Lead is an industrially useful element, but too much exposure is known to be extremely dangerous to human health. Toxicity and poisoning can result with symptoms such as anemia, kidney and brain damage, and death. A particular concern is lead toxicity in children due to the irreversible neurological damage and developmental defects that can occur. The water crisis in Flint, Michigan is a recent and well-known example highlighting these disastrous effects caused by lead concentration far exceeding what is acceptable. In order to prevent such catastrophes, lead detection is critical, but unfortunately, requires highly trained personnel alongside a centralized laboratory. One way of combating this problem is through the application of DNAzymes. DNAzymes are a class of nucleic acids possessing enzymatic activity. Lead sensors have been created with these enzymes as they offer the ability to achieve easy and accurate on-site testing. These enzymes require the presence of lead for activity, leading to the degradation of a reporter substrate and resulting in a measurable fluorescent signal. Though reliable, this method is extremely time consuming. This is because DNAzyme-based sensors suffer from low enzymatic activity, or a small kcat, equating to a slow response. To combat this problem, I have proposed incorporating CRISPR-Cas12 as a signal amplifier. CRISPR-Cas12 is an emerging biosensor currently impacting clinical diagnostics, most notably in COVID-19 testing. Cas12 has shown to be a robust and reliable sensor, possessing a kcat over 100 times greater than that of a regular DNAzyme. Despite the impressive impact on detection sciences, CRISPR-based sensors have been limited to nucleic acid biomarkers. To create a sensor incorporating both systems, I have designed my DNAzyme system to activate Cas12 in the presence of lead. Success will not only enhance lead detection, but expand the scope of CRISPR-Cas12 technology, broadening both bioanalytical chemistry and biotechnology.
Author(s): Alexis Rothchild, Biochemistry Major
Advisor(s): Kevin Yehl, Department of Chemistry and Biochemistry
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