese von PHB aus der hemicellulosischen Fraktion verwendet. Die Zucker der hemicellulosischen Fraktion (Xylose, Glucose, Arabinose) konnten durch diese Organismen zur PHB-Synthese genutzt werden. Hierbei stellte sich heraus, dass die beiden Bacillus-Stämme besser zur Produktion von PHB aus dem hemicellulosischen Hydrolysat geeignet waren als die Stämme der DSMZ. Die alternative Umsetzung der im hemicellulosischem Hydrolysat enthaltenen Zucker (Xylose, Glucose und Arabinose) in die wichtigen Grundchemikalien Lactat und Acetat konnte durch die Verwendung von heterofermentativen Milchsäurebakterien verwirklicht werden. Die Bildung dieser wichtigen Grundchemikalien stellt eine interessante Alternative zur PHB-Synthese dar. Die Menge an teuren Zusätzen wie Tomatensaft, welcher für das Wachstum der MSB essentiell war, konnte reduziert werden. Die Glucose der zweiten Fraktion des Weizenstrohs, der cellulosischen Fraktion, konnte ebenfalls durch den Einsatz von Mikroorganismen in PHB umgewandelt werden. Kommerzielle Cellulasen der Firma Novozymes konnten große Mengen an Glucose (≥10 g/l) aus der cellulosischen Fraktion freisetzen. Diese freie Glucose wurde mit Hilfe von Cupriavidus necator DSM 545, Cupriavidus necator NCIMB 11599, Bacillus licheniformis KHC 3 und Bacillus megaterium KNaC 2 zu PHB fermentiert. Wie auch beim hemicellulosischen Hydrolysat konnten hier die beiden Bacillus-Stämme die besten Ergebnisse erzielen. Bei ihnen machte die PHB mehr als die Hälfte der Trockenmasse aus. Die Abtrennung des Zielprodukts ohne die Verwendung von umweltschädlichen Lösungsmitteln wurde durch die Lyse der Zielzellen durch eigens isolierte Enzyme aus Streptomyceten verwirklicht. Die Zelllyse durch die Enzyme aus Streptomyces globisporus subsp. caucasius DSM 40814 und Streptomyces albidoflavus DSM 40233 war erfolgreich und zeigte vor allem bei den Bacillen hohe Wirkung (83 % und 99 % Zelllyse). Bei dem Gram-negativen Organismus Cupriavidus necator DSM 428 konnte die anfangs niedrige Zelllyse von 38 % durch Ultraschallbehandlung auf ca. 75 % erhöht werden.

Core-crosslinked compartmentalized cylinders

We present a detailed study on the preparation of compartmentalized cylindrical nanoparticles via a templated approach: the polybutadiene part of a linear polybutadiene-block-poly(2-vinyl pyridine)-block-poly(tert-butyl methacrylate) block terpolymer, B420V280T790, having a bulk microstructure with PB cylinders covered by a P2VP double helix and embedded in a PtBMA matrix was selectively crosslinked. Subsequent sonication-assisted dissolution and chemical modifications such as quaternization (P2VP to P2VPq) and ester hydrolysis (PtBMA to poly(sodium methacrylate), PMANa) resulted in core-crosslinked cylinders soluble in organic and aqueous media. Different amounts of crosslinker and the influence of the sonication treatment on size and shape of the cylindrical aggregates were investigated. The cylinders always exhibit a compartmentalized corona. Under certain conditions, in particular quaternization of P2VP in mixtures of THF and MeOH, the helical arrangement of the P2VPq shell could be preserved even in solution, whereas in most other cases randomly distributed P2VP/P2VPq patches were observed. In aqueous solution at high pH, intramicellar interpolyelectrolyte complex (im-IPEC) formation occurred between the positively charged P2VPq shell and the negatively charged PMANa corona. We further show that different noble metal nanoparticles can be generated either selectively within the im-IPEC compartments (Pd) or randomly distributed among shell and corona of the cylinders (Au and Pt).

Diagnosis of periprosthetic joint infections in clinical practice

The diagnosis of a periprosthetic joint infection (PJI) can be challenging, either because of the variable clinical presentation or because of previous antimicrobial treatment interfering with the detection of the pathogen. In recent years, various means to diagnose PJI have been analyzed. These include invasive and non-invasive laboratory tests, imaging procedures, and novel techniques such as sonication of implants and the use of molecular microbiology. In this review, both established and novel diagnostic procedures are presented. An algorithm for detecting PJI in patients with acute and chronic symptoms is proposed.