Bomsztyk, KarolSchrimshire, Theodore2023-08-142023-08-142023-08-142023Schrimshire_washington_0250O_25526.pdfhttp://hdl.handle.net/1773/50229Thesis (Master's)--University of Washington, 2023Abstract:Activation of 96-Well Polypropylene Plates with Radio Frequency Plasma Ion Etching to Increase Chromatin Binding Capacity for Immunoprecipitation. Theodore Schrimshire. Chair of the Supervisory Committee: Dr. Karol Bomsztyk. Department of Allergy & Infectious Disease. Background: Chromatin Immunoprecipitation (ChIP) is an assay that can quantify epigenetic alterations. The Bomsztyk lab has developed technology for high-throughput chromatin immunoprecipitation (ChIP) microplate-based assays called PIXUL-Matrix-ChIP. These assays use 96-well polypropylene (PP) sample plates that are treated with 48 hours of UV light irradiation to activate the surface for the absorption of antibodies that immunoprecipitate chromatin. Plasma reactive ion etching (RIE) is a potential alternative treatment method that can activate PP by adding highly polar oxygen species to the surface in a small fraction of the time compared to UV treatment. Methods: This report investigates the use of oxide-plasma treatments to activate the surface of the PP plates to reduce plate treatment times to less than 5 minutes and ideally increase the ChIP assay efficiency. I used plasma etching to increase the molecular polarity of the surface so more antibodies can bind to the PP and increase the capacity for chromatin immunoprecipitation. To assess surface properties after UV vs plasma treatments, I used water contact angle measurements (WCA), X-ray spectroscopy (XPS), and surface roughness estimation. I used chromatin from serum-treated HCT116 in Matrix ChIP to assess the antibody binding capacity of the PP after treatments based on ChIP signal response and background noise. Results: The UV-treated PP had higher hydrophilicity (WCA) and higher oxygen content and surface polarity (XPS) than tested doses of plasma treatment, PP surface qualities produced by 1 min of plasma treatment which were closest to those yielded by 48 hours of UV. The ChIP data for the UV and 1 min plasma treated plates show the expected immunoprecipitation signal for the well-studied gene sites and antibodies (Pol II: EGR1 exon1-5 and 2-3, H3: all three sites, CTCF: EGR1 -15kb) and a low signal at the unspecific background level which is the assay noise (non-immune IgGs: all sites). Among the examined surface qualities, the hydrophilicity and oxygen content are the major factors in the treated PP surface that correlate with the capacity of antibodies absorption which determines the efficiency of chromatin immunoprecipitation. Conclusions: The 1 min plasma-treated plates outperformed 48 hrs UV treatment by 11-20 percent in ChIP assay (noise adjusted signal). While these data show promise, testing other plasma treatment conditions (duration, oxygen concentration, and others) will be required to replace UV treatment in quality and reliability.application/pdfen-USnoneChromatin ImmunoprecipitationPlasma Ion EtchQuantitative BiologyBioengineeringBioengineeringThesis