Integrated Fabrication-Conjugation Approaches for Biomolecular Assembly and Protein Sensing with Biopolymeric-Synthetic Hydrogel Microparticle Platforms and Bioorthogonal Reactions.
Jung, Sukwon.
2015
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Abstract: Hydrogel
microparticles have gained increasing attention as biosensing platforms due to the
advantages of hydrogels and particle-based suspension arrays. In this dissertation, I
examine facile fabrication‒conjugation approaches to construct hydrogel
microparticle platforms that can be utilized for biosensing. Specifically, I exploit
simple micromolding techniques for fabrication of ... read morehighly uniform and chemically
functional biopolymeric-synthetic hydrogel microparticles, and high yield bioorthogonal
conjugation reactions for biomolecule conjugation with the as-prepared microparticles.
Two novel approaches allowing for enhanced conjugation capacity and kinetics as well as
sensing capability of the microparticle platforms are also examined in this
dissertation. First, facile replica molding (RM) technique is employed to fabricate
chitosan‒poly(ethylene glycol) (PEG) hydrogel microparticle platforms. The
results show that highly uniform and well-defined chitosan‒PEG microparticles are
readily fabricated via RM. Fluorescence labeling and FTIR microscopy results indicate
stable incorporation of chitosan moieties with PEG networks in the microparticles while
retaining their chemical reactivity toward amine-reactive chemistries. The utility of
these microparticles as biomolecule conjugation platforms is then investigated via
conjugation of model biomolecules such as fluorescein-labeled single-stranded (ss) DNAs
and red fluorescent proteins (R-phycoerythrin, R-PE) via strain-promoted
alkyne‒azide cycloaddition (SPAAC) reaction. Fluorescence and confocal microscopy
results show highly selective conjugation of biomolecules near the particle surfaces
under mild conditions as well as long-term stability of the conjugation scheme using
SPAAC reaction. In-depth examination of R-PE conjugation kinetics with the
microparticles shows multiple reaction regimes (i.e. rapid initial, intermediate, and
steady final stage) owing to steric hindrance arising from the as-conjugated R-PEs and
small mesh size of the microparticles. Next, the chitosan‒PEG microparticles are
enlisted for target protein (R-PE) capture upon anti-R-PE antibody conjugation via SPAAC
reaction, and the results show selective and rapid target protein capture with the
antibody-conjugated microparticles. Next, I enlist viral nanotemplates to improve the
hindered environment of the chitosan‒PEG microparticles, and thus their protein
conjugation and kinetics as well as sensing capability. Specifically, tobacco mosaic
virus (TMV) templates are assembled with the chitosan‒PEG microparticles via
nucleic acid hybridization in order to provide abundant conjugation sites with minimal
steric hindrance near the particle surfaces. R-PE conjugation results show significantly
enhanced protein conjugation capacity of TMV assembled microparticles (TMV-particles)
compared to planar substrates and the chitosan‒PEG microparticles. In-depth
examination of protein conjugation kinetics via SPAAC and
tetrazine‒trans-cyclooctene (Tz‒TCO) cycloaddition reaction indicates that
the TMV-particles offer less hindered protein conjugation environment over the
chitosan‒PEG microparticles. Target protein capture results with antibody
conjugated TMV-particles also show substantially enhanced capture capacity over the
antibody conjugated chitosan‒PEG microparticles. In addition, protein and
antibody conjugation capacity are readily controlled by simply varying TMV
concentrations, with negligible negative impact of densely assembled TMVs on the protein
conjugation and capture capability. Lastly, I examine a simple and robust
micromolding-based technique utilizing surface tension-induced droplet formation and
polymerization-induced phase separation that allows for fabrication of monodisperse
chitosan‒PEG microspheres with macroporous and/or intriguing core-shell
structures. The utility of these microspheres as platforms for biomolecule conjugation
is then thoroughly examined via conjugation of the model biomolecules via SPAAC and
Tz-TCO reaction. The results show not only programmable protein conjugation but also
enhanced conjugation capacity and kinetics rising from the controlled macroporous
structures. Overall, the results described in this dissertation illustrate facile
fabrication‒conjugation approaches for construction of biosensing platforms via
simple micromolding techniques and efficient bioorthogonal conjugation reactions. I
expect that these approaches can be readily enlisted in a wide range of biosensing
application areas such as medical diagnostics, bioprocess monitoring, and pathogen
detection for biodefense.
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Hyunmin Yi.
Committee: Asatekin Ayse, Qiaobing Xu, and Chang-Soo Lee.
Keyword: Chemical engineering.read less - ID:
- cr56nc34m
- Component ID:
- tufts:21457
- To Cite:
- TARC Citation Guide EndNote
