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

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.

Figure 1

Calibration curves (A) for PFNs showing small changes in sensitivity to sodium through eight days in storage at room temperature. The calculated K_d values (B) for PFNs at each of the eight days. Data represented as mean values with error bars for standard deviations.

Figure 2

Size stability of PFN (A) stored for eight days at room temperature, measured by DLS. Freshly-made PFNs can be separated for size by centrifugation (B) in a manner dependent on centrifugation speed and time. After centrifugation for 9 minutes, PFNs were calibrated to characterize any effect on their sensitivity (C). Data represented as mean values with error bars for standard deviation.

Figure 3

PFN calibration performed in 1–333 mM potassium background (A). K_d values calculated for sodium response in 1–333 mM potassium background (B). Data represented as mean with error bars for standard deviation.

Figure 4. 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.

Figure 5. Reversibility experiment performed with PFNs loaded into a micro-dialysis tube.

Solutions of 10 or 100 mM NaCl were washed through and the mean fluorescent intensity imaged in the dialysis tubing for Rhd18 (green, 580 nm) and CHIII (red, 680 nm) were measured separately (A). The ratio for CHIII:Rhd18 (680:580) was computed in each frame and presented (B) for the same image set, showing the reversibility through three cycles and 19 minutes of imaging.