Directed evolution for the development of conformation-specific affinity reagents using yeast display

Yeast display is a powerful tool for increasing the affinity and thermal stability of scFv antibodies through directed evolution. Mammalian calmodulin (CaM) is a highly conserved signaling protein that undergoes structural changes upon Ca(2+) binding. In an attempt to generate conformation-specific antibodies for proteomic applications, a selection against CaM was undertaken. Flow cytometry-based screening strategies to isolate easily scFv recognizing CaM in either the Ca(2+)-bound (Ca(2+)-CaM) or Ca(2+)-free (apo-CaM) states are presented. Both full-length scFv and single-domain VH only clones were isolated. One scFv clone having very high affinity (K(d) = 0.8 nM) and specificity (>1000-fold) for Ca(2+)-CaM was obtained from de novo selections. Subsequent directed evolution allowed the development of antibodies with higher affinity (K(d) = 1 nM) and specificity (>300-fold) for apo-CaM from a parental single-domain clone with both a modest affinity and specificity for that particular isoform. CaM-binding activity was unexpectedly lost upon conversion of both conformation-specific clones into soluble fragments. However, these results demonstrate that conformation-specific antibodies can be quickly and easily isolated by directed evolution using the yeast display platform.     

Integration Concepts for the Fabrication of LTCC Structures

At the Keck Smart Materials Integration Laboratory at Penn State University, low-temperature co-fired ceramic (LTCC) material systems have been used to fabricate a number of devices for a variety of applications. This article presents an overview of the integration of the concepts and materials that we have used to achieve miniaturization and unique device function. Examples of microwave filters, metamaterial antennas, and a dielectrophoretic cell sorter will be presented, with emphasis on device modeling and design, prototype construction methods, and test results.     

Numerical Simulation of Anisotropic Shrinkage in a 2D Compact of Elongated Particles

Microstructural evolution during simple solid-state sintering of two-dimensional compacts of elongated particles packed in different arrangements was simulated using a kinetic, Monte Carlo model. The model used simulates curvature-driven grain growth, pore migration by surface diffusion, vacancy formation, diffusion along grain boundaries, and annihilation. Only the shape of the particles was anisotropic; all other extensive thermodynamic and kinetic properties such as surface energies and diffusivities were isotropic. We verified our model by simulating sintering in the analytically tractable cases of simple-packed and close-packed, elongated particles and comparing the shrinkage rate anisotropies with those predicted analytically. Once our model was verified, we used it to simulate sintering in a powder compact of aligned, elongated particles of arbitrary size and shape to gain an understanding of differential shrinkage. Anisotropic shrinkage occurred in all compacts with aligned, elongated particles. However, the direction of higher shrinkage was in some cases along the direction of elongation and in other cases in the perpendicular direction, depending on the details of the powder compact. In compacts of simple-packed, mono-sized, elongated particles, shrinkage was higher in the direction of elongation. In compacts of close-packed, mono-sized, elongated particles and of elongated particles with a size and shape distribution, the shrinkage was lower in the direction of elongation. The results of these simulations are analyzed, and the implication of these results is discussed.     

Microwave Dielectric Properties and Low-Temperature Cofiring of BaTe4O9 with Aluminum Metal Electrode

Polycrystalline BaTe₄O₉ ceramic compound was investigated as a promising microwave dielectric compound for low-temperature cofired ceramics (LTCC) applications. The binary phase BaTe₄O₉ was synthesized and subsequently densified over the temperature range of only 500°–550°C, which allows for low-temperature cofiring with aluminum metal. The dielectric properties of BaTe₄O₉ ceramics sintered at 550°C for 2 h were determined in the microwave region of 12–14 GHz. The dielectric constant and Q × f product obtained were 17.5 and 54 700 GHz at 12 GHz, respectively. The temperature coefficient of resonance frequency showed a negative value of −90 ppm/°C. In terms of its evaluation for LTCC, the BaTe₄O₉ composition was found to be chemically compatible and successfully cofired with highly conductive aluminum electrode, while maintaining good electrical performance.     

Initial Attempt to Characterize Oxidation Damage in C/Sic Composite Using an Ultrasonic Guided Wave Method

An ultrasonic guided wave scan system was used to non-destructively monitor damage over time and position in a C/enhanced SiC sample that was creep tested to failure at 1200°C in air at a stress of 69 MPa (10 ksi). The use of the guided wave scan system for mapping evolving oxidation profiles (via porosity gradients resulting from oxidation) along the sample length and predicting failure location was explored. The creep-rupture tests were interrupted for ultrasonic evaluation every two hours until failure at ∼17.5 cumulative hours.     

Influence of the Rare-Earth Element on the Mechanical Properties of RE–Mg-Bearing Silicon Nitride

Silicon nitride bulk ceramics with varying compositions of the grain boundary phase but similar grain sizes were developed, which allows to analyze the influence of the grain boundary chemistry on mechanical properties. Micrographs of the crack path reveal a much weaker interface when the rare-earth element in the grain boundary phase changes from a small to a large rare-earth cation (RE³⁺). Room temperature measurements of toughness and bending strength show that weaker grain boundaries result in a higher toughness, but in a decreased strength of the bulk material. This investigation demonstrates that the mechanical behavior of silicon nitride can be readily tuned by chemical composition changes of the grain boundary phase, but that a compromise between toughness and strength has to be found when designing the material.     

Effect of photoinitiator segregation on polymerization kinetics in lyotropic liquid crystals

Through photopolymerization lyotropic liquid crystalline (LLC) phases may be templated onto organic polymers to yield highly complex nanostructures. In order to understand the unique polymerization behavior controlling structural development in LLC media, the polymerization kinetics in these systems have been studied using several commercially available photoinitiators. Although monomer segregation and diffusional restrictions largely govern the kinetics in these systems, the initiation may also be influenced by changing LLC order and composition. Nonpolar monomers, which partition to the oil soluble domains of the LLC phase typically display the fastest rate of polymerization in micellar aggregates. The rate decreases in phases with larger nonpolar domains due to decreasing localized double bond concentration. Polar monomers exhibit the opposite behavior. However, the segregation of photoinitiator may contribute to significantly different trends in polymerization behavior. Relatively mobile initiators, displaying favorable interaction with water, yield a trend in polymerization that is governed primarily by monomer and diffusional effects. When bulkier, hydrophobic initiators are used, the polymerization appears much less dependent on these effects. Rather than the decreasing rate usually observed at higher surfactant concentrations, polymerization of oil soluble monomers with the less mobile initiators shows the opposite trend of increasing rate at higher surfactant concentration. This behavior likely results from increasing initiator efficiency of the bulky, hydrophobic initiator in the surfactant rich environment.     

Effects of microreactor wall heat conduction on the reforming process of methane

In this paper, the effect of wall conduction of an autothermal tubular methane microreformer is investigated numerically. It is found that the axial wall conduction can strongly influence the performance of the microreactor and should not be neglected without a careful a priori investigation of its impact. By increasing the wall thermal conductivity, the maximum wall surface temperature is decreased. Due to the complex exothermic–endothermic nature of the chemistry of reforming, the axial variation of the wall temperature is not monotonic. Methane conversion and hydrogen yield are strongly dependent on the wall inner surface temperature, hence the heat conduction through the channel wall. The equivalence ratio and the wall thickness also significantly affect the reforming effectiveness and must be carefully considered in reactor optimization. Furthermore, it is found that exothermic oxidation reaction mechanisms, especially partial oxidation, are responsible for syngas (hydrogen and carbon monoxide) production near the inlet. Farther downstream, in the oxygen deficient region, endothermic steam reforming is the main hydrogen producing mechanism. By increasing the thermal conductivity, steam reforming becomes stronger and partial oxidation becomes weaker. For all investigated inlet conditions, the highest hydrogen yield is obtained for no or very low conductive walls.     

Adsorption of aroma chemicals on cotton fabric from aqueous systems

The adsorption of aroma chemicals on cotton fabric was studied relative to the surfactant concentration, surfactant type, water solubility, and fiber morphology. The adsorption increased with increasing surfactant concentration to a maximum near the critical micelle concentration, then decreased with further increases in surfactant concentration. The adsorption also was found to be highly dependent on the fiber surface area and pore structure; dramatic differences were observed between untreated and mercerized cotton fabric and are believed to be due to morphological differences. Cationic and anionic surfactants increased the aroma chemical adsorption, which varied with surfactant type, with cetyltrimethylammonium chloride (CTAC)>sodium dodecyl sulfate (SDS)>H₂O. Water solubility also influenced adsorption; in most cases, adsorption increased with water solubility. In addition, adsorption was also influenced by chemical structure and hydrophobic interactions. The adsorption of aroma chemicals on cotton fabric can be attributed to the aqueous solution being physically held in capillaries and pore structures within the fibular structure of cotton fiber and also to molecular interactions among the aroma chemical molecules, surfactants, and cotton substrate.