A selective turn-on fluorescent sensor for imaging copper in living cells

Abstract

We present the synthesis, properties, and biological applications of Coppersensor-1 (CS1), a new water-soluble, turn-on fluorescent sensor for intracellular imaging of copper in living biological samples. CS1 utilizes a BODIPY reporter and thioether-rich receptor to provide high selectivity and sensitivity for Cu+ over other biologically relevant metal ions, including Cu2+, in aqueous solution. This BODIPY-based probe is the first Cu+-responsive sensor with visible excitation and emission profiles and gives a 10-fold turn-on response for detecting this ion. Confocal microscopy experiments further establish that CS1 is membrane-permeable and can successfully monitor intracellular Cu+ levels within living cells.

Scheme 1

Synthesis of Coppersensor-1 (CS1)

Figure 1

(A) Fluorescence response of 2 μM CS1 to Cu⁺. Spectra shown are for buffered [Cu⁺] of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 μM. Spectra were acquired in 20 mM HEPES, pH 7, with excitation at 540 nm. (B) Fluorescence responses of CS1 to various metal ions. Bars represent the final integrated fluorescence response (F_f) over the initial integrated emission (F_i). Initial spectra were acquired in 20 mM HEPES, pH 7. White bars represent the addition of an excess of the appropriate metal ion (2 mM for Ca²⁺, Mg²⁺, and Zn²⁺, 50 μM for all other cations) to a 2 μM solution of CS1. Black bars represent the subsequent addition of 10 μM Cu⁺ to the solution. Excitation was provided at 540 nm, and the emission was integrated over 550–650 nm.

Figure 2

Confocal fluorescence images of live HEK 293 cells. (A) Cells incubated with 5 μM CS1 for 5 min at 25 °C. (B) Cells supplemented with 100 μM CuCl₂ in the growth media for 7 h at 37 °C and stained with 5 μM CS1 for 5 min at 25 °C. (C) CuCl₂-supplemented cells pretreated with 500 μM of the competing Cu⁺ chelator, 3,6,12,15-tetrathia-9-monoazaheptadecane, for 5 min at 25 °C before staining with 5 μM CS1 for 5 min at 25 °C. (D) Brightfield image of live HEK 293 cells shown in panel B, confirming their viability. Scale bar = 25 μm.