development-of-a-3d-graphene-electrode-dielectrophoretic-device

Development of a 3D Graphene Electrode Dielectrophoretic Device

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The ...

Many animal viruses produce cytopathic effects in their host cells during a productive infection. While some virus infections can be assayed by the production of plaques, many viruses, while producing cytotoxicity, do not easily form plaques, or do not form plaques at all. Additionally, viruses within families such as the parvoviruses may have different preferred forms of titration making comparative virology difficult even among related groups. Porcine parvovirus (PPV), canine parvovirus (CPV), and minute virus of mice (MVM) are usually titrated using different infectivity assays. A direct comparison of infectious virus titer between these parvoviruses was sought, and a tetrazolium salt assay, MTT has been applied to measure cytopathic effect produced by viral infection for different members of the parvovirus family. Infectious PPV measured using the MTT and the TCID50 assays exhibited excellent correlation and titers for CPV and MVM were consistently duplicated using the MTT assay. The MTT assay was also applied to an unrelated virus, Sindbis, which is routinely titrated by plaque assay. MTT titration of Sindbis virus mutants was found to be valuable for preliminary screening. This assay can be adapted, by correlation to an accepted titration method, to any viral system which produces measurable cytopathic effect.

A colorimetric assay for viral agents that produce cytopathic effects.

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non-enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5-5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.

Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma.

In previous work, we identified two trimeric peptide ligands (designated WRW and KYY), which bound specifically to porcine parvovirus (PPV) and demonstrated their ability to capture and remove the virus from solutions containing 7.5% human blood plasma. This article examines the influences of peptide density and the presence of an ethylene oxide spacer arm on the efficiency of virus capture using these two ligands. The WRW peptide bound the most virus from plasma solutions at the lowest peptide density tested (0.008 mmol/g dry resin), and binding was enhanced by the presence of the spacer arm. On the other hand, the KYY peptide bound the most viruses at the same low peptide density, but it performed better in the absence of the spacer arm. Of the two, the binding efficiency of the WRW peptide was more sensitive to peptide density and spacer arm presence. These results indicate that low peptide densities enhance binding selectivity, facilitating specific peptide-virus binding even in the presence of plasma proteins which can theoretically bind nonspecifically.

Influence of peptide ligand surface density and ethylene oxide spacer arm on the capture of porcine parvovirus.

Insulin is an important biotherapeutic protein, and it is also a model protein used to study amyloid diseases, such as Alzheimer's and Parkinson's. The preparation of the protein can lead to small amounts of aggregate in the solution, which in turn may lead to irreproducible in vitro results. Using several pre-treatment methods, we have determined that pH cycling and diafiltration of the insulin removes microaggregates that may be present in the solution. These microaggregates were not detectable with traditional biochemical methods, but using small-angle neutron scattering, we were able to show that pH cycling reduces the radius of gyration of the insulin. Diafiltration removes the aggregates by size and pH cycling dissolves the aggregates by adjusting the pH through the pI of the protein. Pre-treating the insulin with either pH cycling or diafiltration allowed reproducible kinetics of fibrillation for the insulin protein. Microaggregates are a common problem in protein production, formulation, and preparation; here we show that they are the main cause for inconsistent behavior and how pH cycling and diafiltration can mitigate this problem.

Detection and reduction of microaggregates in insulin preparations.

Amyloids are insoluble, fibrous proteins formed through the aggregation of misfolded proteins. They accumulate in the tissue of individuals with degenerative diseases, such as Parkinson's and Alzheimer's. The purpose of this study was to determine whether fibril growth from an initial model fibril seed is unidirectional or bidirectional. The prevailing theory on amyloid formation is that a symmetric fibril elongates equally from both ends. This study provides evidence to the contrary; the process occurs predominately unidirectionally, demonstrating that amyloid fibrils may be asymmetric and propagate mostly in one direction. Alexa Fluor 568 labeled insulin fibrils were seeded into a native insulin solution and allowed to elongate at 65°C while the kinetics of fibril growth was monitored. The resulting elongated fibrils were labeled with thioflavin-T, and the fluorescent images of the fibrils show that a majority of the elongated fibrils propagated along only one end of the seed, with the remaining labeled fibrils having bidirectional elongation or no elongation. Using a crystallographic model, we offer a structural explanation for asymmetric growth of the insulin fibrils. Thus, instead of the current view that fibrils grow symmetrically from both ends of the fibril, this is the first evidence that insulin amyloid fibrils formed in solution are asymmetric and appear to grow from only one end.

Asymmetric amyloid fibril elongation: a new perspective on a symmetric world.

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.

Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability.

Assuring the microbiological safety of biological therapeutics remains an important concern. Our group has recently reported small trimeric peptides that have the ability to bind and remove a model nonenveloped virus, porcine parvovirus (PPV), from complex solutions containing human blood plasma. In an effort to improve the removal efficiency of these small peptides, we created a biased library of hexamer peptides that contains two previously reported trimeric peptides designated WRW and KYY. This library was screened and several hexamer peptides were discovered that also removed PPV from solution, but there was no marked improvement in removal efficiency when compared to the trimeric peptides. Based on simulated docking experiments, it appeared that hexamer peptide binding is dictated more by secondary structure, whereas the binding of trimeric peptides is dominated by charge and hydrophobicity. This study demonstrates that trimeric and hexameric peptides may have different, matrix-specific roles to play in virus removal applications. In general, the hexamer ligand may perform better for binding of specific viruses, whereas the trimer ligand may have more broadly reactive virus-binding properties.

Porcine parvovirus removal using trimer and biased hexamer peptides.

Appropriate surface attachment is essential for growing embryonic stem (ES) cells in an undifferentiated state. It is challenging to identify the optimal surface chemistry of the substrata for ES cell attachment and maintenance. Using a rapid, high-throughput polymerization and screening platform with a comprehensive library of 66 monomer-grafted membrane surfaces, the optimal substrate, N-[3-(dimethylamino)propyl] methacrylamide (DMAPMA) has been identified to support strong attachment, high expansion capacity, and long-term self-renewal of ES cells (up to 7 passages). This monomer-based, chemically defined, scalable, sustainable, relatively inexpensive, covalently grafted, and controllable polymeric substrate provides a new opportunity to manipulate surface chemistry for pluripotent stem culture.

High-throughput screening of substrate chemistry for embryonic stem cell attachment, expansion, and maintaining pluripotency.

In an effort to increase selectivity among proteins with crossflow ultrafiltration, we offer and demonstrate a comprehensive approach to fractionate proteins of similar molecular weight and relatively close pI values. This multidimensional approach involves optimizing membrane charge type and density together with operating conditions such as precise control of pH, ionic strength, and transmembrane pressure for reduced membrane fouling. Each filtration experiment was performed in cross-flow configuration for ∼20 min, allowing fast screening for optimal separation as determined by maximum selectivity, Ψ, and purity, P. Using our comprehensive approach for fractionating mixtures RNase A-lysozyme and BSA-hemoglobin, we obtained values of Ψ = 9.1, P = 95.7%, and Ψ = 6.5, P = 62.1%, respectively.

A multi-dimensional approach for fractionating proteins using charged membranes.

Osmolytes are natural compounds found in the cells of many organisms that stabilize intracellular proteins against environmental stresses. Protecting osmolytes can promote protein folding, whereas denaturing osmolytes have the opposite effect. A variety of osmolytes were tested for their antiviral activity against porcine parvovirus (PPV). PPV is a non-enveloped, icosahedral, single-strand DNA virus. We have discovered two protecting osmolytes, trimethylamine N-oxide (TMAO) and glycine that reduce the infectivity of PPV by four logs (99.99%). We hypothesize that both osmolytes stabilize viral capsid proteins and prevent them from assembling into viable virus particles. The advantage of the antiviral compounds found is that they can be applied post-infection, which increases their potential to serve as a therapeutic drug.

Reduction of porcine parvovirus infectivity in the presence of protecting osmolytes.

Membranes are an accepted technology for water purification. Membrane filtration can remove pathogens, including bacteria and viruses, by size. For small viruses that can have a diameter <25nm, removal by size leads to large membrane areas, high transmembrane pressures, low water flux, and frequent changing of membranes. In this work, we discovered that electrospun nanofibers made of chitosan and functionalized with a quaternary amine (HTCC) have the ability to adsorb a model non-enveloped virus, porcine parvovirus (PPV). To improve the virus removal of HTCC, we added graphene. Graphene both enhanced the ability to form nanofibers with HTCC and improved the virus removal. The hydrophobicity of graphene and the high charge of the HTCC create a system that can bind 95% of PPV. The HTCC/graphene nanofibers could be incorporated into microfiltration membranes and remove virus by adsorption. This would create a low pressure system that is more likely to benefit areas in need of fresh water.

Non-enveloped virus reduction with quaternized chitosan nanofibers containing graphene.

The burden of unsafe drinking water is responsible for millions of deaths each year. To relieve this burden, we are in search of an inexpensive material that can adsorb pathogens from drinking water. In this pursuit, we have studied the natural carbohydrate, chitosan. To impart virus removal features, chitosan has been functionalized with a quaternary amine to form quaternized chitosan N-[(2-hydroxyl-3-trimethylammonium) propyl] chitosan (HTCC). HTCC can be electrospun into nanofibers with the non-ionogenic polyvinyl alcohol (PVA), creating a high surface area mat. High surface area is a major requirement for effective adsorption processes. HTCC is antiviral and antimicrobial, making it a good material for water purification. However, HTCC dissolves in water. We have explored the parameters to crosslink the nanofibers with glutaraldehyde. We have imparted water stability so there is a maximum of 30% swelling of the fibers after 6h in water. The water stable fibers retain their ability to adsorb virus, as shown for an enveloped and nonenveloped virus. HTCC now has the potential to be incorporated into a microfiltration membrane that can remove viruses. This could create an inexpensive, low pressure filtration membrane for drinking water purification.

Article	Year
A colorimetric assay for viral agents that produce cytopathic effects. Journal of virological methods\| PubMed ID: 16516983	2006
Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma. Biotechnology progress\| PubMed ID: 18470993	2008
Influence of peptide ligand surface density and ethylene oxide spacer arm on the capture of porcine parvovirus. Biotechnology progress\| PubMed ID: 19634175	2009
Detection and reduction of microaggregates in insulin preparations. Biotechnology and bioengineering\| PubMed ID: 20717970	2011
Asymmetric amyloid fibril elongation: a new perspective on a symmetric world. Proteins\| PubMed ID: 20941707	2011
Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability. Biophysical journal\| PubMed ID: 21641325	2011
Porcine parvovirus removal using trimer and biased hexamer peptides. Biotechnology journal\| PubMed ID: 21751387	2012
High-throughput screening of substrate chemistry for embryonic stem cell attachment, expansion, and maintaining pluripotency. Macromolecular bioscience\| PubMed ID: 23239629	2013
A multi-dimensional approach for fractionating proteins using charged membranes. Biotechnology and bioengineering\| PubMed ID: 23296474	2013
Reduction of porcine parvovirus infectivity in the presence of protecting osmolytes. Antiviral research\| PubMed ID: 23648707	2013
Non-enveloped virus reduction with quaternized chitosan nanofibers containing graphene. Carbohydrate research\| PubMed ID: 24041631	2013
Virus adsorption of water-stable quaternized chitosan nanofibers. Carbohydrate research\| PubMed ID: 24561959	2014

Caryn Heldt

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