Title

Antibody Functionalization Studies on Gold Substrates for Listeria Monocytogenes Capture and Detection

Author

Robert Busch

Date of Award

1-1-2019

Degree Name

M.S. in Chemical Engineering

Department

Department of Chemical and Materials Engineering

Advisor/Chair

Advisor: Vasquez Erick

Abstract

Sensitive capture and early detection of difficult to eliminate foodborne pathogens, such as Listeria monocytogenes, is critical to prevent bacterial infections and outbreaks. As such, multifunctional materials must be implemented in unique ways to enhance sensitivity and selectivity of detection. Recently, gold nanoparticles (AuNPs) bound with biomolecules have emerged as suitable biosensors exploiting unique surface chemistries and optical properties. Many efforts have focused on antibody bioconjugation to AuNPs resulting in sensitive bioconjugate to detect specific types of bacteria. Unfortunately, bacteria thrive under various harsh environments and an understanding of bioconjugate stability is needed prior to use as a functionally engineeredbiosensor.First, this study shows a method for optimizing Listeria monocytogenes polyclonal antibodies bioconjugation mechanisms to 40nm AuNPs making robust bonds via covalent binding at different pH, from 2 to 11, and MES, MOPs, NaOH, HCl buffer conditions. By fitting Lorentz curves to the Amide I and II regions obtained through FT-IR analyses, the stability of the antibody secondary structure is presented. This work shows an increase in apparent breakdown of the antibody secondary structure during bioconjugation as pH decreases from 7.9 to 2. Adsorption efficiency, measured as the percentage of antibody adsorbed to the AuNP surface, varied from 17% to 27% as pH increase from 2 to 6 before decreasing to 8% and 13% at pH 7.9 and 11, respectively. TEM analysis reveals discrepancies between size and morphological changes due to the corona layer assembly from antibody binding to single nanoparticles versus aggregation or cluster self assembly into large aggregates. Corona layer formation size increases from 3.9 to 5.1 nm from pH 2 to 6, at pH 7.9 there is incomplete corona formation, while at pH 11 there is a corona layer of 6.4 nm. These results indicate that the covalent binding process was more efficient at lower pH values; however, aggregation and deactivation of the antibodies was observed. An optimum bioconjugation condition was determined at pH 6 and MES buffer type demonstrated by indicators of covalent bonding and stability of the antibody secondary structure using FT-IR, the morphological characteristics and corona layer formation using TEM, and low wavelength shifts of UV-Vis after bioconjugation.Surface-chemistry studies on polyclonal antibody onto gold surfaces is translated to larger nanoclusters of magnetic nanoparticles functionalized with a gold-coating and a carboxylic acid group (pAb-AuMNPs). The multifunctional substrate comprised of stable gold surfaces combined with magnetic cores, and end-functionalized with polyclonal antibodies are investigated as a onestep label-free Raman based biosensor. The AuMNPs surface are surface modified with L. monocytogenes targeting antibody molecules (pAb-AuMNPs) via the wet chemistry conditions analyzed on 40 nm gold nanoparticles, allowing the pAb-AuMNPs to capture and aggregate bacteria via specific antigen-antibody interactions. Using the pAb- AuMNPs, a rapid one-step method with a surfaced-enhanced Raman spectroscopy (SERS) signal is described for the detection and manipulation of the targeted pathogen. SERS measurements are recorded for the minimum detectable amount of L. monocytogenes based on the SERS intensity at the 1388cm-1 Raman shift. L. monocytogenes concentrations are in the range of 104-107 cfu mL-1, before and after aggregation. By fitting these concentrations, the limit of detection of this method is ~103 cfu mL-1 with an enhancement factor of 104.4. This method shows high sensitivity and rapid detection time as a single step biosensor and allows magnetic aggregation of antigen-antibody bioconjugates, which can beapplied to many other types of bacteria or antibody-functionalized substrates.

Keywords

Chemical Engineering

Rights Statement

Copyright 2019, author

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