Using the Layer by Layer (LbL) technique combined with microcontact printing, novel pH sensors were developed in order to detect the extracellular pH (pHe) of the cancerous cell lines K562 and HeLa. Key to this process was the utilization of fluorescence microscopy which allowed for direct measurement of the fluorescence intensity observed from the pH sensors made possible by the integration of fluorescent molecules into the polymer layers, namely Fluorescein Isothiocyanate and Rhodamine Isothiocyanate. In this work eight pH sensor types were analyzed and statistically validated. By subjecting the pH sensors to the pH buffers, pH 5, pH 6, pH 7, and pH 8 a standard curve was also able to be developed. Results found that both Nine Layer pH Sensor formulations, with FITC as the pH sensitive fluorophore, showed significant differences between pH value sets, such as between pH 6 and pH 7 and between pH 7 and pH 8, at **p<0.01 using T-Test analysis. These Nine Layer pH Sensors were then deemed suitable for cell seeding analysis since cells are known to exhibit extracellular pH’s between the ranges of pH 6 and pH 8. Cell seeding analysis of pH sensors revealed that no significant difference occurred with either cell type used, K562 or HeLa. In this analysis, cells were cultured over top of the pH sensor and allowed to bind ~2 nm away from the pH sensing fluorescent layer. It was hoped that the proximity of the layer to the cell would allow for a comparison between cell bound fluorescent striped regions and non-cell bound fluorescent striped regions. It was theorized that these cell bound regions would exhibit dimmed fluorescent stripes directly underneath the cells as the non-cell bound regions would exhibit greater fluorescence intensity comparatively. These two cancer cell lines are known to exhibit slightly acidic extracellular pH’s in the range of pH 6.7 to pH 6.8 while being cultured in media with known pH values of pH 7.4 or greater. This difference in pH values would then theoretically result in fluorescence intensity differences on the fluorescent stripes. In analyzing the pH sensors, it was seen that they did not possess the sensitivity needed to detect pHe, as they simply were only able to detect the pH of the bulk fluid and not pH directly surrounding the cells. Any visible fluorescence intensity change resulted from cell autofluorescence. Future work would then center around reducing interference from cell autofluorescence as well as determining the cause for the lack of sensitivity. It seems that manipulation of the polymer layers would possibly allow for greater diffusion of cell metabolites through the polymer matrix thus resulting in greater access to the fluorophore. It also seems that FITC may not be an adequate fluorophore and more sensitive molecule may need to be selected. Key words: Layer by Layer, microcontact printing, extracellular pH (pHe), K562, HeLa, Fluorescein Isothiocyanate, Rhodamine Isothiocyanate, fluorophore, T-Test. / A Thesis submitted to the Department of Chemical and Biomedical Engineering in partial fulfillment of the requirements for the degree of Master of Science. / Summer Semester 2017. / May 22, 2017. / extracellular pH (pHe), Layer by Layer, microcontact printing / Includes bibliographical references. / Jingjiao Guan, Professor Directing Thesis; Bruce Locke, Committee Member; Ravindran Chella, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_552031 |
| Contributors | Bauer, Mark A. (Mark Andrew) (authoraut), Guan, Jingjiao, 1973- (professor directing thesis), Locke, Bruce R. (committee member), Chella, Ravindran (committee member), Florida State University (degree granting institution), FAMU-FSU College of Engineering (degree granting college), Department of Chemical and Biomedical Engineering (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, master thesis |
| Format | 1 online resource (133 pages), computer, application/pdf |
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