2024 AIChE Annual Meeting
Detecting PFAS Using CRISPR-Cas12 As a Signal Enhancer
PFAS (per- and polyfluoroalkyl substances), referred to as “forever chemicals” due to their inability to break down, are becoming widespread contaminates throughout the environment. Concerningly, their exposure is a major public health concern because it is correlated with cancers, liver disease, and birth defects. The current gold standard for detecting PFAS involves liquid chromatography coupled with mass spectrometry (LC-MS). This approach is highly specific and sensitive, but unfortunately is time-consuming and expensive because it requires trained personnel and a centralized laboratory to perform. Therefore, there is significant interest in developing complementary methods to detect PFAS on-site that do not require trained personnel. Aptamer sensors are a class of functional nucleic acid that are emerging as a potential solution. However, the current sensitivity of aptamers is not low enough to detect PFAS at EPA advisory levels. CRISPR-Cas12 is emerging as a powerful tool for detecting nucleic acids at the point-of-care without requiring sophisticated instrumentation. Therefore, we hypothesize that using CRISPR-Cas12 as a signal enhancer of activated aptamer sensors will increase sensitivity for detecting PFAS. Towards this goal, we investigated several aptamer sensor architectures compatible with Cas12 amplification and examined their corresponding thermodynamic landscape for sensor activation. Several DNA designs were tested having varying 3’ and 5’ overhang lengths of 1 to 7 nt. While studies are still ongoing, our results show that sensors with 3’ overhang lengths between 4 to 7 nt had the largest signal to background enhancement compared to sensors with 5’ overhangs. We also investigated buffer compatibility for optimizing aptamer and Cas12 activity. Together, this study is a significant step in designing a more affordable and ultrasensitive biosensor for the detection of PFAS.