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Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Sam R. Nugen
This work is separated into two individual parts. The first part demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme and a microphage storage format.
1a) Polyvinylpyrrolidone (PVP) nanofibers were spun incorporating the enzyme horseradish peroxidase (HRP). Scanning electron microscopy of the spun nanofibers was used to confirm the non-woven structure with an average diameter of 155±34 nm. The HRP containing fibers were tested for a change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP reacting with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. During a 280 day storage study, the loss in activity began to stabilize at approximately 40%.
1b) We then investigated PVP fibers for T7 bacteriophage storage. The bacteriophage was added to mixtures of polyvinylpyrrolidone and water and electrospun the mixture onto a grounded plate. Trehalose and magnesium salts were added to the mixtures to determine their effect on the infectivity of the bacteriophage following electrospinning, and during storage. The fibers were stored at 20 °C in dry conditions for predetermined amounts of time. The loss of T7 infectivity was determined immediately following electrospinning and during storage using agar overlay plating and plaque counting. It was found that the addition of the magnesium salts resulted in less than a 1 log drop of infective T7 as compared to a drop of approximately 4 logs without the salts. The trehalose did not protect the T7 during the electrospinning process, but had a more significant storage effect. None of the electrospinning methods were as effective as lyophilization. The results indicate that the addition of magnesium salts protects the bacteriophage during the relatively violent and high voltage electrospinning process, but is not as effective as a protectant during storage of the dried T7. Conversely, the addition of trehalose into the electrospinning mix has little effect on the electrospinning, but a more significant role as a protectant during storage. Previous studies have attempted to encapsulate bacteriophage in water-soluble nanofibers for delivery and storage, but the treatment has typically resulted almost complete deactivation of the phage. Here we investigated the effect reagents on the activity of T7 during the electrospinning process as well as storage following the electrospinning.
Electrospinning can therefore be seen as a low-cost method for rapid dehydration of viruses.
2) The second part of this work is to produce a conductive polymer electrode. The conducting polymer PEDOT(Poly (3,4-ethylenedixoythiophene) modified with a functional group, gold nanoparticles or nano-structured fibers were investigated for use as electrochemical sensing electrodes. synthesized through a vapor polymerization route. [SN1] 1) Carboxylic functionalized EDOT monomers was synthesized through electrochemical polymerization. 2) Gold nanoparticles were deposited by monomer reduction. 3) PEDOT nanofibers were synthesized by electrospinning followed by vapor phase polymerization. 4) One-step PEDOT fibers were made from wet vapor-phase polymerization. These materials and structures were characterized using TEM, FE-SEM XPS and FTIR. The advantages of these films are the potential to provide bio-probes binding sites and nano-structures for increased sensitivity. The PEDOT matrix is known to improve catalytic oxidation of the ascorbic acid and functional carboxylic groups provide bonding sites. Additionally, the nanometer-sized gold particles or fiber structures have been demonstrated to allow nanomolar sensing of analyte. Thus, these methods should provide a bio-functionalizable surface with the possibility to detect nanomolar levels of a bio-analyte.
Dai, Minhui, "Functionalized Electrospun Nanofibers for Food Science Applications" (2016). Doctoral Dissertations. 714.