Author ORCID Identifier
Date Available
8-2-2022
Year of Publication
2022
Document Type
Master's Thesis
Degree Name
Master of Science in Chemical Engineering (MSChE)
College
Engineering
Department/School/Program
Chemical and Materials Engineering
Advisor
Dr. Barbara L. Knutson
Co-Director of Graduate Studies
Dr. Stephen E. Rankin
Abstract
In this work, the synthesis of protein-functionalized nonporous silica nanoparticles and their application to capture antibodies was demonstrated. Silica nanoparticles were chosen as they offered particle and pore size tunability, low toxicity, thermal stability, ease of synthesis and ease of surface functionalization. A biotin-streptavidin-biotin bridge was constructed on the surface of the particles and used to attach biotinylated Protein A/G (PAG), which has high affinity for antibodies. The biotin-streptavidin-biotin bridge is hypothesized to orient the PAG in a favorable manner for efficient antibody capture. Nonporous silica nanoparticles (approximately 270 nm in diameter) were synthesized via the Stöber method. Following amine functionalization, biotin was covalently attached to the SNPs. Attachment of streptavidin, followed by biotinylated PAG was achieved using the strong biotin-streptavidin interaction. 42.8 mg streptavidin was loaded onto per gram particles, which was estimated to correspond to monolayer surface coverage. Due to their similar size, 1:1 binding between streptavidin and PAG was assumed. The uptake of IgG by PAG attached particles was measured by solution depletion. The equilibrium isotherm was fit to the Langmuir adsorption model by performing nonlinear regression. Maximum uptake capacity, qmax was found to be 110 mg antibody/g PAG particles, and the equilibrium association constant KA was found to be 6.05×107 L/mol. The maximum uptake capacity of IgG is consistent with 1:1 binding with Protein A/G on the SNP surface. Experiments of antibody binding in the presence of bovine serum albumin, which served as a model nonspecific protein, showed insignificant reduction of antibody binding. The biotin-streptavidin-biotin bridge is a versatile approach to attach and orient biomolecules at the surface of SNPs. This technique can be extended to biomolecule capture for a range of affinity pairs, building on the prevalence of biotin and streptavidin functionalization strategies of proteins and nucleic acids. Advantages of nanoparticle-enhanced biomolecule detection and immunoassays can be further explored based on the versatility and specificity of affinity capture using SNPs.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2022.261
Recommended Citation
Afrin, Sayma, "VERSATILE FUNCTIONALIZATION STRATEGY FOR THE CAPTURE OF ANTIBODIES USING SILICA NANOPARTICLES" (2022). Theses and Dissertations--Chemical and Materials Engineering. 142.
https://uknowledge.uky.edu/cme_etds/142
