Jaclyn K. Murton

Neutron Reflectometry and QCM-D Study of the Interaction of Cellulases with Films of Amorphous Cellulose

Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation monitoring (QCM-D) of the interaction of a fungal enzyme extract ( T. viride ) and an endoglucanse from A. niger with amorphous cellulose films. The use of amorphous cellulose is motivated by that the fact that several biomass pretreatments currently under investigation disrupt the native crystalline structure of cellulose and increase the amorphous content. NR reveals the profile of water through the film at nanometer resolution and is highly sensitive to interfacial roughness, whereas QCM-D provides changes in mass and film stiffness. NR can be performed using either H(2)O- or D(2)O-based aqueous reservoirs. NR measurement of swelling of a cellulose film in D(2)O and in H(2)O revealed that D/H exchange on the cellulose chains must be taken into account when a D(2)O-based reservoir is used. The results also show that cellulose films swell slightly more in D(2)O than in H(2)O. Regarding enzymatic digestion, at 20 °C in H(2)O buffer the T. viride cocktail rapidly digested the entire film, initially roughening the surface, followed by penetration and activity throughout the bulk of the film. In contrast, over the same time period, the endoglucanase was active mainly at the surface of the film and did not increase the surface roughness.

Study of enzymatic digestion of cellulose by small angle neutron scattering.

Small angle neutron scattering (SANS) was used to study the structure of Avicel (FD100) microcrystalline cellulose during enzymatic digestion. Digestions were performed in either of two modes: a static, quiescent mode or a dynamic mode using a stirred suspension recycled through a flow cell. The scattering pattern for as-received Avicel in D(2)O buffer is comprised of a low Q power law region resulting from the surface fractal character of the microcrystalline fibers and a high Q roll-off due to scattering from water-filled nanopores with radii approximately 20 A. For digestions in the dynamic mode the high Q roll-off decreased in magnitude within approximately 1 h after addition of enzymes, whereas in the static digestions no change was observed in the high Q roll-off, even after 60 h. These results indicate that only with significant agitation does enzyme digestion affect the structure of the nanopores.

Nanosilica formation at lipid membranes induced by the parent sequence of a silaffin peptide.

Diatoms are unicellular eukaryotic algae found in fresh and marine water. Each cell is surrounded by an outer shell called a frustule that is composed of highly structured amorphous silica. Diatoms are able to transform silicic acid into these sturdy intricate structures at ambient temperatures and pressures, whereas the chemical synthesis of silica-based materials typically requires extremes of temperature and pH. Cationic polypeptides, termed silica affinity proteins (or silaffins), recently identified from dissolved frustules of specific species of diatoms, are clearly involved and have been shown to initiate the formation of silica in solution. The relationship between the local environment of catalytic sites on these peptides, which can be influenced by the amino acid sequence and the extent of aggregation, and the structure of the silica is not understood. Moreover, the activity of these peptides in promoting silicification at lipid membranes has not yet been clarified. In this work, we developed a model system to address some of these questions. We studied peptide adsorption to Langmuir monolayers and subsequent silicification using X-ray reflectivity and grazing incidence X-ray diffraction. The results demonstrate the lipid affinity of the parent sequence of a silaffin peptide and show that the membrane-bound peptide promotes the formation of an interfacial nanoscale layer of amorphous silica at the lipid-water interface.

Synthetic Polypeptide Adsorption to Cu-IDA Containing Lipid Films : A Model for Protein-Membrane Interactions

Adsorption of synthetic alanine-rich peptides to lipid monolayers was studied by X-ray and neutron reflectivity, grazing incidence X-ray diffraction (GIXD), and circular dichroic spectroscopy. The peptides contained histidine residues to drive adsorption to Langmuir monolayers of lipids with iminodiacetate headgroups loaded with Cu2+. Adsorption was found to be irreversible with respect to bulk peptide concentration. The peptides were partially helical in solution at room temperature, the temperature of the adsorption assays. Comparisons of the rate of binding and the structure of the adsorbed layer were made as a function of the number of histidines (from 0 to 2) and also as a function of the positioning of the histidines along the backbone. For peptides containing two histidines on the same side of the helical backbone, large differences were observed in the structure of the adsorbed layer as a function of the spacing of the histidines. With a spacing of 6 A, there was a substantial increase in helicity upon binding (from 17% to 31%), and the peptides adsorbed to a final density approaching that of a nearly completed monolayer of alpha-helices adsorbed side-on. The thickness of the adsorbed layer (17 +/- 2.5 A) was slightly greater than the diameter of alpha-helices, suggesting that the free, unstructured ends extended into solution. With a spacing of 30 A between histidines, a far weaker increase in helicity upon binding was observed (from 13% to 19%) and a much lower packing density resulted. The thickness of the adsorbed layer (10 +/- 4 A) was smaller, consistent with the ends being bound to the monolayer. Striking differences were observed in the interaction of the two types of peptide with the lipid membrane by GIXD, consistent with binding by two correlated sites only for the case of 6 A spacing. All these results are attributed to differences in spatial correlation between the histidines as a function of separation distance along the backbone for these partially helical peptides. Finally, control over orientation was demonstrated by placing a histidine on an end of the sequence, which resulted in adsorbed peptides oriented perpendicular to the membrane.

Magnetic-adhesive based valves for microfluidic devices used in low-resource settings.

Since the introduction of micro total analytical systems (μTASs), significant advances have been made toward development of lab-on-a-chip platforms capable of performing complex biological assays that can revolutionize public health, among other applications. However, use of these platforms in low-resource environments (e.g. developing countries) has yet to be realized as the majority of technologies used to control microfluidic flow rely on off-device hardware with non-negligible size, cost, power requirements and skill/training to operate. In this paper we describe a magnetic-adhesive based valve that is simple to construct and operate, and can be used to control fluid flow and store reagents within a microfluidic device. The design consists of a port connecting two chambers on different planes in the device that is closed by a neodymium disk magnet seated on a thin ring of adhesive. Bringing an external magnet into contact with the outer surface of the device unseats and displaces the valve magnet from the adhesive ring, exposing the port. Using this configuration, we demonstrate on-device reagent storage and on-demand transport and reaction of contents between chambers. This design requires no power or external instrumentation to operate, is extremely low cost (

Population-level coordination of pigment response in individual cyanobacterial cells under altered nitrogen levels

0.20 materials cost per valve), can be used by individuals with no technical training, and requires only a hand-held magnet to actuate. Additionally, valve actuation does not compromise the integrity of the completely sealed microfluidic device, increasing safety for the operator when toxic or harmful substances are contained within. This valve concept has the potential to simplify design of μTASs, facilitating development of lab-on-a-chip systems that may be practical for use in point-of-care and low-resource settings.

Rapid Nucleic Acid Extraction and Purification Using a Miniature Ultrasonic Technique

Cyanobacterial phycobilisome (PBS) pigment-protein complexes harvest light and transfer the energy to reaction centers. Previous ensemble studies have shown that cyanobacteria respond to changes in nutrient availability by modifying the structure of PBS complexes, but this process has not been visualized for individual pigments at the single-cell level due to spectral overlap. We characterized the response of four key photosynthetic pigments to nitrogen depletion and repletion at the subcellular level in individual, live Synechocystis sp. PCC 6803 cells using hyperspectral confocal fluorescence microscopy and multivariate image analysis. Our results revealed that PBS degradation and re-synthesis comprise a rapid response to nitrogen fluctuations, with coordinated populations of cells undergoing pigment modifications. Chlorophyll fluorescence originating from photosystem I and II decreased during nitrogen starvation, but no alteration in subcellular chlorophyll localization was found. We observed differential rod and core pigment responses to nitrogen deprivation, suggesting that PBS complexes undergo a stepwise degradation process.

Nanosilica Formation at Lipid Membranes Induced by Silaffin Peptides

Miniature ultrasonic lysis for biological sample preparation is a promising technique for efficient and rapid extraction of nucleic acids and proteins from a wide variety of biological sources. Acoustic methods achieve rapid, unbiased, and efficacious disruption of cellular membranes while avoiding the use of harsh chemicals and enzymes, which interfere with detection assays. In this work, a miniature acoustic nucleic acid extraction system is presented. Using a miniature bulk acoustic wave (BAW) transducer array based on 36° Y-cut lithium niobate, acoustic waves were coupled into disposable laminate-based microfluidic cartridges. To verify the lysing effectiveness, the amount of liberated ATP and the cell viability were measured and compared to untreated samples. The relationship between input power, energy dose, flow-rate, and lysing efficiency were determined. DNA was purified on-chip using three approaches implemented in the cartridges: a silica-based sol-gel silica-bead filled microchannel, nucleic acid binding magnetic beads, and Nafion-coated electrodes. Using E. coli, the lysing dose defined as ATP released per joule was 2.2× greater, releasing 6.1× more ATP for the miniature BAW array compared to a bench-top acoustic lysis system. An electric field-based nucleic acid purification approach using Nafion films yielded an extraction efficiency of 69.2% in 10 min for 50 µL samples.

Thermally stable nanoporous palladium alloy powders by hydrogen reduction in surfactant templates

Diatoms are unicellular eukaryotic algae found in fresh and marine water. Each cell is surrounded by an outer shell called a frustule that is composed of highly structured amorphous silica. Diatoms are able to transform silicic acid into these sturdy intricate structures at ambient temperatures and pressures, whereas the chemical synthesis of silica-based materials typically requires extremes of temperature and pH. Cationic polypeptides, termed silica affinity proteins (or silaffins) recently identified from dissolved frustules of specific species of diatoms are clearly involved and have been shown to initiate the formation of silica in solution. The relationship between the local environment of catalytic sites on these peptides, which can be influenced by the amino acid sequence and the extent of aggregation, and the observed structure of the silica is not understood. Moreover, the activity of these peptides in promoting silicification at lipid membranes has not yet been clarified. In this work we developed a model system to address some of these questions. We studied peptide adsorption to Langmuir monolayers and subsequent silicification using X-ray reflectivity and grazing incidence X-ray diffraction. The results demonstrate the lipid affinity of the parent sequences of several silaffin peptides. Further, the results show that the membrane-bound peptides promote the formation of interfacial nanoscale layers of amorphous silica at the lipid-water interface that vary in structure according to the peptide sequence.