Phosphate sensing by fluorescent reporter proteins embedded in polyacrylamide nanoparticles.

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.

[1]  Marcus Fehr,et al.  Visualization of maltose uptake in living yeast cells by fluorescent nanosensors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Jean M. J. Fréchet,et al.  A macromolecular delivery vehicle for protein-based vaccines: Acid-degradable protein-loaded microgels , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  S. Daunert,et al.  Phosphate binding protein as the biorecognition element in a biosensor for phosphate. , 2004, Sensors and actuators. B, Chemical.

[4]  M. Delgado,et al.  A tunable hydrogel for encapsulation and controlled release of bioactive proteins. , 2002, Biomacromolecules.

[5]  R. Jerome,et al.  Enzyme immobilization in reactive nanoparticles produced by inverse microemulsion polymerization , 1996 .

[6]  Hong Gu,et al.  Synthesis and Characterization of Ratiometric, pH Sensing Nanoparticles with Covalently Attached Fluorescent Dyes , 2006 .

[7]  L. Looger,et al.  A novel analytical method for in vivo phosphate tracking , 2006, FEBS letters.

[8]  Jim-Min Fang,et al.  Two-arm ferrocene amide compounds: synclinal conformations for selective sensing of dihydrogen phosphate ion. , 2003, Organic letters.

[9]  P. Anzenbacher,et al.  Sensing of aqueous phosphates by polymers with dual modes of signal transduction. , 2004, Journal of the American Chemical Society.

[10]  G. Whitesides,et al.  Enzyme immobilization by condensation copolymerization into crosslinked polyacrylamide gels , 1980 .

[11]  R. Samulski,et al.  Polymeric nanogels produced via inverse microemulsion polymerization as potential gene and antisense delivery agents. , 2002, Journal of the American Chemical Society.

[12]  T. Kohira,et al.  Cooperation between artificial receptors and supramolecular hydrogels for sensing and discriminating phosphate derivatives. , 2005, Journal of the American Chemical Society.

[13]  E. Erbe,et al.  Transmission-electron microscopic observations of freeze-etched polyacrylamide gels☆ , 1978 .

[14]  E. Hirsch,et al.  Percolation and particle nucleation in inverse microemulsion polymerization , 1989 .

[15]  R. Kopelman,et al.  Analytical properties and sensor size effects of a micrometer-sized optical fiber glucose biosensor. , 1996, Analytical chemistry.

[16]  R. Jerome,et al.  Enzyme Immobilization in Nanoparticles Produced by Inverse Microemulsion Polymerization , 1994 .

[17]  A. English,et al.  NAD(P)H Sensors Based on Enzyme Entrapment in Ferrocene-Containing Polyacrylamide-Based Redox Gels , 1998 .

[18]  Sarah M. Buck,et al.  Nanoscale probes encapsulated by biologically localized embedding (PEBBLEs) for ion sensing and imaging in live cells. , 2004, Talanta: The International Journal of Pure and Applied Analytical Chemistry.

[19]  Kristina D. Micheva,et al.  Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. S. Fawcett,et al.  Molecular-Sieve Chromatography of Proteins on Granulated Polyacrylamide Gels , 1966 .

[21]  Amy E Herr,et al.  Photopolymerized cross-linked polyacrylamide gels for on-chip protein sizing. , 2004, Analytical chemistry.

[22]  R. Rüchel,et al.  Scanning electron microscopic observations of polyacrylamide gels. , 1975, Analytical biochemistry.

[23]  Raoul Kopelman,et al.  Fluorescent nano-PEBBLE sensors designed for intracellular glucose imaging. , 2002, The Analyst.

[24]  H. Clark,et al.  Optical nanosensors for chemical analysis inside single living cells. 1. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors. , 1999, Analytical chemistry.

[25]  J. Asua,et al.  Modeling inverse microemulsion polymerization , 1999 .

[26]  E. Kaler,et al.  Kinetics and Mechanism of Microemulsion Polymerization of Hexyl Methacrylate , 1997 .

[27]  Zeev Rosenzweig,et al.  Synthesis of Glyconanospheres Containing Luminescent CdSe−ZnS Quantum Dots , 2003 .

[28]  Philip A. Gale,et al.  Anion Recognition and Sensing: The State of the Art and Future Perspectives. , 2001 .