Zeolite-based chiral ion-exchangers for chromatographic enantioseparations and potential applications in membrane separation processes.

Chiral resolution of racemic compounds represents an important task in research and development and, most importantly, in the large-scale production of pharmaceuticals. Zeolites, which are already frequently utilized for their unique properties, represent materials that can be used for the development of new chiral stationary phases for liquid chromatography, simulated moving bed or enantioselective membranes. The aim of this study was to modify a series of MWW zeolites by a chiral anion-exchange type selector thereby creating a chiral stationary phase for enantiomeric resolution of acidic compounds. To evaluate the applicability of the prepared chiral stationary phase in liquid chromatography, we used N-protected amino acids as model analytes. First, we tested the new sorbents preferential sorption using N-(3,5-dinitrobenzoyl)leucine. We observed outstanding sorption properties of a zeolite-based sorbent (MCM-36), which were comparable to spherical chromatographic silica. This particular material was subsequently packed into a chromatographic column, which was tested under polar organic mode HPLC conditions facilitating baseline resolution of 5 out of 8 N-protected amino acids. Although the chromatographic performance shows several drawbacks (high backpressure, low column efficiency), it clearly documents the potential of the novel materials in chiral separation. To the best of our knowledge, this is the first example of the preparation of the chiral stationary phase based on MWW zeolites ever.

Haemogregarines of the Genera Haemogregarina, Hemolivia, and Hepatozoon Infecting Vietnamese Freshwater Turtles, with Additional Notes on Primer Specificity and Primer-template Mismatches Affecting Diagnostic Success.

The 18S rRNA gene is a widely used molecular marker for haemogregarines. In recent decades, many primers more or less specific to various haemogregarine genera have been designed. This study applied five commonly used primers targeting the 18S rRNA gene of haemogregarines to blood samples from 168 individuals of nine turtle species captured in Northern Vietnam. Three haemogregarine genera, Haemogregarina, Hemolivia, and Hepatozoon, were detected. Selective specificity of primers EF/ER, HemoFN/HemoRN, and Hemo1/Hemo2 to haemogregarine genera was observed and elucidated by primer-template mismatches. In total, 13 out of 168 turtles (prevalence 7.7%) were both microscopically and PCR positive for haemogregarines. Additionally, a single Heosemys grandis turtle was PCR positive but microscopically negative. Numerous turtles carried mixed infections by various haemogregarines; a single turtle was even coinfected by haemogregarines of all three studied genera: Haemogregarina, Hemolivia, and Hepatozoon. Among the detected haemogregarines, some provided sufficient molecular and morphological data for completing their species diagnosis. Two were described as new species: Haemogregarina cyclemydis sp. nov. from Cyclemys pulchristriata and Hemolivia cruciata sp. nov. from Cuora galbinifrons, so far the first Hemolivia from Southeast Asia. Haemogregarina cuorae Chai and Chen, 1990, required a redescription with reassignment to the genus Hepatozoon Miller, 1908.

One or two pups - optimal reproduction strategies of common noctule females.

BACKGROUND: The success of animal reproduction is impacted by a trade-off between energetic costs and mortality associated with immediate vs. future reproductive attempts. The reproductive strategies of European insectivorous bats differ from common mammalian standards due to the use of delayed fertilisation. Phenology of bat reproduction, including length of pregnancy, which may vary in the same species at different latitudes, between years at the same site or between individuals within a colony, is influenced by ecological conditions. To assess factors influencing the course of pregnancy, we evaluated levels of blood progesterone in 20 female common noctule bats Nyctalus noctula. The bats were individually tagged and randomly divided into two groups with different hibernation ending points (i.e. a control group vs. a treatment group with one-week longer hibernation). Following emergence from hibernation, the bats were kept in a wooden box at a stable temperature of 22 °C. RESULTS: The majority of females gave birth to a single neonate (65%), but one female aborted her pups 2 days before the first successful births of other females. Based on development of progesterone concentration, we were able to define a number of different reproduction strategies, i.e. females with single offspring or twins, and females with supposed resorption of one embryo (embryonic mortality after implantation of the developing fertilised egg). Progesterone levels were much higher in females with two embryos during the first part of gestation and after birth. Progesterone levels were at their highest mid-gestation, with no difference between females carrying one or two foetuses. Length of gestation differed significantly between the two groups, with the longer hibernation (treatment) group having a roughly two-day shorter gestation period. CONCLUSIONS: Female N. noctula are able to manipulate their litter size to balance immediate and future reproduction success. The estimated gestation length of approx. 49-days appears to be standard for N. noctula, with females optimising their thermoregulatory behaviour to keep the length of gestation as close to the standard as possible.

Conservative Treatment of Complicated Crown Fracture and Crown-Root Fracture of Young Permanent Incisor-A Case Report with 24-Month Follow-Up.

The complicated crown-root fracture of young permanent teeth is an uncommon traumatic dental injury that is usually treated in a complex way and is demanding not only for the dentist but even for the treated child. In this case report, we present the conservative treatment of a maxillary central incisor in a 10-year-old boy after a traumatic dental injury. Treatment included partial pulpotomy and adhesive fragment reattachment after reflection of the mucoperiosteal flap. The patient was fully asymptomatic at 24-month follow-up, with an aesthetically acceptable outcome. Vital pulp therapy and adhesive fragment reattachment can be a viable treatment option for complicated crown-root fractures, especially when treating immature permanent teeth.

Electric-field-enhanced selective separation of products of an enzymatic reaction in a membrane micro-contactor.

Processes employed in separations of products of enzyme reactions are often driven by diffusion, and their efficiency can be limited. Here, we exploit the effect of a direct current (DC) electric field that intensifies mass transfer through a semipermeable membrane for fast, continuous, and selective separation of electrically charged molecules. Specifically, we separate low-molecular-weight reaction products (phenylacetic acid, 6-aminopenicillanic acid) from the original reaction mixture containing a free enzyme (penicillin acylase). The developed microfluidic dialysis-membrane contactor allows a stable counter-current arrangement of the retentate and permeates liquid streams on which DC electric field is perpendicularly applied. The applied electric field significantly accelerates the transport of electrically charged products through the semipermeable membrane yielding high separation efficiencies at short residence times. The residence time of 5 min is sufficient to reach 100% separation yield in the electric field. The same residence time provides only a 50% yield in the diffusion-controlled experiments. We experimentally demonstrated that a combined microreactor-microextractor with a recycle of the soluble penicillin acylase can continuously produce both the reaction products at high concentrations. The developed membrane-contactor is a versatile platform allowing to tune its characteristics, such as selectivity given by the membrane, or the type of the retentate phase, for a specific application.

Electric field assisted transport of dielectric droplets dispersed in aqueous solutions of ionic surfactants.

Motion of liquid droplets with a surface electric charge can be efficiently controlled by dc electric field. Here, we show that the surface of a dielectric kerosene droplet can be charged by the addition of ionic surfactants to a surrounding aqueous electrolyte. The direction of droplet motion is determined by the polarity of the surfactant charge and the orientation of the imposed electric field. We have found that the effective electrophoretic mobility of dielectric droplets in a confined channel is directly proportional to the logarithm of the surfactant concentration even for values significantly exceeding critical micelle concentration (CMC). We attribute this finding not only to adsorption of ionic surfactants to the surface of dielectric droplets but also to the weakening of electro-osmosis at channel walls due to the increase of ionic strength in the aqueous phase. Our findings can be exploited in microfluidic reactors and separators for on request dosing, sampling, and separation of dielectric fluids.

Optimization of aqueous two-phase systems for the production of 6-aminopenicillanic acid in integrated microfluidic reactors-separators.

Aqueous two-phase systems (ATPSs) were screened for the production of 6-aminopenicillanic acid (6-APA) catalyzed by penicillin acylase, followed by the extractive separation of 6-APA from the reaction mixture. The key point of this study was to find an ATPS exhibiting a large difference in the partition coefficients of the biocatalyst and reaction products. Several ATPSs based on polyethylene glycol (PEG)/phosphate, PEG/citrate, and PEG/dextran were tested. We found that an ATPS consisting of 15 wt% of PEG 4000, 10 wt% of phosphates, 75 wt% of water (pH value 8.0 after dissolution) provided optimal separation of 6-APA from the enzyme. While the 6-APA was mainly found in the top PEG phase, the free enzyme favored the bottom salt-rich phase. This ATPS also fulfils other important requirements: (i) high buffering capacity, reducing an undesirable pH decrease due to the dissociation of phenylacetic acid (the side product of the reaction), (ii) a relatively low cost of the ATPS components, (iii) the possibility of electrophoretic transport of fine droplets as well as the reaction products for both the acceleration of phase separation and the enhancement of 6-APA concentration in the product stream. Extraction experiments in microcapillary and batch systems showed that the transport of 6-APA formed in the salt-rich phase to the corresponding PEG phase could occur within 30 s. The experimental results described form a base of knowledge for the development of continuously operating integrated microfluidic reactors-separators driven by an electric field for the efficient production of 6-APA.

Minimal oscillating subnetwork in the Huang-Ferrell model of the MAPK cascade.

Prompted by the recent growing evidence of oscillatory behavior involving MAPK cascades we present a systematic approach of analyzing models and elucidating the nature of biochemical oscillations based on reaction network theory. In particular, we formulate a minimal biochemically consistent mass action subnetwork of the Huang-Ferrell model of the MAPK signalling that provides an oscillatory response when a parameter controlling the activation of the top-tier kinase is varied. Such dynamics are either intertwined with or separated from the earlier found bistable/hysteretic behavior in this model. Using the theory of stability of stoichiometric networks, we reduce the original MAPK model, convert kinetic to convex parameters and examine those properties of the minimal subnetwork that underlie the oscillatory dynamics. We also use the methods of classification of chemical oscillatory networks to explain the rhythmic behavior in physicochemical terms, i.e., we identify of the role of individual biochemical species in positive and negative feedback loops and describe their coordinated action leading to oscillations. Our approach provides an insight into dynamics without the necessity of knowing rate coefficients and thus is useful prior the statistical evaluation of parameters.

Electrochemical characteristics of ideal polarizable interfaces with limited number of charge carriers.

Recent progress in material chemistry and surface engineering has led to emergence of new electrode materials with unique physical and electrochemical properties. Here, we introduce a physical model describing charging of ideal polarizable electrode-electrolyte interface where the electrode is characterized by a limited capacity to store charge. The analytical model treats the electrode and electrolyte phases as independent nonlinear capacitors that are eventually coupled through the condition of equality of the total stored electrical charge opposite in sign. Gouy-Chapman and condensed layer theories applied to a general 1:n valent electrolyte are used to predict dependencies of differential capacitance of the electrolyte phase and surface concentration of the electrical charge on the applied potential. The model of the nonlinear capacitor for the electrode phase is described by a theory of electron donors and acceptors present in conductive solids as a result of thermal fluctuations. Both the differential capacitance and the surface concentration of the electrical charge in the electrode are evaluated as functions of the applied potential and related to the capacity of the electrode phase to accumulate charge and its ability to form electron donors and acceptors. The knowledge of capacitive properties of both phases allows to predict electrochemical characteristics of ideal polarizable interfaces, e.g., current responses in linear sweep voltammetry. The coupled model also shows significant potential drops in the electrode comparable to those in the electrolyte phase for materials with low charge carrier concentrations.

Local kinetics and thermodynamics of rapid electrochemical reactions.

We introduce and discuss a local kinetic mechanism for an n-electron electrochemical reaction at the interface formed by an electrode and diluted electrolyte. We show that the suggested mechanism is in agreement with the Nernst equation in the thermal equilibrium. We also qualitatively characterize the structure of a flat electrode-diluted electrolyte boundary in the meaning of the spatial distribution of electrochemical reactants and electric potential. As the suggested kinetic mechanism is not limited by the duration of relaxation processes in electric double layers, it is suitable for the understanding and simulation of fast transient processes that appear in modern applications such as nanocolloid dielectrophoresis, AC electrospray, AC electroosmosis, or nanopore biosensing.

Oscillatory flow accelerates autocrine signaling due to nonlinear effect of convection on receptor-related actions.

We study effects of oscillatory convective flow in extracellular space on the velocity of chemical signal propagation having a form of a front wave above a cellular layer. We found that the time-averaged propagation velocity under oscillatory flow for a particular Péclet number amplitude is slower than the velocity under steady laminar flow regime for the same value of the Péclet number, but significantly faster than under no-flow conditions. We derive asymptotic values of the propagation velocity and asymptotic characteristics of the corresponding concentration fronts in high- and low-frequency regimes and show that the reason for the observed velocity increase under the oscillatory flow stems from a nonlinear dependence of the propagation velocity on the Péclet number, particularly from the convex character of the dependence. Our findings suggest that the specific responses of cellular cultures to different flow conditions in the extracellular space (for example, expression of atherosclerosis protective genes under steady laminar flow but not under oscillatory flow) is a consequence of a nonlinear coupling between the extracellular transport and complex intracellular reaction cascades forming a positive feedback loop of the autocrine signaling. This mechanism can operate independently of, or in conjunction with, a direct stress-sensing due to mechanotransduction.

Three-phase slug flow in microchips can provide beneficial reaction conditions for enzyme liquid-liquid reactions.

Here, we introduce a solution to low stability of a two-phase slug flow with a chemical reaction occurring at the phase interface in a microfluidic reactor where substantial merging of individual reacting slugs results in the loss of uniformity of the flow. We create a three-phase slug flow by introducing a third fluid phase into the originally two-phase liquid-liquid slug flow, which generates small two-phase liquid slugs separated by gas phase. Introduction of the third phase into our system efficiently prevents merging of slugs and provides beneficial reaction conditions, such as uniform flow pattern along the whole reaction capillary, interfacial area with good reproducibility, and intensive water-oil interface renewal. We tested the three-phase flow on an enzyme hydrolysis of soybean oil and compared the reaction conversion with those from unstable two-phase slug flows. We experimentally confirmed that the three-phase slug flow arrangement provides conversions and pressure drops comparable or even better with two-phase liquid-liquid arrangements.

Kinetic mechanism for modeling of electrochemical reactions.

We propose a kinetic mechanism of electrochemical interactions. We assume fast formation and recombination of electron donors D- and acceptors A+ on electrode surfaces. These mediators are continuously formed in the electrode matter by thermal fluctuations. The mediators D- and A+, chemically equivalent to the electrode metal, enter electrochemical interactions on the electrode surfaces. Electrochemical dynamics and current-voltage characteristics of a selected electrochemical system are studied. Our results are in good qualitative agreement with those given by the classical Butler-Volmer kinetics. The proposed model can be used to study fast electrochemical processes in microsystems and nanosystems that are often out of the thermal equilibrium. Moreover, the kinetic mechanism operates only with the surface concentrations of chemical reactants and local electric potentials, which facilitates the study of electrochemical systems with indefinable bulk.

Effects of convective transport on chemical signal propagation in epithelia.

We study effects of convective transport on a chemical front wave representing a signal propagation at a simple (single layer) epithelium by means of mathematical modeling. Plug flow and laminar flow regimes were considered. We observed a nonmonotonous dependence of the propagation velocity on the ligand receptor binding constant under influence of the convective transport. If the signal propagates downstream, the region of high velocities becomes much broader and spreads over several orders of magnitude of the binding constant. When the convective transport is oriented against the propagating signal, either velocity of the traveling front wave is slowed down or the traveling front wave can stop or reverse the direction of propagation. More importantly, chemical signal in epithelial systems influenced by the convective transport can propagate almost independently of the ligand-receptor binding constant in a broad range of this parameter. Furthermore, we found that the effects of the convective transport becomes more significant in systems where either the characteristic dimension of the extracellular space is larger/comparable with the spatial extent of the ligand diffusion trafficking or the ligand-receptor binding/ligand diffusion rate ratio is high.

Detection of immunoglobulins in a laser induced fluorescence system utilizing polydimethysiloxane microchips with advanced surface and optical properties.

We developed an automated laser induced fluorescence system utilizing microfluidic chips for detection and quantification of immunoglobulins. Microchips were fabricated from polydimethysiloxane (PDMS) using the so-called "prepolymerization technique." The microchip structure helped minimize the effects of PDMS autofluorescence and light scattering. Furthermore, a thin and uniform PDMS layer forming the top of the microchip enabled proper focusing and collection of the excitation beam and the emitted fluorescence, respectively. The developed system was tested for the detection of mouse immunoglobulins. The capturing antibodies were immobilized on internal microchannel walls in the form of a polyelectrolyte. We clearly show that this immobilization technique, if correctly realized, gives results with high reproducibility. After sample incubation and washing, secondary antibodies labeled by fluorescein isothiocyanate were introduced into microchannels to build a detectable complex. We show that mouse antibodies can be quantified in a wide concentration range, 0.01-100 µg ml(-1). The lower detection limit was below 0.001 µg ml(-1) (6.7 pM). The developed laser induced fluorescence (LIF) apparatus is relatively cheap and easy to construct. The total cost of the developed LIF detector is lower than a typical price of plate readers. If compared to classical ELISA (enzyme linked immunosorbent assay) plate systems, the detection of immunoglobulins or other proteins in the developed PDMS microfluidic device brings other important benefits such as reduced time demands (10 min incubation) and low reagent consumption (less than 1 µl). The cost of the developed PDMS chips is comparable with the price of commercial ELISA plates. The main troubleshooting related to the apparatus development is also discussed in order to help potential constructors.

Study on surface properties of PDMS microfluidic chips treated with albumin.

Electrokinetic properties and morphology of PDMS microfluidic chips intended for bioassays are studied. The chips are fabricated by a casting method followed by polymerization bonding. Microchannels are coated with 1% solution of bovine serum albumin (BSA) in Tris buffer. Albumin passively adsorbs on the PDMS surface. Electrokinetic characteristics (electro-osmotic velocity, electro-osmotic mobility, and zeta potential) of the coated PDMS channels are experimentally determined as functions of the electric field strength and the characteristic electrolyte concentration. Atomic force microscopy (AFM) analysis of the surface reveals a "peak and ridge" structure of the protein layer and an imperfect substrate coating. On the basis of the AFM observation, several topologies of the BSA-PDMS surface are proposed. A nonslip mathematical model of the electro-osmotic flow is then numerically analyzed. It is found that the electrokinetic characteristics computed for a channel with the homogeneous distribution of a fixed electric charge do not fit the experimental data. Heterogeneous distribution of the fixed electric charge and the surface roughness is thus taken into account. When a flat PDMS surface with electric charge heterogeneities is considered, the numerical results are in very good agreement with our experimental data. An optimization analysis finally allowed the determination of the surface concentration of the electric charge and the degree of the PDMS surface coating. The obtained findings can be important for correct prediction and possibly for robust control of behavior of electrically driven PDMS microfluidic chips. The proposed method of the electro-osmotic flow analysis at surfaces with a heterogeneous distribution of the surface electric charge can also be exploited in the interpretation of experimental studies dealing with protein-solid phase interactions or substrate coatings.

Transient behavior of an electrolytic diode.

The transient behavior of an electrolytic diode system was studied. A gel-like electrolytic diode was incorporated in a capillary microfluidic chip. The microfluidic platform guaranteed a constant composition of solutions on the diode boundaries. The current responses of the electrolytic diode to step-like changes of the imposed DC electric voltage were measured. Some of these transients were accompanied by a short-time overshoot of electric current density. In order to explain this phenomenon, a mathematical model of the electrolytic diode system was developed. Dynamical analysis of the model equations confirmed the existence of the electric current overshoots. Because the results of the experimental and the numerical transient studies were quite similar, we have explained the physical meaning of three selected overshoots by means of an analysis of the reaction-transport processes inside the electrolytic diode system. The transient experiments carried out in this study indicate that our physical concept of the electrolytic diode system presented in previous papers is correct.

Microfluidic chip for fast bioassays-evaluation of binding parameters.

A seven channel polystyrene (PS) microchip has been constructed using a micromilling machine and a high-temperature assembling. Protein A (PA) has been immobilized by a passive sorption on the microchannel walls. Two bioaffinity assays with human immunoglobulin G (hIgG) as a ligand have been carried out. (i) PA as the receptor and fluorescently labeled hIgG (FITC-hIgG) as the ligand, (ii) PA as the receptor with hIgG as the quantified ligand and fluorescently labeled goat anti-human IgG (FITC-gIgG) as the secondary ligand. One incubation step of the assays took only 5 min instead of hours typical for enzyme-linked immunosorbent assay applications. Calibration curves of the dependence of a fluorescence signal on the hIgG concentration in a sample have been obtained in one step due to a parallel arrangement of microchannels. A mathematical model of the PA-FITC-hIgG complex formation in the chip has been developed. The values of the kinetic constant of the PA-FITC-hIgG binding (k(on)=5.5 m(3) mol(-1) s(-1)) and the equilibrium dissociation constant of the formed complex (K(d)

Discrete models of autocrine cell communication in epithelial layers.

Pattern formation in epithelial layers heavily relies on cell communication by secreted ligands. Whereas the experimentally observed signaling patterns can be visualized at single-cell resolution, a biophysical framework for their interpretation is currently lacking. To this end, we develop a family of discrete models of cell communication in epithelial layers. The models are based on the introduction of cell-to-cell coupling coefficients that characterize the spatial range of intercellular signaling by diffusing ligands. We derive the coupling coefficients as functions of geometric, cellular, and molecular parameters of the ligand transport problem. Using these coupling coefficients, we analyze a nonlinear model of positive feedback between ligand release and binding. In particular, we study criteria of existence of the patterns consisting of clusters of a few signaling cells, as well as the onset of signal propagation. We use our model to interpret recent experimental studies of the EGFR/Rhomboid/Spitz module in Drosophila development.

Long-range signal transmission in autocrine relays.

Intracellular signaling induced by peptide growth factors can stimulate secretion of these molecules into the extracellular medium. In autocrine and paracrine networks, this can establish a positive feedback loop between ligand binding and ligand release. When coupled to intercellular communication by autocrine ligands, this positive feedback can generate constant-speed traveling waves. To demonstrate that, we propose a mechanistic model of autocrine relay systems. The model is relevant to the physiology of epithelial layers and to a number of in vitro experimental formats. Using asymptotic and numerical tools, we find that traveling waves in autocrine relays exist and have a number of unusual properties, such as an optimal ligand binding strength necessary for the maximal speed of propagation. We compare our results to recent observations of autocrine and paracrine systems and discuss the steps toward experimental tests of our predictions.