Date of Award

5-2023

Document Type

Thesis

Committee Chair/Advisor

Dr. Jordan Gilmore

Abstract

Chronic wounds (wounds that fail to proceed through the normal phases of wound healing and cannot restore to full function in three months) can pose physical and financial burdens on patients. The most common chronic wound is diabetic foot ulcers, which affect 6.3% of the global population [1]. Current diagnostic and tracking methods to monitor wound healing are subjective and involve visual inspection and observation of the wound as it heals. However, when dealing with chronic wounds, the wound healing process is more difficult to observe due to the delayed nature of healing. There is also a need to closely monitor the wound healing process of a diabetic ulcer, as nonhealing of the ulcer to lead to dangerous complications. Therefore, it is important to develop quick, accurate, and objective methods to diagnose and monitor the healing of chronic wounds. In this study, we report the use of biosensors to detect cytokines commonly found in the wound bed, based on impedance measurements.

Many biosensor designs employ immobilization of proteins such as antibodies and other bio-recognizing elements to yield highly specific sensors. Improving the binding strength of such elements to the target analyte increases the signal-to-noise ratio of such platforms. In this study, we report the use of physical adsorption to facilitate immobilization of capture antibodies on the surface of solution blow spun (SBS) nanofiber substrates in a modified enzyme-linked immunoassay (ELISA) approach. A multi-layered, conductive nanofiber composite was used as the base biosensor. Then, a flexible conductive silver ink was screen printed on Polyethylene Terephthalate (PET) film to serve as the conductive base material. Poly (l-lactide acid) (PLA) and Multi-walled Carbon Nanotubes (MWCNT) were solution blow spun over the silver interdigitated electrode pattern to provide a hydrophobic scaffold for antibody immobilization. Physical adsorption via soaking was used to fix antibodies to the SBS PLA/MWCNT samples. Fluorescently tagged antibodies were used to verify protein adsorption along with bicinchoninic acid assay. For impedance sensing applications, an LCR meter was used to measure the impedance and phase angle of the electrode.

Results show that Donkey anti-goat capture antibodies conjugated with Alexa Fluor 594 were successfully immobilized on SBS PLA/MWCNT nanofiber substrate. Qualitative fluorescent signal verified the presence of proteins on the surface. Increasing the soak time also caused an increase in fluorescent signal. BCA results showed that SBS scaffolds can adsorb protein (albumin), which is essential for proper biosensor immobilization techniques. The incorporation of carbon also generally decreased the adsorption of PLA scaffolds. However, including more PLA in the composite allowed for more protein immobilization. Additionally, SBS PLA/MWCNT remained conductive post immobilization, indicating that an electrical signal could still be obtained from a functionalized electrode.

The protocol employed allows for in situ nanofiber fabrication and immobilization of antibodies for increased biosensor specificity. Results demonstrate the ability to fabricate electrodes for protein adsorption via physical adsorption on SBS MWCNT electrodes. Future efforts will be focused on optimizing electrical signal-to-noise ratio and making the biosensors more uniform, which can potentially help reduce variability during testing.

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