Poster J14 - Developing in-vitro Nucleic Acid-Quantum Dot Complexes to Detect Cadmium with Potential Multiplexing Capabilities

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Era Srivastava (she/her/hers)

Undergraduate Research Assistant
Case Western Reserve University
Bloomingdale, Illinois, United States

Co-Author(s)

Sara Desai, -

Undergraduate Research Assistant
Case Western Reserve University, United States
Kevin WR Liu

Research Assistant
Case Western Reserve University
Cleveland, Ohio, United States
MT

Meghna Thakur

Research Scientist
U.S. Naval Research Laboratory, United States
GE

Gregory Ellis

Research Biologist
U.S. Naval Research Laboratory, United States
IM

Igor Medintz

Research Biologist
U.S. Naval Research Laboratory, United States

Primary Investigator(s)

Divita Mathur, PhD (she/her/hers)

Assistant Professor
Case Western Reserve University
Cleveland, Ohio, United States

Introduction: Exposure to metal contaminants can have severe effects on human health. Current biosensors that detect the presence of metals in fluids are largely cell-based with inefficient response times, portability issues, and lack multiplexing capabilities. Studies show that bacteria are capable of detecting and responding to metals. Bacterial operons are primarily responsible for such “sensing” mechanisms and can be harnessed and coupled with enzymatic activity to report the presence of metals in cell-free systems (Beabout et al., 2020). Quantum dots (QDs) are semiconductor fluorescent nanoparticles that have diverse optical properties, allowing them to be distinguishable from each other. When excited, QDs transfer energy to other “acceptor” fluorescent molecules in close proximity, which then emit energy in the form of fluorescence. By linking a QD and acceptor fluorophore through DNA containing an enzyme recognition sequence, we can observe changes in fluorescence over time in the presence of the enzyme (Mathur et al., 2022). QDs’ unique emission spectra allows for the possibility of creating a multiplexed sensor with the ability to detect multiple analytes by pairing different QDs with their respective acceptor molecules. Thus, by coupling bacterial operons with QD-based sensors, we aim to create a cell-free, multiplexing system that can efficiently detect heavy metals in fluids. In this poster, I discuss the validation of a QD525-Cy3 sensor that is responsive to enzymatic activity, the optimization of the bacterial operon reporter and repressor construct system, as well as initial assessments of sensor performance in presence of cadmium (Cd2+).

Materials and

Methods: Reporter and repressor gene blocks were amplified through polymerase chain reactions (PCR), and then purified. The concentrations of the PCR products were obtained using a NanoQuant, and then diluted to the necessary concentration for further use. Our collaborators have previously designed the PDD (Peptide-DNA-Dye) portion of the sensor: the peptide-PNA (peptide nucleic acid) strand hybridizes with the QD, a DNA strand that serves as a linker between the two fluorophores, and the DNA strand functionalized with the acceptor dye molecule. The PDD is assembled in a thermocycler to ensure proper annealing of the three components. Then, the QD and assembled PDD constituents are mixed together and left to assemble at room temperature for an hour. Once ready to use, the sensor constituents are all pipetted and scanned using a Tecan Plate Reader. This includes the cell-free extract myTXTL, the QD-PDD, the reporter and repressor genes, metal(s), and buffer. Aside from the myTXTL and QD-PDD, the concentrations of each of these components may vary depending on the experiment. We collect spectral emission data ranging from 400-800 nm, as well as kinetic data, measuring fluorescence at the expected donor and acceptor peaks to see FRET efficiency and acceptor/donor fluorescence ratios over time. This data is then analyzed using Origin. For trials with metals, we calculate the fold-change ratio, to see if the presence of the target metal changes our sensor activity. We determined our baseline value for this ratio to be 1.25 to provide distinguishable data.

Results, Conclusions, and Discussions: We first validated the QD-PDD complex designed by Mathur et al. (2022). We assembled a complex of QD525 (donor) and Cy3 (acceptor), achieving high FRET ratios (Acceptor peak/Donor peak fluorescence) of 0.831. After verifying the construction of a QD-PDD complex, we used commercially sourced BamHI to test its ability to be separated by the cleaving of DNA at the enzyme recognition sequence encoded in the PDD, and produce a change in FRET. Our findings supported the literature: we observed A/D ratios decrease from 0.910 to 0.153 after 4 hours in the presence of BamHI. Next, we tested the sensor in a cell-free system with a reporter gene encoding BamHI, determining an optimal gene concentration of 0.05 nM. We optimized the repressor concentration to effectively inhibit reporter activity, finding 29 nM to be optimal. We then tested sensor activity in presence of Cd2+, testing a range of concentrations between 0.25 - 10 µM. We found that the sensor is able to report on concentrations of 0.5, 5, and 10 µM. Our sensor can detect Cd2+ at the short-term US Military Exposure Guideline level for Cd2+ (0.5 µM). Further testing must be done to achieve lower detection limits and meet the World Health Organization recommended limits of exposure (0.027 µM). However, because our sensor was unable to produce fold-changes of 1.25 or higher for concentrations within this range, further testing is required to validate the sensor’s capabilities. We also aim to run tests with non-target metals, such as Zn2+, Ca2+, and Fe2+. Another future direction is to explore the quantification of results, reporting on the concentration of the target metal by relating it to how fast the A/D ratio changes. Additionally, we plan to optimize corresponding reporters and repressors for other QDs with their respective acceptor molecules to create a multiplexing system. Previous research has shown that QD425-ATTO425 and QD625-AF647 are two potential pairs. Once we develop a biosensor for metal detection, the same concept can be extended to other targets. By altering the bacterial operons to respond to other molecules of interest, we can create sensors for detecting substances like neurotransmitters.

Acknowledgements (Optional): I would like to thank undergraduate student Sara Desai, who contributed an equal amount of time in the lab carrying out the experiments, data collection, and analysis throughout this project. I would also like to thank collaborators Kevin Liu, Drs. Meghna Thakur, Gregory Ellis, Igor L. Medintz, and Divita Mathur for their contributions to the design and development of the sensors, as well as their continued guidance. Lastly, I would like to thank Case Western Reserve University for funding this research and the Biomedical Engineering Summer Internship Program for providing travel support for this conference through the National Institute of Biomedical Imaging and Bioengineering.