Per- and Polyfluorinated substances (PFAS) pose a significant health risk to the United States, since these chemicals are persistent, toxic, bio-accumulative, and ubiquitous. PFAS are commonly found in ground and surface waters, which can lead to elevated levels of PFAS in agricultural products. To protect the safety of the nation's food and water supply, more frequent and widespread PFAS testing is necessary. Currently, methods to detect these chemicals are primarily laboratory-based and the rapid methods that exist have significant limitations. Here, we will develop a rapid, quantitative, sensitive test for PFAS that will detect many of these chemicals simultaneously. The test will be based on a competitive immunoassay using peroxisome proliferator-activated receptor alpha (PPARα), which binds to PFAS. The immunoassay will be developed on the LightDeck platform, which uses waveguide technology to perform rapid, easy-to-use immunoassays with a fluorescent readout that is digitally processed to provide a numeric result to users. The primary objective is the development of an immunoassay based on PPARα binding to PFAS.The test is expected to have a lower limit well below the actionable regulatory guidelines for these chemicals. This test will be used in agriculture, water utilities, and manufacturing industries to ensure the safety of the US water supply.The goal of this project is the creation of an immunoassay to detect PFAS with the necessary sensitivity and assay range. Here, we plan to use a competitive immunoassay that uses a capture reagent containing a PFAS which competes with the PFAS present in the sample when binding to a detection reagent. In a competitive immunoassay, the antigen present within a sample competes with an assay reagent for binding sites on an antibody or another bioreceptor. The binding of antigen to the bioreceptor is then quantified using a reporter molecule (i.e., fluorophore), whose signal is proportional to the concentration of antigen.Objective 1, Identify and Procure Reagents: In this objective, reagents will be identified and purchased for use in an immunoassay.Objective 2, Perform conjugation chemistries to create the capture and detection reagents used in the immunoassay: In this objective, protein bioconjugates will be generated for use in assay development to develop the capture and detection reagents necessary for the immunoassay.Objective 2.1: Conjugation of PFOA to BSA. These assays will be performed as competitive assays, where a PFAS-protein conjugate is printed on the surface of the waveguide in a microarray as a capture reagent. In this Phase I project, perfluorooctanoic acid (PFOA) will be conjugated to a carrier protein. Carrier proteins, such as bovine serum albumin (BSA) or ovalbumin (OVA), are commonly used for immobilization of small molecule antigens.Objective 2.2: Conjugation of PPARα to a fluorescent dye. In the simplest assay format, PPARα will be directly labeled with the fluorescent dye. In humans and animals, one reason that PFAS are dangerous is that behave as peroxisome proliferator-response elements (PPREs) that can be ligated by PPARα, which demonstrates that there is expected to be good binding of PFAS to PPARα in this immunoassay. PPARα can be used as a detection reagent in a competitive immunoassay if it is labeled with a fluorophore. In this assay format, PPARα serves as the detection bioreceptor, binding to PFAS and allowing for quantification.Objective 2.3: Conjugation of anti-PPARα to a fluorescent dye. In humans and animals, one reason that PFAS are dangerous is that they behave as peroxisome proliferator-response elements (PPREs) that can be ligated by PPARα, which demonstrates that there is expected to be good binding of PFAS to PPARα in this immunoassay. As an alternative to fluorophore labeled PPARα, anti-PPARα antibodies can bind to PPARα to serve as a detection mechanism. Depending on the assay format, either PPARα or anti-PPARα antibodies need to be conjugated to a fluorescent dye for optical readout.Objective 2.4: Characterization of protein bioconjugates. Establishing well-defined characterization methods for protein bioconjugates is important to ensure assay repeatability and reproducibility.Objective 3, Demonstrate and screen competitive immunoassay formats: Formats for the competitive immunoassay will be explored using the protein bioconjugates prepared in Objective 2. Among the assay formats to be screened are a multi-step turn-off competitive, a single-step turn-off competitive, and a turn-off bioreceptor competitive assay. Each of the assay formats will be initially evaluated to determine a final assay format. Assays will be evaluated based on sensitivity, reproducibility, and ease-of-use, with the best performing assay format optimized in Objective 4.Objective 3.1: Multi-Step Turn-Off Competitive Assay. The first assay format is a multi-step turn-off competitive assay. In this format, BSA-PFOA bioconjugates will be immobilized on the assay surface. Sample containing PFAS will then be added to the PPARα detection reagent off-cartridge, before mixing and incubating the sample to allow for interaction between PFAS and PPARα.Objective 3.2: Single-Step Turn-Off Competitive Assay. If the multi-step assay demonstrated in Objective 3.1 demonstrates feasibility, then a condensed version of the assay will be screened.Objective 3.3: Turn-Off Bioreceptor Competitive Assay. In an alternative assay format, the specificity of the bioreceptor PPARα towards PFOA will be leveraged to further simplify the assay. The fluorescence intensity is measured. Rather than relying on a fluorescent dye-labeled antibody for the detection and readout, the PPARα-dye serves as both the detection and reporter reagents.Objective 4, Optimize Best Performing Competitive Immunoassay: The best performing assay format from Objective 3 will be optimized. In Phase I, optimization of cartridges will have the goal of demonstrating feasibility of the assay format and that the assay sensitivity is tunable.Objective 4.1: Demonstrate Tunability of Assay Cartridge Reagents. The best performing assay format tested in Objective 3 will be used moving forward for the final stages of assay development. In this Objective, the assay tunability using reagents on the assay cartridge will be demonstrated.Objective 4.2: Demonstrate Assay Tunability of Assay Detection Reagents. The best performing assay conditions from Objective 4.1, based on sensitivity and reproducibility, will be used in this Objective.Objective 5, Evaluate analytical performance of immunoassay by measuring the limit of detection, limit of blank, and assay range: In this Objective, the analytical performance of the assay cartridges, developed in Objective 4, will be evaluated. The assay will be evaluated for analytical sensitivity (i.e., limit of blank (LOB), limit of detection (LOD), lower limit of quantification (LLOQ), upper limit of quantification (ULOQ), and range), analytical precision, and accuracy. Analytical sensitivity and precision will be evaluated using spiked laboratory water samples, while accuracy will be measured using certified reference standards. The goal is to achieve a LOD of ≤1000 ppt with a stretch goal of a LOD of ≤10 ppt and percent coefficients of variation (%CVs) of ≤20% over the linear dynamic range of the assay. For this Phase I feasibility study, a LOD of hundreds of ppt is acceptable, but the optimized assay developed in Phase II will have a goal LOD of ≤10 ppt.