A highly sensitive and selective nanosensor for near-infrared potassium imaging

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Science Advances  17 Apr 2020:
Vol. 6, no. 16, eaax9757
DOI: 10.1126/sciadv.aax9757
  • Fig. 1 Design and sensing mechanism of the K+ nanosensor.

    (A) Schematic illustration for the synthesis of the nanosensor. The NaYF4:Yb/Tm@NaYF4:Yb/Nd (UCNP) core was synthesized and coated with a dense silica layer and a successive mesoporous silica shell. Etching away of the dense silica layer forms a hollow cavity that allows the loading of PBFI. The nanosensor was lastly coated with the K+-selective filter membrane. (B) Schematics showing a magnified view of the nanosensor [from the red dotted box in (A)] and its K+ sensing mechanism. The filter membrane layer allows only K+ to diffuse into and out of the nanosensor, thus excluding the interference from other cations. Once diffused into the nanosensor, K+ will bind to PBFI immediately. Upon NIR irradiation, the upconverted UV light from the UCNPs excites PBFI, leading to the emission of K+-bonded PBFI.

  • Fig. 2 Structural characterization of the K+ nanosensor.

    (A to C) High-angle annular dark-field images of UCNP@dSiO2 (A), UCNP@dSiO2@mSiO2 (B), and UCNP@hmSiO2 (C). (D) Scanning electron microscopy (SEM) image of the shielded nanosensor. (E) SEM image of the shielded nanosensor immersed in an aqueous solution containing 150 mM Na+, 150 mM K+, 2 mM Ca2+, 2 mM Mg2+, 50 μM Fe2+, 2 mM Zn2+, 50 μM Mn2+, and 50 μM Cu2+. (F) EDS elemental line scanning profiles along the white line in (E) reveal that only K+ signals are present in the mesopores and hollow cavities of the shielded nanosensors.

  • Fig. 3 Spectroscopic features, sensitivity, selectivity, and kinetics of the nanosensor.

    (A) Normalized absorption and emission spectra showing the spectral overlap between the emission of the UCNPs (blue line) and the absorption of the PBFI (black line). The red line is the upconverted emission spectrum of the nanosensor. a.u., arbitrary units. (B) [K+]-dependent upconverted luminescence (UCL) spectra of the nanosensor at varied [K+]. (C) Intensity ratio of the 540-nm UCL emission to 475-nm emission (I540 nm/I475 nm) is linearly correlated to [K+]. (D) Fluorescence intensities of the unshielded and shielded nanosensors at 540 nm increase by 5- and 12-fold, respectively, in response to [K+] that varies within the physiological range of 0 to 150 mM (n = 5). F is the fluorescence intensity at a certain [K+], and F0 is the emission intensity at 0 mM K+. (E) [K+] in aqueous solutions decrease upon application of unshielded, shielded nanosensor, or shielded UCNPs. Initial concentration of K+ in the solutions was 5 mM (n = 5). (F) Variations in fluorescence intensity of the shielded nanosensor upon stepwise and alternate additions of different concentrations of KCl or NaCl. (G) Selectivity of the shielded and unshielded nanosensors toward [K+] against various other physiological cations. (H) Time-dependent fluctuations of the nanosensor luminescence intensity upon the addition of K+ and EDTA into the solution. (I) Fluorescence intensity of the shielded nanosensor during K+-rich and K+-absent cycles (n = 5).

  • Fig. 4 Imaging of K+ efflux in HEK 293 cells.

    (A) Schematics showing the detection of K+ efflux by a streptavidin-conjugated nanosensor, which is tethered to a biotin-modified cell. (B) Confocal microscopy images showing the fluorescence (at 400 to 500 nm and 500 to 600 nm) of nanosensor-labeled HEK 293 cells at different time points after treatment with the K+ efflux stimulator (a mixture of 5 μM nigericin, 5 μM bumetanide, and 10 μM ouabain). (C) Time courses of nanosensor fluorescence variations and calculated time dependence of K+ efflux rate after treatment with K+ efflux stimulator. (D) Time-dependent fluorescence fluctuations of shielded nanosensor-labeled HEK 293 cells after treatments with different concentrations (20, 40, 60, 80, and 100%) of K+ efflux stimulator. Results from five independent experiments were summarized as mean ± SEM in (C) and (D).

  • Fig. 5 Nanosensor-assisted imaging of extracellular K+ waves across the cortical surface in a CSD model of mice.

    (A) Schematic of the electrophysiological recording (left) and optical imaging (right) of CSD in the intact mouse brain. Left: The mouse skull was thinned to visualize the cortical surface. CSD was evoked by either KCl incubation or pinprick. A single electrode was inserted to detect CSD by measuring local field potential. Right: CSD-associated K+ release was visualized by detecting the increased fluorescence from the nanosensor. (B) Surface fluorescence image showing nanosensor-stained brain parenchyma. Insets show the time courses of the nanosensor’s fluorescence signal (red line) at the red region of interest (ROI) and the field potential (black line) recorded near the red ROI. (C) Fluorescence images of the brain cortex at the indicated time points after initiating spreading depression. Nanosensor fluorescence is represented as pseudo-color. Color circles represent the locations of the spreading wavefront at different time points after KCl triggering. White arrows depict the direction of the wave propagation. (D) Time course of nanosensor fluorescence intensity at the locations indicated by colored circles in (C). Similar results were replicated in five individual mice.

  • Fig. 6 Extracellular potassium burst in larval zebrafish brain upon PTZ treatment.

    (A) PTZ treatment induced increases in both neuronal calcium activity (middle) and extracellular potassium concentration (right). Left: Imaged brain areas include the left and right telencephala (Tel-l and Tel-r, respectively), the left and right habenulae (Hb-l and Hb-r, respectively), the pineal body (P), and the left and right optic tecta (OT-l and OT-r, respectively). Middle: Neuronal calcium activity was monitored by using a genetically expressed calcium indicator, jRGECO1a. The measured response amplitude is coded in red and mapped back to the imaged brain region. Scattered activity spots are marked as white, and their neighboring zones are marked as gray. Four ROIs (yellow) are selected. Right: Extracellular potassium concentration was monitored by using the potassium nanosensor. The measured response amplitude is coded in green and mapped back to the imaged brain region. (B) Neuronal calcium activity (red) and extracellular potassium concentrations observed for the four representative ROIs are marked in (A). After PTZ application, both neuronal calcium activity and extracellular potassium concentration have increased at several activity spots, including the pineal body and the anterior optic tecta, as represented by ROI1. At neighboring zones of the activity spots, neuronal calcium activity change is absent or minimal, while the extracellular potassium concentration continues to increase (ROI2 and ROI3). However, in the area far from the activity spots (ROI4), neither neuronal calcium activity nor extracellular potassium concentration has increased.

Supplementary Materials

  • Supplementary Materials

    A highly sensitive and selective nanosensor for near-infrared potassium imaging

    Jianan Liu, Limin Pan, Chunfeng Shang, Bin Lu, Rongjie Wu, Yun Feng, Weiyu Chen, Rongwei Zhang, Jiwen Bu, Zhiqi Xiong, Wenbo Bu, Jiulin Du, Jianlin Shi

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