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Protein Assembly for Biosensing and Drug Delivery Devices.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Protein Assembly for Biosensing and Drug Delivery Devices./
作者:	Hartzell, Emily J.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2021,
面頁冊數:	154 p.
附註:	Source: Dissertations Abstracts International, Volume: 82-10, Section: B.
Contained By:	Dissertations Abstracts International82-10B.
標題:	Bioengineering. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28315611
ISBN:	9798597004747

Protein Assembly for Biosensing and Drug Delivery Devices.
Hartzell, Emily J.

Protein Assembly for Biosensing and Drug Delivery Devices. - Ann Arbor : ProQuest Dissertations & Theses, 2021 - 154 p.

Source: Dissertations Abstracts International, Volume: 82-10, Section: B.

Thesis (Ph.D.)--University of Delaware, 2021.

This item must not be sold to any third party vendors.

Nature provides an abundance of proteins to preform specific tasks that have been perfected through evolution. Many of these natural functions are quite valuable for the construction and customization of biosensing and drug delivery devices. These include: self-assembly of stable nanoscale structures, specific and selective binding to target molecules, optical signaling, therapeutic action, and stimuli responsive behavior. Efforts towards incorporating proteins into biosensing and drug delivery devices have created a protein conjugation toolbox, which includes a variety of chemical and biological methods. The method chosen for assembly is critical for device performance, as the desired protein function must be preserved while providing a stable linkage to the device. We can further expand the utility of these devices by assembly of proteins onto nanoscale architecture, conditionally controlling the assembly, as well as programming device disassembly in response to environmental stimuli.Nanoscale devices have unique properties bestowed by their size that make them particularly useful for biosensing and drug delivery applications. They are small enough to engage with biomolecular targets, while being large enough to escape renal clearance and be retained in the leaky vasculature of tumor tissue. Self-assembling protein nanoparticle platforms are particularly valuable scaffolds for such devices as their multiunit makeup allows the attachment of a high density of functional components, which can provide signal amplification, avidity for targeting, and a high therapeutic payload. We explored using the SpyCatcher/SpyTag conjugation system to modify Hepatitis B viral-like particles (HBV VLPs). This platform allowed us to modularly and site-specifically customize HBV VLPs for applications in both biosensing and drug delivery. The combination of HBV VLP architecture and SpyCatcher location within the VLP created a platform, which outperformed similar SpyCatcher nanoparticle devices. For the biosensing application, we were able to achieve over 1,500-fold amplification in nanoluciferase signal, which allowed detection of low nanomolar levels of thrombin with the naked eye. For the delivery application, we demonstrated efficient killing of inflammatory breast cancer cells with delivered yeast cytosine deaminase and 5-fluoro-cytosine prodrug.While the robust and user-friendly SpyCatcher/SpyTag conjugation system has been used for the assembly of functional proteins onto both protein-based and synthetic biosensing and drug delivery devices, one of the main limitations is that the reaction is unregulated. The ability to spatially and temporally control protein conjugation using light is of great interest for the biofabrication of hydrogels and solid state-based bioassays. We developed a means of conditionally controlling the SpyCatcher reaction with blue light by caging the SpyTag within the Avena sativa second light oxygen voltage (AsLOV2) photoreceptor domain. We identified three variants with rate constants 15-fold higher in the light state compared to the dark state. These three variants had maximal switching in different concentration ranges. Our blue light inducible SpyTag system (BLISS) was used to demonstrate the site-specific photopatterning of different proteins onto nickel coated surfaces.While conditional assembly creates exciting opportunities for developing biosensing and drug delivery devices, conditional disassembly in response to environmental signals is important for drug delivery applications. To expand our HBV-SpyCatcher drug delivery system, we desired to incorporate a previously studied redox sensitive PDZ affinity domain. We were able to streamline a process for loading and conjugating the redox sensitive module to our HBV VLPs for release of protein cargo. The efficiency of the streamlined conjugation was improved 5-fold, and the release of cargo from HBV VLPs was specific to the reducing conditions, showing promise for future delivery studies. Altogether, these studies demonstrate how protein assembly techniques can enhance and promote new function for biosensing and drug delivery devices.

ISBN: 9798597004747Subjects--Topical Terms:

657580
Bioengineering.
Subjects--Index Terms:

Biosensing

Protein Assembly for Biosensing and Drug Delivery Devices.
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520 $a Nature provides an abundance of proteins to preform specific tasks that have been perfected through evolution. Many of these natural functions are quite valuable for the construction and customization of biosensing and drug delivery devices. These include: self-assembly of stable nanoscale structures, specific and selective binding to target molecules, optical signaling, therapeutic action, and stimuli responsive behavior. Efforts towards incorporating proteins into biosensing and drug delivery devices have created a protein conjugation toolbox, which includes a variety of chemical and biological methods. The method chosen for assembly is critical for device performance, as the desired protein function must be preserved while providing a stable linkage to the device. We can further expand the utility of these devices by assembly of proteins onto nanoscale architecture, conditionally controlling the assembly, as well as programming device disassembly in response to environmental stimuli.Nanoscale devices have unique properties bestowed by their size that make them particularly useful for biosensing and drug delivery applications. They are small enough to engage with biomolecular targets, while being large enough to escape renal clearance and be retained in the leaky vasculature of tumor tissue. Self-assembling protein nanoparticle platforms are particularly valuable scaffolds for such devices as their multiunit makeup allows the attachment of a high density of functional components, which can provide signal amplification, avidity for targeting, and a high therapeutic payload. We explored using the SpyCatcher/SpyTag conjugation system to modify Hepatitis B viral-like particles (HBV VLPs). This platform allowed us to modularly and site-specifically customize HBV VLPs for applications in both biosensing and drug delivery. The combination of HBV VLP architecture and SpyCatcher location within the VLP created a platform, which outperformed similar SpyCatcher nanoparticle devices. For the biosensing application, we were able to achieve over 1,500-fold amplification in nanoluciferase signal, which allowed detection of low nanomolar levels of thrombin with the naked eye. For the delivery application, we demonstrated efficient killing of inflammatory breast cancer cells with delivered yeast cytosine deaminase and 5-fluoro-cytosine prodrug.While the robust and user-friendly SpyCatcher/SpyTag conjugation system has been used for the assembly of functional proteins onto both protein-based and synthetic biosensing and drug delivery devices, one of the main limitations is that the reaction is unregulated. The ability to spatially and temporally control protein conjugation using light is of great interest for the biofabrication of hydrogels and solid state-based bioassays. We developed a means of conditionally controlling the SpyCatcher reaction with blue light by caging the SpyTag within the Avena sativa second light oxygen voltage (AsLOV2) photoreceptor domain. We identified three variants with rate constants 15-fold higher in the light state compared to the dark state. These three variants had maximal switching in different concentration ranges. Our blue light inducible SpyTag system (BLISS) was used to demonstrate the site-specific photopatterning of different proteins onto nickel coated surfaces.While conditional assembly creates exciting opportunities for developing biosensing and drug delivery devices, conditional disassembly in response to environmental signals is important for drug delivery applications. To expand our HBV-SpyCatcher drug delivery system, we desired to incorporate a previously studied redox sensitive PDZ affinity domain. We were able to streamline a process for loading and conjugating the redox sensitive module to our HBV VLPs for release of protein cargo. The efficiency of the streamlined conjugation was improved 5-fold, and the release of cargo from HBV VLPs was specific to the reducing conditions, showing promise for future delivery studies. Altogether, these studies demonstrate how protein assembly techniques can enhance and promote new function for biosensing and drug delivery devices.
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