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Polymer-free optode nanosensors for dynamic, reversible, and ratiometric sodium imaging in the physiological range.

Ruckh TT, Mehta AA, Dubach JM, Clark HA - Sci Rep (2013)

Bottom Line: Ratiometric fluorescent measurements demonstrated that the nanosensors are selective for sodium over potassium by ~1.4 orders of magnitude, have a dynamic range centered at 20 mM, and are fully reversible.The ratiometric signal changes by 70% between 10 and 100 mM sodium, showing that they are sensitive to changes in sodium concentration.These nanosensors will provide a new tool for sensitive and quantitative ion imaging.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, MA 02115 [2].

ABSTRACT
This work introduces a polymer-free optode nanosensor for ratiometric sodium imaging. Transmembrane ion dynamics are often captured by electrophysiology and calcium imaging, but sodium dyes suffer from short excitation wavelengths and poor selectivity. Optodes, optical sensors composed of a polymer matrix with embedded sensing chemistry, have been translated into nanosensors that selectively image ion concentrations. Polymer-free nanosensors were fabricated by emulsification and were stable by diameter and sensitivity for at least one week. Ratiometric fluorescent measurements demonstrated that the nanosensors are selective for sodium over potassium by ~1.4 orders of magnitude, have a dynamic range centered at 20 mM, and are fully reversible. The ratiometric signal changes by 70% between 10 and 100 mM sodium, showing that they are sensitive to changes in sodium concentration. These nanosensors will provide a new tool for sensitive and quantitative ion imaging.

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Calibration experiment performed with PFNs loaded into a micro-dialysis tube at unknown concentrations and imaged on a confocal microscope.Solutions of 0–1000 mM NaCl were washed through, and the mean fluorescent intensity for Rhd18 (green, 580 nm) and CHIII (red, 680 nm) were measured simultaneously (A). The ratio for CHIII:Rhd18 (680:580) was computed in each frame and presented (B) for the same image set.
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f4: Calibration experiment performed with PFNs loaded into a micro-dialysis tube at unknown concentrations and imaged on a confocal microscope.Solutions of 0–1000 mM NaCl were washed through, and the mean fluorescent intensity for Rhd18 (green, 580 nm) and CHIII (red, 680 nm) were measured simultaneously (A). The ratio for CHIII:Rhd18 (680:580) was computed in each frame and presented (B) for the same image set.

Mentions: PFN responses when both fluorophores are simultaneously excited were measured on a confocal microscope by first loaded dialysis tubing with PFNs and then imaging the PFNs in 0–1000 mM sodium (Video S1). Both fluorophores responded dynamically to changes in sodium concentration (Figure 4A), and the ratio of the two fluorophores, measured as 680:580, changed by 87% between 0 mM NaCl and 100 mM NaCl (Figure 4B). PFNs were also responsive to physiologically-relevant changes as well; the fluorescence ratio changed by nearly 60% between 8 and 100 mM sodium. The calibration data also fit the Hill equation (Figure S4) and produced a Kd of 10.9 mM for the ratio of 680:580 nm. This Kd value is lower than the values measured in calibrations performed on a plate reader, but this is likely a consequence of exciting both fluorophores simultaneously since CHIII has some absorption from 555 nm laser excitation and the 580 Rhd18 emission.


Polymer-free optode nanosensors for dynamic, reversible, and ratiometric sodium imaging in the physiological range.

Ruckh TT, Mehta AA, Dubach JM, Clark HA - Sci Rep (2013)

Calibration experiment performed with PFNs loaded into a micro-dialysis tube at unknown concentrations and imaged on a confocal microscope.Solutions of 0–1000 mM NaCl were washed through, and the mean fluorescent intensity for Rhd18 (green, 580 nm) and CHIII (red, 680 nm) were measured simultaneously (A). The ratio for CHIII:Rhd18 (680:580) was computed in each frame and presented (B) for the same image set.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842545&req=5

f4: Calibration experiment performed with PFNs loaded into a micro-dialysis tube at unknown concentrations and imaged on a confocal microscope.Solutions of 0–1000 mM NaCl were washed through, and the mean fluorescent intensity for Rhd18 (green, 580 nm) and CHIII (red, 680 nm) were measured simultaneously (A). The ratio for CHIII:Rhd18 (680:580) was computed in each frame and presented (B) for the same image set.
Mentions: PFN responses when both fluorophores are simultaneously excited were measured on a confocal microscope by first loaded dialysis tubing with PFNs and then imaging the PFNs in 0–1000 mM sodium (Video S1). Both fluorophores responded dynamically to changes in sodium concentration (Figure 4A), and the ratio of the two fluorophores, measured as 680:580, changed by 87% between 0 mM NaCl and 100 mM NaCl (Figure 4B). PFNs were also responsive to physiologically-relevant changes as well; the fluorescence ratio changed by nearly 60% between 8 and 100 mM sodium. The calibration data also fit the Hill equation (Figure S4) and produced a Kd of 10.9 mM for the ratio of 680:580 nm. This Kd value is lower than the values measured in calibrations performed on a plate reader, but this is likely a consequence of exciting both fluorophores simultaneously since CHIII has some absorption from 555 nm laser excitation and the 580 Rhd18 emission.

Bottom Line: Ratiometric fluorescent measurements demonstrated that the nanosensors are selective for sodium over potassium by ~1.4 orders of magnitude, have a dynamic range centered at 20 mM, and are fully reversible.The ratiometric signal changes by 70% between 10 and 100 mM sodium, showing that they are sensitive to changes in sodium concentration.These nanosensors will provide a new tool for sensitive and quantitative ion imaging.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, MA 02115 [2].

ABSTRACT
This work introduces a polymer-free optode nanosensor for ratiometric sodium imaging. Transmembrane ion dynamics are often captured by electrophysiology and calcium imaging, but sodium dyes suffer from short excitation wavelengths and poor selectivity. Optodes, optical sensors composed of a polymer matrix with embedded sensing chemistry, have been translated into nanosensors that selectively image ion concentrations. Polymer-free nanosensors were fabricated by emulsification and were stable by diameter and sensitivity for at least one week. Ratiometric fluorescent measurements demonstrated that the nanosensors are selective for sodium over potassium by ~1.4 orders of magnitude, have a dynamic range centered at 20 mM, and are fully reversible. The ratiometric signal changes by 70% between 10 and 100 mM sodium, showing that they are sensitive to changes in sodium concentration. These nanosensors will provide a new tool for sensitive and quantitative ion imaging.

Show MeSH