Quantum Dots-based Nanobiosensors for Simultaneous Dynamic Measurements of Multiple Intracellular Ion Concentrations

摘要

Cells maintain their homeostatic functions by dynamically regulating the concentrations of intracellular ions via ionic channels. Using two recently developed QD-based nanobiosensors for Cl~- and Na~+ that emit separate wavelengths, herein we report the fluorescence microscopy measurements of the concentrations of intracellular chloride ([Cl']i) and sodium ([Na~+]_i) simultaneously in intact cells using a 405 nm excitation wavelength. The CI-QD_(525) and Na-QD_(630nm) were constructed by conjugating the chloride ion receptor, MEPTU, and sodium ion receptor, 12-crown-4, within the FRET distances of their respective QD_(525nm) and QD_(630nm). This enables FRET energy transferred from the QD (donor) to the receptor complexes (acceptor). The fluorescence intensities of Cl-QD_(525) and QD_(630nm) determined by photon counting were inversely proportional to the increased concentrations of the respective Cl~- and Na~+ according to Stern-Volmer. By co-loading C1-QD_(525)™ and Na-QD_(630)™ into HEK-293F or T84 cells, we have determined the dynamic responses of [Cl~-]_i and [Na~+]_i by pharmacologically manipulating the chloride and sodium channels using their respective agonists and antagonists. The measurements of the dynamics of [Cl~-]_i and [Na~+]_i have not heretofore been possible without these QD-based nanobiosensors. The predictable physio-pharmacological responses elicited by these agents indicate that the regulatory mechanisms of [Cl~-]_i and [Na~+]_i are independent but coupled. Investigations into the mechanisms of the [Cl~-]_i and [Na~+]_i signal transduction pathways and for translational ion channel drug discovery can now be performed using this assay.