A Molecular Hybrid of the GFP Chromophore and 2,2'-Bipyridine: An Accessible Sensor for Zn2+ Detection with Fluorescence Microscopy

Abstract

The few commercially available chemosensors and published probes for in vitro Zn²⁺ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2'-bipyridine chelator moiety (GFZnP BIPY) for Zn²⁺ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging.

Keywords: 2,2′-bipyridine; DFT; NMR; chelator; chemosensor; fluorescence; two-photon imaging; zinc.

Figure 1

(A) Illustrative structure of the Green Fluorescent Protein (GFP), where its chromophore is highlighted in red [32]. (B) The planned sensor and its mechanism of action. The rings filled with green show the parts of the sensors resembling the GFP chromophore; the dark green lines represent the ionophore part.

Scheme 1

Simple synthesis of GFZnP BIPY chemosensor in Knoevenagel condensation.

Figure 2

(A) UV-VIS and fluorescence spectra of the reported probe without Zn²⁺ (blue) and in the presence of 1 mM Zn²⁺ (red) in HEPES (pH 7.4). (B) Fluorimetric titration of GFZnP BIPY with Zn²⁺. The fluorescence intensity (blue dots) is plotted against the free Zn²⁺ concentration on a logarithmic scale (red line: fitted logistic curve) and (C) on a linear scale (red line: fitted 1:1 complex model, Equation (5)). (D) Fluorescence Job’s plot of GFZnP BIPY with Zn²⁺.

Figure 3

(A) ¹H NMR spectra of solutions of GFZnP BIPY in DMSO-d₆ containing different amounts of Zn²⁺. (B) Zoomed-in region showing well-resolved peaks of the two formed complexes at 1 equiv. with added Zn²⁺. (C) Optimized structure of GFZnP BIPY–Zn²⁺ complex (M06-2X/6-311++G(2d,2p)//PCM(water)). Gray and white colors mark carbon and hydrogen respectively, while red, blue and light blue mark oxygen, nitrogen and zinc atoms, respectively.

Figure 4

(A) The cation and (B) anion selectivity of GFZnP BIPY. Normalized fluorescence intensity of the probe in the presence of 1 mM of different interfering ions (turquoise), Zn²⁺ (red), or both of them simultaneously (blue); inset: photographs of vials containing the same solutions under UV light (above) and ambient light (below). Error bars represent the standard deviation of triplicate measurements. (C) Normalized fluorescence intensity of the probe in buffers at different pH levels in the presence of Zn²⁺ (1mM, red) or EGTA (zinc-free, blue). The pink background shows the physiologically relevant pH range.

Figure 5

(A) Two-photon action cross section spectrum of GFZnP BIPY in the presence of Zn²⁺ ions (red) and without Zn²⁺ (blue). (B) Comparison of the one-photon and two-photon absorption spectra of GFZnP BIPY in the presence of Zn²⁺. (C) Two-photon image of HEK 293 cells stained with GFZnP BIPY (D) after the addition of Zn²⁺ and (E) after the withdrawal of Zn²⁺. (F) Mean two-photon fluorescence intensity of whole images containing the shown cell samples plotted against the time from the start of the experiment. GM: Goeppert Mayer unit.