Linear Polymer Affinity Agents for the Intrinsic SERS Detection of Food Safety Targets

Abstract

This dissertation explores the use of polymer affinity agents for the surface-enhanced Raman spectroscopy (SERS) detection of food safety targets. First, current molecular motifs used as affinity agents in intrinsic surface-enhanced Raman spectroscopy (SERS) sensors are reviewed. By comparing antibody, aptamer, small molecule, and polymer affinity agents, the largely unresearched potential of polymer affinity agents (chemical customization, tunable length, ease of production, opportunity for multiplexing) is highlighted. The first proof of concept work of this dissertation targets the detection of the bioterror agent, ricin B-chain (RBC) in water and liquid food matrices. An N-acetyl-galactosamine glycopolymer capture layer was designed and applied to create a SERS sensor. The sensing scheme’s detection limit (20 ng/mL) is well below that of the predicted oral exposure limit. The RBC was detected in two types of juice, and a computed normal Raman spectrum of the glycomonomer supports polymer–RBC intermolecular interactions at the functional group level. Subsequent work focuses on the translation of this sensing scheme from the detection of proteins to the detection of small molecules relevant to food safety. Because interactions between a small molecule target and a polymer affinity agent are less specific than those that were leveraged in the RBC work, the development of a rapid affinity agent screening method was deemed necessary. A potent carcinogenic metabolite of a fungal pathogen that can infect food and feedstocks, aflatoxin B1 (AFB1), was used as a model target. Seven homopolymers of nitrogen-inclusive poly(N-(2-aminoethyl) methacrylamide) (pAEMA) and their oxygen analogs, poly(2-yydroxyethyl methacrylate) (pHEMA) were synthesized to be evaluated as AFB1 affinity agents based on hypothetical hydrogen bonding interactions and optimal polymer length. An isothermal titration calorimetry (ITC) method was development for rapid affinity agent screening and good agreement was observed between the ITC results and follow-on SERS sensing experiments. A final polymer series (poly(N-acryloyl glycinamide), pNAGA) was designed for the capture of AFB1 and was used to explore the influence of polymer molecular weight (2.0 – 5.2 kDa), attachment chemistry (thiol vs. trithiocarbonate), and order of addition (pre- vs. post- functionalization of the substrate) on the sensitivity of AFB1 detection. The best polymer chain length (pNAGA22), anchoring chemistry (thiol), and polymer/toxin assembly scheme (in-solution) allowed detection of 10 ppb AFB1 in water (below the FDA regulatory limit of 20 ppb), a hundred-fold improvement over SERS sensing without the pNAGA affinity agent. This dissertation concludes with the advantages, disadvantages, and future perspectives of polymers used as analytical affinity agents. Adjustment of surface attachment moieties, the use of crosslinkers with target affinity, and application to other signal transduction mechanisms are emphasized a potential areas for continuing work.