Lead is a useful element, particularly in industrial settings, but too much exposure can cause lead poisoning and prove to be very dangerous. Symptoms include anemia, kidney and brain damage, and even death. Especially concerning to young children are the neurological and developmental defects caused by lead exposure. The water crisis in Flint, Michigan highlighted such devastating effects, where the lead concentration in drinking water far exceeded what is acceptable by the Environmental Protection Agency (EPA), thus poisoning an entire community. In order to prevent events like this from happening in the future, environmental monitoring for the detection of lead is imperative. One major way to monitor lead is through the application of DNAzymes. DNAzymes are a class of nucleic acids possessing enzymatic activity. Lead sensors based on these enzymes have been engineered by requiring the presence of Pb2+ for enzymatic activity, leading to degradation of a reporter substrate and resulting in a measurable increase in fluorescence signal. Throughout my findings so far, I have designed a lead DNAzyme sensor and screened conditions for optimal activity. These conditions include changing the enzyme and substrate concentration and quantifying the resulting detection limit for lead. These kinetic activity assays are carried out using a microplate reader to measure fluorescence as a function of time. Though this method for lead detection is reliable, it is time consuming. This is due to DNAzymes’ poor enzymatic activity, demonstrated by its low kcat. To combat this problem, I propose incorporating CRISPR-Cas12 as a signal amplifier, where the DNAzyme sensor will activate Cas12, an enzyme possessing a much faster kcat, around 100 times faster. Cas12 is an emerging biosensor in clinical diagnostics, most notably for Covid testing. However, CRISPR technology has been limited to detecting nucleic acid biomarkers. By combining the two enzymes in a single assay, not only will the detection of lead be greatly enhanced, but CRISPR-Cas12 sensors will be expanded to detect a new class of molecules, heavy metals. Success in this work would be a major breakthrough in the fields of bioanalytical chemistry and biotechnology.
Authors: Alexis Rothchild, Kevin Yehl
Advisor: Kevin Yehl, Chemistry and Biochemistry
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