Cell signaling involves the conversion of an externally sensed stimulus (input) into a change in cellular behavior or morphology that can be measured (output). The output is most commonly cell proliferation, differentiation, growth, apoptosis, or necrosis. This conversion involves the interaction of a network of different classes of signaling molecules (proteins-e.g., kinases, proteases, transcription factors; nucleic acids-e.g., mRNA transcripts, inorganic metabolites). Monitoring such signaling events has applications ranging from the detection of bacterial and viral biothreat agents, to the diagnosis of disease and drug development. Currently, technologies to monitor the activities of signaling molecules belong to one of two categories: (1) offline, where the signaling molecule is monitored upon its removal from the cell or (2) real-time, which monitors the signaling molecule in situ as the signal is being propagated. Each of these approaches has its own inherent limitations. Offline detection renders a “static” picture of the signaling landscape as detection is achieved after the fact. Real-time technologies, while able to monitor signaling events as they occur, are limited to probing only a single class of event at any given time because of their almost exclusive reliance on such low resolution materials as organic fluorophores or pairs of fluorescent proteins engaged in FRET. Thus, technologies which can specifically detect and resolve multiple signaling events in real-time (i.e., multiplex detection) are needed.

Luminescent semiconductor nanocrystals or quantum dots (QDs) are a relatively new class of fluorescence probe with unique properties that make them superior alternatives to traditional organic dyes. Their advantageous properties include (1) the ability to excite multiple populations of QDs at a single wavelength far removed from their emission wavelength and (2) their ability to be implemented in multiplexed FRET-based assays. Building on this latter property as well as on our more recent work on intracellular delivery of QDs, this proposal will seek to address a key technological gap in the field of cellular signaling: the development of the tools to enable the real-time detection and resolution of multiple cellular signaling events. We will develop and characterize signaling sensors aimed at each of the main classes of signaling molecules within mammalian cells; cell surface receptors, protein kinases, proteases, transcription factors, mRNA transcripts, and secondary messengers (e.g., calcium).

This proposal has the potential to meet the current and future needs of such customers as researchers involved in gene expression profiling of host responses to pathogen infection, to those monitoring viral infection of cells. Ultimately, these tools will allow not only the real-time detection and monitoring of signaling events within both eukaryotic and prokaryotic cells, but they will also be amenable to the development of sensors for use within artificial cell platforms.