Collaborative Research: Multimodal Mapping of Gene Isoforms by Electromicrofluidic Manipulation

Cells produce different versions of genes from the same genetic material. This ability is crucial for the functioning of organisms in various situations. However, current methods for studying different gene versions are limited and cannot precisely analyze their expression within the organism. Additionally, studying different protein versions is challenging because existing tools may not distinguish between them. While there are techniques available to measure the abundance of gene versions, they require isolating and separating cells, which results in the loss of important information about cell interactions and the environment. In this project, the investigators aim to develop a new technology called electromicrofluidics (EMF), which combines electricity, engineered gels, and cell culture on a microchip to automatically study gene versions at the single-cell level. This technology will provide insights into how these different gene versions function in complex biological processes involving multiple cells, such as development, growth, and repair. In addition to creating a novel biosensing approach, the research will be integrated across several educational fronts, including graduate program development, active learning through student-designed projects, social media dissemination, undergraduate and minority research opportunities,and outreach programs. The research aims to establish an EMF based biosensing platform for comprehensive gene expression profiling. This platform incorporates electroalignment-enhanced anisotropic gel polymerization and in situ immunolabeling and amplification to enable simultaneous analysis of multiple gene expression modes. The investigators will explore different gel layer configurations suitable for studying protein isoforms and (ribonucleic acid) RNA splicing variants. This approach allows for accurate profiling while minimizing sample loss and contamination during transfer. By implementing electroalignment-enhanced anisotropic gel polymerization, the lateral diffusion of molecules is constrained, resulting in improved spatial resolution and sensitivity. This enhancement facilitates in situ single-cell analysis, enabling the study of spatial coordination and cell heterogeneity. Furthermore, the use of EMF technology automates the assay procedures, enhancing reproducibility and facilitating wider adoption of the platform. Initially, 3D gel assays will be developed to analyze protein isoforms and RNA splice variants. Subsequently, these assays will be integrated into a versatile platform capable of simultaneously detecting multiple protein isoforms and RNA splicing variants. The project will focus on mapping the Neurogenic locus notch homolog protein (NOTCH) family members in migrating cell monolayers to evaluate their regulatory functions in collective cell migration. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.