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Innovative approaches for multiplext detection: DNAzyme and bacteria-based arrays for environmental monitoring

Morrison, Kevin Scott
http://orcid.org/0000-0003-2280-714X
http://rave.ohiolink.edu/etdc/view?acc_num=miami1713531681849758

Abstract Details

2024, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
Detection of environmental pollutants, both inorganic and organic, has been an ongoing area of research for nearly three-quarters of a century. In recent years there has been growing research in the discovery of complementary methods for detection of environmental pollutants that are decentralized, rapid, and can be used onsite. A promising approach is the application of biosensors. Two common sensor classes are DNAzymes and Bacteria-Based Sensors (BB sensors). DNAzyme sensors have been discovered for the detection of many different metals. However, DNAzyme sensors have historically had issues with non-specific activity due to the presence of other metal ions with similar valency as their native metal cofactor. Further, they are limited in multiplex detection of environmental pollutants due to the limited number of orthogonal fluorophores. Moreover, there are limited sources in the literature for the design of DNAzyme sensors for the detection of organic pollutants, e.g., per- and polyfluoroalkyl substances (PFAS). In contrast, BB sensors have been developed to detect a broad range of compounds from metals to organic substances. Although many BB sensors have been developed for the detection of organic pollutants, there have been limited reports in the literature of BB sensors designed for the detection of PFAS. This dissertation aims to leverage the nonspecific activity of DNAzyme sensors by using a pattern-based readout to identify metal pollutants. Towards this goal, we investigated the DNAzyme cross-reactivity on five DNAzymes (17E, GR-5m EtNa, Ag10c, and NaA43) with eight different metals, and analyzed the data through several statistical methods. Through this approach, we were able to correctly identify four validation solutions, of which, one validation solution consisted of 100 μM Zn2+, which is a known interferent for 17E. Following this, the dissertation explored a new DNAzyme reporter architecture, DzNanoporeSeq, where sequencing is used as a signal readout instead of fluorescence. DzNanoporeSeq showed high accuracy for determination of validation solutions when compared to the fluorescent substrate architecture. This can especially be observed for the 5 nM Pb2+, where DzNanoporeSeq determined this solution to be 4.95 ± 0.12 nM, while the fluorescent reporter architecture determined this solution to be 14.53 ± 1.22 nM. With the promise of multiplex detection of metals by DzNanoporeSeq, this dissertation sought to explore the possibility of constructing an array for the detection of PFAS but using bacterial biosensors. To achieve this, a commercially available E. coli K12 MG1655 promoter library was mixed for each promoter strain to be present in the same quantity, and then screened using fluorescence activated cell sorting (FACS) for the determination of promoters that would “turn-on” and “turn-off” due to PFAS. However, this research did not elucidate promoters sensitive to PFAS.
Kevin Yehl (Advisor)
Richard Page (Committee Chair)
Jason Boock (Committee Member)
Cory Rusinek (Committee Member)
Neil Daneilson (Committee Member)
182 p.
Analytical Chemistry; Chemistry
DNAzymes, Metal Sensing, Pattern-Based Sensing, Sensor Arrays, Bacterial Biosensors, PFAS, FACS, Nanopore Sequencing

Recommended Citations

Citations

  • Morrison, K. S. (2024). Innovative approaches for multiplext detection: DNAzyme and bacteria-based arrays for environmental monitoring [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1713531681849758

    APA Style (7th edition)

  • Morrison, Kevin. Innovative approaches for multiplext detection: DNAzyme and bacteria-based arrays for environmental monitoring. 2024. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1713531681849758.

    MLA Style (8th edition)

  • Morrison, Kevin. "Innovative approaches for multiplext detection: DNAzyme and bacteria-based arrays for environmental monitoring." Doctoral dissertation, Miami University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=miami1713531681849758

    Chicago Manual of Style (17th edition)

miami1713531681849758
© 2024, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.