2.3. Selectivity of EW2 towards FA
To evaluate the selectivity of probe EW2 towards FA, the fluorescence response towards a range of competing biological species was also examined, as shown in Figure 2. Here, the probe EW2 is seen to be highly selective towards FA, with a distinct fluorescence enhancement at 547 nm, over other analytes, such as Br−, Cl−, F−, I−, NO2−, NO3−, SCN−, OAc−, HSO3−, OH−, t-BuO−, OCl−, O2, H2O2, HS−, cysteine (Cys), homocysteine (Hcys), glutathione (GSH), 4-hydroxybenzaldehyde, 4-nitrobenzaldeyde, acetaldehyde, terephthaldehyde, and glyoxal. This result clearly indicates the excellent selectivity of the probe EW2 towards FA over other competing species for possible biological applications.
Figure 2. The selectivity of probe EW2 (10 μM) against various analytes (50 μM): (a) fluorescence spectra and (b) a bar graph of the fluorescence intensities at 547 nm for (1) the probe alone, (2) Br−, (3) Cl−, (4) F−, (5) I−, (6) NO2−, (7) NO3−, (8) SCN−, (9) OAc−, (10) HSO3−, (11) OH−, (12) t-BuO−, (13) OCl−, (14) O2, (15) H2O2, (16) HS−, (17) Cys, (18) Hcys, (19) GSH, (20) 4-hydroxybenzaldehyde, (21) 4-nitrobenzaldeyde, (22) acetaldehyde, (23) terephthaldehyde, (24) glyoxal, and (25) FA in PBS buffer.
In addition, an experiment was performed in which FA was inhibited by the addition of NaHSO3. A progressive decrease in fluorescence was observed when FA (50 μM) was pretreated with increasing concentrations (50, 100, and 200 μM) of NaHSO3, followed by incubation with 5 μM EW2. These results demonstrate that the emission enhancement is diminished by the reaction of FA with NaHSO3.
2.4. Proposed Sensing Mechanism
To further verify the reaction product derived from the probe after the addition of FA, the final solution was characterized by electrospray ionization (ESI) mass spectrometry. A new peak was observed at m
= 395.58 upon the addition of FA. The species observed is formulated as [3 + H+
, thus confirming the formation of the hydrazone product via the reaction with FA (Scheme 2
Scheme 2. The mechanism of the condensation reaction between probe EW2 and FA.
2.5. Detection of Formaldehyde in Food Samples
To evaluate the performance of the probe EW2 in the detection of FA in food, various samples (dried shiitake mushroom and onions) were prepared as explained in the Experimental Section. The food samples were then treated with EW2 (5 μM) and the corresponding emission intensities were recorded. The mushroom and onion extracts responded to the probe with intensity enhancements, thus revealing the presence of certain amounts of FA.
To determine the amount of FA in the food extracts, the test samples were spiked with various amounts of FA (5, 10, and 15 µM), and the fluorescence intensity of all these samples were obtained. The level of FA in the dried shiitake mushroom was found to be 5.2 µM, while that in onions was 4.8 µM. FA in these samples can be detected with probe EW2, and the recovery shows over 80 %. These results demonstrate that the EW2 probe can be used for the quantitative detection of FA in food.
2.6. Cell Imaging of the Probe
The probe EW2 was used to image FA in MCF7 cells. Negative control cells were treated with either FA alone or EW2 alone, with both controls affording no fluorescence (Figure 3). However, when the MCF7 cells were pre-treated with 50 μM FA for 30 min followed by 5 μM EW2 for an additional 40 min, strong fluorescence signals were observed. These results indicate that the EW2 probe can be used to detect FA in living cells. Moreover, the MCF7 cells treated with various concentrations of FA (5, 50, and 500 μM) revealed a significant dose-dependent turn-on fluorescence. These results confirm the ability of the probe EW2 to assay the FA quantitatively in living cells.
Figure 3. The fluorescence imaging of FA in live MCF7 cells using the probe EW2. The MCF7 cells were incubated with (a) 5, (b) 50, and (c) 500 μM FA for 30 min followed by 5 μM of probe EW2 for 40 min. The image in (d) is the bright-field image of the cells in (c) overlaid with an image stained with 5 μg/mL Hoechst 33342. The scale bar represents 50 μm in all images. (e) The mean fluorescence intensities of representative images with 5 μM EW2 with varying concentrations of FA.
2.7. Cytotoxicity Studies
The feasibility of using probe EW2 to image FA in living cells was investigated. The cytotoxicity of EW2 was first evaluated via the MTT assay, as shown in Figure 4. The MCF7 cells were treated with various concentrations (0–30 μM) of EW2. The results indicate that probe EW2 possesses low toxicity towards living cells and is suitable for imaging living organisms under the selected conditions. In addition, cell viability was verified using Hoechst 33342 staining. As shown in Figure 3d, a bright-field image was acquired and overlaid with a fluorescence image of the cells stained with Hoechst 33342. The result clearly reveals the presence of intact and viable nuclei after the treatment of probe EW2.
Figure 4. The cytotoxicity of probe EW2 (0–30 μM) towards MCF7 cells.
2.8. Inhibition Studies of the Probe
The inhibiting effect of NaHSO3 on FA was then investigated. In this set of experiments, the cells were treated with NaHSO3 as a negative control, and their fluorescence in the green channel was examined. This negative control afforded no fluorescence. However, when the cells were pre-treated with 500 µM FA for 20 min followed by 5 µM of probe EW2 for 40 min, an intense green fluorescence was observed. By contrast, no fluorescence was observed for the cells that were pre-treated with 500 µM FA and NaHSO3 for 20 min followed by 5 µM of EW2 for 40 min. Thus, the scavenging effect of NaHSO3 upon FA was demonstrated, confirming that the probe can be used to monitor exogenous FA in living cells.
2.9. Colocalization Experiment
Colocalization experiments were performed to investigate the specificity of the probe EW2 towards the Golgi apparatus afforded by the introduction of a phenyl sulfonamide group. In this experiment, the MCF7 cells were labelled with EW2 for 30 min and followed by staining of the Golgi apparatus with a BODIPY TR Ceramide for 15 min. The resulting green fluorescence signal from the EW2 exhibits a good degree of overlap with the red fluorescence of the BODIPY TR Ceramide. The imaging for the detection of FA in the Golgi apparatus suggests that it is the organelle where the toxic substance FA is detoxified. Moreover, the probe EW2 exhibits much higher specificity towards the Golgi apparatus than other FA probes 
2.10. Imaging Endogenous FA in Cells
Endogenous FA production is triggered by endoplasmic reticulum stress, which is known to cause various diseases, including neurodegeneration, atherosclerosis, type-2 diabetes, liver disease, and cancer 
. To induce FA production, the cells were treated with thapsigargin (TG) prior to being stained with EW2 
. As the concentration of TG increased, the green fluorescence intensity became stronger , demonstrating that EW2 is also highly useful for imaging FA produced in cells.