(H-315) Suppressing Non-Specific Binding of Proteins onto SWNT Sensors for Enhanced Quantification of Extracellular NO Concentration

Introduction: Nitric Oxide (NO) is a reactive nitrogen species that plays key roles in the vascular, nervous, and immune systems. Although its presence is well-known, its effects on disease progression are not fully understood. For instance, the presence of NO has shown both positive and negative effects on cancer [1]. Thus, in order to better understand disease and produce more effective methods for diagnosis, treatment, and prevention, an improved NO sensor is needed. The problems with the previous NO sensor technology are its lack of specificity and its absence of spatial and temporal information. Recently it was discovered that a real-time NO sensor could be made by wrapping a Single-Walled Carbon Nanotube (SWNT) with (AT)₁₅, a single-stranded DNA (ssDNA), [2], [3]. However, when tested with cells in growth media, the sensor’s signal was attenuated by the proteins present. It is hypothesized that by functionalization of SWNT sensors with Polyethylene Glycol (PEG), the interactions between the sensor and the surrounding proteins can be minimized, thus making a more suitable NO sensor for in vivo, real-time, extracellular measurements.

Materials and Methods: Three different SWNT-based sensors were tested, each with a different ratio of Biotinylated (AT)₁₅ to (AT)₁₅ to PEGylated (AT)₁₅, and the ratios were 1:1:0, 1:1:1, and 2:1:1. The sensors were immobilized onto a sensory platform, avidin coated glass slides, and the fluorescence intensity of each was recorded using a custom-built, nIR hyperspectral microscope. The emission spectra of each sensor before and after the addition of protein containing media were recorded. Additionally, in vitro NO sensing capabilities of each sensor were tested.

Results, Conclusions, and Discussions: The heat maps shown in Figure 1 indicate that the 2:1:1 ratio sensor results in the highest fluorescence intensity. It is hypothesized that having 50% of the biotinylated (AT)₁₅, 25% of the (AT)₁₅ and 25% of the PEGylated (AT)₁₅ results in the optimal SWNT attachment to the surface and that the PEG causes additional SWNT, beyond those adhered through the avidin-biotin interaction, to stay on the platform. The 2:1:1 ratio sensor also has the lowest signal attenuation with the addition of proteins at a 17.85% decrease. Also, in vitro tests showed that PEGylated SWNT sensors showed enhanced NO sensing capabilities. Since the 2:1:1 ratio sensor showed even distribution with the highest fluorescence intensity and the least amount of signal attenuation in the presence of protein, it would be the most suitable sensor for testing in cell culture.

References (Optional): [1] Xu, W., Liu, L., Loizidou, M. et al. The role of nitric oxide in cancer. Cell Res 12, 311–320 (2002).

[2]Iverson, N. M., Strano, M. S., and Wogan, G. N. 2015. In vivo delivery of nitric oxide-sensing, single-walled carbon nanotubes. Curr. Protoc. Chem. Biol. 7:93-102.

[3] Joseph A. Stapleton, Eric M. Hofferber, Jakob Meier, Ivon Acosta Ramirez, and Nicole M. Iverson ACS Applied Nano Materials 2021 4 (1), 33-42