Phosphate sensing by fluorescent reporter proteins embedded in polyacrylamide nanoparticles
ABSTRACT Phosphate sensors were developed by embedding fluorescent reporter proteins (FLIPPi) in polyacrylamide nanoparticles with diameters from 40 to 120 nm. The sensor activity and protein loading efficiency varied according to nanoparticle composition, that is, the total monomer content (% T) and the cross-linker content (% C). Nanoparticles with 28% T and 20% C were considered optimal as a result of relatively high loading efficiency (50.6%) as well as high protein activity (50%). The experimental results prove that the cross-linked polyacrylamide matrix could protect FLIPPi from degradation by soluble proteases to some extent. This nanoparticle embedding method provides a novel promising tool for in vivo metabolite studies. It also demonstrates a universal method for embedding different fragile bioactive elements, such as antibodies, genes, enzymes, and other functional proteins, in nanoparticles for, for example, sensing, biological catalysis, and gene delivery.
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ABSTRACT: Little is known about regulatory networks that control metabolic flux in plant cells. Detailed understanding of regulation is crucial for synthetic biology. The difficulty of measuring metabolites with cellular and subcellular precision is a major roadblock. New tools have been developed for monitoring extracellular, cytosolic, organellar and vacuolar ion and metabolite concentrations with a time resolution of milliseconds to hours. Genetically encoded sensors allow quantitative measurement of steady-state concentrations of ions, signaling molecules and metabolites and their respective changes over time. Fluorescence resonance energy transfer (FRET) sensors exploit conformational changes in polypeptides as a proxy for analyte concentrations. Subtle effects of analyte binding on the conformation of the recognition element are translated into a FRET change between two fused green fluorescent protein (GFP) variants, enabling simple monitoring of analyte concentrations using fluorimetry or fluorescence microscopy. Fluorimetry provides information averaged over cell populations, while microscopy detects differences between cells or populations of cells. The genetically encoded sensors can be targeted to subcellular compartments or the cell surface. Confocal microscopy ultimately permits observation of gradients or local differences within a compartment. The FRET assays can be adapted to high-throughput analysis to screen mutant populations in order to systematically identify signaling networks that control individual steps in metabolic flux.New Phytologist 02/2008; 180(2):271-95. DOI:10.1111/j.1469-8137.2008.02611.x · 6.55 Impact Factor
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ABSTRACT: Numerical simulations and experimental measurements were performed to determine the effect of smooth-walled fractures on particle swarms. The numerical simulations were based on a particle interaction approach that included a first order approximation for particle-wall interactions. Transparent cubic samples (100 mm x 100 mm x 100 mm) containing synthetic fractures, with uniform aperture distributions, were used to quantify the effect of aperture on swarm formation, swarm velocity, and swarm geometry using optical imaging. A series of experiments were performed to determine how swarm movement and geometry are affected as the walls of the fracture are brought closer together from 50 mm to 1 mm. The simulations and experimental data confirm that swarm velocity decreases with decreasing aperture because of a significant increase in drag on the particles from the walls. However, the experiments showed that by increasing the swarm particle number density, the swarm cohesiveness and velocity were maintained over longer distances in the fracture.