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The motivation behind this work stems from Jedlicka’s work on
Chinese hamster ovary (CHO) cells and her observation that these cells
proliferated differently depending on the glass chemistry on which the cells
were growing. It is well established that proteins form the bonds between
cells and glass substrates and so this work was aimed at discovering whether
proteins also react differently to different glass surfaces. It is believed that
the bond formed between glass and protein is a hydroxyl-amine interaction
via hydrogen bonding. The scope of this work deals with silica glass in
various forms including slides, cane, fiber, micron-sized spheres and
Cab-o-Sil®. These forms are subjected to surface treatments such as ethanol
cleaning, HF acid etching, water plasma treatments and 1000°C thermal
treatments. A select few proteins are chosen as a tool to probe the surfaces of
the silica glass and single crystal quartz surfaces. These proteins are human
serum albumin (HSA), streptavidin, mouse immunoglobulin G (IgG), biotin,
and anti-mouse IgG. A few characterization techniques are employed in an
attempt to examine protein adsorption and its feasibility as a surface probe.
These techniques include sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), bicinchoninic acid (BCA) assay, glancing
incidence X-ray analysis (GIXA), fluorescence spectrometry, atomic force
microscopy (AFM), and chemical force microscopy (CFM). The main goal of
this work is to determine which of the above techniques in conjunction with
protein adsorption is the most promising as a surface characterization
technique.
It was determined that CFM is the most promising surface
characterization technique utilizing proteins as surface probes. It is possible
to attach a wide variety of molecules to a standard contact mode tip including
proteins. Streptavidin, –COOH, and –CH3 functionalized tips were utilized
in the CFM study. The overall adhesion forces between silica and tip were
greatest for –COOH, then streptavidin and least for –CH3. This ordering is a
broad generalization however, since the adhesion forces depend greatly on
surface treatment and can either be very consistent across the surface or vary
significantly. SDS-PAGE on streptavidin was preventatively difficult, but
IgG electrophoresis was possible and did show some promising results. BCA
assay and fluorimetry both utilized “depleted” protein solutions and thus it
was very difficult to uncover trends in the data. GIXA showed that the
protein layer thickness was monolayer in nature. AFM allowed proteins to be
imaged while in the tris-buffered saline and the features were on the order of
ten protein agglomerations. The GIXA and AFM data agree very well. CFM
is able to discern between the various surface treatments. With the eventual
development of carbon nanotube growth onto a contact mode tip and the
subsequent application of a single chemical molecule onto the end of the tube,
CFM will become an even stronger technique for surface characterization. |
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