Javier Sotres

Biofuel cell as a power source for electronic contact lenses.

Here we present unequivocal experimental proof that microscale cofactor- and membrane-less, direct electron transfer based enzymatic fuel cells do produce significant amounts of electrical energy in human lachrymal liquid (tears). 100 μm diameter gold wires, covered with 17 nm gold nanoparticles, were used to fashion three-dimensional nanostructured microelectrodes, which were biomodified with Corynascus thermophilus cellobiose dehydrogenase and Myrothecium verrucaria bilirubin oxidase as anodic and cathodic bioelements, respectively. The following characteristics of miniature glucose/oxygen biodevices operating in human tears were registered: 0.57 V open-circuit voltage, about 1 μW cm(-2) maximum power density at a cell voltage of 0.5 V, and more than 20 h operational half-life. Theoretical calculations regarding the maximum recoverable electrical energy can be extracted from the biofuel and the biooxidant, glucose and molecular oxygen, each readily available in human lachrymal liquid, fully support our belief that biofuel cells can be used as electrical power sources for so called smart contact lenses.

A soluble and highly functional polyaniline–carbon nanotube composite

A completely soluble polyaniline-multi-wall carbon nanotube (CNT–PANI) composite with drastically enhanced conductivity, improved thermal stability, and luminescent behaviour, has been synthesized. The presence of straight multi-wall carbon nanotubes during the polymerization of aniline induces the formation of a more planar conformation of polyaniline which acts as coating layer for the carbon nanotubes and leads to favourable interaction between the constituents. The polyaniline-coated multi-wall carbon nanotubes align into bundles and form a three-dimensional network in the overall composite. A highly functional carbon nanotube composite completely soluble in n-methylpyrrolidinone (NMP) exhibiting all the favourable processing and transformation possibilities of PANI has been obtained. These findings have important consequences for practical technological applications, especially for the development of opto-electronic devices.

Biofuel Cell Based on Microscale Nanostructured Electrodes with Inductive Coupling to Rat Brain Neurons

Miniature, self-contained biodevices powered by biofuel cells may enable a new generation of implantable, wireless, minimally invasive neural interfaces for neurophysiological in vivo studies and for clinical applications. Here we report on the fabrication of a direct electron transfer based glucose/oxygen enzymatic fuel cell (EFC) from genuinely three-dimensional (3D) nanostructured microscale gold electrodes, modified with suitable biocatalysts. We show that the process underlying the simple fabrication method of 3D nanostructured electrodes is based on an electrochemically driven transformation of physically deposited gold nanoparticles. We experimentally demonstrate that mediator-, cofactor-, and membrane-less EFCs do operate in cerebrospinal fluid and in the brain of a rat, producing amounts of electrical power sufficient to drive a self-contained biodevice, viz. 7 μW cm−2 in vitro and 2 μW cm−2 in vivo at an operating voltage of 0.4 V. Last but not least, we also demonstrate an inductive coupling between 3D nanobioelectrodes and living neurons.

The biological response to three different nanostructures applied on smooth implant surfaces

OBJECTIVE To evaluate the biological effects of three calcium phosphate (CaP) coatings with nanostructures on relatively smooth implant surfaces. MATERIAL AND METHODS Stable CaP nanoparticle suspensions of different particle sizes and structures were coated onto implants by immersion and subsequent heat treatment. An uncoated implant was used as the control. After topographical and chemical characterizations, implants were randomly inserted into rabbit tibiae for removal torque (RTQ) testing. To confirm the biological reaction, implants were placed in the bilateral femurs of three rabbits. RESULTS The topographical characterization showed that each surface had different nanostructural characteristics and X-ray photon spectroscopy showed various CaP compositions. The control and test groups had different nanotopographies; however, the differences among the test groups were only significant for Surfaces B and C and the rest were insignificant. The RTQ tests showed significantly higher values in two test groups (Surface A and Surface C). Histologically, no adverse effects were seen in any group. Histomorphometrical evaluation showed comparable or better osseointegration along the implant threads in the test groups. CONCLUSION The three different CaP coatings with nanostructures on the implant surfaces had enhancing effects on osseointegration. Along with the surface nanotopography, the CaP chemistry might have influenced the biological outcomes.

Interfacial Behavior and Activity of Laccase and Bilirubin Oxidase on Bare Gold Surfaces

Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL⁻¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm⁻², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O₂) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s⁻¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s⁻¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes.

AFM Imaging and Analysis of Electrostatic Double Layer Forces on Single DNA Molecules

Electrical double layer (EDL) forces develop between charged surfaces immersed in an electrolyte solution. Biological material surrounded by its physiological medium constitutes a case where these forces play a major role. Specifically, this work is focused on the study of the EDL force exerted by DNA molecules, a standard reference for the study of single biomolecules of nanometer size. The molecules deposited on plane substrates have been characterized by means of the atomic force microscope operated in the force spectroscopy imaging mode. Force spectroscopy imaging provides images of the topography of the DNA molecules, and of the EDL force spectrum. Due to the size of the molecule being much smaller than that of the tip, both the tip-substrate and tip-molecule interactions need to be considered in the analysis of the experimental results. We solve this problem by linearly superposing the two contributions. EDL force images are presented where DNA molecules are clearly resolved. The lateral resolution of the EDL force is discussed and compared with that of the topography. The method also allows the estimation of the DNA surface charge density, thereby obtaining reasonable values.

In vitro characterization and osteoblast responses to nanostructured photocatalytic TiO2 coated surfaces

The aims of the study were to characterize a nanostructured photoactive titanium dioxide (TiO(2)) coating and to compare the cellular response of human osteoblasts before and after ultraviolet (UV) irradiation of the coating. A specific nanostructured TiO(2) powder (Degussa P-25), which consists of approximately 80% anatase and 20% rutile, was spin-coated onto commercially pure titanium discs, and was heat-treated thereafter. After topographical, chemical and photocatalytic property characterizations, human osteoblasts were cultured on the coated discs before and after UV irradiation. Cell morphology was evaluated by scanning electron microscopy (SEM), and cell viability was analysed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. From the contact angle analysis, the wettability significantly improved after UV irradiation. The cultured cells were flattened with numerous elongated lammellipodia; however, no morphological differences were indicated between -UV and +UV surfaces. The MTT assay analysis showed that -UV surface presented significantly higher viability compared to the +UV surface except for one cell population group at 3h where there were no differences. The nanostructured photoactive TiO(2) surface improved its hydrophilicity by UV irradiation, however no enhancing effect in cell response was confirmed at the time tested compared to the non-irradiated surface.

Tau aggregation followed by atomic force microscopy and surface plasmon resonance, and single molecule tau-tau interaction probed by atomic force spectroscopy.

Intracellular neurofibrillary tangles, composed mainly of tau protein, and extracellular plaques, containing mostly amyloid-beta, are the two types of protein aggregates found upon autopsy within the brain of Alzheimers disease patients. Polymers of tau protein can also be found in other neurodegenerative disorders known as tauopathies. Tau is a highly soluble protein, intrinsically devoid of secondary or tertiary structure, as many others proteins particularly prone to form fibrillar aggregations. The mechanism by which this unfolded molecule evolves to the well ordered helical filaments has been amply studied. In fact, it is a very slow process when followed in the absence of aggregation inducers. Herein we describe the use of surface plasmon resonance, atomic force microscopy, and atomic force spectroscopy to detect tau-tau interactions and to follow the process of aggregation in the absence of aggregation inducers. Tau-tau interactions are clearly detected, although a very long period of time is needed to observe filaments formation. Tau oligomers showing a granular appearance, however, are observed immediately as a consequence of this interaction. These granular tau oligomers slowly evolve to larger structures and eventually to filaments having a size smaller than those reported for paired helical filaments purified from Alzheimers disease.

Influence of pH on the build-up of poly-L-lysine/heparin multilayers

The effect of pH on the build-up of polyelectrolyte multilayers, PEMs, composed by poly-L-lysine and heparin onto two different substrates, silica and gold, has been studied by means of ellipsometry and quartz crystal microbalance with dissipation, QCM-D. Ellipsometry results indicate that the dry mass grows exponentially with the number of layers, and that this amount is larger as the pH values are raised. From QCM-D data the viscoelastic properties of the multilayered structure have been obtained. These data reflect that PEMs become more viscoelastic as the pH values are increased for silica substrates, while for gold the highest viscoelastic behavior is obtained at neutral pH and the elastic behavior becomes dominant as the pH is further increased or decreased. By combining these two surface techniques it has been also possible to determine the solvent content in the multilayers and reach a deeper understanding of the internal structure.

The influence of nanoparticles on enzymatic bioelectrocatalysis

In nearly all papers concerning enzyme–nanoparticle based bioelectronic devices, it is stated that the presence of nanoparticles on electrode surfaces per se enhances bioelectrocatalysis, although the reasons for that enhancement are often unclear. Here, we report detailed experimental evidence that neither an overpotential of bioelectrocatalysis, nor direct electron transfer and bioelectrocatalytic reaction rates for an adsorbed enzyme depend on the size of nanoparticles within the range of 20–80 nm, i.e. for nanoparticles that are considerably larger than the enzyme molecules.