Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Agricultural and Biological Engineering

Committee Chair

Bernard Y. Tao

Committee Member 1

Frederick S. Gimble

Committee Member 2

Jenna L. Rickus

Committee Member 3

Kari L. Clase

Committee Member 4

Nathan S. Mosier


In the past two decades, crosslinking has been a popular choice in synthesizing bioconjugatesinthefieldsofbiotechnology,medicaldiagnostics,andgeneticscreening.However, their efficacyin synthesizing the desired bioconjugates is scarcely analyzed and documented. The scope of this investigation encompassed studying covalent bioconjugation techniques, and determining the structural characteristics of the bioconjugates formed via several crosslinking strategies. The major goal of the study was to determine relatively inexpensive, less complex, and universal bioconjugation techniques with the flexibility to control the distance between wild-type enzymes. In this investigation, three different crosslinkers were studied to determine the efficacy of crosslinking biological molecules. In the first aim of the study, an adapted strategy from the conventional 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) was developed to form a novel phosphoramidated single stranded DNA (ssDNA) bioconjugate. Matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) was used to determine the molecular weights, and the stability of the bioconjugates formed. A reversed phase high performance liquid chromatography(RP-HPLC)analytical method was developed to determine the conjugate product yields. The analytical procedure’s accuracy, precision, linearity, limit of detection (LOD), and limit of quantitation (LOQ) were determined. Afterwards, the yield of the phosphoramidated ssDN Abioconjugate was determined using the developed analytical procedure. In our attempts to determine suitable crosslinkers, another crosslinker (BS3) was also investigated to accomplish bioconjugation between ssDNA, and a small peptide. However, mass spectrometry results indicated conjugate molecular weights evidencing only partial bioconjugate formation.

In the next aim, the successful adapted EDC crosslinking technique was applied to two wild-type enzymes to synthesize enzyme-single stranded DNA (enzyme-ssDNA) bioconjugates, in an aqueous system. Like so, glucose oxidase-ssDNA4 (GOD-ssDNA4), and horseradish peroxidase-ssDNA3 (HRP-ssDNA3) conjugates were synthesized separately. These enzymessDNA conjugates were analyzed, and purified using size exclusion chromatography (SEC). Fluorescence spectroscopy was used to determine the presence of ssDNA in the GOD-ssDNA4 bioconjugates, while MALDI-TOF spectrometry was used to determine the molecular weights of the HRP-ssDNA3 bioconjugates. Altogether, size exclusion chromatography, fluorescence spectroscopy, and MALDI-TOF results suggest that the adapted EDC crosslinking method was successful in producingthe expected enzyme-ssDNA conjugates. In order to spontaneously assemble the GOD-ssDNA4, and HRP-ssDNA3 bioconjugates, DNA hybridization was utilized. However, high annealing temperatures used in the general heating-cooling protocols has the potential to degenerate these enzyme conjugates. Therefore, possibilities of accomplishing DNA hybridization at lowered annealing temperatures was investigated as well. After facilitating DNA hybridization between GOD-ssDNA4 and HRPssDNA3, the final enzymatic macromolecule was analyzed on size exclusion chromatography. Based on the structural evidence obtained from mass spectrometry, UV-Vis spectrophotometry, reversed phase chromatography, size exclusion chromatography, and fluorescence spectroscopy, we analyzed the desired bioconjugates at each successive stage in this bottom-up-synthesis.