An environmentally sensitive zinc-selective two-photon NIR fluorescent turn-on probe and zinc sensing in stroke

Abstract

A two-photon near infrared (NIR) fluorescence turn-on sensor with high selectivity and sensitivity for Zn²⁺ detection has been developed. This sensor exhibits a large Stokes' shift (∼300 nm) and can be excited from 900 to 1000 nm, with an emission wavelength of ∼785 nm, making it ideal for imaging in biological tissues. The sensor's high selectivity for Zn²⁺ over other structurally similar cations, such as Cd²⁺, makes it a promising tool for monitoring zinc ion levels in biological systems. Given the high concentration of zinc in thrombi, this sensor could provide a useful tool for in vivo thrombus imaging.

Keywords: Molecular imaging; Near-infrared; Stroke; Thrombus.

Graphical abstract

Fig. 1

(A) Structures of 2-(2′-hydroxyphenyl)benzoxazole (HBO) derivatives 1a and 1b and their zinc complexes. (B) Fluorescence spectra of 1a, 1b (10 μM) and their zinc complexes (10 μM) 1a-Zn²⁺ and 1b-Zn²⁺ in dichloromethane (DCM) (excited at 450 nm for 1a and 1a-Zn, and 460 nm for 1b and 1b-Zn). (C) The corresponding images taken under a ultraviolet (UV) lamp (365 nm) in a dark room.

Fig. 2

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS)⁺ of 1b-Zn²⁺.

Fig. 3

Normalized fluorescence emission of (A) 1b-Zn (10 μM) complex excited at 490 nm and (B) 1a-Zn (10 μM) complex excited at 450 nm in different solvents. DCM: dichloromethane.

Fig. 4

(A) Fluorescence spectra of 1b (10 μM) in HEPES buffer (10 mM, pH = 7.2) containing 50% EtOH upon addition of 5.0 equiv. of different metal ions excited at 460 nm. (B) Selective and competitive experiments of 1b (10 μM) in EtOH:HEPES (10 mM) = 1:1 (V/V) (pH = 7.2). Black bars: fluorescence intensity of 1b (10 μM) in the presence of 5.0 equiv. of corresponding metal ions only; Red bars: fluorescence intensity of 1b (10 μM) in the presence of 5.0 equiv. of corresponding metal ions and Zn²⁺ ions.

Fig. 5

Fluorescence images of Zn²⁺ detection in Hela cells were collected on an Olympus spectral confocal microscopy (Olympus Life Science, Waltham, MA, USA) excited with a 488 nm laser. (A–C): cells exposed to 10 μM dye 1b for 30 min (control group). (D–F): cells exposed to 30 μM Zn²⁺ for 30 min, then washed with phosphate buffered saline (PBS) 3 times and further incubated with 10 μM dye 1b in PBS for another 30 min. We employed identical confocal settings as those used for compound 1a, which exhibited a very strong green signal in our previous publication [54]. The images were collected at bright field (A and D), green channel (B and E) and near infrared (NIR) channel (C and F, 700–800 nm).

Fig. 6

Fluorescence images of Zn²⁺ detection in C. elegans were collected on an Olympus FV1000 multiphoton confocal microscope (Olympus Life Science, Waltham, MA, USA) excited with a 950 nm laser, and the near infrared (NIR) emission window was from 700 to 800 nm. 10 μM dye 1b was used in this experiment. We employed identical confocal settings as those used for compound 1a [54]. (A–D): C. elegans were cultured in a zinc free medium. (E–H): C. elegans were cultured in a zinc rich medium (10 μM).

Fig. 7

In vitro thrombus imaging using 10 μM dye 1b with and without Zn²⁺ (10 μM). (A) Bright filed. (B) Near infrared (NIR) channel. (C) Green channel. In Fig. 7A, (a) Human plasma + 1b. (b) Human plasma + 1b + thrombin. (c) Human plasma + 1b + Zn²⁺. (d) Human plasma + 1b + Zn²⁺ + thrombin. The samples were imaged with Ex/Em = 465/560 nm (Green channel) or Ex/Em = 465/760 nm (NIR channel) using an IVIS® spectrum imaging system (PerkinElmer, Inc. Waltham, MA, USA).

Fig. 8

In vivo stroke imaging in C57BL/6J mice by a two-photon microscope (excited with a 900 nm laser). After the injury, we can see the vessel was blocked.