ABSTRACT The ability to probe the temporal profile of the protein secretion behavior of individual immune cells will impact future immunology, cell biology, and even infectious disease diagnosis. Knowledge of the ordering and timing of cytokines (water-soluble proteins essential for intercellular signaling) secreted by activated T cells can additionally provide the means to discriminate subsets of differentiated T cells by function. Here, the temporal information is one of the pieces of the whole puzzle in monitoring the behavior of the immune system. The other critical piece is the cytokine-mediated interplay between different cell types, which involves spatial transport of cytokines between cells. Putting both pieces of the puzzle together allows us to capture the full picture of the cytokine release dynamics and cytokine-mediated interactions of cells, which allows us to fully understand the intercellular signaling processes underlying immunity. However, no study has yet obtained such a picture due to the lack of a technology for real-time sensing of intercellular cytokine-mediated signaling processes at high spatial resolution. This research aims to develop a novel label-free imaging technique to fully understand cellular behaviors during cytokine-mediated activation and communication at a single-cell level. Our approach will employ biosensors consisting of plasmonic nanoantenna structures, each specifically targeting a particular cytokine species. We will integrate these biosensors in a microfluidic system incorporating an array of sample/reagent-flow channels and single-cell trapping microwells. The microfluidic sensor integration will provide the ability to capture, manipulate, and activate single cells for cell-to-cell communications on a single chip and to obtain the spatiotemporal profile of cellular cytokine secretion processes in real time, both in a massively, parallel manner. We will also develop a theoretical algorithm that allows us to extract the quantitative values of the local cytokine concentration distributions from measured image intensities. SA 1: We will create highly ordered, high-density plasmonic nanoantenna biosensor arrays, each functionalized by highly selective aptamers against targeted cytokines. SA 2: We will integrate the aptamer-conjugated plasmonic nanoantenna arrays into a single-cell manipulation microfluidic system and achieve real-time single-cell secretion imaging at high throughput. SA 3: We will develop a two-mode (fluorescence and dark-field) microscopy imaging technique to image spatiotemporal cytokine secretomic profile patterns and cell surface sytokine binding sites. Using this technique, we will study the IL-6-mediated dynamic intercellular communication between individual human hepatoma Hep3b cells and CD 4+ T cells.

Narrative This proposal develops a nanoplasmonic-based single-cell imaging technology that enables probing the spatiotemporal profile of protein molecules secreted by individual immune cells. Implementing this technology in studying cellular cytokine secretion behaviors is relevant to the public health because it provides scientific insight into inflammation-related immunology and cell biology governing immune dysregulations and cancer diseases. The technology can be a versatile tool for cellular paracrine/autocrine signaling, cellular immunophenotyping coupled with protein expression profiling, gene expression analysis, molecular-level analysis of infectious diseases, and human leukocyte antigen (HLA) testing.