Mesoporous silica networks with improved diffusion and interference-rejecting properties for electroanalytical sensing

Mesoporous silica materials characterized by well-ordered microstructure and size- and shape-controlled pores have attracted much attention in the last years. These systems can be used for the development of functional thin films for advanced applications in catalysis and electrocatalysis, sensors and actuators, separation techniques, micro- and nano-electronic engineering [1-2]. In this work, \u201cinsulating\u201d and mesoporous silica films were prepared by spin coating a home-made silica sol on a cleaned ITO glass support. The mesoporosity was controlled by the use of Polystyrene (PS) latex beads with different dimensions (30-60-100 nm) as template. The number of successive multi-layer depositions was varied (1-2-3-5 layers) and after the template removal, stable, homogeneous and reproducible transparent films were obtained, characterized by an interconnected porous structure. The morphological features and the physicochemical and optical properties of the films and/or sol-precursors were studied by DLS, FE-SEM, AFM, UV-vis transmittance spectroscopy and wettability analyses. Moreover, a deep electrochemical characterization was also performed by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). In particular, the use of two redox mediator probes [(K4Fe(CN)6) and (Ru(NH3)6Cl3)], presenting opposite charge and different diffusional behaviour, allowed the comprehension of the mass transport and charge transfer phenomena, evidencing the effects of spatial confinement and charge selection. In the case of \u201cinsulating\u201d films prepared without the use of PS latexes, we proved an experimental evidence for theoretical models [3] concerning electroinactive layer-modified electrodes, with a scan-rate-dependent variation of the CV shape due to a progressive increase in the diffusion coefficient inside the insulating layer. A complex balance between diverging effects (higher hydrophilicity and insulating behavior effects of silica) when increasing the numbers of layers was also observed [4]. In the case of mesoporous layers, a better electrochemical response of smaller pores and of thicker layers was found, due to two main cooperative phenomena: i) a diffusion modification from fully planar to radial-convergent at the pore-silica interface due to surface porosity; ii) the presence of pores in a hydrophilic matrix which leads to a capillary pull effects, stronger in the case of smaller hydrophilic pores. The easiness of preparation and the interesting properties of these devices pave the way towards their use in many fields, particularly trace electroanalysis in real matrices. In fact, for example, the porous and properly charged network is able to exclude interfering macromolecules (mucin in our case), preventing electrode biofouling and enhancing the performances of the sensor towards dopamine detection. References [1] M. Ogawa, Chem. Rec. 17 (2017) 217-232. [2] A. Walcarius, Chem. Soc. Rev. 42 (2013) 4098-4140. [3] D. Menshykau, R.G. Compton, Langmuir 25 (2009) 2519\u20132529. [4] V. Pifferi, L. Rimoldi, D. Meroni, F. Segrado, G. Soliveri, S. Ardizzone, L. Falciola, Electrochem. Commun. 81 (2017) 102-105

Xylan-binding xylanase Xyl30 from Streptomyces avermitilis: cloning, characterization, and overproduction in solid-state fermentation

A DNA fragment from the lignocellulolytic actinomycete Streptomyces avermitilis CECT 3339 was cloned using a DNA probe from the xylanase gene xysA of Streptomyces halstedii. The nucleotide sequence analysis revealed two potential ORFs, xyl30 and hd30, encoding a deduced multimodular F/10 xylanase with a binding domain and a secreted glycoxyl hydrolase, respectively. In Streptomyces lividans carrying the subcloned DNA fragment, two xylanase activity bands with estimated molecular masses of 42.8 and 35 kDa (named Xyl30 forms "h" and "l", respectively), were detected by zymograms and SDS-PAGE. The two xylanases had identical N-terminal sequences, suggesting that Xyl30 "l" derived from Xyl30 "h" by C-terminal processing in the culture supernatant. No transcripts of hd30 were detected by RT-PCR. Characterization of the partially purified Xyl30 "h" confirmed the presence of a modular endoxylanase containing a xylan-binding domain, which after processing in the culture supernatant loses the aforementioned domain and thus its capacity to bind xylan (Xyl30 "l"). Xyl30 "h" achieved maximal activity at pH 7.5 and 60 degrees C, retaining more than 50% of its activity from pH 3 to 9 and more than 40% after a 1-h incubation at 70 masculineC. Moreover, in the recombinant host strain up to 400 U xylanase/g medium (dry weight) was produced in solid-state fermentation (SSF) using cereal bran as substrate. The high production yields of this enzyme and its biochemical features make it a good candidate for use in industrial applications

The interplay between pore size and wettability in solid-templated silica films

In recent years, the attention to templated SiO2 nanomaterials is particularly focused to film structures, for several different applications. Beside the use of MCM-41 mesoporous silica types, solid templating agents such as polystyrene (PS) latex have been adopted in order to create a morphologically different porosity network, characterized no more by cylindrical pores, but by spherical and possibly interconnected cavities. In the present work, a silica sol was prepared and deposited on conductive glass in a mixture with a PS latex suspension. A careful adjustment of the preparation procedure was conducted and allowed stable, reproducible and electrochemically performing mesoporous thin films to be synthesized. Especially, the sol composition, the controlled ageing procedure, the right template to silica precursor ratio and the deposition procedure parameters revealed to be pivotal for the obtainment of homogeneous and transparent silica thin films. In this context, the physicochemical properties of the films as well as the SiO2 sol were studied. For example, Dynamic Light Scattering (DLS) analyses allowed the dimensions of the silica nuclei promoting the obtainment of the deposited layers to be quantified as a function of the sol ageing. Both the number of successive deposited layers and the dimension of the template (30, 60, 100 nm) were varied. In this sense, aspects pertaining the morphological features of the silica films were deeply characterized by FE-SEM and AFM analyses. Multi-layered depositions allowed an interconnected porous structure to be created, while the influence of the template diameter affected not only the morphology of the film, but above all the diffusion properties of electrochemically active species to the conductive surface of the substrate. In this regard, the electrochemical properties of the prepared devices were compared to thin films in which no templating agent was adopted, revealing large differences both by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). Electrochemical results were also interpreted in the light of water contact angle measurements. The wettability of the samples surface revealed to be a crucial parameter for the sensing properties of the films and was found to be in complete agreement with roughness data provided by AFM measurements, according to the Wenzel model. Moreover, the study of the optical properties by UV-vis transmittance spectroscopy revealed nonreflective properties with respect to the pristine substrate, thus opening the path to other possible applications, e.g. in the field of optical devices

Electrochemical characterization of insulating silica-modified electrodes : Transport properties and physicochemical features

The effect of depositing different numbers of insulating layers from a silica sol onto an ITO support was investigated to elucidate the changes occurring to diffusion and transfer mechanisms compared with bare electrodes. The electrochemical studies highlighted unexpected trends, which were discussed with respect to literature models and interpreted in the light of the physicochemical characterization (by FE-SEM, AFM, UV-vis transmittance) and particularly the hydrophilicity of the layers

Strategies for penicillin V dendronization with cationic carbosilane dendrons and study of antibacterial properties.

Strategies to synthesize a cationic carbosilane dendron containing the antibiotic penicillin V potassium salt (PenVK) at the focal point are discussed. The preparation of such a compound requires the use of systems with no donor atoms such as N or S in their framework, because their presence favours the rupture of the penicillin beta-lactam ring. The antibacterial activity of the new dendron containing ammonium groups, at the periphery, and the PenV moiety, at the focal point, against gram-positive Staphylococcus aureus strains was evaluated. These results were compared with those obtained for free PenVK, a related cationic dendron without a penicillin moiety at the focal point, and also compared with an equimolar mixture of this last dendron with free PenV. The data obtained indicate that, on one hand, the conjugation or interaction of PenV with cationic dendrons reduces its activity in comparison with free PenVK. On the other hand, the penicillin dendron is able to release the antibiotic in the presence of esterease, due to the breaking of the ester bond in this derivative.Ministerio de Economía y Empres

Self-cleaning properties in engineered sensors for dopamine electroanalytical detection

Fouling and passivation are the major drawbacks for a wide applicability of electroanalytical sensors based on nanomaterials, especially in biomedical and environmental fields. The production of highly engineered devices, designed ad hoc for specific applications, is the key factor in the direction of overcoming the problem and accessing effective sensors. Here, the fine-tuning of the system, composed of a highly ordered distribution of silver nanoparticles between a bottom silica and a top titania layer, confers multifunctional properties to the device for a biomedical complex challenge: dopamine detection. The crucial importance of each component towards a robust and efficient electroanalytical system is studied. The total recovery of the electrode performance after a simple UV-A cleaning step (self-cleaning), due to the photoactive interface and the aging resistance, is deeply investigated

Structure-activity relationship study of cationic carbosilane dendritic systems as antibacterial agents

This work focuses on the antibacterial activity against Gram-positive Staphylococcus aureus and Gramnegative Escherichia coli and the hemolytic properties of two types of ammonium cationic carbosilane systems: dendrimers and dendrons. The effects of: i) the generation, ii) the type of peripheral groups near the cationic charges (a SiMe2 moiety or a S atom depending on the synthetic procedure, hydrosilylation or thiol-ene addition, respectively), iii) the core of dendrimers (polyphenoxo vs. Si atom) and iv) the focal point of dendrons (-N3, -NH2, -OH) have been assessed. The structure-activity relationship analysis indicates the importance of an adequate balance between the hydrophilic and lipophilic fragments of these molecules to reach the best antibacterial activity. Regarding hemolysis, lowest toxicity values were registered for dendritic systems with a sulfur atom close to the surface and, in the particular case of dendrons, for those with a hydroxyl focal point. One dendrimer and one dendron, both bearing a sulfur atom close to the surface, scored best in the activity-toxicity relationship analysis and were chosen for resistance assays. No changes in the inhibitory and bactericidal capacity in the case of the dendron and only a slight increase of these values for the dendrimer were observed after 15 subculture cycles. Furthermore, these two compounds stayed active towards different strains of resistant bacteria and avoid formation of biofilm at concentrations over the minimum inhibitory concentration (MIC).Ministerio de Economía y Empres

DNA recognition and transcriptional regulation by the WhiA sporulation factor

Sporulation in the filamentous bacteria Streptomyces coelicolor is a tightly regulated process involving aerial hyphae growth, chromosome segregation, septation and spore maturation. Genetic studies have identified numerous genes that regulate sporulation, including WhiA and the sigma factor WhiG. WhiA, which has been postulated to be a transcriptional regulator, contains two regions typically associated with DNA binding: an N-terminal domain similar to LAGLIDADG homing endonucleases, and a C-terminal helix-turn-helix domain. We characterized several in vitro activities displayed by WhiA. It binds at least two sporulation-specific promoters: its own and that of parABp2. DNA binding is primarily driven by its HTH domain, but requires full-length protein for maximum affinity. WhiA transcription is stimulated by WhiG, while the WhiA protein binds directly to WhiG (leading to inhibition of WhiG-dependent transcription). These separate activities, which resemble a possible feedback loop, may help coordinate the closely timed cessation of aerial growth and subsequent spore formation

Structure of a Wbl protein and implications for NO sensing by M. tuberculosis

Mycobacterium tuberculosis causes pulmonary tuberculosis (TB) and claims ~1.8 million human lives per annum. Host nitric oxide (NO) is important in controlling TB infection. M. tuberculosis WhiB1 is a NO-responsive Wbl protein (actinobacterial iron-sulfur proteins first identified in the 1970s). Until now, the structure of a Wbl protein has not been available. Here a NMR structural model of WhiB1 reveals that Wbl proteins are four-helix bundles with a core of three α-helices held together by a [4Fe-4S] cluster. The iron-sulfur cluster is required for formation of a complex with the major sigma factor (σA) and reaction with NO disassembles this complex. The WhiB1 structure suggests that loss of the iron-sulfur cluster (by nitrosylation) permits positively charged residues in the C-terminal helix to engage in DNA binding, triggering a major reprogramming of gene expression that includes components of the virulence-critical ESX-1 secretion system

Adult-onset KMT2B-related dystonia

KMT2B-related dystonia (DYT-KMT2B, also known as DYT28) is an autosomal dominant neurological disorder characterized by varying combinations of generalized dystonia, psychomotor developmental delay, mild-to-moderate intellectual disability and short stature. Disease onset occurs typically before 10 years of age. We report the clinical and genetic findings of a series of subjects affected by adult-onset dystonia, hearing loss or intellectual disability carrying rare heterozygous KMT2B variants. Twelve cases from five unrelated families carrying four rare KMT2B missense variants predicted to impact protein function are described. Seven affected subjects presented with adult-onset focal or segmental dystonia, three developed isolated progressive hearing loss, and one displayed intellectual disability and short stature. Genome-wide DNA methylation profiling allowed to discriminate these adult-onset dystonia cases from controls and early-onset DYT-KMT2B patients. These findings document the relevance of KMT2B variants as a potential genetic determinant of adult-onset dystonia and prompt to further characterize KMT2B carriers investigating non-dystonic features