Enzyme driven bacterial detection and decontamination on surfaces
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Authors
Xu, Shirley
Issue Date
2024-08
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemical engineering
Alternative Title
Abstract
Bacteria are ubiquitous in our everyday lives and are often transmitted through surfaces. The most common places for bacterial transmission are crowded places, such as public transportation, classrooms, healthcare facilities, and food production establishments. The transmission of bacteria on these surfaces often lead to infections and sicknesses, especially in hospitals. Additionally, bacteria have been shown to remain on surfaces after several hours making transmission an even greater concern. The most common method of disinfection is through the use of chemicals. However, these chemicals are often toxic and corrosive to surfaces. As a result, enzymatic decontamination methods have been developed to provide a safe alternative to harsh chemicals. In the first part of this thesis, a surface coating was fabricated to contain an embedded enzyme using a one-step reaction. We analyzed the thermostability, reusability, and activity of these coatings. Additionally, we showed that these coatings contained a broad antibacterial and antiviral spectrum that efficiently kill pathogens in a short period of time. The other part of this thesis will be to deepen our understanding of pathogens on surfaces, where bacterial detection systems utilizing the cell binding domain (CBD) of cell lytic enzymes, which are capable of targeting bacteria with high specificity, were established. We first identified and characterized various CBDs for targeting Gram-positive bacteria. We then applied these CBDs for bacterial detection by coupling them with enzymes such as horseradish peroxidase (HRP) for signal amplification giving a limit of detection (LOD) of 105-106 cells/mL. We also coupled these CBDs with copolymeric dyes for enhanced fluorescent on the surface of the cell compared to monomeric dyes.
Furthermore, we utilized CBDs and a split fluorescent protein (FP) system for multimerization of CBD on the surface of cells for bacterial detection. Here, we designed a self-complementing FP complex where a multimeric FP chain fused with specific CBDs ((FP-CBD)n) is assembled inside the cell resulting in a daisy chained fusion protein with 1, 3, or 7 CBDs linked together. We demonstrated the functionality of these ((FP-CBD)n) for bacterial detection through complexing with HRP and saw a degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet within 15 min on a polystyrene surface.
Lastly, we expanded the use of ((FP-CBD)n) proteins by combining with a zymogen-based cascade for signal amplification. These ((FP-CBD)n) proteins were conjugated with an initiator enzyme, enteropeptidase, which activates a cascade consisting of trypsinogen and chymotrypsinogen. With this combined CBD-zymogen based system, a LOD of 105 cells/mL could be achieved. Overall, experiments in this thesis provided us with valuable methods for the detection and disinfection of pathogens on surfaces.
Description
August2024
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY