3.2. Optical properties of the probe ER-Tf
Firstly, we investigated the absorption spectra of the probe ER-Tf treated with or without O2•−. The probe ER-Tf showed no significant absorption peak in the visible region in PBS (20 mM, 5% MeOH) (Fig. 1A). However, after 20 µM O2•− was added to 5µM ER-Tf solution, an absorption peak could be clearly found at 405 nm, indicating that the probe ER-Tf reacted with O2•−. Under 405 nm excitation, the probe ER-Tf showed nearly no fluorescence (Fig. 1B). Upon the introduction of O2•−, the fluorescence peak at 462 nm appeared and increased continuously along with the gradual change of O2•− concentration from 0 µM to 90 µM (Fig. 1C). Meanwhile, the intensity of the fluorescence at 462 nm displayed the satisfactory linear relationship (R2 = 0.9909) with O2•− concentration in the range 5–60 µM, and the detection limit was calculated to 65 nM (Fig. 1D). In addition, after ER-Tf reacted with O2•−, the fluorescence intensity reached the maximum at 462 nm within 7 min (Figure S1). Hence, ER-Tf could be used as a sensitive fluorescent probe to detect O2•−.
Whereafter, we assessed the selective property of the probe ER-Tf to O2•−. As shown in Fig. 2, the fluorescence spectra of ER-Tf showed negligible change in the following the addition of different biologically related species including the other ROS (H2O2, ClO−, etc), reactive nitrogen species, Cys, GSH and a battery of ions. By contrast, when the probe ER-Tf was added to 90 µM O2•−, a strong fluorescent peak at 462 nm can be clearly obtained, manifesting that ER-Tf had a desirable selectivity to O2•−. Furthermore, ER-Tf exhibited excellent fluorescence response to O2•− at pH 4.0–9.0. Considering that the normal pH at ER is about 7.2 (Figure S2), ER-Tf can potentially be used as the selective fluorescent probe for the detection of cellular O2•− at ER.
The response mechanism to O2•− of ER-Tf was then explored by HRMS test. As shown in the HRMS data of the reaction product between ER-Tf with O2•−(Figure S3), besides the peak at 535.0391 corresponding to ER-Tf (Cald. for [M + H]+, 535.0451), an obvious peak at 403.0966 and 425.0785, corresponding the compound ER-Cou (Cald. for [M + H]+, 403.0958; Cald. for [M + Na]+, 425.0778), indicating that the emissive compound ER-Cou was generated after the reaction of ER-Tf and O2•− (Figure S3). We proposed that O2•− firstly attacked S atom of triflate group, and then peroxytriflate free radical was removed to provide the emissive compound ER-Cou by protonation (Scheme 2).
Then, we tried to study the ability of ER-Tf to detect O2•− at ER. The MTT assay showed that ER-Tf showed very low cytotoxity below the concentration of 15 µM (Figure S4). When the HeLa cells were only treated with 5 µM ER-Tf for 20 min, almost no fluorescence was found under the excitation at 405 nm. Nevertheless, after the cells were firstly incubated with 30 µM or 60 µM O2•−, the bright fluorescence can be found in blue channel (Fig. 3A). It indicated that ER-Tf can visualize O2•− at ER. Besides, the fluorescence intensity of the cells treated with 60 µM O2•− was more intense than that of the cells incubated with 30 µM O2•−, suggesting that the probe ER-Tf could evaluate the concentration of cellular O2•− (Fig. 3B). Hence, ER-Tf could act as a sensitive fluorescent probe to detect O2•− in living cells.
Subsequently, in order to explore the ER-targeting ability of the probe ER-Tf, we selected the commercial ER-specific dye (ER-tracker) as a reference to compare the positioning ability of the probe ER-Tf. The specific operation of the experiment was as follows: Firstly, the cells were treated with 5 µM ER-Tf and incubated at a constant temperature for 20 min, and then washed off the medium and incubated with 50 µM O2•−, finally treated with 1 µM ER-tracker. Then we observe the positioning of the probe ER-Tf under a confocal microscope, the blue channel signal generated by the reaction of ER-Tf with O2•− can better overlap with the red channel signal generated by the ER-tracker, and the Pearson correlation coefficient is as high as 0.89 (Fig. 4).Therefore, the co-localization experiment showed that ER-Tf possessed ER-targeting property.
Then, we assessed the ability of ER-Tf to image endogenous O2•− in living cells. Rotenone, as a mitochondrial electron transport chain complex I inhibitor, which can lead to apoptosis and increase the production of ROS . When HeLa cells were coated with 5µM ER-Tf for 20 min, almost no fluorescence was observed in the blue channel (Fig. 5). While HeLa cells were firstly treated with 10 µM or 20µM rotenone for 30 min and then stained with 5 µM ER-Tf for 20 min, the bright fluorescence could be found in blue channel. It indicates that HeLa cells produce O2•− when stimulated by rotenone. In addition, we evaluate the changes of endogenously generated cellular O2•− during ferroptosis. Erastin is a famous iron ion activator, which can inhibit the uptake of cystine by cystine/glutamate reverse transporter (system XC), and finally lead to ferroptosis. Erastin was employed as an activator to investigate the subtle changes of O2•− in living cells during ferroptosis . After treatment of HeLa cells with 10 µM erastin for 2 h and then 5µM ER-Tf for 20 min, the fluorescence intensity of blue channel showed nearly no change (Fig. 5). It suggests that the O2•− level at ER showed no remarkable change during ferroptosis. Hence, ER-Tf can be used for the fluorescence imaging of endogenously generated O2•− at ER.