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Developing a Biosensor for PFAS Detection
PFAS are a large and diverse family of synthetic chemicals that have been manufactured for industrial and consumer products since the early 1930s. (NIEHS) These chemicals have at least one fully fluorinated carbon bond, which makes it harder for them to degrade in the environment. Originally developed for their grease-resistant properties, PFAS are found in a range of products, including nonstick cookware, plastic food wrap, and even firefighting foams. (U.S. EPA) Due to the inability to degrade, they can lead to serious health concerns, with research showing links to various cancers and organ failures.
While related chemicals like PFOA and PFOS have largely been phased out of commercial use in the United States, other variations, such as GenX, persist and carry the same health implications as their predecessors. Current regulations from the FDA are largely voluntary phase-outs rather than hard bans. The present limit for PFOA—4 parts per trillion—is set based on the most precise detection capabilities currently available (U.S. EPA). This overwhelming application has almost entirely contaminated drinking water, soil, and even the atmosphere, to the point where nearly 97% of Americans have traces of them in their blood (NHANES), hence, why there is a need for organizations like the EPA to set a regulation aimed at reducing the environmental and health risks caused by these chemicals.
This issue highlights an urgent need for low-cost, effective biosensors and stronger regulations related to PFAS contamination. Areas of poverty or rural areas, such as communities in Latin America, have high levels of PFAS in local water systems and wells. This comes as a result of pesticides that are used in agriculture, which leak into irrigation systems and community water systems (UC Berkeley Public Health). These areas typically lack the financial resources to afford expensive PFAS detection technologies. Methods to develop inexpensive and fast-acting PFAS detection methods could help these rural communities that lack proper access to avoid unnecessary consumption and eventually suffer both environmental and health risks. Current methods of detecting PFAS in the environment remain rather expensive and unaffordable to the average person. In addition to being expensive, current methods require complex and specific expertise to perform, usually being extremely time-consuming as well. The most popular current methods are LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry), GC-MS (Gas Chromatography-Mass Spectrometry), and IMS (Ion Mobility Spectrometry). Each of these methods is used differently depending on the situation; however, for each of them, testing a sample to detect PFAS would cost hundreds of dollars. An amount inaccessible to many. New methods are being developed as time continues to pass, though these are still very much concepts and not ready for widespread use.
Current methods for detecting harmful PFAS chemicals often fall short, as they struggle with accuracy, availability, feasibility, and reliability. We were inspired by a biosensor concept from Dr. Berger that uses the Human Fatty Acid Binding Protein (FABP), which changes its fluorescence in the presence of PFOA (a common PFAS). However, this protein is not specific and can be triggered by other fat-like molecules, leading to a high rate of false positives.
Our goal was to engineer a more precise, reliable, and affordable sensor. We used the chemical structure of PFOA to screen seven databases for predicted proteins that would bind to PFOA specifically. After filtering the top candidates based on cost and scientific relevance, we tested the five most promising proteins in the lab with a DSF assay.
Our experiments revealed that two (TYMS and PDGFRA) of the five proteins showed a significant change in stability when they came into contact with PFAS. The most promising of these was Human Thymidylate Synthase. This breakthrough is a critical step toward developing a highly accurate and dependable biosensor for identifying these persistent contaminants. We are now conducting further research to fully develop this technology into a viable product that can benefit the general public.
Thymidylate synthase (TYMS) is a crucial enzyme for DNA synthesis and repair. Via reductive methylation of dUMP, where 5,10-methylenetetrahydrofolate acts as the methyl donor, deoxythymidylate (dTMP) is synthesized. Because of this dTMP production, TYMS is the only enzyme to produce thymine through de novo means. By producing dTMP, TYMS ensures the supply of deoxythymidine triphosphate (dTTP) for DNA replication and repair.
Structurally, TYMS functions as a dimer, where each monomer has two binding sites, one for dUMP and the other for 5,10-methylenetetrahydrofolate. Its full catalytic site (composed of residue subunits) switches between an active and inactive conformation controlled by a Cysteine residue.
It is this role specific to TYMS in helping make a necessary DNA precursor that makes the enzyme a cancer target for chemotherapies today. Rapidly dividing tumor cells depend on the continuous production of dTMP, and drugs that inhibit TYMS block DNA replication, triggering apoptosis. This makes TYMS highly valuable for cell synthesis.
In addition to its role in DNA synthesis, TYMS is actively linked to the folate cycle. While TYMS uses 5,10-methylenetetrahydrofolate as the methyl donor in dTMP production, the reaction simultaneously generates the folate precursor, dihydrofolate (DHF). DHF is reduced to tetrahydrofolate (THF) by dihydrofolate reductase (DHR) for the cycle to continue. THF is then converted into one-carbon donors, such as 5-methyltetrahydrofolate (5-MTHF), an essential methyl donor for several methylation reactions. These methylation pathways promote the production of several neurotransmitters such as dopamine and serotonin, which help regulate mood, behavior, and emotions.