Poster J16 - Colocalization of Intracellular Organelles and Single Walled Carbon Nanotubes Sensors

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sofia E. Morales-Mendivelso (she/her/hers)

Undergraduate Research Assistant at University of Nebraska-Lincoln
University of Nebraska-Lincoln
Lincoln, Nebraska, United States

Primary Investigator(s)

NI

Nicole Iverson

Primary Investigator
University of Nebraska Lincoln
Lincoln, Nebraska, United States

Introduction: Nitric oxide (NO) is a key signaling molecule known to regulate the immune system and plays a significant role in many inflammatory diseases, including cancer. NO levels can impact tumor progression, making its detection crucial. Single walled carbon nanotubes (SWNT) can be made into NO sensors by wrapping them with (AT)15. The (6,5) SWNT that we are using are excited at 561 nm and emit light at 990 nm. However, upon contact with NO, the SWNT’s fluorescence quenches, enabling NO detection. Unfortunately, since the NO sensor is a “turn off” sensor, we are unable to track the SWNT when it interacts with NO and signal quenching occurs. The overarching goal of this project is to add a fluorescent tag to the DNA that wraps the SWNT to enable tracking of SWNTs independent of NO levels, allowing researchers to track SWNT and determine the specific organelles that are producing NO during different cellular functions and in different disease states.

Materials and

Methods: The (6,5) SWNT were wrapped with two different DNA strands at various ratios, one that was fluorescently tagged and one that was not tagged. The non-tagged DNA was (AT)15, which provided the NO sensor specification. Multiple strands of DNA were fluorescently tagged with 6FAM or CY5 and used to wrap the SWNT. SWNT wrapping was performed by using bath sonication for 20 minutes, followed by four rounds of tip sonication for 10 minutes each with a pulse of 59 seconds on and 10 seconds off, and finally two centrifugation for 90 minutes. Following each centrifugation, the supernatant was retained, and the pellet was discarded. The concentration of wrapped SWNT in the supernatant was determined with UV-Vis as previously described (Attal et al., 2006). Different concentrations of wrapped SWNT (1mg/L, 5mg/L, and 10mg/L) were added to MDA-MB 231 (triple negative breast cancer) cells, which are known for their high NO levels, prior to 3 or 24 hours of incubation. The samples were tested on a custom built hyperspectral microscope to analyze DNA wrapping efficiency and sensor after cellular internalization. ImageJ and MATLAB were used to analyze fluorescent intensity, cell viability, and co-localization of the SWNT with fluorescently tagged organelles.

Results, Conclusions, and Discussions: Results show that high concentrations (10mg/L) of the fluorescently tagged SWNT were toxic for the cells within 3 hours, while lower concentrations resulted in low fluorescent intensity when only incubated for 3 hours prior testing. Therefore, low concentrations of SWNT (1mg/L and 5mg/L) were incubated with cells for 24 hours. Both CY5 (sample data shown below) and 6FAM labeled SWNT were successfully internalized by the cells without causing toxicity after 24 hours. Sample data for one of the CY5 studies is shown below, demonstrating the co-localization of SWNT with the CY5.

Acknowledgements (Optional): I would like to thank the NIH (R35GM138245) and NSF (2050587) for the funding.