Progress 01/01/05 to 12/31/07
Outputs
OUTPUTS: This project employs funcionalized diblock copolymers containing metal ions that are capable of selectively adsorbing recombinant proteins and tobacco viruses. This research addresses NRI issues in Food Safety as well as in Agricultural and Food Security, specifically the development of methods for detecting and controlling food borne pathogens, and the early detection of pathogen pests and other threats to homeland security. The specific scientific challenges in this research were 1. The use of a block copolymer template for the creation of regularly-shaped metal loaded domains for the purpose of interacting with tobacco viruses. 2. The simultaneous visualization of nanoscale polymer pattern as well as bound virus. Our main reserch activities were 1. The synthesis and processing into thin films of Nickel functionalized block copolymers. 2. Recombinant protein and plant virus confinement on nanostructured polymer pattern. Hydrophilic/hydrophobic block copolymers were
synthesized and their hydrophilic block was loaded with metal ions. Protein binding ability was examined by exposing the metal-loaded copolymer film to histidine-tagged green fluorescent protein (hisGFP), washing with detergent to remove non-specifically bound protein, and measuring the surface fluorescence. Dissemination: Our USDA sponsored research was featured on PBS Nightly business report. It was part of the September 24 2007 broadcast.
PARTICIPANTS: Arthur V. Cresce, Graduate Student Angela L. Fu, Graduate Student Daniel S. Janiak, Graduate Student Peter Kofinas, PI, University of Maryland
Impacts
Outcomes: Our experimental results showed that nickel ions on the copolymer surface had superior hisGFP binding ability to copper and iron. Further investigation comparing the binding of hisGFP and GFP lacking the histidine tag, showed that the block copolymer was binding only hisGFP. It was concluded that the chelation between nickel on the polymer surface and histidine groups on the protein is the dominant binding force in the hisGFP-copolymer system. The block copolymer pattern was also exposed to solutions of tagged tobacco mosaic virus (TMV). A staining technique was developed to simultaneously visualize virus and block copolymer structure in TEM. Electron Microscopy revealed virus particles associated with block copolymer microphase separated domains, even after extensive washes with Tween. In contrast, virus associated with block copolymers lacking Ni were readily removed by Tween. The cylinder long axis of the microstructure was oriented using a hot press and a
cooled channel die for quenching. When exposed to flowing solutions of TMV, the block copolymer-Ni surface exhibited an ability to retain TMV in a partially aligned state, when the direction of flow coincided with the long axis of the microphase separated cylindrical morphology. These results suggest that Coulombic interactions provide a robust means for the binding of virus particles to block copolymer surfaces. Impacts: Exploiting the microphase separation of block copolymers is a facile method of synthesizing nanometer-sized structures, such as ordered arrays of metal nanoparticles. The interactions of recombinant proteins with block copolymer surfaces displaying nanoscale order could be used to investigate intercellular signaling, and for creating arrays of nanoreactors for lab-on-a-chip applications in the detection of food pathogens.
Publications
- "Selective adsorption of histidine-tagged green fluorescent protein by a norbornene diblock copolymer" A.V. Cresce, A.T. Lewandowski, W.E. Bentley, and P. Kofinas ACS National Meeting Proceedings, Polymeric Materials Science and Engineering, 93,195, 2005.
- "Block Copolymer Nanotemplating of Tobacco Mosaic and Tobacco Necrosis Viruses" A.V. Cresce, J.N. Culver, W.E. Bentley, and P. Kofinas. Acta Biomaterialia, submitted, to be reviewed in 2008.
- "Nanopatterning of Recombinant Proteins Using Block Copolymer Templates" A.V. Cresce, W.E. Bentley, and P. Kofinas. Macromolecules, 39(17), 5826-5829, 2006.
|
Progress 01/01/06 to 12/31/06
Outputs
Block copolymers were exposed to nickel salts while in solution, resulting in high specificity in targeting the nickel ion to the receptive block of each block copolymer. Transmission electron micrographs showed that nanoparticle formation is not observed. Rather, the receptive block complexes the nickel ion and confines it within its poly(4-vinylpyridine) with apparently minimal leakage between blocks. The binding between tobacco viruses and the copolymer occurs because of unlike-charge Coulombic interaction. The viruses studied (Tobacco mosaic and Tobacco necrosis virus) carry a net negative surface charge at neutral pH. The block copolymers, loaded with metal ions carry a net positive charge. It is assumed that this interaction is the dominant mechanism of virus binding to the metal-loaded block copolymer surface. Tween detergent was used to examine the strength of the binding of the virus to the copolymer surface by disrupting weak functional group interactions
between the virus and the copolymer. Since it is theorized that the interaction between the virus and copolymer is Coulombic, viruses should remain on the surface after washing with Tween. The purpose of experiments presented here was to provide a better understanding of the nature of the virus-copolymer interaction. It was necessary to resolve the tendency of rodlike virus particles to lie randomly on the copolymer surface rather than self-orienting to maximize contact with the attracting block of the copolymer surface. To have any chance of large-scale ordering of the virus, the surface of the block copolymer itself must have a degree of long-range microstructural order. This was accomplished through the use of applied stress above the glass transition temperature. Tween detergent was again used to disrupt weak bonding between the copolymer and the virus.
Impacts
This study is unique in its use of a block copolymer template for the creation of regularly-shaped metal loaded domains for the purpose of interacting with tobacco viruses. Block copolymers present a unique advantage in that they inherently form periodic domains of nanometer size. Control over domain morphology can be exerted through simple choice of block length and composition, allowing a wide range of available morphologies and functional groups.
Publications
- Nanopatterning of Recombinant Proteins Using Block Copolymer Templates. A.V. Cresce, W.E. Bentley, and P. Kofinas. Macromolecules, 39(17), 5826-5829, 2006.
|
Progress 01/01/05 to 12/31/05
Outputs
The goal of this research is to investigate methods of nanoscale confinement and patterning of biological entities such as proteins and viruses by using the templating abilities of block copolymers. This study utilizes proven polymerization and characterization techniques to understand and control the interactions between polymer surface and biomolecule. It has been shown through initial experiments that it is possible to form microphase separated domains from a diblock copolymer that are able to template the formation of metal nanoparticles which chelate histidine-tagged green fluorescent protein (hisGFP). Ring-opening metathesis polymerization (ROMP) was used to synthesize norbornene-norbornene dicarboxylic acid (NOR/NORCOOH) diblock copolymers exhibiting a spherical microphase separation. When mixed with nickel ions, this block copolymer was shown to template the formation of spherical nanoparticles of metal. Initial fluorescence spectroscopy results indicate that
exposed metal nanoparticles on the polymer surface are able to strongly chelate hisGFP. The protein binding affinity was tested through the use of strong biological detergent washes. Preliminary experiments have also shown that a microphase separated block copolymer surface will bind tobacco mosaic virus (TMV). Ring-opening metathesis polymerization (ROMP) allowed control over the polymerization such that diblocks of various block ratios were produced with high yield and low polydispersity (PDI). Preliminary tests were performed with a NOR/NORCOOH copolymer with a block ratio of 400:50 NOR:NORNORCOOH. Gel permeation chromatography (GPC) was able to show that the copolymer was within 5 percent of the target molecular weight (46773 g/mol) for a 400:50 NOR/NORCOOH diblock copolymer. The GPC also gave a PDI of 1.21. Transmission electron microscopy (TEM) allowed direct observation of the copolymer microphase separated morphology. The 400:50 NOR/NORCOOH block ratio produced spherical
domains of NORCOOH with an average diameter of 30 nm. Once the polymer had undergone film formation through solution casting, the surface of the copolymer was doped with metal by exposing it to an aqueous solution of metal salts. Histidine-tagged green fluorescent protein (hisGFP) was chosen to test the affinity of the surface to tagged proteins. Fluorescence spectroscopy showed that the NOR/NORCOOH copolymer surface was able to chelate some hisGFP through interaction of surface metal groups and the histidine affinity tag of the hisGFP. Tobacco mosaic virus (TMV) was selected to test the viability of binding other biological species to a copolymer surface. To test this system, the sPSTB copolymer exhibiting cylindrical morphology was selected. The polystyrene blocks of this triblock copolymer were partially sulfonated to give the polystyrene hydrophilic properties. This sulfonated block was used to template the formation of metal nanoclusters in the sPSTB copolymer. When examined in
the TEM, it was shown that the metal-loaded polymer had the capacity to adsorb the TMV on its surface.
Impacts
This study could result in the production of rapid and efficient protein separation devices for use in purification and structural studies of proteins and viruses. This research addresses NRI issues in Food Safety as well as in Agricultural and Food Security, specifically the development of methods for detecting and controlling food borne pathogens, and the early detection of pathogen pests and other threats to homeland security. There is much practical significance that can be taken from this project. The polymer surface, functioning as a simple hexahistidine-tagged protein chelator, can serve as a fast protein separation device for protein purification, and could possibly be integrated into a detection system designed to identify and quantify biological molecules and entities. Nanopatterning of protein particles will allow for greater ease and speed in structural studies of proteins, especially x-ray crystallography which relies on protein crystals to derive
structural data. A similar situation exists for nanopatterned viruses, allowing for for x-ray analysis of the virus particle. A patterned and virus-specific surface will also allow for the study of the virus capsid protein groups, a critical piece of information for infection studies.
Publications
- "Separation of histidine-tagged green fluorescent protein by a norbornene diblock copolymer" Arthur V. Cresce, Angela T. Lewandowski, William E. Bentley, and Peter Kofinas, 2005 BMES Annual Fall Meeting, Baltimore, MD
- "Selective adsorption of histidine-tagged green fluorescent protein by a norbornene diblock copolymer" Arthur V. Cresce, Angela T. Lewandowski, William E. Bentley, and Peter Kofinas ACS National Meeting Proceedings, Polymeric Materials Science and Engineering, 93,195, 2005.
|
|