A metal-organic framework showing selective and sensitive detection of exogenous and endogenous
4.3 Results and discussion
4.3.6 Sensing of FA in HEPES buffer medium
For the carcinogenic effect of FA, its real time detection has become a highly demanding task. As a result, a huge number of fluorescent probes based on small organic molecules have been developed in the last few years. 34-38, 52 Unfortunately, the number of existing MOF-based fluorescent probes for formaldehyde is very less (Table 4.1).41-44 Al(III) based MOFs are always preferable to MOFs containing other metal ions for their high hydrolytic stability, non-toxic nature and bio-friendly behavior.46 All the above facts motivated us to examine the efficacy of the new hydrazine functionalized Al(III) based MIL-53 compound for sensitive and selective sensing of FA in both aqueous and physiological media (HEPES buffer, pH = 7.4).
Fluorescence experiments were performed to evaluate the detection performance of FA by the presented material. Prior to the introduction of FA, the hydrazine functionalized 3′ was in fluorescent “turn-off” state due to photo-induced electron transfer (PET) from the hydrazine moiety to the phenyl ring.34 But, after the addition of FA, the hydrazine functionality is transformed to hydrazone moiety, which suppresses the PET process and restores the fluorescence property of the fluorogenic core. Consequently, a drastic enhancement in fluorescent emission intensity was detected for the material in the presence of FA.
For time-dependent study, 500 µL of 20 mM FA was introduced to the suspension of 3′ in HEPES buffer medium and fluorescence spectra were collected upon excitation at 330 nm at a regular time interval of 1 min up to 10 min. Figure 4.10a reveals that saturation of the emission intensity occurred within 60 s with 4-fold increment as compared to the
initial intensity. Another fluorescence experiment was performed by replacing the HEPES buffer medium with aqueous medium (Figure 4.10b). In this case, the fluorescence intensity also attained saturation within 60 s but a remarkably higher fold increment (7 fold) was noticed for the aqueous medium than the HEPES buffer. It can be accounted for the higher stability of the hydrazone moiety at lower pH (water at pH = 7.0) than higher pH (HEPES at pH = 7.4) of the medium.52 Though the fold increment in fluorescence intensity in aqueous medium is reasonably superior to HEPES buffer medium, we have employed HEPES buffer for the subsequent fluorescence experiments as the biological conditions are more accurately mimicked by the later medium.
Figure 4.10 Increment in emission intensity of 3′ after the introduction of 20 mM formaldehyde (500 μL) from 1 min to 10 min in (a) HEPES buffer and (b) aqueous medium. Inset plot shows the enhancement in emission intensity as a function of time (monitored at 436 nm).
An ideal fluorescent sensor should exhibit high selectivity towards the desired analyte over other competing species. Hence, we have examined the selectivity of the MOF probe towards FA with respect to other common aldehyde compounds (oxaldehyde, acetaldehyde, butyraldehyde, propionaldehyde, valeraldehyde, crotonaldehyde, benzaldehyde, 4-chlorobenzaldehyde and anisaldehyde) in both HEPES buffer and aqueous media. From Figure 4.11 and 4.12, it becomes obvious that the fluorescence intensity (monitored at 436 nm) of the Al-MIL-53-N2H3 probe changed hardly in the presence of other aldehyde compounds. Only FA was capable of enhancing the emission intensity of the compound. As the bulky and polar hydrazine functionality is grafted with
the BDC ligand, a highly polar environment has been generated in the MOF structure.
Moreover, the interconnection of infinite trans chains of corner-sharing (via OH groups) [AlO4(OH)2] octahedra by the BDC-N2H3 ligands forms a 3D framework containing 1D channels. Furthermore, the hydrazine groups attached with the coordinated BDC-N2H3
ligands point towards the interior of the pores, which is not possible in discrete H2BDC- N2H3 ligand. On the other hand, as FA is the smallest and highest electrophilic aldehyde among other competitive aldehydes, it can easily diffuse through the porous channel of the MOF material and react with the nucleophilic hydrazine functional group present to form hydrazone moiety. Other aldehyde molecules having larger sizes are unable to diffuse through the pore channel. These competitive aldehydes can hardly react with the hydrazine functional group owing to their lower electrophilic nature as compared to FA. A careful inspection of Figure 4.11 reveals that both oxaldehyde and acetaldehyde show similar enhancement in emission intensity which is lower than the emission intensity of 3′ in presence of FA. This is due to the larger size and lower electrophilic nature of acetaldehyde and oxaldehyde as compared to FA. Therefore, the selective response of FA towards 3′
can be assigned to the highly polar environment and porous structure of the MOF material as well as the smallest size and highest electrophilic character of FA over other aldehydes.71
Figure 4.11 Relative fluorescence turn-on signal of 3′ towards the addition of different aldehydes (20 mM, 500 μL) in HEPES buffer.
Figure 4.12. Relative fluorescence turn-on response of 3′ towards different aldehydes in aqueous medium.
Another set of fluorescence experiments was performed to examine the selectivity of the probe towards FA in the presence of other aldehyde compounds in both HEPES buffer and aqueous media. For these experiments, FA was added to the suspension of 3′
which already contained other competing aldehydes. From Figure 4.13 and 4.14, it becomes clear that FA is capable to enhance the emission intensity of the suspension of the probe in both HEPES buffer and aqueous media, even though other aldehydes exist in the system. Hence, we can conclude that the present probe has shown remarkable selectivity towards FA even in the existence of other competing aldehyde compounds.
Figure 4.13 Relative fluorescence turn-on signal (monitored at 436 nm) of 3′towards formaldehyde (20 mM, 500 μL) in presence of other possibly intrusive aldehydes (20 mM, 500 μL) in HEPES buffer.
Figure 4.14 Relative fluorescence turn-on response of 3′ towards formaldehyde in presence of other potentially interfering aldehydes in aqueous medium.
To quantify the detection process of FA by the probe, a concentration-dependent fluorescence titration experiment was performed. Figure 4.15a reveals that with incremental introduction of FA in the HEPES buffer suspension of 3′, the fluorescence intensity also enhanced gradually. Similar outcome was observed when the concentration- dependent fluorescence titration experiment was conducted with the aqueous suspension of the MOF material (Figure 4.15b).
Figure 4.15 Enhancement of the fluorescence emission intensity of 3′ upon gradual addition of 20 mM formaldehyde solution in (a) HEPES buffer and (b) aqueous medium.
In order to determine the limit of detection (LOD) of 3′ towards FA, the fluorescence intensity of the HEPES buffer suspension of the compound was monitored upon incremental addition of very low concentrations of FA solution. When the emission intensity of 3′ versus the concentration of FA was plotted, a linear curve (R2 = 0.99) having a slope (m) of 200262.98 was obtained (Figure 4.16a). The standard deviation (σ) was determined from eight blank measurements with the MOF probe. The LOD value was calculated by employing the formula: LOD = 3σ/m. The calculated LOD value of 3′ for FA sensing in HEPES buffer was 8.37 μM (0.25 ppm). It is worthy to note that the LOD value of the probe for FA in the aqueous medium (Figure 4.16b) was estimated to be 2.14 μM (64.33 ppb). These values fall within the permissible limit of formaldehyde concentration in drinking water and food stuffs as set by WHO/EPA (86 μM). Moreover, the LOD values are lower than the intracellular concentration of FA (100-400 µM).4, 17 It is interesting to note that the structural integrity of the MOF material was retained throughout the sensing event (Figure 4.7).
Figure 4.16 Change in the fluorescence intensity of 3′ in as a function of concentration of formaldehyde in (a) HEPES buffer and (b) aqueous medium.
To examine the retention of shape and size of the particles of the material in HEPES buffer, 3′ was soaked in HEPES buffer medium for 12 h (as the cells were loaded with 3′
for 10 h and afterward FA was treated and incubated for 1 h before imaging experiment).
Then, 3′ was collected by filtration and dried in a conventional oven at 60 °C for 12 h. The FE-SEM images of the HEPES buffer soaked material (Figure 4.17a) reveal that the overall morphology of the material is very similar with the activated material (Figure 4.10). Hence, no alteration in shape and size of the particles of the material was detected
even after soaking the material in HEPES buffer. Moreover, we have recorded the XRPD pattern of 3′ obtained after soaking in HEPES buffer. Figure 4.17b suggests that framework topology of 3′ is unchanged even after soaking in HEPES buffer for 12 h.
Figure 4.17 (a) FESEM images of 3′ after soaking in HEPES buffer for 12 h. (b) XRPD pattern of 3′ after soaking in HEPES buffer for 12 h.