With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Yi Xiao and her research team in the Department of Chemistry and Biochemistry at Florida International University (FIU) seek to develop a widely usable method for the rapid generation of DNA-based bioreceptors, termed aptamers, which selectively recognize small molecules targets. Aptamers have shown promise as key sensing components for point-of-use, small-molecule biosensing detectors that recognize target molecules by aptamer binding to the targets, making them potentially valuable for scientific, clinical, and forensic applications. However, current methods for aptamer generation are labor intensive, time consuming, and typically yield aptamers that are unable to clearly tell the difference between molecules of similar shape and size. The proposed approach produces high-quality aptamers with superior recognition properties and significantly accelerates the aptamer isolation process. Moreover, this technique might replace existing methods for aptamer generation. In parallel with these research efforts, Professor Xiao, in partnership with Hispanic-serving and historically black colleges and universities, as well as student training programs at FIU, plans a multi-level outreach effort focusing on underrepresented minority (URM) students. These activities will provide students with opportunities to participate in scientific research and encourage them to pursue careers in science, technology, engineering, and mathematics (STEM) fields. In pursuit of this, Dr. Xiao gives science seminars and presentations, organizes summer science camps, and provides summer internships. Leveraging the high structural specificity and efficiency of flap endonuclease 1 (FEN1), Professor Xiao and her research team seek to develop an innovative and generalizable nuclease-assisted aptamer isolation technique, termed NA-SELEX, for the rapid isolation of high-affinity, small-molecule-binding aptamers. To establish this technology, a model SELEX system is first used to systematically study and optimize FEN1-based strand separation. The feasibility and generalizability of NA-SELEX is then demonstrated by isolating high-affinity aptamers for different small-molecule targets with clinical and forensic relevance. Finally, the sensing capabilities of the resulting aptamers is assessed by fabricating electrochemical aptamer-based sensors for analyte detection in biological samples. This new approach has the potential to efficiently generate high-affinity, customizable aptamers on demand for any small-molecule target and greatly facilitate the development of sensing elements for the detection of small molecules for various applications such as food safety, environmental monitoring, therapeutic treatment, and medical diagnostics. In addition, an educational outreach program at FIU increases the exposure of graduate, undergraduate, and high school URM students to STEM fields and promotes an inclusive environment for URMs engaged in STEM.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.