4.3. Aldehyde Visualization in Cells

In addition to HPLC with fluorimetric detection, fluorescent probes were designed and synthesized for real-time visualization of aldehydes in cells such as FP1 and FAP-1 for formaldehyde detection [169,170]. These formaldehyde probes are based on the 2-aza-Cope sigmatropic rearrangement, which yields highly fluorescent signal for the selective and sensitive detection of aldehydes in cells [169,170]. Recently, a novel technique based on real-time imaging of aldehydes in cells using multicolor fluorogenic hydrazone transfer (“DarkZone”) was developed (Figure 5). This approach used a cell permeable DarkZone dye (7-(diethylamino)coumarin; DEAC) as a quenched hydrazone, which lights up when the quencher-aldehyde is replaced by the target aldehyde. The fluorescence signals are then detected by flow cytometry or microscopy without the need for washing or cell lysis. This strategy is useful for determining the aldehyde load associated with human diseases [171]. Recently, a novel fluorescent probe to visualize specific and total biogenic carbonyls was developed based on the pattern and fluorescence spectral profile unique to the target carbonyl compound. The probe is based on an N-aminoanthranilate methyl ester moiety [96]. These techniques offer real time monitoring of total aldehydes in cells and identification of specific aldehydes based on their unique fluorescence excitation and emission spectra. Overall, real-time imaging of aldehyde production in cells using aldehyde-specific probes allows elucidation of the roles and functions of these compounds in cellular processes and their involvement in disease pathogenesis. These techniques, however, lack the selectivity and specificity for the identification of specific carbonyls in cells as no structural information can be obtained. Finally, these techniques are not applicable to biological matrices such as blood, urine, CSF or saliva.

Real-time imaging of total aldehydic load in cells. Cellular aldehyde labeling fluorescence images and flow cytometry data. Hela cells were exposed to varying concentrations of: (a) formaldehyde; (b) glycolaldehyde; (c) acrolein; and (d) acetaldehyde along with 20 μM of the dye AFDZ and 10 mM catalyst (2,4-dimethoxyaniline) with images taken after 1 h of incubation. Note that 50 μM was used with acrolein and 100 μM for the other aldehydes tested. (e) K562 cells pretreated with 250 μM daidzin and incubated with 40 μM of AFDZ dye, 10 mM catalyst (2,4-dimethoxyaniline), and with/without 20 mM ethanol. (f) Flow cytometry data monitoring the production of aldehyde over time in K562 cells with/without ethanol. The fluorescence intensities were compared to that obtained from t = 0 without added ethanol and daidzin. Scale bars (20 μM) are shown. Reprinted from [171] (Copyright 2016, American Chemical Society).