(656e) Fabrication of Chitosan-Poly(ethylene glycol) Hybrid Microparticles Via Replica Molding and Its Application Toward Facile Biomolecular Conjugation Via Copper-Free Click Chemistry Conference: AIChE Annual MeetingYear: 2013Proceeding: 2013 AIChE Annual MeetingGroup: Materials Engineering and Sciences DivisionSession: Biomaterials for Biosensing Time: Thursday, November 7, 2013 - 1:42pm-2:00pm Authors: Jung, S., Tufts University Yi, H., Tufts University Facile conjugation of biological molecules on robust platforms is important in many application areas such as therapeutics and diagnostics. One such example is nano and microparticles based on polysaccharide chitosan due to the abundant primary amine groups that offer covalent binding sites via nucleophilic reactions. However, poor physical properties of chitosan such as high viscosity, poor solubility in solvents and low mechanical strength due to its rigid crystalline structure have limited its utility in the fabrication of biofunctionalized platforms. While the chitosan-based particles can be formed with water-in-oil emulsion method, the shape of the particles is usually limited to spheres. Meanwhile, standard amine-reactive conjugation chemistries for the biomolecular conjugation (e.g. glutaraldehyde or homobifunctional N-hydroxysuccinimidyl (NHS) ester) have drawbacks such as limited stability, nonselective conjugation, and self-blocking of active amine sites by the chemical agents. In addition, reactive Cu(I) catalysts in the Cu-catalyzed click chemistry can cause structural damage to the biomolecules during the conjugation reaction. In this work, we exploited replica molding (RM) to fabricate non-spherical, shape-controlled hybrid microparticles containing chitosan moieties for facile biomolecular conjugation in a simple, robust, and inexpensive manner. We also exploited Cu-free click chemistry between strain-promoted alkyne of dybenzylcyclooctyne (DBCO) and azide for selective and efficient conjugation of biomolecules with the chitosan-containing microparticle platforms under mild reaction conditions. Specifically, we utilized preparticle solution composed of chitosan oligomer and poly(ethylene glycol) diacrylate (PEGDA) to fabricate chitosan-PEG hybrid microparticles with uniform and well-defined dimensions and shapes via RM. The incorporation and distribution of chitosan molecules within the PEG networks as well as the chemical reactivity of chitosan’s amine groups were confirmed via fluorescent labeling. The results show that the chitosan molecules are mostly located near the particle surfaces, while particles with high PEG concentration yielded retention of chitosan molecules within the core area. Next, the primary amines in the chitosan-PEG microparticles were exploited for conjugation of biomolecules such as single-stranded (ss) DNAs and fluorescent proteins (R-phycoerythrin, R-PE) via Cu-free click chemistry. The results show selective conjugation of ssDNAs and R-PE near the particle surface where mass transfer limitation is minimal for rapid biosensing. In this presentation, our recent results on protein conjugation kinetics of the Cu-free click chemistry will be highlighted. We then examined antibody-conjugated chitosan-PEG microparticles as a protein sensing platform through Cu-free click chemistry. The results show that the target proteins are selectively captured with the antibody-conjugated particles, and subpicomolar quantity of the proteins can be readily detected with standard imaging conditions. In this presentation, our recent results on substantially improved antibody conjugation capacity via genetically modified tobacco mosaic virus (TMV1cys) as a nanotubular template will also be introduced. Combined these results demonstrate a facile fabrication-conjugation strategy of hybrid bio-/synthetic polymer microparticles via RM and Cu-free click chemistry, enabling efficient use of precious biomolecules. We envision that our methodologies may find wide application areas where efficient fabrication and conjugation of precious materials are highly desired.