A new class of highly active solid base catalysts for biodiesel production was developed by creating hierarchically porous aluminosilicate geopolymer with affordable precursors and modifying the material with varying amounts of calcium. For the catalysts containing ≥8 wt% Ca, almost 100% conversion has been achieved in one hour under refluxing conditions with methanol solvent, and the high catalytic activity was preserved for multiple regeneration cycles. Temperature-programed desorption studies of CO2 indicate that the new base catalyst has three different types of base sites on its surface whose strengths are intermediate between MgO and CaO. The detailed powder X-ray diffraction (PXRD) and X-ray photoelectron spectroscopic (XPS) studies show that the calcium ions were incorporated into the aluminosilicate network of the geopolymer structure, resulting in a very strong ionicity of the calcium and thus the strong basicity of the catalysts. Little presence of CaCO3 in the catalysts was indicated from the thermogravimetric analysis (TGA), XPS and Fourier transform infrared spectroscopy (FT-IR) studies, which may contribute to the observed high catalytic activity and regenerability. The results indicate that new geopolymer-based catalysts can be developed for cost-effective biodiesel production.

Electrophoretic and dielectrophoretic approaches to separations can provide unique capabilities. In the past, capillary and microchip-based approaches to electrophoresis have demonstrated extremely high-resolution separations. More recently, dielectrophoretic systems have shown excellent results for the separation of bioparticles. Here we demonstrate resolution of a difficult pair of targets: gentamicin resistant and susceptible strains of Staphylococcus epidermidis. This separation has significant potential implications for healthcare. This establishes a foundation for biophysical separations as a direct diagnostic tool, potentially improving nearly every figure of merit for diagnostics and antibiotic stewardship. The separations are performed on a modified gradient insulator-based dielectrophoresis (g-iDEP) system and demonstrate that the presence of antibiotic resistance enzymes (or secondary effects) produces a sufficient degree of electrophysical difference to allow separation. The differentiating factor is the ratio of electrophoretic to dielectrophoretic mobilities. This factor is 4.6 ± 0.6 × 109 V m−2 for the resistant strain, versus 9.2 ± 0.4 × 109 V m−2 for the susceptible strain. Using g-iDEP separation, this difference produces clear and easily discerned differentiation of the two strains.

To achieve improved sensitivity in cardiac biomarker detection, a batch incubation magnetic microbead immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I. A sandwich immunoassay was performed in a simple micro-centrifuge tube allowing full dispersal of the solid capture surface during incubations. Following magnetic bead capture and wash steps, samples were analyzed in the presence of a manipulated magnetic field utilizing a modified microscope slide and fluorescent inverted microscope to collect video data files. Analysis of the video data allowed for the quantitation of myoglobin, heart-type fatty acid binding protein and cardiac troponin I to levels of 360 aM, 67 fM, and 42 fM, respectively. Compared to the previous detection limit of 50 pM for myoglobin, this offers a five-fold improvement in sensitivity. This improvement in sensitivity and incorporation of additional markers, along with the small sample volumes required, suggest the potential of this platform for incorporation as a detection method in a total sample analysis device enabling multiplexed detection for the analysis of clinical samples.

New and important separations capabilities are being enabled by utilizing other electric field-induced forces besides electrophoresis, among these is dielectrophoresis. Recent works have used experimentally simple insulator-based systems that induce field gradients creating dielectrophoretic force in useful formats. Among these, juxtaposing forces can generate gradient-based steady-state separations schemes globally similar to isoelectric focusing. The system of interest is termed gradient insulator-based dielectrophoresis and can create extremely high resolution steady-state separations for particles four nanometers to ten micrometers in diameter, including nearly all important bioparticles (large proteins, protein aggregates, polynucleotides viruses, organelles, cells, bacteria, etc.). A theoretical underpinning is developed here to understand the relationship between experimental parameters and resolution and to identify the best expected resolution possible. According to the results, differences in particles (and bioparticles) as small as one part in 10⁴ for diameter (subnanometer resolution for a one micrometer particle), one part in 10⁸ for dielectrophoretic parameters (dielectrophoretic mobility, Clausius-Mossotti factor), and one part in 10⁵ for electrophoretic mobility can be resolved. These figures of merit are generally better than any competing technique, in some cases by orders of magnitude. This performance is enabled by very strong focusing forces associated with localized gradients.

In proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the β barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.

Most current approaches for quantification of RNA species in their natural spatial contexts in single cells are limited by a small number of parallel analyses. Here we report a strategy to dramatically increase the multiplexing capacity for RNA analysis in single cells in situ. In this method, transcripts are detected by fluorescence in situ hybridization (FISH). After imaging and data storage, the fluorescence signal is efficiently removed by photobleaching. This enables the reinitiation of FISH to detect other RNA species in the same cell. Through reiterative cycles of hybridization, imaging and photobleaching, the identities, positions and copy numbers of a large number of varied RNA species can be quantified in individual cells in situ. Using this approach, we analyzed seven different transcripts in single HeLa cells with five reiterative RNA FISH cycles. This approach has the potential to detect over 100 varied RNA species in single cells in situ, which will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.