Progress 09/01/05 to 08/31/09
Outputs
OUTPUTS: The goal of this research is to elucidate the mechanism of plant virus recognition in molecularly imprinted polymers (MIPs) using already utilized techniques. The development of a virus imprinted MIP, which would apply to the indentification, classification, and removal of viruses. The separation of viruses and virus-like particles from various me dia represents an enormous challenge. Since virus MIPs must function in aqueous environments, our approach em- ploys a more exible non-covalent imprinting method which starts from a readily available polymer and utilizes an aqueous environment for both MIP synthe sis and testing. Crosslinked polymers imprinted against Tobacco mosaic virus (TMV) via non-covalent interactions were synthesized using poly (allylamine hydrochloride) (PAA), epichlorohydrin (EPI), and TMV. The role of MIP hydrogel electrostatic charge density on the recognition and selectivity properties of protein-imprinted hydrogels was also explored, and the effect of variations of the template extraction protocol on the MIP recognition properties were also studied in depth. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The TMV imprinted polymer exhibited an increase affinity to the target virus compared to the control polymer and demonstrated a preferential affinity (imprinting factor of 2.1), based on shape, to the target virus compared to a non-target virus, Tobacco necrosis virus (TNV). In contrast, there was no significant increase in binding of the control polymer to either target or non-target virus. Once it was determined that virus imprinted polymers can be successfully synthesized having preferential binding to a targeted virus, the synthesis procedure was optimized to obtain better binding characteristics to the targeted virus. Efforts were made to avoid polymer-template aggregation in the MIP pre polymerization mixture, and determine a proper wash solution by the ability to remove the templated virus from the crosslinked polymer. TMV imprinted hydrogels were synthesized using an optimized procedure and binding test performed on these MIPs to determine binding capacity, and more importantly, imprinting factor. The highest imprinting factor of 2.3 resulted from the MIP composed of 35 % PAA at pH 7, 15 %, ethylene glycol diglycidyl ether (EGDE), and 0.4 mg/mL TMV. The TMV imprinted hydrogels exhibited a lower binding capacity to TNV than when exposed to TMV. These results show that using optimized procedures, TMV MIPs with better shape selectivity can be achieved. The protein-imprinted MIP gels exhibited template recognition properties that were dependent on both the monomer charge density and on whether the chosen monomer carried a positive or negative charge. In addition, we found that common agents used in template extraction may be responsible for the specific and selective binding properties exhibited by molecularly imprinted polymers in many published studies.
Publications
- 5. Optimization of Virus Imprinting Methods to Improve Selectivity and Reduce Non-Specific Binding. L.D. Bolisay, J.N. Culver, and P. Kofinas. Biomacromolecules, 8(12), 3893-899, 2007.
- 6. Molecular Imprinting of Peptides and Proteins in Aqueous Media. D. S. Janiak, and P Kofinas. Analytical and Bioanalytical Chemistry, 389(2), 399-404, 2007.
- 7. Molecular Imprinted Polymers for Tobacco Mosaic Virus Recognition. L.D. Bolisay, J.N. Culver, and P. Kofinas. Biomaterials, 27(22), 4165-4168, 2006.
- 8. Molecularly imprinted polymers for selective recognition of signal peptides. D. S. Janiak, and P. Kofinas. American Chemical Society National Meeting, Polymer Materials Science and Engineering, 96, 780, 2007.
- 9. A Study of homogeneity and template removal during virus imprinted polymer synthesis. L. D. Bolisay, J. N. Culver, and P. Kofinas. American Chemical Society National Meeting, Polymer Materials Science and Engineering, 96, 787, 2007.
- 10. 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. Abstracts of Papers of the American Chemical Society. AUG 28;230:U3560-U3560, 2005.
- 11. Virus recognition using molecularly imprinted polymer hydyrogels. L. D. Bolisay, J. N. Culver, and P. Kofinas. Abstracts of Papers of the American Chemical Society. AUG 28;230:U4273-U4273, 2005.
- 1. Effects of Charge Density on the Recognition Properties of Molecularly Imprinted Polyampholyte Hydrogels. D.S. Janiak, O.B. Ayyub, and P. Kofinas. Polymer, 51, 665-670, 2010.
- 2. Effects of Charge Density on the Recognition Properties of Molecularly Imprinted Polymeric Hydrogels. D.S. Janiak, O.B. Ayyub, and P. Kofinas. Macromolecules, 42(5), 1703-1709, 2009.
- 3. Block Copolymer Nanotemplating of Tobacco Mosaic and Tobacco Necrosis Viruses. A.V. Cresce, J.N. Culver, W.E. Bentley, and P. Kofinas. Acta Biomaterialia, 5(3), 893-902, 2009.
- 4. Towards oriented assembly of proteins onto magnetic nanoparticles. C-W Hung, T. Holoman, P. Kofinas, and W.E. Bentley. Biochemical Engineering Journal, 38(2), 164-170, 2008.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: We examined the interaction between a block copolymer template and a tobacco virus. A poly(styrene-b-4-vinylpyridine) block copolymer was loaded with nickel, and cast from a selective solvent mixture to form a cylindrical microstructure (PS/P4VP-Ni). The nickel ions were confined within the P4VP block of the copolymer. The binding of Tobacco mosaic virus and Tobacco necrosis virus on microphase separated PS/P4VP-Ni was examined. 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 PS/P4VP 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, resulting in PS/P4VP cylinders that had a strong anisotropic directional preference. When exposed to flowing solutions of TMV, the PS/P4VP-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 PS/P4VP-Ni cylinders. PARTICIPANTS: Linden D. Bolisay, Graduate Student Arthur V. Cresce, Graduate Student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Microphase separation of the PS/P4VP block copolymer cast from a mixed 91.5 % CHCl3 \ 8% THF selective solvent mixture resulted in a cylindrical microstructure with the nickel residing in the P4VP block. Nickel ion added in solution is confined within the block copolymer's P4VP microstructure by metal chelation to the pyridine nitrogen. A staining technique was developed to simultaneously visualize virus and block copolymer structure in TEM using a 1% solution of uranyl acetate. It was shown that TMV and TNV viruses remain on the surface of nickel-loaded PS/P4VP block copolymers after detergent Tween washes. The cylinder long axis of the microstructure was oriented using a hot press and a cooled channel die for quenching, resulting in PS/P4VP cylinders that had a strong anisotropic directional preference. This microstructure, when exposed by microtoming, was seen in the electron microscope to be able to bind both TMV and TNV virions when loaded with nickel. Without nickel, the binding ability of the surface was no different than that of the Spurr's epoxy used to fix the PS/P4VP for microtoming. The biodetergent Tween was used to test binding because of its known ability to disrupt weak, nonspecific binding in biological systems. In Tween washes of up to 2 hours, TMV remained visible on the surface in increasingly diminished, but still significant, amounts. TMV, because of its size, was seen to cross several cylinders and often formed aggregates with other nearby TMV, although this did not seem to have an affect on the binding ability of the PS/P4VP surface. It was also observed that when exposed to flowing solutions of TMV, the PS/P4VP-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 PS/P4VP-Ni cylinders. It is therefore concluded that unlike-charge Coulombic interactions is the dominant mechanism in binding of the negatively charged TMV and TNV to the positively charged PS/P4VP-Ni block copolymer surface. The viability of virus nanopatterning by the PS/P4VP-Ni surface is validated by these results.
Publications
- Block Copolymer Nanotemplating of Tobacco Mosaic and Tobacco Necrosis Viruses A.V. Cresce, J.N. Culver, W.E. Bentley, and P. Kofinas. 2009 Acta Biomaterialia, in press. DOI:10.1016/j.actbio.2008.10.013
- Imprinted Polymer Hydrogels for the Separation of Viruses L.D. Bolisay, and P. Kofinas. 2009 Macromolecular Symposia, in press.
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: Molecular imprinting is a technique that creates synthetic materials containing highly specific receptor sites that have an affinity for a target molecule. The goal of this work is to synthesize molecular imprinted polymers (MIPs) targeted for a specific plant or insect virus. The relevance of this study to agriculture and food systems relates to the identification, classification, and removal of viruses. This is currently a very difficult task, but the need is widespread in diverse sectors, including agriculture, homeland security, animal health, and crop protection. When large particles like viruses are imprinted, special consideration must be taken to ensure the formation of complementary cavities. Factors that influence imprint formation, include uniformity of the pre-crosslinked mixture and release of the virus template after crosslinking. Objectives: 1. MIP hydrogels will be synthesized, having nanoscale imprinted cavities of the size and shape of specific model plant
and insect viruses. Methods will be developed for the detection, and quantification of each molecular imprint. 2. The MIP's total virus infectivity, specificity, and binding and dissociation kinetics with the nanoscale virus template will be evaluated. Specific virus recognition will be optimized. 3. The contribution of template and matrix functional groups on the hydrogel, in the recognition ability of the MIP will be determined.
PARTICIPANTS: Linden D. Bolisay, Graduate Student Brendan Casey, Graduate Student Chi-Wei Hung , Graduate Student Peter Kofinas PI William E. Bentley Co-PI James N. Culver Co-PI
Impacts
Methodologies and key results: Experiments using TMV- imprinted and non-imprinted polymer hydrogels in TMV solutions showed a two-fold increase in affinity of MIPs to TMV compared to non-imprinted control polymers (NIPs). The TMV-imprinted hydrogels were subjected to the same experiment but placed in solutions of Tobacco Necrosis Virus (TNV), an icosahedral virus used as the non-targeted virus, and showed no increased affinity when compared to NIPs. Polymer-virus aggregates formed when poly (allylamine hydrochloride) (PAA) was mixed with TMV at low polymer concentrations ( < 0.0001 % w/v ), but such aggregates were prevented at high polymer concentrations ( > 25 % w/v). Various wash protocols were compared for their ability to remove the virus template from the crosslinked MIP, with sodium hydroxide (1 M) exhibiting the best performance. Based on these results, optimized MIPs targeted for TMV virus were synthesized, exhibiting a high affinity to TMV (imprinting factor
of 2.3) and low affinity to Tobacco necrosis virus, (TNV), the non-target virus. Impacts: This research has demonstrated that molecular imprinting of viruses can be used to selectively induce binding capacity of target viruses based on shape differences of their virions. MIPs against plant and insect viruses may be an attractive and inexpensive alternative to existing techniques. Acting as a viral "sponge" the MIP could be designed to selectively remove one or more viral species.
Publications
- "Molecular Imprinted Polymers for Tobacco Mosaic Virus Recognition" L.D. Bolisay, J.N. Culver, and P. Kofinas. Biomaterials, 27(22), 4165-4168, 2006.
- "Virus recognition using molecularly imprinted polymer hydyrogels" L.D. Bolisay, J.N. Culver, and P. Kofinas Polym. Prepr. (Am. Chem. Soc. Div. Polym. Chem.), 46, 2, 1184, 2005.
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Progress 09/01/05 to 08/31/06
Outputs
The aim was to create MIPs targeted for TMV, which would preferentially bind TMV over other viruses of different shapes. Experiments using TMV- imprinted and non-imprinted polymer hydrogels in TMV solutions showed a two fold increase in affinity to TMV compared to non-imprinted control polymers (NIPs). The TMV-imprinted hydrogels were subjected to the same experiment but placed in solutions of Tobacco Necrosis Virus (TNV), an icosahedral virus used as the non-targeted virus, and showed no increased affinity when compared to NIPs. Optimization has been performed to increase the affinity to the target molecule even further. MIPs using different crosslinkers as well as with varying degree of crosslinking have been synthesized to determine influence on virus affinity of the MIP to virus target molecule. The amount of virus template removal has also been investigated. This research has demonstrated that molecular imprinting of viruses can be used to selectively induce
binding capacity of target viruses based on shape differences of their virions.
Impacts
Molecular imprinting is a technique used to create synthetic polymers with a high affinity for a specific target molecule. The goal in this work is to synthesize molecular imprinted polymers (MIPs) targeted for a specific plant or insect virus. The relevance of this study relates to the identification, classification, and removal of viruses. This is currently a very difficult task, but the need is widespread in diverse sectors, including agriculture, homeland security, human and animal health, crop protection, and biologics production. Molecularly imprinted polymers against plant and insect viruses may be an attractive and inexpensive alternative to existing techniques. Acting as a viral sponge the MIP could be designed to selectively remove one or more viral species.
Publications
- Molecular Imprinted Polymers for Tobacco Mosaic Virus Recognition. L.D. Bolisay, J.N. Culver, and P. Kofinas. Biomaterials, 27(22) , 4165-4168, 2006.
- Virus recognition using molecularly imprinted polymer hydyrogels. L.D. Bolisay, J.N. Culver, and P. Kofinas Polym. Prepr. (Am. Chem. Soc. Div. Polym. Chem.), 46, 2, 1184, 2005.
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