R.T. Busch, J. Weis, F. Karim, C. Zhao, Y. Sun, E.S. Vasquez
University of Dayton,
Keywords: gold nanoparticles, listeria monocytogenes, antibodies, biosensors, TEM, ATR FT-IR, UV-Vis, DLS, Zeta(ζ) Potential
Summary:Identifying and detecting low levels of foodborne pathogens is an increasing priority in manufacturing settings.1 Antibodies have become an ideal targeting mechanism due to their increasing availability, quality and specificity. To utilize antibodies in biosensors favorable substrate interactions must be developed.2 Gold nanoparticles (Au NPs) have emerged as an ideal vessel for biosensor purposes due to their unique surface characteristics, optical properties, stability and consistency.3 To detect pathogens at low levels using Au NPs, exploring antibody efficiency and stability during biofunctionalization is vital. Covalently binding the antibodies to carboxyl functionalized Au NPs is achieved through a well-established carbodiimide reaction.4 The effectiveness of this reaction is highly dependent on the surface conditions of the particle and subsequently the wet chemistry reaction conditions.4,5 In this study, we determine the balance between efficiency and stability of the bound antibody as a function of pH and buffer solutions to carboxyl functionalized Au NPs using Listeria monocytogenes targeting Antibodies (Immunoglobulin G class). Initially, zeta(ζ) potential measurements were used to describe the surface charges of the particles prior to and after binding the antibody at each pH condition (HCl-2, MES-4.5, MES-6, MOPs-7.5, NaOH-11). The surface charge was correlated to DLS and UV-Vis data which describes the aggregation and size increase of the nanoparticles at the different reaction steps. TEM images were taken to analyze discrepancies between size and morphological changes due to the corona layer assembly from antibody binding versus aggregation. UV-Vis (Figure 1) was further utilized to describe the efficiency of antibody uptake following each reaction step. Binding efficiency and morphological features were coupled with ATR FT-IR (Figure 2) characterization to understand the relationship between efficiency and stability of the antibody coated nanoparticles under different solution conditions.6,7 The covalent binding process was determined to be more efficient at lower pH values; however, aggregation and deactivation of the antibodies was observed. Overall, this work demonstrates the exquisite balance to bind Listeria monocytogenes antibodies to carboxyl functionalized Au NPs at different pH conditions.