3.1 Detection of CEs with the Probe PCeEr.
Initially, The fluorescence spectra of PCeEr to CEs were investigated at 37°C in aqueous buffered solution (10 mM PBS, pH = 7.4). As shown in Fig. 1a, free PCeEr itself was almost non-fluorescent due to the static quenching process. However, a significant fluorescence emission with the peak at 522 nm (about 11 fold enhancement) was also observed after increasing concentrations of CEs. These changes in photophysical properties were caused by the CEs-triggered cleavage of the carboxylic ester bond and the release of methylfluorescein fluorophore with a stable angle structure. Moreover, the fluorescence response of PCeEr toward CEs with different concentrations, as shown in Fig. 1b. The intense fluorescence emission with the peak at 522 nm exhibited good linearity over a range of CEs concentrations (0–80 µM), and the detection limit (S/N = 3) for PCeEr was calculated to be 3.9 nM. It showed that the efficacy of PCeEr for imaging intracellular CEs activity.
Figure 2a showed the kinetics test of probe reacting with CEs by incubation time. Notably, after the addition of CEs to the substrate solution, the fluorescence intensity appeared a quick response in about 5 min, and fluorescence enhancement kept unchanged when it reached a plateau until 30 min later with the excitation wavelength 465 nm, which validated that the cleavage of the carboxylic ester bond of the designed dye. Thereafter, we evaluated the different pH responses of PCeEr. It was clearly found that the probe processed good stability in the range of pH 4.0–8.0, as shown in Fig. 2b. In response to CEs, the noticeable fluorescence emission peak gradually increased at 522 nm in the pH range of 4.0–6.0 (Fig. 1B). Considering that probe can well respond to CEs at pH 7.2, the PCeEr can potentially be applied to monitor cellular CEs in ER. In contrast, it can be seen that the fluorescence of PCeEr without CEs (control experiment) barely changed during 50 min in buffer solution (pH = 7.4), displaying that good stability of the probe in bio-systems.
Subsequently, the selectivity of PCeEr toward CEs was then evaluated by measuring the fluorescence spectroscopy with a variety of other potential interfering agents including H2O2, HClO, arginine, cysteine (Cys), bovine serum albumin (BSA), acetylcholinesterase (AchE) and vitamin C (VC). As depicted in Figure S5, after addition of these related substances, the fluorescence spectra of PCeEr displayed almost no obvious changes. In sharp contrast, with addition of CEs, the fluorogenic response of probe elicited significant enhancement in emission at 522 nm, verifying that the PCeEr possessed high specificity for CEs over other competitive biological molecules. These clearly indicated that the probe was selectively catalyzed by CEs in complex biological systems.
3.2 Application of PCeEr to imaging CEs in living cells.
With the PCeEr in hand, we investigated the cellular location of CEs in living cells by confocal microscopy. Before imaging, cytotoxicity evaluation to living A549 cells through standard MTT assay, and the data (Figure. S6) demonstrated that the probe had no marked toxicity to live cells at concentrations below 20 mM at our experimental conditions. With the satisfactory outcome, We then checked the endoplasmic reticulum targeting property of PCeEr by conducting colocalization experiments using commercial ER tracker. As presented in the Fig. 3 (a-c), the colocalization fluorescence images were finely overlapped between the green fluorescence of PCeEr and red fluorescence of the ER-tracker, and with a the Pearson’s colocalization coeffcient (R) value of 0.93.
In addition, we carried out whether probe was able to image endogenous CEs in ER, the A549 cells were performed at different times (Fig. 4). Upon pre-incubated with different times, the green fluorescence intensity increased clearly within 30 min, indicating that the reaction occurred in the ER. Next, to identify the probe have the ability of ER-targetable for visualizing endogenous CEs in different cell types, three tumor cell lines (A549, HeLa and B16F1) were incubated with the probe PCeEr. As illustrated in Fig. 3 (a-i), the cancer cells A549, HeLa and B16F1 treated with PCeEr showed strong fluorescence on green channel under the same conditions, suggesting that a higher level of CEs in tumor cells. Combined together, the efficient fluorescent probe could been specifically distributed in cellular ER by imaging.