Sensitive biosensor for choline and acetylcholine involving fast immobilization of a bienzyme system on a disposable membrane

A biosensor was prepared for the determination of choline or acetylcholine by co-immobilizing choline oxidase and cholinesterase on a chemically preactivated membrane ready for use. This rapid procedure allows the coupling to be performed in a few minutes. The determination is based on the electrochemical detection of enzymatically generated hydrogen peroxide. This sensor has a detection limit of 5 × 10^?8 M. The response was obtained in 2 min and was linear up to 2 × 10^?5 M.     

A molecular dynamics simulation of a homogeneous organic-inorganic hybrid silica membrane

A new molecular dynamics simulation method was successfully applied to construct a homogeneous organic-inorganic hybrid silica membrane using the hybrid-pcff (h-pcff) potential function. Analysis suggested that the hybrid BTESE silica membrane provided a looser network and larger cavity size for the enhancement of gas permeability and selectivity.     

The concentration dependence of the diffusion and permeability in a homogeneous membrane

For the sorption and diffusion coefficient dependence on the concentration of the penetrant the transport properties of a homogeneous medium are calculated. The diffusion current is assumed to be proportional to the negative gradient of the chemical potential. This is in contrast with the first Fick's law that assumes this current to be proportional to the negative gradient of the concentration of the penetrant. The difference between the two cases depends on the concentration dependence of the sorption coefficient. In a homogeneous membrane the chemical potential formulation leads to an equation which is very similar to the Fickian expression. The apparent diffusion coefficient, however, depends not onlly on the transport resistance but also on the deviation of the sorption coefficient from constancy.     

Nanofiltration membrane behavior in a supercritical medium

The permeability values of “TN” organomineral nanofiltration membranes to water, L, and to supercritical CO₂, G, were compared. The resulting values for G were an order of magnitude higher than for L. The difference may be directly related to the viscosity difference between the two fluids. Temperature- and pressure-related variations in G were also analyzed; for this purpose, Poiseuille's model satisfactorily accounts for experimental behavior, while Knudsen's model is unsuitable. A hysteresis effect was observed on the isotherms corresponding to variations in G versus pressure, suggesting partially irreversible CO₂ adsorption on the micropore walls, that would diminish the radius. This phenomenon could be enhanced by an increased fluid density and viscosity.     

Phase-separated bienzyme compartmentalization as artificial intracellular membraneless organelles for cell repair

Implanting artificial organelles in living cells is capable of correcting cellular dysfunctionalities for cell repair and biomedical applications. In this work, phase-separated bienzyme-loaded coacervate microdroplets are established as a model of artificial membraneless organelles in endothelial dysfunctional cells for the cascade enzymatic production of nitric oxide(NO) with a purpose of correcting cellular NO deficiency. We prepared the coacervate microdroplets via liquid-liquid phase separat...     

Ionic mass transport through a homogeneous membrane in the presence of a uniform electric field

The time-dependent theoretical solution to ionic mass transport through a uniform membrane under the influence of a uniform electric field is derived. Equivalent lag times and steady-state fluxes are then determined. The enhancement ratio of ionic flow (at a given voltage) to passive flow is shown to be proportional to the voltage for large augmenting voltages, and to decrease exponentially for large retarding voltages. Positive enhancement factors can be as large as 120 for triply charged ions, with one volt imposed across a membrane. The lag times decrease with increasing voltage magnitudes, and can (for triply charged ions, with one volt imposed across a membrane) be as small as 1/20 of the passive lag time.     

Phase behavior of selected artificial lipids

The flexibility of biomembranes is based on the physical-chemical properties of their main components - glycerophospholipids. The structure of these modular amphiphilic molecules can be modified through organic synthesis making it possible to study specific physical-chemical effects in detail. In particular, the roles of the hydrophobic tails of the phospholipids and their hydrophobic/hydrophilic interfacial backbone on the phase behaviour are highlighted. The spatial orientation of the glycerol backbone changes from sn-1,2 to sn-1,3 phospholipids leading to an increase of the in-plane area of the molecule. The larger distance between the hydrophobic tails can lead to membrane leaflet interdigitation. The introduction of methyl side groups in the hydrophobic tails increases the fluidity of the bilayer. Depending on the position of the methyl branches partial interdigitation is observed. In the case of bolaamphiphiles, methyl side groups have a similar effect on the fluidity, but interdigitation cannot occur.     

Development of a bienzyme reactor sensor system for the determination of ornithine

A bienzyme reactor sensor system with amperometric detection was developed for the determination of ornithine. The system based on the immobilized enzymes (ornithine carbamyl transferase and pyruvate oxidase) consisted of a buffer tank, a peristaltic pump, an enzyme reactor, an oxygen electrode and a recorder. Then, 0.1 M MOPS buffer, containing pyruvic acid (0.5 mM) and carbamyl phosphate (0.5 mM), was continuously transferred into the system at 35 °C. Phosphate ion was formed enzymatically by transformation of ornithine in the presence of carbamyl phosphate. Pyruvate oxidase is activated by the presence of phosphate. Therefore, ornithine was determined from the oxygen consumed upon oxidation of pyruvic acid catalyzed by pyruvate oxidase in the presence of phosphate ion. The limit of detection was 0.05 mM and the response was linear to 3 mM (R²=0.9905). The variation coefficients were 4.9 (n=15) and 3.9% (n=15) for 1.1 and 3.0 mM standard ornithine, respectively. Good comparative results (R²=0.9238) were observed between ornithine contents in prawn muscle determined by the proposed system and by the HPLC. One assay was completed within 4 min. The immobilized enzymes were stable for 2 months at 4 °C and more than 150 samples could be continually determined using this enzyme reactor.     

Development of a hollow fiber membrane module for using implantable artificial lung

This study examined the effects of multiple mechanical forces on gas exchange and hemolysis in intravascular lung assist devices (IVLAD). Specific attention was paid to on the effect of membrane vibration. This study adhered to the recommended practice for the assessment of hemolysis described by the American Society of Testing and Materials (ASTM). The results showed a higher oxygen (O₂) transfer rate and carbon dioxide (CO₂) removal rate in each excited frequency bandwidth than those without vibration. The maximum oxygen transfer and carbon dioxide removal rate occurred at frequency band of 7 Hz. The gas exchange improved maximum 52%. The plasma-free hemoglobin was 11.2 ± 0.57 and 14.4 ± 0.74 mg/100 ml by exciting a piezo-vibrator with a sinusoidal wave magnitude of DC10 V and DC50 V, respectively. The NIHO value was determined to be 59 ± 2.76 and 95 ± 4.32 mg/100 ml by exciting a piezo-vibrator with a sinusoidal wave magnitude of DC10 V and DC50 V, respectively. In conclusion, piezoelectric lead zirconate titanate (PZT) materials are exciting systems for improving the oxygen transfer efficiency and blood suitability of hollow fiber membrane in the development of new IVLAD.     

Vectorial properties of small vesicles

³¹P-NMR spectra of suspensions of small phospholipidic vesicles (SUVs) often give two peaks, assigned to the outside and inside leaflets of the membranes. We now show that SUVs formed from polyprenyl phosphates, postulated as 'primitive' membranes, can exhibit this phenomenon. At pH 7.35, stable SUVs could not be obtained from phytanyl phosphate, as vesicles spontaneously grew too much. Phytanyl phosphate + 5 mol% phytanol produced stable SUVs at the same pH, in which, however, ³¹P-NMR showed a single symmetrical peak. At pH 8.9, where dianion phosphates are predominant (they may occupy principally the outer leaflet), ³¹P-NMR showed two signals of the phosphate both in phytanyl phosphate and phytanyl phosphate/5 mol% phytanol SUVs. This asymmetry of the membrane implies a difference in the ionisation state of the phosphate groups on both sides of the membrane. The resulting gradient of electrochemical properties implies the presence of vectorial properties, a factor that may lead to the 'self-complexification' of these vesicles towards proto-cells.     

Osmotic behavior of a Nafion membrane in methanol-water electrolyte solutions

In this paper, an experimental study was carried out in order to investigate the osmotic transport of methanol-water electrolyte solutions through a Nafion membrane. The experimental data indicated that the Nafion membrane showed the typical anomalous osmotic behavior of charged membranes. The influence of some relevant parameters, such as electrolyte concentration difference, weight fraction of methanol on solution, and nature of cation was considered. The results showed that the osmotic volume flow was decreased with the presence of methanol on solvent, but did not alter the anomalous osmotic behavior of the membrane.     

Construction of a pH-responsive artificial membrane fusion system by using designed coiled-coil polypeptides

In many viruses, pH-responsive coiled-coil domains in the specific fusion proteins play important roles in membrane fusion and the infection of viruses into host cells. To investigate the relationship between the conformational change of the coiled coil and the fusion process, we have introduced a de novo designed polypeptide as a model system of the coiled-coil domain. This system enables the systematic study of the dynamics of pH-responsive coiled-coil polypeptide-membrane interactions. First, we designed and synthesized pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptides. Then the relationship between the pH-induced conformational change of the polypeptide and the membrane's interactive properties was studied by physicochemical methods. Structural changes in the designed polypeptides were examined by means of circular dichroism measurements. And finally, the behavior of the membrane fusion was investigated by leakage of liposomal contents, turbidity analysis, dynamic light scattering, and lipid mixing experiments. Our data show that coiled-coil formation under acidic pH conditions enhances polypeptide-induced membrane fusion. The results in this study demonstrate that an artificial membrane fusion system can be constructed on a molecular level by the use of a pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptide.     

Electric mass transport through homogeneous and surface-modified heterogeneous ion-exchange membranes at a rotating membrane disk

Polarization characteristics of the homogeneous MF-4SK perfluorinated sulfonated cation-exchange membrane and the heterogeneous MK-40 sulfonic acid membrane with its surface modified by a homogeneous film of Nafion are studied at a rotating membrane disk in 0.1 and 0.001 M sodium chloride solutions. Partial current-voltage curves (CVC) are obtained for sodium and hydrogen ions, and limiting current densities in the electromembrane systems (EMS) under study are calculated as a function of the rotation rate of the membrane disk. Contribution from different mechanisms (electrodiffusion, electroconvection, dissociation of water, and the effect of the limiting-current exaltation) to the total ion flow is estimated experimentally and theoretically under conditions that the diffusion layer in the EMS has stabilized in thickness. It is established that surface modification of the heterogeneous MK-40 membrane with a 7 μm layer of a modifying agent almost completely eliminates the dissociation of water molecules, and the properties of the heterogeneous MK-40 membrane approximate those of the homogeneous Nafion membrane. From IR spectra and potentiometric titration curves of the MK-40 and MF-4SK membranes, it is shown that the acidity of the sulfonate groups in these membranes is nearly identical, but a difference in the dissociation rate of water at these membranes is determined by a different character of charge-density distribution and potential near the membrane-solution interphase boundary. By means of the theory of the overlimiting state in EMS, the internal parameters of the systems under investigation are calculated: distribution of space-charge density and electric-field potential in the diffusion layer and in the membrane. Partial CVC are calculated for H⁺ ions for the space-charge region in the phase of the MF-4SK and MK-40/Nafion ion-exchange membranes. Partial CVC with similar characteristics are compared for the heterogeneous monopolar MK-40 and the bipolar MB-2 membranes, which contain sulfonate groups. It is concluded that the membrane surface layer, where the space charge is localized, plays a dominant role in speeding up the dissociation of water in EMS.     

Development of a bienzyme system for the electrochemical determination of nitrate in ambient air

This work reports the development of a bienzyme system consisting of salicylate hydroxylase (SHL) and nitrate reductase (NaR) for the electrochemical determination of nitrate. This method measures the concentration of nitrate directly under ambient air without suffering from oxygen interferences. The determination is based on the detection of NADH consumption, and the principle is as follows: NADH initiates the irreversible decarboxylation and hydroxylation of salicylate by SHL in the presence of oxygen to produce catechol, which results in a detectable signal due to its oxidation at the working electrode; the second enzyme, NaR, in the presence of nitrate, reduced the availability of NADH, and consequently, the current difference after the injection of nitrate is proportional to its concentration. This method shows high performance characteristics for nitrate determination with a broad detection range between 10 μM and 1,000 μM, a short measuring time of around 5 min, and a simple operation without sample pretreatment by inert gas purge or oxygen scavenger.

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Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

Effect of a homogeneous magnetic field on the mechanical behavior of soft magnetic elastomers under compression

The mechanical properties of new magnetic composite materials were studied. The above materials represent rubbery silicon matrices filled with magnetic microparticles of metallic iron or magnetite. In homogeneous magnetic fields with an intensity of up to 0.4 T, the shear modulus of the composites was abnormally high (up to 10 000%). The variation of elastic properties of new materials on the type and volume content of the magnetic filler was investigated. In the presence of a sufficiently strong magnetic field, the above composites were shown to behave as elastoplastic materials with strengthening.     

pH-dependent behavior of surface-immobilized artificial leucine zipper proteins

The coiled-coil protein motif occurs in over 200 proteins and has generated interest for a range of applications requiring surface immobilization of the constituent peptides. This paper describes an investigation of the environment-responsive behavior of a monolayer of surface-immobilized artificial proteins, which are known to assemble to form coiled-coil structures in bulk solution. An extended version of the quartz crystal microbalance (QCM-D) and surface plasmon resonance (SPR) are independently employed to characterize the adsorption of the proteins to a gold surface. The data suggest that the molecules arrange in a closely packed layer orientated perpendicular to the surface. QCM-D measurements are also employed to measure pH-induced changes in the resonant frequency (f) and the energy dissipation factor (D) of a gold-coated quartz crystal functionalized with the formed monolayer. Exposure of the protein monolayer to a pH 4.5 solution results in a shift of 43 Hz in f and a shift of -0.7 x 10(-6) in D as compared to pH 7.4. In contrast, increasing the pH to 11.2, results in f and D shifts of -17 Hz and 0.6 x 10(-6), respectively. The magnitude of the observed shifts suggests that the proteins form a rigid layer at low pH that can be hydrated to a fluid layer as the pH is increased. These observations correlate with spectroscopic changes that indicate a reduction in the helical content of the protein in bulk solutions of high pH.     

Amperometric bienzyme screen-printed biosensor for the determination of leucine

Leucine plays an important role in protein synthesis, brain functions, building muscle mass, and helping the body when it undergoes stress. Here, we report a new amperometric bienzyme screen-printed biosensor for the determination of leucine, by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and leucine dehydrogenase (LDH) on a screen-printed electrode with NADP⁺ and p-hydroxybenzoate as the cofactors. The detection principle of the sensor is that LDH catalyzes the specific dehydrogenation of leucine by using NADP⁺ as a cofactor. The product, NADPH, triggers the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a change in electron concentration at the working carbon electrode, which is detected by the potentiostat. The sensor shows a linear detection range between 10 and 600 μM with a detection limit of 2 μM. The response is reproducible and has a fast measuring time of 5–10 s after the addition of a given concentration of leucine.