PhD Thesis Proposal: Gabrielle Moss

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"Methods for Wash-Free Real-Time Magnetic Nanoparticle-Based Cytokine Biosensing for In Vitro Applications"

Abstract

Magnetic nanoparticle (MNP)-based aggregation assays have been engineered to sense clinically-relevant biomolecules including H1N1 virus, thrombin, staphylococal toxins, and SARS-CoV2 spike and nucleocapsid proteins in vitro. Wherein functionalized MNP (fMNP)-target binding is transduced via magnetic particle spectroscopy (MPS) and can be modeled via Brownian and Néel relaxation. We are developing an fMNP-based aggregation assay to sense tumor necrosis factor (TNF)-alpha in a 3D melanoma tissue culture to aid in the development of immunotherapies for cancer treatment. TNF-alpha is an inflammatory cytokine, whose production can be linked to cell death. An increase in TNF-alpha concentrations in the 3D melanoma tissue culture, following immunotherapy exposure, suggests a positive therapeutic outcome. The 3D melanoma tissue culture is a dynamic environment, with salt and off-target protein concentrations similar to those found in vivo. fMNP-based biosensors suffer from inaccuracies due to interactions with salts and off-target proteins. Off-target proteins tend to nonspecifically adsorb to nanoparticle (NP) surfaces. The adsorbed off-target proteins tend to mask targeting ligands and lead to a loss of efficacy. Salts tend to alter NP stability. Wherein higher salt concentrations destabilize NPs, leading to aggregation. fMNP-based biosensors for 3D tissue culture applications must be engineered to produce accurate readouts in environments having pH as well as off-target protein and salt concentrations within the physiologically relevant range.

For my proposal defense, I will outline several methods to overcome biosensing inaccuracies stemming from interactions with physiological components to enable wash-free real-time fMNP-based cytokine biosensing in 3D tissue cultures. To date we have: (1) demonstrated the effects of off-target proteins and salts on fMNP targeting efficacy using a model system, (2) developed an approach to localize the fMNPs within the 3D tissue culture for monitoring the target concentration profile over time, and (3) developed software to calibrate fMNP MPS signal to target concentrations. Moreover, we are working towards (4) developing a site-directed method for MNP antibody functionalization to promote optimal targeting efficiency, (5) software to predict protein diffusion and consumption throughout the 3D tissue culture, and (6) digital image processing methods to better investigate fMNP-target binding with cryogenic transmission electron microscope images.

Thesis Committee

• Solomon Diamond (chair)
• Jifeng Liu
• Geoffrey Luke
• Kathryn Miller-Jensen (Yale University)

Contact

For more information, contact Thayer Registrar at thayer.registrar@dartmouth.edu.