FESEM results
Electron microscopy images indicated that synthesized MOFs were semi-spherical, and adding NH2 and COOH functional groups did not affect the MOF's morphology (Fig. 1, A, B, and C). The EDS element analysis results confirmed the presence of essential functional group elements (Fig. 1, D, E, and F). Based on Fig. 1, adding functional groups didn't change UiO-66 clusters' morphology and/or size significantly in this study, which is consistent with previous reports[23]. In this case, other functional groups' physicochemical properties may determine the differences observed in droplet and fibril dimensions.
XRD patterns
X-ray diffraction (XRD) analysis determined the crystal structure of UiO-66 (Fig. 1G). UiO-66 matched literature patterns with peaks at 7.4, 8.5, 14.8, 18.5, 25.4, and 31.1, confirming successful synthesis [17]. UiO-66-(COOH)2 showed a similar XRD pattern to UiO-66 [28, 29]. The NH2-grafted framework also matched the original UiO-66, indicating that NH2 groups did not modify crystal structure [23, 25].
FTIR spectrum
The Fourier transform infrared (FTIR) spectra of the synthesized MOF samples (Fig. 1H) reveal distinct bands attributable to specific chemical structures and functional groups. For UiO-66, the band at 1568 cm− 1 indicates coordination of -COOH with Zr4+. Bands at 1506 cm− 1 and 1395 cm− 1 correspond to vibrations of the benzene ring and carboxylate group of the organic linker [17]. Additional bands at 1019 cm− 1, 760–780 cm− 1, and 660 cm− 1 arise from C-H vibrations. UiO-66-NH2 and UiO-66-(COOH)2 exhibit similar spectra, with extra bands due to grafted functional groups. The band at 1620 cm− 1 in UiO-66-NH2 originates from the N-H bending of aromatic amines. The 1252 cm− 1 band indicates C-N stretching[23, 25]. For UiO-66-(COOH)2, the band at 1712 cm− 1 confirms the presence of uncoordinated -COOH groups. The FTIR data confirms the complete, uncorrupted synthesis of the MOF samples with full retention of chemical structures. [28, 29]
BSA – MOF interaction
In order to investigate the strength of interaction between MOFs and BSA an experiment was design as follow. BSA was incubated overnight with varying concentrations of UiO-66, UiO-66-NH2, and UiO-66-(COOH)2 MOFs. Subsequently, the samples were centrifuged at 18,000 rpm for 5 minutes. The rationale was that if robust interactions such as bond formation occurred between any of the MOFs and BSA, then after centrifugation BSA would co-sediment with the MOFs due to the high density of the MOF metal clusters. In this case, increasing MOF concentration would increase co-sedimentation, which could be quantified by measuring BSA concentration in the supernatant using UV-Vis spectroscopy. The concentrations used for each of MOFs UiO-66, UiO-66-(COOH)2, UiO-66-NH2 was as mentioned in Table 1.
Table 1
Different concentrations of MOFs in MOF-BSA interaction experiment.
Mg.ml− 1 0.04 | 0.12 mg.ml− 1 | mg.ml− 1 0.2 | 0.28 mg.ml− 1 | mg.ml− 1 0.36 | Mg.ml− 1 0.44 |
In the next step, intrinsic fluorescence emission and ANS-binding assays were performed to evaluate structural changes in the protein. Intrinsic tryptophan fluorescence was measured at 340 nm, corresponding to the emission peak wavelength of tryptophan residues in BSA. This allowed detection of changes in the local environment of aromatic amino acids like tryptophan. Additionally, ANS binding assays were carried out, with fluorescence recorded at 440 nm. Since hydrophobic dye ANS binds to exposed hydrophobic regions on protein surfaces, changes in ANS fluorescence indicate alterations of surface hydrophobicity.
The results of UV-vis spectroscopy, intrinsic fluorescence, and ANS-binding assays after incubating BSA with UiO-66, UiO-66-(COOH)2, and UiO-66-NH2 followed by centrifugation to remove MOFs are shown in Fig. 2. The UV-vis absorption profiles indicate no decrease compared to BSA alone. Rather, in most cases, a slight increase in UV-vis absorbance is observed upon addition of MOFs.
The intrinsic fluorescence results reveal decreased emission intensities for BSA incubated with the NH2-functionalized MOF at all concentrations tested. A similar effect is seen for some concentrations of non-functionalized UiO-66. In contrast, the COOH-functionalized MOF generally increases or does not affect intrinsic fluorescence, except at the highest 0.44 mg/mL concentration.
Finally, ANS fluorescence is enhanced in the presence of UiO-66-NH2, reflecting greater exposure of hydrophobic regions. However, the differences in ANS emission are not significant. These results demonstrate that MOFs neither form strong bonds with BSA proteins and nor cause significant permanent conformational change in protein structure.
The BSA phase separates into liquid droplets.
Increasing NaCl concentration from 50 to 300 mM led to larger BSA droplets (Fig. 3A), with 300 mM NaCl resulting in significantly larger droplet diameters (Fig. 3B). Since the total BSA concentration was held constant, this suggests that more BSA protein partitioned from solution into droplets at 300 mM NaCl to form the larger condensed phases observed.
FITC-labeled BSA has been used to confirm the protein nature of droplets (Fig. 3C). Fluorescence microscopy using the FITC-BSA showed intense fluorescence within the liquid droplets, verifying that condensed protein accumulation had occurred. Furthermore, larger droplets exhibited higher fluorescence intensity compared to smaller droplets.
Kinetics of droplet size evolution
To examine the dynamic nature of liquid-liquid phase separated proteins, it was necessary to track droplet size over time and visualize growth [30].
Optical microscopy visualized droplets at 0, 2, 6, and 24 hours after inducing phase separation (Fig. 4). Over 24 hours, the number of droplets decreased while the size of remaining droplets increased, consistent with the coalescence and Ostwald ripening phenomenon [31, 32]. By 24 hours, only a sparse population of enlarged droplets remained. Relating LLPS droplet size to subsequent fibril length provided insights into their relationship under different conditions [33].
To have a better quantitative comparison about the impact of UiO-66, UiO-66-NH2 and UiO-66-(COOH)2 on the size of droplets, their diameters were measured by ImageJ software plotted in Fig. 4B. The result was in consistent with fluorescence microscopy results.
Conformational changes due to protein interaction with MOFs and LLPS
Intrinsic and ANS fluorescence probed BSA tertiary structure and surface hydrophobicity changes after phase separation (Fig. 6A and B). Also, UiO-66-NH2 slightly decreased ANS emission and increased intrinsic fluorescence, but these changes were not significant which implies that MOFs did not cause any significant conformational change. Circular dichroism (Fig. 6C) monitored BSA secondary structure changes after separation. The signal appearance remained constant despite a slight intensity decrease, likely due to the presence of PEG. [34]
Fibril formation kinetics
After phase separation (7 days of incubation at room temperature), Thioflavin T (ThT) stained samples were observed by fluorescence microscopy (Fig. 6A). Fibril length measurement demonstrated that UiO-66-NH2 significantly decreased BSA fibril length in comparison with other synthesized MOFs and the sample without MOF (Fig. 6B).
Fibrils from the smallest droplets induced by UiO-66-NH2 were significantly shorter than those of the control. UiO-66-NH2 had lower ThT emission than the control, probably correlating with its smaller and fewer droplets. However, non-functionalized UiO-66 had a more significant ThT increase versus control, indicating fluorescence depends on both droplet size and number.
Zeta potential and contact angels’ results
In this case, the functional groups' physico-chemical properties determined the differences in droplet and fibril dimensions (Table 2). Measuring zeta potential and contact angle revealed that UiO-66-NH2 had the highest contact angle and, thus, the greatest hydrophobicity, and UiO-66-(COOH)2 had the highest zeta potential[35–38].
The zeta potential results at pH = 7.4, as shown in Table 2, indicate that the potential of frameworks with a carboxyl functional group is more negative. As seen in Table 1, despite the significant difference in the potential of the carboxyl group, adding the NH2 functional group did not cause a significant change compared to the state without the functional group. Table 1 also indicates that the contact angle for UiO-66, UiO-66-NH2, and UiO-66-(COOH)2 were obtained as 26.19, 42.98, and 24.9, respectively.
Table 2
Results of zeta potential and contact angle measurements.
MOFs | Zeta potential (mV) | Contact angle (rad) |
UiO-66 | -18.53 | 26 |
UiO-66-(COOH)2 | -27.2 | 25 |
UiO-66NH2 | -19.46 | 43 |