Bottom-up Layer-by-Layer Assembling of Antibacterial Freestanding Nanobiocomposite Films.

In this study, freestanding nanobiocomposite films were obtained by the sequential deposition of biopolymer-capped silver nanoparticles (AgNPs) and hyaluronic acid (HA). At first, dispersions of AgNPs decorated with chitosan (CS) or aminocellulose (AC) were synthesized by applying high intensity ultrasound. These polycationic nanoentities were layer-by-layer assembled with the HA polyanion to generate stable 3D supramolecular constructs, where the biopolymer-capped AgNPs play the dual role of active agent and structural element. SEM images of the assemblies revealed gradual increase of thickness with the number of deposited bilayers. The composites of ≥50 bilayers were safe to human cells and demonstrated 100% antibacterial activity against Staphylococcus aureus and Escherichia coli. Moreover, the films containing CSAgNPs brought about the total prevention of biofilm formation reducing the cells surface adherence by up to 6 logs. Such nanobiocomposites could serve as an effective barrier to control bacterial growth on injured skin, burns, and chronic wounds.

Bacteria-responsive multilayer coatings comprising polycationic nanospheres for bacteria biofilm prevention on urinary catheters.

This work reports on the development of infection-preventive coatings on silicone urinary catheters that contain in their structure and release on demand antibacterial polycationic nanospheres. Polycationic aminocellulose conjugate was first sonochemically processed into nanospheres to improve its antibacterial potential compared to the bulk conjugate in solution (ACSol). Afterward the processed aminocellulose nanospheres (ACNSs) were combined with the hyaluronic acid (HA) polyanion to build a layer-by-layer construct on silicone surfaces. Although the coating deposition was more effective when HA was coupled with ACSol than with ACNSs, the ACNSs-based coatings were thicker and displayed smoother surfaces due to the embedment of intact nanospheres. The antibacterial effect of ACNSs multilayers was 40% higher compared to ACSol coatings. This fact was further translated into more effective prevention of Pseudomonas aeruginosa biofilm formation. The coatings were stable in the absence of bacteria, whereas their disassembling occurred gradually during incubation with P. aeruginosa, and thus eradicate the biofilm upon release of antibacterial agents. Only 5 bilayers of HA/ACNSs were sufficient to prevent the biofilm formation, in contrast to the 10 bilayers of ACSol required to achieve the same effect. The antibiofilm efficiency of (HA/ACNSs)10 multilayer construct built on a Foley catheter was additionally validated under dynamic conditions using a model of the catheterized bladder in which the biofilm was grown during seven days. STATEMENT OF SIGNIFICANCE: Antibacterial layer-by-layer coatings were fabricated on silicone that efficiently prevents Pseudomonas aeruginosa biofilm formation during time beyond the useful lifetime of the currently employed urinary catheters in medical practice. The coatings are composed of intact, highly antibacterial polycationic nanospheres processed from aminated cellulose and bacteria-degrading glycosaminoglycan hyaluronic acid. The importance of incorporating nanoscale structures within bacteria-responsive surface coatings to impart durable antibacterial and self-defensive properties to the medical indwelling devices is highlighted.

Quorum-Quenching and Matrix-Degrading Enzymes in Multilayer Coatings Synergistically Prevent Bacterial Biofilm Formation on Urinary Catheters.

Bacteria often colonize in-dwelling medical devices and grow as complex biofilm communities of cells embedded in a self-produced extracellular polymeric matrix, which increases their resistance to antibiotics and the host immune system. During biofilm growth, bacterial cells cooperate through specific quorum-sensing (QS) signals. Taking advantage of this mechanism of biofilm formation, we hypothesized that interrupting the communication among bacteria and simultaneously degrading the extracellular matrix would inhibit biofilm growth. To this end, coatings composed of the enzymes acylase and α-amylase, able to degrade bacterial QS molecules and polysaccharides, respectively, were built on silicone urinary catheters using a layer-by-layer deposition technique. Multilayer coatings of either acylase or amylase alone suppressed the biofilm formation of corresponding Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. Further assembly of both enzymes in hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase position in the layers. Hybrid coatings, with the QS-signal-degrading acylase as outermost layer, demonstrated 30% higher antibiofilm efficiency against medically relevant Gram-negative bacteria compared to that of the other assemblies. These nanocoatings significantly reduced the occurrence of single-species (P. aeruginosa) and mixed-species (P. aeruginosa and Escherichia coli) biofilms on silicone catheters under both static and dynamic conditions. Moreover, in an in vivo animal model, the quorum quenching and matrix degrading enzyme assemblies delayed the biofilm growth up to 7 days.

Enzymatic functionalization of cork surface with antimicrobial hybrid biopolymer/silver nanoparticles.

Laccase-assisted assembling of hybrid biopolymer-silver nanoparticles and cork matrices into an antimicrobial material with potential for water remediation is herein described. Amino-functional biopolymers were first used as doping agents to stabilize concentrated colloidal dispersions of silver nanoparticles (AgNP), additionally providing the particles with functionalities for covalent immobilization onto cork to impart a durable antibacterial effect. The solvent-free AgNP synthesis by chemical reduction was carried out in the presence of chitosan (CS) or 6-deoxy-6-(ω-aminoethyl) aminocellulose (AC), leading to simultaneous AgNP biofunctionalization. This approach resulted in concentrated hybrid NP dispersion stable to aggregation and with hydrodynamic radius of particles of about 250 nm. Moreover, laccase enabled coupling between the phenolic groups in cork and amino moieties in the biopolymer-doped AgNP for permanent modification of the material. The antibacterial efficiency of the functionalized cork matrices, aimed as adsorbents for wastewater treatment, was evaluated against Escherichia coli and Staphylococcus aureus during 5 days in conditions mimicking those in constructed wetlands. Both intrinsically antimicrobial CS and AC contributed to the bactericidal effect of the enzymatically grafted on cork AgNP. In contrast, unmodified AgNP were easily washed off from the material, confirming that the biopolymers potentiated a durable antibacterial functionalization of the cork matrices.

Enzyme multilayer coatings inhibit Pseudomonas aeruginosa biofilm formation on urinary catheters.

Bacteria use a signaling mechanism called quorum sensing (QS) to form complex communities of surface-attached cells known as biofilms. This protective mode of growth allows them to resist antibiotic treatment and originates the majority of hospital-acquired infections. Emerging alternatives to control biofilm-associated infections and multidrug resistance development interfere with bacterial QS pathways, exerting less selective pressure on bacterial population. In this study, biologically stable coatings comprising the QS disrupting enzyme acylase were built on silicone urinary catheters using a layer-by-layer technique. This was achieved by the alternate deposition of negatively charged enzyme and positively charged polyethylenimine. The acylase-coated catheters efficiently quenched the QS in the biosensor strain Chromobacterium violaceum CECT 5999, demonstrated by approximately 50% inhibition of violacein production. These enzyme multilayer coatings significantly reduced the Pseudomonas aeruginosa ATCC 10145 biofilm formation under static and dynamic conditions in an in vitro catheterized bladder model. The quorum quenching enzyme coatings did not affect the viability of the human fibroblasts (BJ-5ta) over 7 days, corresponding to the extended useful life of urinary catheters. Such enzyme-based approach could be an alternative to the conventional antibiotic treatment for prevention of biofilm-associated urinary tract infections.

Building an antifouling zwitterionic coating on urinary catheters using an enzymatically triggered bottom-up approach.

Catheter associated urinary tract infections are common during hospitalization due to the formation of bacterial biofilms on the indwelling device. In this study, we report an innovative biotechnology-based approach for the covalent functionalization of silicone catheters with antifouling zwitterionic moieties to prevent biofilm formation. Our approach combines the potential bioactivity of a natural phenolics layer biocatalytically conjugated to sulfobetaine-acrylic residues in an enzymatically initiated surface radical polymerization with laccase. To ensure sufficient coating stability in urine, the silicone catheter is plasma-activated. In contrast to industrial chemical methods, the methacrylate-containing zwitterionic monomers are polymerized at pH 5 and 50 °C using as an initiator the phenoxy radicals solely generated by laccase on the phenolics-coated catheter surface. The coated catheters are characterized by X-ray photoelectron spectroscopy (XPS), Fourier transformed infrared (FTIR) analysis, atomic force microscopy (AFM), and colorimetrically. Contact angle and protein adsorption measurements, coupled with in vitro tests with the Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus in static and dynamic conditions, mimicking the operational conditions to be faced by the catheters, demonstrate reduced biofilm formation by about 80% when compared to that of unmodified urinary catheters. The zwitterionic coating did not affect the viability of the human fibroblasts (BJ-5ta) over seven days, corresponding to the extended useful life of urinary catheters.

Self-assembly of collagen building blocks guided by electric fields.

Show me the way: protein building blocks are programmed to assemble hierarchically and yield a defined fiber morphology of micrometric length and precise nanometric diameter. The key step of this method is to align the building blocks with an AC field prior to assembly. The resulting protein nanofibers are straightforwardly integrated with the circuitry for potential applications in bionanotechnology.

Sonochemical coating of textiles with hybrid ZnO/chitosan antimicrobial nanoparticles.

Textiles are good substrates for growth of microorganisms especially under moisture and temperature conditions found in hospitals. Microbial shedding from the body occurs continuously at contact of the patient with textile materials used in medical practices, contributing to the occurrence of hospital acquired infections. Thus, the use of efficient antimicrobial textiles is necessary to prevent the transfer of pathogens and the infection incidence. In this work, hybrid antimicrobial coatings were generated on cotton fabrics by means of a one-step simultaneous sonochemical deposition of ZnO nanoparticles (NPs) and chitosan. The process was further optimized in terms of reagents concentration and processing time in order to improve the antibacterial properties of the fabric and ensure their biocompatibility. The highest antibacterial activity of the fabrics against two medically relevant bacterial species was achieved in a 30 min sonochemical coating process using 2 mM ZnO NPs suspension. When chitosan was simultaneously deposited with the same amount of ZnO, the obtained hybrid NPs coating displayed higher by 48 and 17% antibacterial activity against Staphylococcus aureus and Escherichia coli, respectively. The presence of biopolymer also improved the durability of the antimicrobial effect of the coatings by 21% for Staphylococcus aureus and 40% for Escherichia coli, evaluated after applying multiple washing cycles at hospital laundering regimes. Finally, 87% biocompatibility improvement supported by fibroblast viability was observed for the hybrid ZnO/chitosan coating compared to the steady decrease of cells viability over one week in contact with the fabrics coated with ZnO alone.

Exceptional oxidation activity with size-controlled supported gold clusters of low atomicity.

The catalytic activity of gold depends on particle size, with the reactivity increasing as the particle diameter decreases. However, investigations into behaviour in the subnanometre regime (where gold exists as small clusters of a few atoms) began only recently with advances in synthesis and characterization techniques. Here we report an easy method to prepare isolated gold atoms supported on functionalized carbon nanotubes and their performance in the oxidation of thiophenol with O2. We show that single gold atoms are not active, but they aggregate under reaction conditions into gold clusters of low atomicity that exhibit a catalytic activity comparable to that of sulfhydryl oxidase enzymes. When clusters grow into larger nanoparticles, catalyst activity drops to zero. Theoretical calculations show that gold clusters are able to activate thiophenol and O2 simultaneously, and larger nanoparticles are passivated by strongly adsorbed thiolates. The combination of both reactants activation and facile product desorption makes gold clusters excellent catalysts.

Improving immunosensor performance through oriented immobilization of antibodies on carbon nanotube composite surfaces.

We report the straightforward oriented covalent attachment of antibodies (Abs) on the surface of carboxylated multiwalled carbon nanotube-polystyrene (MWCNT-PS) materials. The combination of this composite material, applied as a robust electrochemical transducer platform, and its covalent functionalization with Abs in a controlled way by means of a two-step process, could contribute to the development of highly sensitive immunosensor devices. Using the simple and versatile carbodiimide chemistry, Abs were attached to the carboxylic groups of the MWCNT-PS composite surfaces via their superficial amine groups. By taking into account the Ab isoelectric point and the net charge of the composite surface, we engineered an immobilization process to achieve the oriented binding of the Ab molecules by favoring an ionic pre-adsorption step before covalent binding occurred. Thus, the antigen binding capacity of the attached Abs was enhanced by up to 10 times with respect to the capacity estimated for a random spatial distribution of these molecules. The proposed strategy would also serve as a model for the efficient biofunctionalization of other carboxylated carbon-based polymer composite materials with potential applications in the biosensor field.

Carbon nanotube composite peptide-based biosensors as putative diagnostic tools for rheumatoid arthritis.

This work reports on the fabrication and performance of a simple amperometric immunosensor device to be potentially used for the detection of serum anti-citrullinated peptide antibodies (ACPAs), which are specific for rheumatoid arthritis (RA) autoimmune disease. Sera of RA patients contain antibodies to different citrullinated peptides and proteins such as fibrin or filaggrin. Herein, a chimeric fibrin-filaggrin synthetic peptide (CFFCP1) was used as a recognition element anchored to the surface of a multiwalled carbon nanotube-polystyrene (MWCNT-PS) based electrochemical transducer. The transducer fabrication process is described in detail together with its successful electrochemical performance in terms of repeatability and reproducibility of the corresponding amperometric response. The resulting immunosensor approach was initially tested in sera of rabbits previously inoculated with the synthetic peptide and eventually applied to the detection of ACPAs in human sera. A comparative study was carried out using control serum from a blood donor, which demonstrated the selectivity of the immunosensor response and its sensitivity for the detection of anti-CFFCP1 antibodies present in RA patients.

Synthesis and stabilization of subnanometric gold oxide nanoparticles on multiwalled carbon nanotubes and their catalytic activity.

Small gold nanoclusters in a very narrow size distribution (1.1 ± 0.5 nm) have been stabilized onto multiwalled carbon nanotubes (MWCNT). Theoretical studies supported by XPS and (16)O(2)/(18)O(2) isotopic exchange experiments have shown that, on small gold nanoparticles (0.9-1.5 nm), dissociation of molecular O(2) and formation of a surface oxide-like layer is energetically favorable and occurs at room temperature, while O(2) recombination and desorption involves a larger activation barrier. CO titration experiments and theoretical studies demonstrate that the reactivity of the oxidized particles toward CO does not only depend on particle size but also on oxygen coverage. The oxidation-reduction process described is reversible, and the oxidized nanoparticles are active in the epoxidation of styrene with air.

Plasma-activated multi-walled carbon nanotube-polystyrene composite substrates for biosensing.

Carbon nanotube-polymer composites have shown to be suitable materials for the fabrication of electrochemical transducers. The exposed surface of these materials is commonly passivated by a very thin layer of the polymer component that buries the conductive carbon particles. Working with multi-walled carbon nanotube-polystyrene (MWCNT-PS) composite structures, it was previously described how a simple low power oxygen plasma process produced an effective etching of the composite surface, thereby exposing the conductive surface of CNTs. This work shows how this plasma process not only gave rise to a suitable composite conductive surface for electrochemical sensing but simultaneously exposed and created a high density of oxygen-containing functional groups at both the CNT and the PS components, without affecting the material's mechanical stability. These chemical groups could be effectively modified for the stable immobilization of biological receptors. A detailed chemical characterization of the plasma-activated composite surface was possible using x-ray photoelectron spectroscopy. The material reactivity towards the tethering of a protein was studied and protein-protein interactions were then evaluated on the modified composite transducers by scanning electron microscopy. Finally, an amperometric immunosensor approach for the detection of rabbit Immunoglobulin G target analyte was described and a minimum concentration of 3 ng ml(-1) was easily measured.

Neutron activation of engineered nanoparticles as a tool for tracing their environmental fate and uptake in organisms.

Studies regarding the environmental impact of engineered nanoparticles (ENPs) are hampered by the lack of tools to localize and quantify ENPs in water, sediments, soils, and organisms. Neutron activation of mineral ENPs offers the possibility of labeling ENPs in a way that avoids surface modification and permits both localization and quantification within a matrix or an organism. Time-course experiments in vivo also may be conducted with small organisms to study metabolism and exposure, two aspects currently lacking in ecotoxicological knowledge about ENPs. The present report explains some of the prerequisites and advantages of neutron activation as a tool for studying ENPs in environmental samples and ecologically relevant organisms, and it demonstrates the suitability of neutron activation for Ag, Co/Co3O4, and CeO2 nanoparticles. In a preliminary experiment with the earthworm Eisenia fetida, the dietary uptake and excretion of a Co nanopowder (average particle size, 4 nm; surface area, 59 m2/g) were studied. Cobalt ENPs were taken up to a high extent during 7 d of exposure (concentration ratios of 0.16-0.20 relative to the ENP concentration in horse manure) and were largely retained within the worms for a period of eight weeks, with less than 20% of absorbed ENPs being excreted. Following dissection of the worms, 60Co was detected in spermatogenic cells, cocoons, and blood using scintillation counting and autoradiography. The experimental opportunities that neutron activation of ENPs offer are discussed.

Steam purification for the removal of graphitic shells coating catalytic particles and the shortening of single-walled carbon nanotubes.

Purification and shortening of single-walled carbon nanotubes (SWNTs) is carried out by treatment with steam. During the steam purification the graphitic shells coating the catalytic metal particles are removed. Consequently, the exposed catalytic particles can be easily dissolved by treatment with hydrochloric acid. No damage to the carbon nanotube tubular structure is observed, even after prolonged treatment with steam. Samples are characterized by HRTEM, TGA, magnetic measurements, Raman spectroscopy, AFM, and XPS.

Composite planar electrode for sensing electrochemical oxygen demand.

This work reports on the development of a graphite-polystyrene composite electrode of planar configuration, containing silver(II) oxide and copper(II) oxide catalysts (AgO-CuO), for the measurement of electrochemical oxygen demand (EOD). Optimisation studies of the composite composition as well as conditions for its processing on planar substrates and generation of an appropriate electrochemical active area resulted in the scalable fabrication of robust composite electrodes. These were evaluated with glucose as target analyte. They showed competitive low limits of detection in a linear concentration range from 5 mgL(-1) to 1400 mgL(-1) of O(2). Besides, they were stable for at least one year. The determination of EOD in wastewater samples coming from production lines of parenteral food and winemaking was successfully carried out.

Scalable fabrication of immunosensors based on carbon nanotube polymer composites.

In this work we present the fabrication and characterization of immunosensors based on polystyrene (PS)-multiwalled carbon nanotube (MWCNT) composites. The electrochemical properties of the sensors have been investigated and show that the surface area is increased upon addition of the MWCNT-PS layer. Furthermore, a plasma activation process is used to partially remove the PS and expose the MWCNTs. This results in a huge increase in the electrochemical area and opens up the possibility of binding biomolecules to the MWCNT wall. The MWCNTs have been functionalized covalently with a model antibody (rabbit IgG). The biosensors have been tested using amperometric techniques and show detection limits comparable to standard techniques such as ELISA.