Electrokinetics of the silica and aqueous electrolyte solution interface: Viscoelectric effects.

The manipulation of biomolecules, fluid and ionic current in a new breed of integrated nanofluidic devices requires a quantitative understanding of electrokinetics at the silica/water interface. The conventional capacitor-based electrokinetic Electric Double Layer (EDL) models for this interface have some known shortcomings, as evidenced by a lack of consistency within the literature for the (i) equilibrium constants of surface silanol groups, (ii) Stern layer capacitance, (iii) zeta (ζ) potential measured by various electrokinetic methods, and (iv) surface conductivity. In this study, we consider how the experimentally observable viscoelectric effect - that is, the increase of the local viscosity due to the polarisation of polar solvents - affects electrokinetcs at the silica/water interface. Specifically we consider how a model that considers viscoelectric effects (the VE model) performs against two conventional electrokinetic models, namely the Gouy-Chapman (GC) and Basic Stern capacitance (BS) models, in predicting four fundamental electrokinetic phenomena: electrophoresis, electroosmosis, streaming current and streaming potential. It is found that at moderate to high salt concentrations (>5×10(-3)M) predictions from the VE model are in quantitative agreement with experimental electrokinetic measurements when the sole additional adjustable parameter, the viscoelectric coefficient, is set equal to a value given by a previous independent measurement. In contrast neither the GS nor BS models is able to reproduce all experimental data over the same concentration range using a single, robust set of parameters. Significantly, we also show that the streaming current and potential in the moderate to high surface charge range are insensitive to surface charge behaviour (including capacitances) when viscoelectric effects are considered, in difference to models that do not consider these effects. This strongly questions the validity of using pressure based electrokinetic experiments to measure surface charge characteristics within this experimentally relevant high pH and moderate to high salt concentration range. At low salt concentrations (<5×10(-3)M) we find that there is a lack of consistency in previously measured channel conductivities conducted under similar solution conditions (pH, salt concentration), preventing a conclusive assessment of any model suitability in this regime.

Reconstituted Keratin Biomaterial with Enhanced Ductility.

Nowadays the waste from protein fibres represents an important renewable source for a new generation of biomaterials and promising competitors for carbohydrate based biomaterials. Regenerated keratin biomaterials are biodegradable in vivo and in vitro, biocompatible, and support cell attachment and proliferation; however, their major drawback has been their weak mechanical properties such as ductility. The following study was conducted in an attempt to improve the ductility of reconstituted keratin films obtained from Australian merino wool fibres. Keratin was extracted from wool fibres according to an established protocol proposed by Yamauchi, and then dialyzed and desalted by multiple diafiltration wash cycles. The resulting keratin film was transparent, biodegradable, and, opposite to its predecessors, mechanically durable, possessing a Young modulus about 12.5 MPa with 35% extensibility. The polypeptide chains were found to rearrange themselves in the ß-sheet state in this keratin film, which was shown to be semi-crystalline. This film, unlike its predecessors, did not support human cell proliferation. These properties of the diafiltered keratin film have led us to think that diafiltration resulted in producing a totally new keratin film, which is envisaged to find applications in various areas.

Stationary chemical gradients for concentration gradient-based separation and focusing in nanofluidic channels.

Previous work has demonstrated the simultaneous concentration and separation of proteins via a stable ion concentration gradient established within a nanochannel (Inglis Angew. Chem., Int. Ed. 2001, 50, 7546-7550). To gain a better understanding of how this novel technique works, we here examine experimentally and numerically how the underlying electric potential controlled ion concentration gradients can be formed and controlled. Four nanochannel geometries are considered. Measured fluorescence profiles, a direct indicator of ion concentrations within the Tris-fluorescein buffer solution, closely match depth-averaged fluorescence profiles calculated from the simulations. The simulations include multiple reacting species within the fluid bulk and surface wall charge regulation whereby the deprotonation of silica-bound silanol groups is governed by the local pH. The three-dimensional system is simulated in two dimensions by averaging the governing equations across the (varying) nanochannel width, allowing accurate numerical results to be generated for the computationally challenging high aspect ratio nanochannel geometries. An electrokinetic circuit analysis is incorporated to directly relate the potential drop across the (simulated) nanochannel to that applied across the experimental chip device (which includes serially connected microchannels). The merit of the thick double layer, potential-controlled concentration gradient as a particle focusing and separation tool is discussed, linking this work to the previously presented protein trapping experiments. We explain why stable traps are formed when the flow is in the opposite direction to the concentration gradient, allowing particle separation near the low concentration end of the nanochannel. We predict that tapered, rather than straight nanochannels are better at separating particles of different electrophoretic mobilities.

Temperature-dependent dynamics of bovine casein micelles in the range 10-40 °C.

Milk is a complex colloidal system that responds to changes in temperature imposed during processing. Whilst much has been learned about the effects of temperature on milk, little is known about the dynamic response of casein micelles to changes in temperature. In this study, a comprehensive physico-chemical study of casein micelles in skim milk was performed between 10 and 40 °C. When fully equilibrated, the amount of soluble casein, soluble calcium and the pH of skim milk all decreased as a function of increasing temperature, whilst the hydration and volume fraction of the casein micelles decreased. The effect of temperature on casein micelle size, as determined by dynamic light scattering and differential centrifugation, was less straightforward. Real-time measurements of turbidity and pH were used to investigate the dynamics of the system during warming and cooling of milk in the range 10-40 °C. Changes in pH are indicative of changes to the mineral system and the turbidity is a measure of alterations to the casein micelles. The pH and turbidity showed that alterations to both the casein micelles and the mineral system occurred very rapidly on warming. However, whilst mineral re-equilibration occurred very rapidly on cooling, changes to the casein micelle structure continued after 40 min of measurement, returning to equilibrium after 16 h equilibration. Casein micelle structure and the mineral system of milk were both dependent on temperature in the range 10-40 °C. The dynamic response of the mineral system to changes in temperature appeared almost instantaneous whereas equilibration of casein was considerably slower, particularly upon cooling.

Evaporative concentration of skimmed milk: effect on casein micelle hydration, composition, and size.

Understanding the effect of evaporative concentration on casein micelle composition is of high importance for milk processing. Alterations to the hydration, composition and size of casein micelles were investigated in skimmed milk evaporated to concentrations of 12-45% total solids content. The size of casein micelles was determined by dynamic light scattering, and the water content and composition determined by analysis of supernatants and pellets obtained by ultracentrifugation. The mass balance and hydration results showed that during the evaporation process, while micelles were dehydrated, water was removed preferentially from the serum. The amount of soluble casein and calcium in the serum decreased as a function of increasing solids content, indicating a shift of these components to the micelles. The formation of a small proportion of micelle aggregates at high concentrations appeared dependent on the time kept at these concentrations. Upon redilution with water, casein micelles were immediately rehydrated and aggregates were broken up in a matter of minutes. Soluble calcium and pH returned to their original state over a number of hours; however, only a small percentage of original soluble casein returned to the serum over the 5h period investigated. These results showed that casein micelles are significantly affected by evaporative concentration and that the alterations are not completely and rapidly reversible.

Chitosan microparticles encapsulating PEDF plasmid demonstrate efficacy in an orthotopic metastatic model of osteosarcoma.

The major stumbling block for most therapies against deep-seated disease, including tumours, is inefficient drug delivery. Such a concern is particularly important for osteosarcoma, the predominant form of bone cancer, and the largest cancer of its type in the paediatric age group. Pigment epithelium-derived factor (PEDF) is the most potent anti-angiogenic factor found endogenously in the body, with an increasing number of reports pointing to its direct antitumour activity. In this report, when a plasmid expressing PEDF (pPEDF) was encapsulated within two types of chitosan microparticles, anti-invasion and increased adhesion of the osteosarcoma cell line SaOS-2 was noted. Microparticles were formulated using two methods of complex coacervation and were approximately 400-600 nm in diameter. The plasmids were strongly attached to the particles which were polymorphic in shape as determined by electron microscopy. Preliminary experiments with the green fluorescent protein (GFP) reporter plasmid revealed that cells were efficiently transfected with the particles, with particles outlasting transfection with lipofectamine cationic liposomes at 5 days. In vivo, the better pPEDF microparticle resulted in a decrease in primary tumour growth, reduced bone lysis and reduced establishment of lung metastases in a clinically relevant orthotopic model of osteosarcoma. Thus, this new mode of localised gene delivery may hold promise for molecular therapy of osteosarcoma.

Soluble manganese(IV) as a chemiluminescence reagent for the determination of opiate alkaloids, indoles and analytes of forensic interest.

We present the results of our investigations into the use of soluble manganese(IV) as a chemiluminescence reagent, which include a significantly faster method of preparation and a study on the effect of formaldehyde and orthophosphoric acid concentration on signal intensity. Chemiluminescence detection was applied to the determination of 16 analytes, including opiate alkaloids, indoles and analytes of forensic interest, using flow injection analysis methodology. The soluble manganese(IV) reagent was less selective than either acidic potassium permanganate or tris(2,2'-bipyridyl)ruthenium(III) and therefore provided a more universal chemiluminescence detection system for HPLC. A broad spectral distribution with a maximum at 730+/-5nm was observed for the reaction between the soluble manganese(IV) and a range of analytes, as well as the background emission from the reaction with the formaldehyde enhancer. This spectral distribution matches that reported for chemiluminescence reactions with acidic potassium permanganate, where a manganese(II) emitting species was elucidated. This provides further evidence that the emission evoked in reactions with soluble manganese(IV) also emanates from a manganese(II) species, and not bimolecular singlet oxygen as suggested by previous authors.

The central domain of Escherichia coli TyrR is responsible for hexamerization associated with tyrosine-mediated repression of gene expression.

TyrR from Escherichia coli regulates the expression of genes for aromatic amino acid uptake and biosynthesis. Its central ATP-hydrolyzing domain is similar to conserved domains of bacterial regulatory proteins that interact with RNA polymerase holoenzyme associated with the alternative sigma factor, sigma(54). It is also related to the common module of the AAA+ superfamily of proteins that is involved in a wide range of cellular activities. We expressed and purified two TyrR central domain polypeptides. The fragment comprising residues 188-467, called TyrR-(188-467), was soluble and stable, in contrast to that corresponding to the conserved core from residues 193 to 433. TyrR-(188-467) bound ATP and rhodamine-ATP with association constants 2- to 5-fold lower than TyrR and hydrolyzed ATP at five times the rate of TyrR. In contrast to TyrR, which is predominantly dimeric at protein concentrations less than 10 microm in the absence of ligands, or in the presence of ATP or tyrosine alone, TyrR-(188-467) is a monomer, even at high protein concentrations. Tyrosine in the presence of ATP or ATPgammaS promotes the oligomerization of TyrR-(188-467) to a hexamer. Tyrosine-dependent repression of gene transcription by TyrR therefore depends on ligand binding and hexamerization determinants located in the central domain polypeptide TyrR-(188-467).

Particulate hematite diffusion in sodium polyacrylate solutions. The effect of ionic strength.

The diffusion coefficients of hematite particles in polyelectrolyte solution have been investigated using dynamic light scattering. Two apparent diffusion coefficients, a fast and a slow diffusional mode, are observed for the hematite particles in high-molecular-weight sodium polyacrylate solution at pH 10.5. The slow diffusion coefficient (Dslow) shows a decrease with increase in polyelectrolyte concentration. The fast diffusion coefficient (Dfast) shows an increase to a maximum with increasing polyelectrolyte concentration and then a rapid decrease as the polyelectrolyte concentration increases further. With an increase in ionic strength from 10(-4) to 0.1 M NaNO3, the maximum value of Dfast increased in magnitude, while the polyacrylate concentration at which the maximum occurs is seen to increase. The dependence of Dfast on the measurement angle indicates that it is coupled to the fluctuations of the chains. The observed behavior is attributed to the hematite probe particle sensing both macroscopic (viscous) and elastic fluctuations associated with the polyelectrolyte motion.