Characterization of guanosine based hydrogels and application of hydrogels in microchip electrophoresis for sequence based separation of ssDNA

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Stevens, Wyatt Palmer
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Information about the bulk properties of the binary G-gels led to consideration of possible applications for them. Previous work in the McGown group had found that sequence-based separation of single stranded DNA was possible in capillary zone electrophoresis (CZE) by using buffers with high ionic strength. Attempts had been made to adapt the results from CZE to microchip electrophoresis using similar buffers but the results were largely unsuccessful. When the DNA mixtures were introduced into the microchip for separation, any indications of peak resolution were irreproducible. It was believed that the DNA was travelling too fast through the channel to allow sufficient time for separation. This was supported by the time it would take for separation to occur in CZE in the 50 cm capillaries, on the order of 20 to 50 min, compared to 5 to 10 min on the 8 cm microfluidic channel. To overcome this limitation, the present work explored the use of hydrogels in the separation channel of the microfluidic chip in order to increase the viscosity of the medium and thereby lower the velocity of the DNA to provide more time for the separation to occur. The first hydrogels we explored were the G-gels, but issues with these gels led us to investigate other hydrogels, along with various buffers and additives to optimize the separation. Different arrangements of gel in the microfluidic channels and combinations of applied voltage across the injection and separation channels were tried. It was found that using linear polyacrylamide gel prepared in potassium phosphate buffer in the separation channel with a TBE buffer with added Triton X-165 in the injection channel led to the best conditions for separation of the ssDNA in the PMMA microchip.
Hydrogels are a class of materials based on polymers which can absorb many times their weight in water. Hydrogels are a relatively new type of material, with the first real hydrogel having been characterized in 1960. Since then, research into hydrogels has grown rapidly because their properties can be easily modified through covalent derivatization of the polymer backbone or changing other factors such as pH, ionic strength and concentration of gelator. One hydrogel that was known before the introduction of hydrogels as a class of materials is the guanosine-based hydrogel (G-gel) which was first described for a solution of high concentration guanosine monophosphate disodium salt and low pH. Since then, interest in these G-gels has grown due to their unique structural properties and their potential applications in the biomedical fields and smart materials.
Previous work in the McGown group on G-gels found that in some formulations, G-gels formed by binary mixtures of guanosine monophosphate and guanosine exhibit thermoassociative properties. At refrigerator temperatures these gels are liquids, while at higher temperatures they transition to the gel phase. To understand the factors that determine the thermoresponsiveness of these binary G-gels, the components of the G-gels as well as pH and added ions were varied. It was found the ratio of the two guanosine compounds and the total guanosine concentration were the dominant factors in determining if a G-gel would exhibit the unexpected thermoassociative behavior. These prior studies provided insight into thermoresponsiveness of the binary G-gels but as a function of their composition, but information about their bulk mechanical properties were left unknown.
In the present work, oscillatory rheological experiments were used to investigate the mechanical properties of the binary G-gels of varying composition. These rheological experiments focused on three aspects: how the gels respond to different frequencies of oscillation, how much stress could be applied to the gels before their three-dimensional structure is destabilized, and how easily the gels could reform when recovering from such high applied stresses. In these studies, the guanosine monophosphate to guanosine ratio and total concentration of guanosine compound were varied along with concentration and type of cation used to stabilize the G-gel. The rheological results for these different G-gels provide a better understanding of how the G-gel composition affects their bulk properties and provide a basis for comparison with other hydrogels.
May 2019
School of Science
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Rensselaer Polytechnic Institute, Troy, NY
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