Diamond-nanoneedle-assisted Intracellular Sensing and Its Application for Probing Innate Immune Response in Living Neurons
DescriptionMost of cellular behaviors and functions are associated with intracellular signaltransduction. Understanding such signaling cascades and network is one of the core topics inmodern biology, and is also crucial for the development of many therapeutic reagents.Therefore, great efforts have been made to develop novel tools for probing and analyzingintracellular signaling in living cells with sufficient sensitivity, specificity andspatial/temporal resolution. In this proposed study, we aim to develop a minimally invasivemethod for in situ probing of innate immune response in living primary neurons. Thebiosensing device will be based on a diamond-nanoneedle array functionalized withaptamer-based molecular sensors. We have shown that nanostructures of similar morphologygreatly facilitate effective intracellular delivery by temporarily disrupting cell membrane(Wang et al, Nature Commun 2014). Grounded on these preliminary results, the proposednovel biosensing device will significantly benefit investigations of neuronal signaling from atleast four perspectives. First, the nanoscale device will be applied to access cytosolic domainof neuronal cell by a carrier-free, nanoneedle-assisted, and well-controlled mechanicalprocedure, which dramatically reduce the toxicity issue and potential interference fromcarrier elements. Second, the aptamer-based molecular sensing strategy takes advantage ofthe highly specific nucleotide-protein interaction, and provides the capability for real-timedetection of signaling cascades and dynamics upon activation. Third, this device willintegrate our nanoneedle-based intracellular delivery with molecular sensing, and enablesunprecedented studies of cellular host-defense in response to artificially introduced pathogenmolecular such as DNAs or RNAs, which is especially important for the study of neuronalinnate immunity. Fourth, a reversible application procedure will guarantee retrieval ofbiosensors after cytosolic insertion, enabling “protein-fishing” of relevant messengerelements, released upon activation of many signaling pathways, for further multimodalanalysis. With all of these great features, the diamond-nanoneedle-assisted intracellularsensing will particularly be used to investigate the innate immune response in primaryneurons, especially the host-defense-related gene activation by exogenous DNAs or RNAs.This is especially significant given the increasingly recognized role of innate immunity in allkinds of attacks on the integrity of the central nervous system (CNS), including brain injuries,neurodegenerative diseases, brain tumors and viral/bacterial infections. It is expected thatsuccess with this project not only provide a novel tool for dissecting intracellular signaling,but can also resolve valuable insights into neuronal innate immunity, which is potentiallybeneficial for the treatment of brain trauma and neurodegenerative diseases.?
|Effective start/end date||1/01/16 → 30/12/19|
- biosensor,biomedical devices and sensors,immnosensing,Geometry-Force control,