Grant: Collaborative Health Research Projects (NSERC partnered)

We propose the development of a cell biosensor which reports on the effects of external stimuli on cellular functions and signalling activities. This biosensor is based on surface plasmon resonance (SPR) for which proof of concept in cell signal monitoring has recently been demonstrated by our group. This biosensor will allow cellular signalling to be recorded label free in real-time to address the following needs, for which current technologies are lacking in performance and/or flexibility: 1) screening compound libraries in transfected cell models to detect positive responses and thus facilitate the pre-selection of promising compounds; 2) discrimination of specific intracellular signaling pathways; 3) working with primary cell cultures isolated from various tissues and expressing targets of interest, enabling the assessment of tissue selectivity of compounds; 4) monitoring of cell membrane receptor response desensitization. SPR sensing is based on resonant coupling of light to plasma oscillations in a thin metal film, at a metal-dielectric interface. The dielectric interface, air or water, is the sensing medium, where changes close to the surface (within ~250 nm) such as the attachment of biomolecules or conformation or structural changes in cells are directly detected; SPR sensing is therefore markerless. We propose to expend this work through the following objectives: Obj. 1: Construction of a SPR-based detection platform for cell signal monitoring and simultaneous fluorescence imaging. This specialized SPR platform will have a sensing interface (excitation wavelength, metal/dielectric materials, optics, and detection hardware), environmental controls, and fluid handling capability optimized for measurements on living cells. By taking advantage of the surface plasmon enhanced fluorescence phenomenon, the platform will be capable of simultaneous fluorescence imaging. Obj. 2: Assignment of pathways and cellular events by SPR in transfected and primary cell cultures with an emphasis on delta opioid receptor (DOR) signalling in a pathophysiological relevant context. Here, we will design a series of experiments targeting the contribution of receptor-activated signalling pathways to the ensuing cellular events. Using SPR, the experiments will examine movements of molecular, organelle, or cytoskeleton components, occurring within the cell as a result of receptor-induced individual signalling pathways.