Robust hydrogel microsphere platforms for improved biomolecular conjugation and protein sensing
Liu, Eric.
2019
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Hydrogel microparticles
have recently emerged as materials with immense potential for biosensing due to their
hydrophilic nature and tunable properties. In this dissertation, I examine methods for
the fabrication of chemically functional hydrogel microspheres and rapid conjugation
chemistries for biomolecular conjugation towards the detection of biological molecules
of interest. Specifically, ... read moreI examine the characteristics of highly uniform, chemically
functional microspheres with tunable properties fabricated using a straightforward
micromolding-based technique and a rapid capillary microfluidics-based technique. I then
examine the utility of these microspheres as biosensing platforms by investigating the
diffusion and reaction of biomolecules within their polymer networks via rapid and
orthogonal conjugation chemistries. First, I examine the fabrication of highly uniform,
carboxylate functional poly(acrylamide-co-acrylic acid) (p(AAm-co-AA)) microspheres
using a straightforward micromolding-based technique involving surface tension-induced
droplet formation and interfacially initiated free radical polymerization. Brightfield
micrographs show that this technique readily fabricates (p(AAm-co-AA)) microspheres are
highly monodisperse and display hydrogel-like swelling behavior, thus accommodating
monomer systems with slow polymerization rates along with a wide range of fabrication
parameters and prepolymer conditions. To examine the functionality of the microspheres,
I enlist small model fluorescent molecule fluorescein glycine amide (FGA) for labeling
and kinetic studies via carbodiimide chemistry. Brightfield and epifluorescence
micrographs show the uniform dimensions of the microspheres and demonstrate controllable
functional group incorporation simply by changing the prepolymer compositions. Next, I
examine a range of carbodiimide concentrations for more efficient activation of the
microspheres' functional groups. Finally, I carry out protein conjugation studies with
large fluorescent model protein R-phycoerythrin (R-PE) to examine the mesh size of
p(AAm-co-AA) microspheres. The epifluorescence and confocal micrographs show the
complete penetration of R-PE into the centers of the microspheres by 30 min without the
need for porogens, indicating the highly macroporous nature of the microspheres and a
significant improvement in the mesh size of these microspheres toward the diffusion of
large biomolecules within their polymer networks. Next, I examine the characteristics of
primary amine (via chitosan) and carboxylate (via acrylic acid) poly(ethylene) glycol
(PEG)-based microspheres fabricated using a rapid capillary microfluidic method.
Brightfield micrographs of the as-prepared microspheres show the strength of this
microfluidic technique in rapidly fabricating highly monodisperse microspheres by
utilizing water-in-oil-in-water double emulsions with a thin oil layer, thus
significantly reducing the process complexity and cost. Again, I utilize small
fluorescent dyes to examine the functionality of the microspheres, with epifluorescence
micrographs showing uniform functional group distribution. I then use large model
fluorescent proteins R-PE and green fluorescent protein modified for maximum
fluorescence under UV light (GFPuv) to examine the diffusive and kinetic behavior of
protein conjugation with these microspheres via tetrazine-trans-cyclooctene (Tz-TCO) and
carbodiimide chemistries. Epifluorescence and confocal micrographs of R-PE conjugation
with primary amine microspheres show the rapid rate of reaction of TCO-modified proteins
with Tz-activated primary amine microspheres and the slower diffusion of proteins into
the centers of the microspheres likely due to the smaller mesh size from crosslinking
chitosan and a rapid reaction-based steric crowding effect resulting in diffusion
limited behavior. In contrast, the epifluorescence and confocal micrographs of R-PE
conjugation with carboxylate microspheres show rapid diffusion of R-PE into the centers
of the microspheres with continued reaction, indicating that both diffusion and reaction
govern the behavior of protein conjugation likely due to the increased mesh size from
acrylic acid and slower reaction rate. Finally, epifluorescence micrographs of the one
pot conjugation of TCO-modified R-PE and unmodified GFPuv with primary amine and
carboxylate microspheres respectively via simple size-based encoding demonstrate the
orthogonality of the two conjugation chemistries and the utility of capillary
microfluidics-based microspheres toward protein conjugation. Finally, I examine the
capillary microfluidics triple emulsion-based fabrication of primary amine functional
multicompartmental microspheres (multigels) composed of a functional spherical PEG core
and a separate outer PEG shell region surrounding the core. Epifluorescence micrographs
of small fluorescent molecule labeling studies indicate the uniform distribution of
functional groups throughout the cores with minimal fluorescence in the shell regions,
indicating minimal diffusion of the functional groups out of the cores during the
fabrication process. Next, epifluorescence micrographs of R-PE conjugation studies with
the multigels via Tz-TCO chemistry confirm the rapid conjugation of large proteins into
the polymer networks of the primary amine functional cores with minimal nonspecific
adsorption in the core and shell regions. Finally, I examined GFPuv and R-PE conjugation
with the multigels with shell regions prepared with varying PEGDA content.
Epifluorescence micrographs show the uniform fluorescence of the cores for multigels
with 10-20% PEG shells by 30 min but significantly retarded or complete lack of
fluorescence for multigels prepared with 30% and 40% PEG shells respectively, indicating
the potential for the shell regions to act as size-selective barriers by simply tuning
the prepolymer compositions. Combined, the results encompassed in this dissertation
highlight the potential of the robust micromolding-based and rapid capillary
microfluidic-based techniques for fabricating highly porous, chemically functional
hydrogel microspheres with uniform dimensions and tunable properties for biomolecular
conjugation via rapid and orthogonal carbodiimide and Tz-TCO chemistries toward the
detection of biological molecules of interest. I envision that these
fabrication-conjugation approaches can be readily utilized for a broad range of
biosensing applications including bioprocess monitoring, basic research, and
diagnostics.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Hyunmin Yi.
Committee: Ayse Asatekin, Qiaobing Xu, and Jin Ryoun Kim.
Keyword: Chemical engineering.read less - ID:
- g158bw17f
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