ELECTROSPUN FIBERS FOR SPECIFIC CYTOTOXIC CONTAMINATION DETECTION AND FILTRATION FROM WATER

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007350
• Project No.: NYC-329402
• Project Start Date: Oct 13, 2015
• Project End Date: Sep 30, 2018

Project Director
Frey, MA.

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Fiber Science & Apparel Design

Non Technical Summary
We are creating water filters capable of capturing residual chemicals from chemotherapy drugs. These chemicals enter the water system in urine and are not adequately removed by current sewage treatment methods. The DNA breaking and mutating properties of cytotoxic drugs that are commonly used to treat cancer specifically pose a major threat; they are carcinogenic to people at low dosages, cause birth defects, and immune dysfunction.(Pharma-Cycle, 2012; Rowney et al., 2009) When cytotoxic drugs are excreted by patients a portion goes un-metabolized (Ensslin et al., 1994; Herman, 2013; Johnson et al., 2008), and studies have shown the presence of these drugs and their metabolites in patients' urine(Jardine et al., 1978) and water supplies.(Bell et al., 2011; Rowney et al., 2009) This is of concern for people and the environment as cytotoxic drugs target rapidly growing cells, putting women and children particularly at risk.(Rowney et al., 2009) Low concentrations of these "emerging pollutants," as the EPA calls them, in the environment and difficulty in detecting them at nano-gram levels make removal at water treatment plants insufficient and may result in undetected contaminates.(Kern et al., 2010; Metcalfe et al., 2003) Previous work on PLA-b-PEG nanofibers has allowed for determination of the proper percentage of PEG to result in the most hydroxyl groups at the fiers surface for functionalization. We plan to capture CP on nanofabrics fiber's functionalized surface for florescence detection and upon its success, a prototype nanofiber membrane device will be developed for filtration media. Unlike current nanofiltration media, the nanofiber membrane produced in this study would specifically capture CP on the filter's surface and filter it out at a higher percentage.

Animal Health Component

0%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
112	0210	1000	40%
314	5350	2000	40%
723	5370	1150	20%

Knowledge Area
112 - Watershed Protection and Management; 314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
5350 - Domestic and community water supply facilities and systems; 0210 - Water resources; 5370 - Sewage and waste disposal facilities and systems;

Field Of Science
1000 - Biochemistry and biophysics; 2000 - Chemistry; 1150 - Toxicology;

Keywords

environmental toxicology

water purification

nanofiber

cytotoxic

microfluidic

Goals / Objectives
Fibers will be made by electrospinning copolymers in homogenous solutions at a constant elevated temperature. X-ray photon spectroscopy analysis will be used to estimate the concentration of specific targeting antibodies present at the fiber surface, allowing for comparison of attachment methods. Scanning electron microscopy will be used to analyze the nanofibers diameters and porosity, this in turn will affect the capture of contaminates. Solutions with known concentration of Chlorpheniramine will be made and the concentration will be verified using either liquid chromatograph-mass spectrometry (Llewellyn et al., 2011; Wang et al., 2009) and/or gas chromatograph-mass spectrometry (Takamoto et al., 2004). The concentration of the solution will be re-evaluated to determine the amount of target solution captured by nanofiber fabrics. The antibody labeled nanofabric developed above will be placed into microfluidic channels. Target compound will be captured from aqueous solutions flowing through the channels. Specific detection of the target compound captured within the channel will be achieved with dye-encapsulated liposomes. Antibodies (H-1 receptor antibodies for Chlorpheniramine (Male et al., 2012), and polyclonal antibodies for Cyclophosphamide(CUSABIO BIOTECH CO.) will also be tagged with the dye-encapsulated liposomes to form a sandwich assay with the target compound bound to the fiber. Flourescence intensity will be measured as a function of chlorpheniramine/cyclophosphamide concentration in the starting solution.Antibodies for capture will be added to PEG in PLA/PLA-b-PEG nanofabrics using two methods (1) Botinylation and (2) PEGlation, to create fabrics to capture model compound Chlorpheniramine.By varying nanofabric morphology, porosity, and size, an ideal fabric will be determined to promote the greatest percent of Chlorpheniramine captured from aqueous solutions. Physical test conditions (temperature, pH, and chemical concentration in aqueous media). Fabrics immersed in test solutions will need to be immersed for an ideal amount of time. A set of time intervals will be used to test the amount of Chlorpheniramine absorbed on the fabrics. A microfluidic assay for detection and quantification of the target compound will be developed. Lastly tests will be performed using CP in the microfluidic sandwich assay and upon successful detection, a three layer nanofiltration membrane of PLA/PLA-b-PEG/-antibody and common nanofiltration media will be developed.

Project Methods
Fibers will be made by electrospinning copolymers in homogenous solutions at a constant elevated temperature. X-ray photon spectroscopy analysis will be used to estimate the concentration of specific targeting antibodies present at the fiber surface, allowing for comparison of attachment methods. Scanning electron microscopy will be used to analyze the nanofibers diameters and porosity, this in turn will affect the capture of contaminates. Solutions with known concentration of Chlorpheniramine will be made and the concentration will be verified using either liquid chromatograph-mass spectrometry (Llewellyn et al., 2011; Wang et al., 2009) and/or gas chromatograph-mass spectrometry (Takamoto et al., 2004). The concentration of the solution will be re-evaluated to determine the amount of target solution captured by nanofiber fabrics. The antibody labeled nanofabric developed above will be placed into microfluidic channels. Target compound will be captured from aqueous solutions flowing through the channels. Specific detection of the target compound captured within the channel will be achieved with dye-encapsulated liposomes. Antibodies (H-1 receptor antibodies for Chlorpheniramine(Male et al., 2012), and polyclonal antibodies for Cyclophosphamide(CUSABIO BIOTECH CO.)) will also be tagged with the dye-encapsulated liposomes to form a sandwich assay with the target compound bound to the fiber. Flourescence intensity will be measured as a function of chlorpheniramine /cyclophosphamide concentration in the starting solution.

Progress 10/13/15 to 09/30/18

Outputs
Target Audience:This work specifically targets wastewater treatment and detection of emerging pollutants including biproducts of chemotherapy drugs. Since October 1st, 2015, results were shared with the scientific community (academic and professional) via 6 peer reviewed publications, 3 conference papersand 7 presentations at conferences. Conferences including PITTCON '16 in Atlanta, GA, Gordon Conference on Biosensors in Newport, RI, the international 75th anniversary meeting of the Fiber Society in Mulhouse, FR, Tech Connect World Innovation Conference and Expo, June 2017 and The U.S. 75th meeting of the Fiber Society in October of 2016 attracted broad audiences including international, industrial and government scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the duration of this project, 1 Ph.D. student completed her dissertation, 3 post-doctoral researchers learned new techniques and broadened their research portfolios and 8 undergraduate students participated as research assistants. How have the results been disseminated to communities of interest?The PI for this project served as the keynote speaker for the 2018 Expanding Your Horizons conference at Cornell University, demonstrating electrospinning and dye absorption for an audience of approximately 500 middle school girls. Feedback strongly indicated that conference participants understood the concepts presented and potential uses for nanofibers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? PLA/PLA-b-PEG-biotin was determined to be the optimum version of these nanofibers based on maximizing biotin at the nanofiber surface, maximizing protein binding at the nanofiber surface and minimizing diffusion of biotin off of the fiber in aqueous systems. However, a significant fraction of biotin, the expensive and active ingredient in the system, was encapsulated within the fibers and unavailable for protein binding. Therefore, a new approach to activating nanofibers for protein binding was developed.Oxygen plasma treatment was used tocreate carboxyl groups atthe surface of electrospun poly(lactic acid) (PLA) nanofibers to increase both reactivity and hydrophilicity of the nanofibers.Well known Sulfo-NHS/EDC (Sulfo-N-hydroxysulfosuccinimide/1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride) chemistry was added at the carboxyl groups on PLA nanofibers. Subsequently, bovine serum albumin (BSA) protein was successfully immobilized on the reactive sites on the PLA surfaces. Additionally, two new methods of utilizing the nanofibers for protein capture have been developed. First, a rapid prototyping method for microfluidic devices based on double stick tape sandwiched between plexiglass sheets has been developed. Channel designs can be developed quickly in computer aided design tools and cut into the double stick tape using a laser cutter. Nanofiber mats are deposited on the channels which are then sandwiched between two plexiglass plates and clamped together using binder clips.Fluids can be pumped through the device using a syringe pump. In a second utilization method circles of the nanofiber membranes are cut to fit into standard filter housings allowing direct filtration of specific proteins from solutions of mixed proteins.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Najafi, M, Chery J., Frey W. M., (2018). Functionalized Electrospun Poly(Vinyl Alcohol) Nanofibrous Membranes with Poly(Methyl Vinyl Ether-Alt-Maleic Anhydride) for Protein Adsorption, Mater., 11 (6), https://doi.org/10.3390/ma11061002.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Najafi, M, Chery J., Frey W. M., (2018). Development of Electrospun Composite Nanofibers for Proteins Separation, 77th International Fiber Society Conference, UC Davis, 2018.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Xiao, M., Chery, J., Frey, M.,(2018) Functionalization of Electrospun Poly(vinyl alcohol) (PVA) Nanofiber Membranes for Selective Chemical Capture. ACS Applied Nano Materials. 1 pp.722-729, http://dx.doi.org/10.1021/acsanm.7b00180.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mesbah Najafi, (2018) Plasma Surface Modification of Electrospun Poly(lactic acid) (PLA) Nanofbers for Protein Immobilization, 77th International Fiber Society Conference, UC Davis, 2018.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:This work specifically targets wastewater treatment anddetection of emerging pollutants including biproducts of chemotherapy drugs. Results were shared with the scientific community (academic and professional) via peer reviewed publications and presentations at conferences. Conferences attracted broad audiences including international, industrial and government scientists. Presentation of this work was made at Tech Connect World Innovation Conference and Expo, June 2017 and The 75th meeting of the Fiber Society in October of 2016. Multiple presentations (oral and poster) were made at conferences to take advantage of broad audiences for both dissemination of and feedback on the research as well as efficiency in travel time and costs. Results of this work were also presented at Regensburg University in Regensburg, Germany and Luxembourg University, Luxembourg in group meetings with collaborators contributing analytical and physical materials expertise. Changes/Problems:Additional strategies for creating capture sites on fiber surfaces were deemed necessary to improve the presentation of the capture probes at the fiber surface and the stability of the fibers in aqueous systems. Strategies including custom polymerization and oxygen plasma treatment have been employed to improve the fiber properties and performance. We will focus additional effort on binding chemistries other than biotin during this years. This represents a significant change in strategy from the one step method of functional fiber production used to date to requiring post-treatment to create surface functionality. The improvement in surface function and performance will be balanced against the additional time, steps and costs involved. What opportunities for training and professional development has the project provided?One doctoral student has completed her Ph.D. based in part on this project. Two post-doctoral research associates participated, part-time, in this project learning new techniques and bringing expertise in polymerization andfiber science to the project. Two undergraduate research assistants participated in this project during the academic semesters and as summer REU students supported through the NSF funded CCMR-REU research Experience for Undergraduates program (DMR-1460428 and DMR-1719875). How have the results been disseminated to communities of interest?Progress of research was presented at the 75th anniversary meeting of the Fiber Society hosted by the Cornell University Department of Fiber Science & Apparel Design in October, 2016. Results were also presented at the TechConnect World Innovation Conference and Expo in Washington, DC in June, 2017. Ongoing work and brainstorming was also discussed with collaborators at Regensburg University and Luxembourg University. What do you plan to do during the next reporting period to accomplish the goals?Improvement of specific capture on nanofiber surfaces is now targeted. Although the PLA/PEG/biotin systems have been successful, biotin availability and stability on the fiber surface can still be improved. Several new methods for creating specific binding sites for our target chemicals on the nanofiber surfaces are planned. First, binding sulfo-NHS chemistry to the nanofiber surface as a bridge for protein binding will be attempted. Since this chemistry is notoriously finicky, activating the nanofiber surface with oxygen plasma to facilitate binding of the sulfo-NHS will be attempted. Click chemistry has also been suggested as more robust than the sulfo-NHS system. This would require some modification of our nanofiber surface, potentially using plasma, to create an azide site.

Impacts
What was accomplished under these goals? Significant advances were made in preparation of nanofibers with surface available biotin for biohazard capture. The effects of both solvent and copolymer block lengths on the stability of electrospun poly(lactic acid)/poly(lactic acid)-b-poly- (ethylene glycol) (PLA/PLA-b-PEG) and PLA/PLA-b-PEG-Biotin fibers in water. By tailoring the block length of copolymers PLA-b-PEG, water stability of electrospun fibers is improved over fibers reported previously. The solvent used also influenced the stability and hydrophilicity of resulting fibers. Fibers formed using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) have greater water stability, but less PEG at the surface of fibers than fibers spun from dimethylformamide (DMF). Attaching biotin to the end of PLA(3600)-b-PEG(2000) and spinning from DMF resulted, initially, in 7.6% of the total biotin incorporated into the fiber, assuming every PEG terminal had one biotin attached (1.1 mg of biotin per gram of fiber) available at the fibers' surface. In addition, PLA/PLA(3600)-b-PEG(2000)-Biotin spun from DMF hindered biotin migration to the aqueous phase, leaving 2% of the incorporated biotin remaining at the surface of fibers after 7 days of water exposure. Additionally, longterm shelf life of the PLA/PEG nanofibers was confirmed by monthly material characterization over the course of 9 months. Samples were stored in a controlled environment at 72 degrees F and 65% relative humidity for a period of 9 months. Some shift in surface wetability was seen after 2 weeks, however, no further changes in surface, composition or structure were measurable over the remainder of the testing period.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Xiao, M., Gonzalez, E., Monterroza, A. M., & Frey, M. (2017). Fabrication of thermo-responsive cotton fabrics using poly(vinylcaprolactam-co-hydroxyethyl acrylamide) copolymer. Carbohydrate Polymers, 174, 626-632. doi: 10.1016/j.carbpol.2017.06.092
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Xiao, M., Chery, J., Keresztes, I., Zax, D. B., & Frey, M. W. (2017). Direct characterization of cotton fabrics treated with di-epoxide by nuclear magnetic resonance. Carbohydrate Polymers, 174, 377-384. doi: 10.1016/j.carbpol.2017.06.077
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Shepherd, L. M., Gonzalez, E., Chen, E. X., & Frey, M. W. (2017). Increasing Stability of Biotin Functionalized Electrospun Fibers for Biosensor Applications. Acs Applied Materials & Interfaces, 9(2), 1968-1974. doi: 10.1021/acsami.6b14348
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Gonzalez, E., & Frey, M. W. (2017). Synthesis, characterization and electrospinning of poly(vinyl caprolactam-co-hydroxymethyl acrylamide) to create stimuli-responsive nanofibers. Polymer, 108, 154-162. doi: 10.1016/j.polymer.2016.11.053
• Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Shepherd, L.M., THE LIFECYCLE OF ADVANCED FIBERS: (I) A NEW METHOD FOR THE FABRICATION OF FIEBRS, (II) THE FUNCTIONALIZATION OF ELECTROSPUN PLA/PLA-B-PEG FIBERS, AND (III) THE DEGRADATION OF CELLULOSE FIBERS BY OXYGEN PLASMA, Ph.D. Dissertation, Cornell University, 2017

Progress 10/13/15 to 09/30/16

Outputs
Target Audience:Methods and early results were presented to a broad range of scientific audiences at PITTCON '16 in Atlanta, GA, Gordon Conference on Biosensors in Newport, RI, the 75th anniversary meeting of the Fiber Society in Mulhouse, FR. L. Shepherd, "Increasing Surface Available Biotin and Improving WaterStability of PLA/PLA-b-PEG Nanofibers", Mulhouse, FR M.W. Frey, 'Electrospun nanofibers for analyte purification', Gordon Research Conference on Bioanalytical Sensors, Newport, RI, June 2016. M.W. Frey, 'Nanofiber chemistry and synthesis and the impact on analytical systems', PITTCON, Atlanta, March 2016. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate students were trained and mentored in laboratory methods, data collection and analysis by post-doctoral researchers and advanced graduate students. A Ph.D. student associated with this project attended a leadership training course offered by the Cornell University graduate school. Post-docs associated with this project attended teaching, grant writing and other professional development workshops offered by the Cornell University graduate school. One undergraduate associated with this project participated in the Cornell Center for Materials Science (CCMR) research experience for undergraduates (REU) program (DMR-1460428 and DMR-1120296) How have the results been disseminated to communities of interest?Results have been disseminated via conference presentations and peer reviewed publications as detailed above. What do you plan to do during the next reporting period to accomplish the goals?The effects of both solvent and copolymer block lengths on the stability of electrospun poly(lactic acid)/poly(lactic acid)-b-poly- (ethylene glycol) (PLA/PLA-b-PEG) and PLA/PLA-b-PEG-Biotin fibers in water will be a focus in the coming year. By tailoring the block length of copolymers PLA-b-PEG, water stability of electrospun fibers may be improved over fibers reported previously. The solvent used may also influenced the stability and hydrophilicity of resulting fibers. Fibers formed using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) have greater water stability, but less PEG at the surface of fibers than fibers spun from dimethylformamide (DMF). Attaching biotin to the end of PLA(3600)-b-PEG(2000) and spinning from DMF is extpected to result in improved stabilityof the fibers and.hinder biotin migration to the aqueous phase.

Impacts
What was accomplished under these goals? Biotin surface functionalized hydrophilic non-water-soluble biocompatible poly(lactic acid) (PLA) nanofibers are created for their potential use as biosensors. Varying concentrations of biotin (up to 18 weight total percent (wt %)) were incorporated into PLA fibers together with poly(lactic acid)-block-poly(ethylene glycol) (PLA-b-PEG) block polymers. While biotin provided surface functionalization, PLA-b-PEG provided hydrophilicity to the final fibers. Morphology and surface-available biotin of the final fibers were studied by Field Emission Scanning Electron Microscopy (FESEM) and competitive colorimetric assays. The incorporation of PLA-b-PEG block copolymers not only decreased fiber diameters but also dramatically increased the amount of biotin available at the fiber surface able to bind avidin. Finally, fiber water stability tests revealed that both biotin and PLA-b-PEG, migrated to the aqueous phase after relatively extended periods of water exposure. The functional hydrophilic nanofiber created in this work shows a potential application as a biosensor for point-of-care diagnostics as demonstrated in prototype devices.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Edurne Gonz�lez, Margaret W. Frey, Synthesis, characterization and electrospinning of poly(vinyl caprolactam-co-hydroxymethyl acrylamide) to create stimuli-responsive nanofibers, Polymer, 108(13) 154-162, ISSN 0032-3861, http://dx.doi.org/10.1016/j.polymer.2016.11.053.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Gonz�lez, Edurne, Shepherd, Larissa M, Saunders, Laura, & Frey, Margaret W. (2016). Surface Functional Poly (lactic Acid) Electrospun Nanofibers for Biosensor Applications. Materials, 9(1), 47.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Larissa M. Shepherd, Edurne Gonz�lez, Esther X. Chen, and Margaret W. Frey, Increasing Stability of Biotin Functionalized Electrospun Fibers for Biosensor Applications, ACS Applied Materials & Interfaces Article ASAP, DOI: 10.1021/acsami.6b14348
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: L. Shepherd, "Increasing Surface Available Biotin and Improving WaterStability of PLA/PLA-b-PEG Nanofibers", Mulhouse, FR
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: M.W. Frey, 'Electrospun nanofibers for analyte purification', Gordon Research Conference on Bioanalytical Sensors, Newport, RI, June 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: M.W. Frey, 'Nanofiber chemistry and synthesis and the impact on analytical systems', PITTCON, Atlanta, March 2016.