Biophotonics: Gold Superparticles as Raman-active Nanoprobes of Molecular Transport Across Cell Membranes

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0086804WeiThe objective of this proposal is to develop a minimally intrusive, Raman-based sensor for monitoring changes in intracellular ion and neurotransmitter concentrations. These will be used to address outstanding questions in transporter function. Gold-silica 'superparticles' (shell of gold particles around a silica core) 100-200 nm in diameter will be prepared by self-assembly techniques and used as substrates for surface-enhanced Raman scattering (SERS). The surfaces of the superparticles will be functionalized with non-fluorescent receptors designed to recognize inorganic or organic cations. These nanoprobes will be introduced into cells via picoliter injection and will be monitored for SERS using near-infrared laser excitation. Sampling will be performed using a micro-Raman instrument with high spectral resolution coupled to an inverted microscope stage. The vibrational spectra will enable specific binding events to be detected by direct assignment of analyte group frequencies or by modulation of receptor spectral profiles. Detection limits can be in the low nanomolar range with resolutions on the order of milliseconds.Raman-active nanoprobes will be employed to investigate two transport systems of fundamental importance to neuron activity. First, the influx of Zn2+ ions will be monitored by SERS in a time-dependent fashion to provide insight into the mechanism of Zn2+ transport by Ca2+ ion channels. This will be correlated to electrophysiological measurements of total ion influx by patch clamp recording techniques. Second, novel reverse transport activity by the serotonin transporter (SERT) will be investigated by monitoring intracellular levels of serotonin and the amphetamine--like drug MDMA. Third, the mode of SERT transport will be elucidated by correlating intracellular neurotransmitter concentrations with changes in ion current caused by Na+ co--transport, again by the concurrent use of SERS and patch clamping techniques. This may reveal valuable insights into the mode of action behind amphetamine and related psychoactive drugs. These studies are representative of the myriad molecular transport issues that can be addressed by intracellular monitoring, such as actively coupled transport of carbohydrates and amino acids, efflux pumps in multidrug resistant cells, and viral infections.The mutual exchange of ideas from traditionally unrelated disciplines will provide fertile ground for student training. Students will be exposed to issues in supramolecular and materials chemistry (organic synthesis, colloid science, design principles in molecular recognition), photonics (microscope and laser optics, Raman theory and applied spectroscopy, signal processing), and molecular and cellular neuroscience (cell transfection and cultures, transporter function, electrophysiology). Their intimate involvement in the proposed project will make them experts in one or more of these areas, and have a working knowledge of the others by their active participation in project-related discussion groups and conferences.