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Micropatterns for surface potential mapping of biomolecules by kelvin probe force microscopy

Student thesis: Doctoral ThesisDoctor of Philosophy

Current methods to detect and quantify electrostatic properties of biomolecules, nowadays, still facing many challenges. For instance, they are not sensitive enough to meet the needs of more precise measurements, they require the use of external markers or antibodies, or rely on indirect calculations or models such is the case for zeta potential and electrophoretic mobility. However, Kelvin probe force microscopy (KPFM) have gained attention due to its expanding possibilities to asses electrostatic properties of biomolecules. Among the advantages that KPFM possess over other methods, stands out its high sensitivity and spatial resolution. Moreover, it does not require the use of external labels. In the interest of establishing a method for mapping surface potential of biomolecules by means of Kelvin probe force microscopy, two techniques of poly-dimethylsiloxane (PDMS) soft lithography for micro-patterning (micro channel filling and micro contact printing) are studied. Similarly, the possibility of patterning biomolecules on different substrates (mica, glass and silicon dioxide) is explored. Primarily, poly-L-lysine was used as a model biomolecule due to the exposure of amino functional groups. Different physicochemical conditions on poly-Llysine micro patterns were tested in order to assess the suitability of soft lithography combined with KPFM to measure electrostatic properties of biomolecules. In this sense, continuous KPFM scans, immersions in water, lift height and time dependence were investigated. Moreover, due to the importance of the understanding of the effects of exposure of biomolecules to elevated levels of sugars, immersions in D-ribose were performed. A concentration dependent effect was observed, affecting drastically the surface potential of polyL-lysine. Electrostatically driven patterning of colloidal gold nanoparticles was also achieved on pol-L-lysine micro patterns, resulting in a good method for marking the presence of pol-Llysine and opening the possibility to improve some properties of nanoparticle systems. Besides microchannel filling, micro contact printing of poly-L-lysine was successfully achieved on mica, glass and silicon dioxide, resulting in better quality micro patterns and understanding of the influence of using different substrates on surface potential. On this regard, continuous KPFM scans revealed a surface charge dynamic on mica, whereas on glass and silicon dioxide surface potential became more stable. Insulin, BSA and β-lactoglobulin were successfully patterned on mica by micro contact printing and imaged by KPFM. Response to pH and immersions in water was investigated showinga clear reversible shift on surface potential. Similarly, cross-patterning of different proteins on the same substrate for surface potential oneto-one comparison was successfully achieved. The classic case of avidin-biotin complex was also investigated byboth fluorescence optical microscopy and KPFM. Finally, in an attempt to stretch the applications of KPFM for surface potential mapping of biomolecules, insulin amyloid fibrils were co-fibrillated with highly charge nanoparticles. Surface potential maps of amyloid structures were achieved and the effect of continuous scanning on surface potential was assessed.
Original languageEnglish
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Award date2018

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