Ionic selectivity of nystatin A1 confined in nanoporous track-etched polymer membrane.

The hybrid biological/polymeric solid-state nanopore membrane offers several opportunities to combine the advantage of biological channel (selectivity) and material (robustness). Based on this technology, the challenge is to obtain selective ionic exchange membranes, with no energy intake. The direct insertion of an ionic channel inside a nanopore should be a promise solution. Here, the authors report a hybrid nanopore based on nystatin A1 confinement in commercial nanopore membrane. Ionic transport and selectivity studies show that the hybrid nanopores exhibit mainly an anionic behaviour, on the contrary to biological conditions. However, the order of magnitude between the different ratios of permeation of several cationic species is retained even if the blocking of divalent cation is not totally proved.

Slow translocation of polynucleotides and their discrimination by α-hemolysin inside a single track-etched nanopore designed by atomic layer deposition.

We report the formation of a hybrid biological/artificial nanopore by the direct insertion of non-modified α-hemolysin at the entrance of a high aspect ratio (length/diameter) biomimetic nanopore. In this robust hybrid system, the protein exhibits the same polynucleotide discrimination properties as in the biological membrane and the polynucleotide dwell time is strongly increased. This nanopore is very promising for DNA sequencing applications where the high DNA translocation velocity and the fragility of the support are the main bottlenecks.

Enhanced potassium selectivity in a bioinspired solid nanopore.

Biological ion channels present unique ionic properties. They can be highly permeable to ions, while selecting only one type of ions, without external energy supply. An important research field has been developed to transfer these properties to solid state nanoporous membranes in order to develop artificial biomimetic nanofilters. One of the promising ways to develop biomimetic structures is based on the direct insertion of the gramicidin A, i.e. an ionic channel, inside a nanopore. Experiments have recently proved the feasibility of such a hybrid membrane with very interesting results regarding the ionic selectivity. Here, we propose to interpret these experiments using theoretical molecular dynamic simulations which allow us to analyze more profoundly the structures of the proteins confined inside the nanopore and the relation between their conformation and the observed ionic properties.

Avidin adsorption on silicone elastomer: effects of stretching and polyelectrolyte coatings.

Zeta potential and avidin adsorption of silicone elastomer (SE) and SE coated with polyethyleneimine (PEI) and heparin is studied under controlled hydrodynamic conditions. Zeta potential (ζ) of SE depends on the duration of contact with buffer (0.02M MOPS-KCl, pH 7.2): ζ descends from 12mV to a plateau value of -32mV during 5h and decreases further if SE is stretched (30% elongation). The time variation of ζ is attributed to surface charging of the core polymer PDMS. Coating the negatively charged SE with PEI results in ζ=42mV; a layer of heparin on top of PEI yields ζ=-46mV. On the negatively charged surfaces, avidin adsorption levels off at 5mV. Desorption of avidin from SE is negligible even when SE with adsorbed avidin is stretched, or when the stretching force is removed after adsorption of avidin. The results can be employed for designing biomaterials with properties such as surface charge and protein adsorption adjustable by stretching; the strong attachment of avidin can allow for further surface modification.

Controlling potassium selectivity and proton blocking in a hybrid biological/solid-state polymer nanoporous membrane.

Specific separations of protons and cations are usually performed by electromembrane processes, which require external electric energy. An easier process would be using a membrane able to separate both entities by passive diffusion. Presently, such synthetic nanoporous membranes do not exist. Here, we report the production of a robust hybrid biological/artificial solid-state membrane, which allows selective permeation of alkali metal cations without competing or concurrent permeation of protons. This membrane is simple to prepare and is based on the hydrophobic nature of the polymeric pore walls, and the confined gramicidin A molecules within. This work opens a new route for separation in the domain of nanobiofiltration, especially for tunable nanodevices based on differential ion conduction, with a fundamental understanding of the confinement mechanism.

Thin phosphatidylcholine films as background surfaces with further possibilities of functionalization for biomedical applications.

Non-specific adsorption is a crucial problem in the biomedical field. To produce surfaces avoiding this phenomenon, we functionalized thin (7-180 nm) poly(methylhydrosiloxane) (PMHS) network films at room temperature (≈20°C) with phospholipids (PL) bearing a phosphorylcholine head. Regardless of their mode of preparation (casting or immersion), all surfaces appeared to be very hydrophilic with a captive air-bubble contact angle stabilized around 40°. The thin films were protein-repellent in phosphate saline buffer pH 7.4 according to analysis by normal scanning confocal fluorescence. Neither was any adsorption or spreading of l-α-phosphatidylcholine liposomes on such films observed. In addition, amino functional groups could be easily attached to the surface remaining available for further functionalization.

Intake of specific nutrients and foods and hearing level measured 13 years later.

Only a few studies have investigated the impact of nutrients and food groups on hearing level (HL) with a population-based approach. We examined the 13-year association between intake of specific nutrients and food groups and HL in a sample of French adults. A total of 1823 subjects, aged 45-60 years at baseline, participating in the Supplementation with Antioxidant Vitamins and Minerals 2 cohort were selected. Nutrient and food intake was estimated at baseline among participants who had completed at least six 24 h dietary records. HL was assessed 13 years after baseline and was defined as the pure-tone air conduction of the worse ear at the following thresholds: 0·5, 1, 2 and 4 kHz. The relationship between quartiles of energy-adjusted nutrient and food intake and HL was assessed by multivariate linear regression analyses, in men and women separately. Intakes of retinol (P-trend » 0·058) and vitamin B12 (P-trend=0·068) tended to be associated with better HL in women. Intakes of meat as a whole (P-trend=0·030), red meat (P-trend=0·014) and organ meat (P-trend=0·017) were associated with better HL in women. Higher intake of seafood as a whole (P-trend=0·07) and of shellfish (P-trend=0·097) tended to be associated with better HL in men. Consumption of meat is therefore associated with a better HL in women. Further research is required to better elucidate the mechanisms behind the associations between diet and hearing.

Nanopore sensors: from hybrid to abiotic systems.

The use of nanopores of well controlled geometry for sensing molecules in solution is reviewed. Focus is concentrated especially on synthetic track-etch pores in polymer foils and on biological nanopores, i.e. ion channels. After a brief section about multipore sensors, specific attention is provided to works relative to a single nanopore sensor. The different strategies to combine the robustness of supports with the high selectivity of the biological channels are reviewed. The scope ranges from keeping the membrane natural environment of biological channels in supported and suspended bilayer membranes, to considering completely abiotic designed nanopores created through synthetic materials. The α-hemolysine channel and the mechanosensitive channel of large conductance with their modifications are especially considered in the first strategy, the conical functionalized nanopores created in polymer foils in the second one. The different attempts of reading macromolecules are also discussed. A third hybrid strategy, which was not extensively explored, consists in the inclusion of a biological structure into a well-designed nanopore through the support, as recently with gramicidin.

Interface of covalently bonded phospholipids with a phosphorylcholine head: characterization, protein nonadsorption, and further functionalization.

Surface anchored poly(methylhydrosiloxane) (PMHS) thin films on oxidized silicon wafers or glass substrates were functionalized via the SiH hydrosilylation reaction with the internal double bonds of 1,2-dilinoleoyl-sn-glycero-3-phosphorylcholine (18:2 Cis). The surface was characterized by X-ray photoelectron spectroscopy, contact angle measurements, atomic force microscopy, and scanning electron microscopy. These studies showed that the PMHS top layer could be efficiently modified resulting in an interfacial high density of phospholipids. Grafted phospholipids made the initially hydrophobic surface (θ = 106°) very hydrophilic and repellent toward avidin, bovine serum albumin, bovine fibrinogen, lysozyme, and α-chymotrypsin adsorption in phosphate saline buffer pH 7.4. The surface may constitute a new background-stable support with increased biocompatibility. Further possibilities of functionalization on the surface remain available owing to the formation of interfacial SiOH groups by Karstedt-catalyzed side reactions of SiH groups with water. The presence of interfacial SiOH groups was shown by zeta potential measurements. The reactivity and surface density of SiOH groups were checked by fluorescence after reaction of a monoethoxy silane coupling agent bearing Alexa as fluorescent probe.

Adsorption of alexa-labeled Bt toxin on mica, glass, and hydrophobized glass: study by normal scanning confocal fluorescence.

We studied the kinetics of adsorption of alexa-labeled Bt toxin Cry1Aa, in monomer and oligomer states, on muscovite mica, acid-treated hydrophilic glass, and hydrophobized glass, in the configuration of laminar flow of solution in a slit. Normal confocal fluorescence through the liquid volume allows the visualization of the concentration in solution over the time of adsorption, in addition to the signal due to the adsorbed molecules at the interface. The solution signal is used as calibration for estimation of interfacial concentration. We found low adsorption of the monomer compared to oligomers on the three types of surface. The kinetic adsorption barrier for oligomers increases in the order hydrophobized glass, muscovite mica, acid-treated hydrophilic glass. This suggests enhanced immobilization in soil if toxin is under oligomer state.

Adsorption rate dependence on convection over a large length of a sensor to get adsorption constant and solute diffusion coefficient.

We propose a representation of initial adsorption kinetic constant as a function of convection in a slit flow cell device, averaged over some restricted length of a wall acting as a sensor. The complete domain from transport-control to surface reaction control is included. The intercepts with axes give access to adsorption constant and solute diffusion coefficient. It is shown that, provided the close entrance is avoided, the function for the restricted length is very close to the function for local values.

Fate of prions in soil: adsorption kinetics of recombinant unglycosylated ovine prion protein onto mica in laminar flow conditions and subsequent desorption.

Prions can be disseminated in soils. Their interaction with soil minerals is a key factor for the assessment of risks associated with the transport of their infectivity. We did not examine here the infectivity itself but the adsorption kinetics of an ovine recombinant prion protein (ovine PrPrec), as a noninfectious model protein, on muscovite mica, a phyllosilicate with surface properties analogous to soil clays, in conditions of laminar flow through a channel. The protein was labeled with (125)I, and its adsorption examined between pH 4.0 and 9.0. At wall shear rate 100 s(-1), we found the process to be controlled mainly by transport at the beginning of the adsorption process. Additional experiments at 1000 s(-1) (pH 5 and 6) determined that the diffusion coefficient was in accordance with the hydrodynamic radius measured by size exclusion chromatography. The pseudo-plateau of the interfacial concentration with time was compatible with more than a monolayer and suggests the presence of aggregates. Desorption was not observed in the presence of buffer between pH 4 and 9 and sheep plasma, while the addition of alkaline detergent or 10(-1) M NaOH allowed an almost complete removal from the interface. The ensemble of results suggests that the largely irreversible adsorption of the ovine PrPrec onto mica is mainly due to electrostatic attraction between the protein and the highly negatively charged mica surface. Possible consequences for the mode of dissemination of prion proteins in soils are indicated.

Streaming potential in cylindrical pores of poly(ethylene terephthalate) track-etched membranes: variation of apparent zeta potential with pore radius.

Streaming potential variation with pressure measured through poly(ethylene terephthalate) track-etched membranes of different pore sizes led to the determination of an apparent interfacial potential zetaa in the presence of 10-2 M KCl. The variation of zetaa with the pore radius r0 is interpreted by (i) the electric double layer overlap effect and (ii) the presence of a conductive gel layer. We propose a method which integrates both effects by assuming a simple model for the conductive gel at the pore wall. We observed the following three domains of pore size: (i) r0 > 70 nm, where surface effects are negligible; (ii) approximately 17 nm < r0 < 70 nm, where the pore/solution interface could be described as a conductive gel of thickness around 1 nm; (iii) r0 < approximately 17 nm, which corresponds to the region strongly damaged by the ion beam and is not analyzed here. The first one (zeta = -36.2 mV) corresponds to the raw material when etching has completely removed the ion beam predamaged region, which corresponds to the second intermediate domain (zeta = -47.3 mV). There the conductance of the gel layer deduced from the treatment of streaming potential data was found to be compatible with the number of ionic sites independently determined by the electron spin resonance technique.

Adsorption of alpha-chymotrypsin onto mica in laminar flow conditions. Adsorption kinetic constant as a function of tris buffer concentration at pH 8.6.

We examined the adsorption kinetics of alpha-chymotrypsin (pH 8.6, 10(-2) to 0.5 M Tris buffer) on muscovite mica in conditions of laminar flow through a slit. The range of buffer concentrations is between two limits: (i) no adsorption in 1 M Tris and (ii) no desorption in 10(-3) M Tris. Studying the dependence of adsorption kinetics on the wall shear rate leads to the determination of the interfacial adsorption kinetic constant ka and the diffusion coefficient. The obtained value for the diffusion coefficient is close to the one expected from the molecular size of alpha-chymotrypsin. The interfacial adsorption kinetic constant of alpha-chymotrypsin decreases when ionic strength increases, while the initial desorption constant (over a part of all the adsorbed population) shows the contrary. Although alpha-chymotrypsin is almost at its isoelectric point, the effect of ionic strength on the adsorption kinetics suggests the importance of electrostatic interactions between the protein and mica. We observed an increase in the adsorption rate, at a surface coverage near 0.14 microg cm(-2), for adsorption in 10(-2) M Tris and the low wall shear rates (<300 s(-1)). This change in the adsorption rate suggests a structural transition, that we assume again to be due to electrostatic interactions, but between proteins. The large dipole moment of the protein may induce this transition, illustrated here by the ferroelectric/antiferroelectric pattern. The variation of the zeta potential with interfacial concentration seems to be in agreement with such a model.

Kinetics of adsorption, desorption, and exchange of alpha-chymotrypsin and lysozyme on poly(ethyleneterephthalate) tracked film and track-etched membrane.

Adsorption kinetics of (125)I-radiolabeled alpha-chymotrypsin at pH 8.6 was studied in a laminar regime between two walls of poly(ethyleneterephthalate) tracked films and membranes. Adsorption kinetics in the presence of solution (10 microg/mL), desorption by rinsing with buffer, and the following exchange of proteins by flowing unlabeled solution were measured. At pH 8.6, alpha-chymotrypsin is almost neutral and can be mostly removed from the film surface, contrary to positive lysozyme adsorbed at pH 7.4. Results suggest that alpha-chymotrypsin is irreversibly adsorbed in pores, while desorption and exchange occur on membrane flat faces. A method is proposed to determine adsorption kinetics in the pores. Kinetics of desorption and exchange of alpha-chymotrypsin from the film surface can be described by stretched exponential functions in the examined time domain with the same exponent, beta approximately 0.62, which does not depend also on the former adsorption duration. However, the mean residence time at the interface is about 2.5 times greater in the presence of only the buffer than that in the presence of solution. This effect could be explained by a fast exchange at the arrival of unlabeled solution for a part of the adsorbed population.