Assembly of Quorum Sensing Pathway Enzymes onto Patterned Microfabricated Devices

dc.contributor.advisorBentley, Williamen_US
dc.contributor.authorLewandowski, Angelaen_US
dc.contributor.departmentChemical Engineeringen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.description.abstractI report patterned protein assembly onto microfabricated devices using our unique assembly approach. This approach is based on electrodeposition of the aminopolysaccharide chitosan onto a selected electrode pattern of the device, and covalent conjugation of a target protein to chitosan upon biochemical activation of a genetically fused C-terminal pentatyrosine "pro-tag." With this approach, assembly is "spatially selective", occurring only at selected electrode patterns, and the entire process occurs under mild experimental conditions. Additionally, assembly is reversible and the devices reusable, as the deposited chitosan can be removed by simple incubation in dilute acid. Finally, the protein is covalently and robustly linked to chitosan through the pro-tag versus the native tyrosines, and thus our approach confers "orientational control". I have examined patterned assembly of metabolic pathway enzymes onto both flat microfabricated chips and into 3-dimensional microfluidic devices. The assembled enzymes retain reproducible catalytic activities and protein recognition capabilities for antibody binding. Additionally, catalytic activity is retained over multiple days, demonstrating enzyme stability over extended time. Finally, substrate catalytic conversion can be controlled and manipulated through the assembly patterned area, or in the case of microfluidic devices, through the substrate flow rate over the assembled enzyme. I specifically examined the patterned assembly of Pfs and LuxS enzymes, members of the bacterial autoinducer-2 (AI-2) biosynthesis pathway. AI-2 is a small signaling molecule that mediates interspecies bacterial communication termed type II "quorum sensing", which is involved in regulating the pathogenesis of a bacterial population. Significantly, this is the first time that Pfs and LuxS have been assembled onto devices. More significantly, Pfs and LuxS have both been assembled onto the same chip; that is, the quorum sensing pathway has been assembled onto a single device. This device could be used to screen inhibitors of AI-2 biosynthesis and discover novel "anti-pathogenic" drugs. In summary, I have demonstrated patterned enzyme assembly onto microfabricated devices. The assembled enzymes retain reproducible catalytic activities and are capable of recognizing and binding antibodies. Importantly, patterned device-assembly of multiple enzymes representing a metabolic pathway is possible. I envision many potential biosensing, bioMEMS, drug screening, and metabolic engineering applications.en_US
dc.format.extent2471949 bytes
dc.subject.pqcontrolledEngineering, Chemicalen_US
dc.subject.pquncontrolledimmobilized enzymeen_US
dc.titleAssembly of Quorum Sensing Pathway Enzymes onto Patterned Microfabricated Devicesen_US


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