3.1. Structural characterization of MIL-101/agarose NCH
To investigate the morphology and particle size of MIL-101/agarose NCH, TEM analysis was used. As depicted in Fig. 1A, MIL-101 (Fe) was dispersed well throughout the agarose matrix without aggregation and showed an intact crystal morphology with a size of lower than 200 nm. Zeta potential measurements in Fig. 1B indicated a negative zeta potential of -21.5 mV for MIL-101/agarose NCH. The negative zeta potential observed in the MIL-101/agarose NCH is mainly due to the presence of deprotonated carboxylate groups in the MIL-101 (Fe) structure. Furthermore, the hydroxyl groups present in the agarose component may also contribute to enhancing the negative surface charge. This combination of functional groups from both the MOF structure and the agarose results in the overall negative zeta potential seen in the NCH.
To further verify the successful synthesis of MIL-101/agarose NCH, FT-IR spectroscopy was conducted (Fig. 2A). For as-prepared MIL-101 (Fe), the absorption band at around 747, 1017, 1393, 1507 and 1600 cm-1 belong to C-H bending vibrations in benzene, symmetric vibration of carboxyl group (-COO-), asymmetric vibration of carboxyl group (-COO-), C=O bonds in free carboxylic groups, respectively. The C-H bending vibrations in benzene are indicated by the peak at 747 cm−1. Symmetric and asymmetric vibrations of carboxyl groups (-COO-) are assigned to peaks at 1393 and 1507 cm−1, respectively, while the peak at 1600 cm-1 is related to C=O bonds in free carboxylic groups, demonstrating the dicarboxylate linker. These FT-IR peaks confirm the organic carboxylate bridging ligand structure and ultimately confirm that the orange powder is crystal pure MIL-101 (Fe) [19]. The FT-IR spectra of agarose reveals specific absorption peaks at 3439 cm−1 (OH stretching of the hydroxyl group), 1646 cm−1 (C=O stretching vibration), 1073 cm−1 (C-O glycosidic bonding), and 930 cm−1 (vibration of C-O-C bridge of 3, 6-anhydro-L-galactopyranose) [22]. In the case of MIL-101/agarose NCH, similar bands with MIL-101 (Fe) and agarose with slightly different positions and intensity are shown, indicating the successful incorporation of MIL-101 (Fe) into the agarose matrix. Also, XRD analysis was performed on the samples to verify the crystallinity and phase purity of the materials (Fig. 2B). The typical characterization peaks of MIL-101 (Fe) can be seen at 9.1°, and 10.14° related to (311) and (511) crystal planes at fairly high intensity similar to that observed in previous studies [23]. Pure agarose shows a single peak at 2ϴ = 19.24° [24]. By comparing the XRD patterns of MIL-101/agarose NCH with MIL-101 (Fe) and agarose alone, the appearance of the above-mentioned peaks evidenced the successful fabrication of the MIL-101/agarose NCH.
3.2. Sensing mechanism
The performance of the synthesized nanoprobe has been analyzed after ensuring the preparation process. The interaction between the nanoprobe and deferiprone has been distinguished by studying the spectra and analytical response of the nanoprobe. A quenching process was observed in the fluorescence intensity of MIL-101/agarose NCH system after deferiprone adding which can be attributed to the interactions between iron centers in MIL-101 (Fe) and deferiprone. Probably these interactions led to form a non-fluorescent complexes that do not fluoresce upon excitation, thus reducing the overall observed fluorescence intensity. The porous structure of MIL-101 (Fe) supports these interactions by facilitating the diffusion of deferiprone into the framework where it can directly interact with iron centers. The embedding in agarose does not impede this interaction but rather contributes to a controlled environment where deferiprone can efficiently access the MIL-101 (Fe) pores. This structural arrangement ensures that the quenching mechanisms are not merely surface phenomena but are deeply integrated into the material's framework, enhancing the sensitivity and specificity of the system towards deferiprone. Fluorescence descending of proposed probe proportional to increase in deferiprone concertation, make it a potent tool for the determination of deferiprone in various matrices.
3.3. Optimization of reaction conditions
Optimizing experimental conditions is crucial for probe design as it directly affects its performance. In initially experiments, it was found that the fluorescent intensity was influenced by three factors: pH, MIL-101/agarose NCH concentration, and incubation time. Deferiprone concentration of 1 µg mL-1 was selected for optimization procedure. The pH level of the reaction environment has a significant impact on the complexation reactions between metal ions and ligands [25]. The effect of pH values was investigated on the response of the system using phosphate buffer (PBS, 0.10 mol.L-1) ranging from 2.0 to 8.0. The response of the nanoprobe was increased along with increasing pH in which the highest fluorescence response (ΔF) was obtained at pH 4.0 and then, the fluorescence response values gradually decreased in upper pH values as illustrated in Fig. 3 A. The highest fluorescence response observed at pH 4.0 due to high interaction of NCH with negative surface and deferiprone with pKa1 of 4.0 [26]. To determine the optimal amount of MIL-101/agarose NCH, different amounts were used during the design process. It was observed that the highest fluorescence response of the nanoprobe was obtained at 20.0 μL of MIL-101/agarose NCH, and thus 20.0 μL was selected as the optimal volume, as illustrated in Fig. 3B. The decreasing in fluorescence after this value can be related to probe self-quenching at high concentration. The time taken for the reaction of deferiprone molecules and MIL-101/agarose NCH to reach equilibrium is called the incubation time. For this purpose, MIL-101/agarose NCH was added to a solution containing deferiprone and PBS (pH 4.0) for various time periods. As the incubation time increased from 1 to 20 minutes, the response of the nanoprobe showed a decreasing pattern (Fig. 3C). So, fluorescence intensity was recorded immediately after solution preparation. The obtained optimal conditions for executing the reactions are as follows: pH:4.0, MIL-101/agarose concentration: 20 μL, and incubation time: immediately.
3.4. Interference study with coexisting substances
The established fluorescent method needs to be investigated for its specificity and selectivity to study the effect of any potential interference caused by co-administered drugs with deferiprone. To evaluate selectivity, system responses towards potential available interfering substances including losartan, dexamethasone, amoxicillin, ampicillin, alprazolam, glucose, ibuprofen, clonazepam, phenytoin, carbamazepine, phenobarbital, acetaminophen, nicotinamide, caffeine, ascorbic acid, diclofenac, chlordiazepoxide, metoprolol, dextromethorphan, cetirizine, metronidazole, aspirin, and FeCl3 were assessed in EBC media. The system response was evaluated for a deferiprone and all interfering substances with concentration of 1.0 µg mL−1. As shown in Fig.4, the interferences had a negligible effect on the response of probe. Therefore, it is suggested that this method could be best performed for deferiprone tracing in the EBC of patients receiving these drugs.
3.5. Analytical figures of merit
The optimal condition was used to obtain the concentration-dependent behavior of the method. Fig. 5 shows that the fluorescence peak of MIL-101/agarose NCH at around 410 nm with excitation at 360 nm which was gradually decreased as the deferiprone concentration increased. The results indicated a linear relationship between the intensity and deferiprone concentration within the range of 0.005–1.5 μg mL−1, as shown in the inset of Fig. 5. The equation for regression was ΔF (F–F0) = 103.96 CDEF + 8.2813 (R2 = 0.9992). Where, CDEF was the concentration of deferiprone and F0 and F were the fluorescence intensity in the absence and presence of deferiprone at 410 nm, respectively. The calculated limit of detection (LOD) based on 3Sb/m (Sb: blank’s standard deviation; m: calibration slope) was 0.003 μg mL−1. The precision of the proposed method was evaluated by conducting repeat tests on the same and different days during the investigation. The (RSDs%) for 5 determinations of deferiprone (1.0 μg mL−1) were 0.3% and 0.4% for intra-day and inter-day measurements, respectively. The respectable repeatability and reproducibility of the method confirmed the applicability of the nanoprobe for deferiprone monitoring. The analytical comparison of the validated method with other reported techniques in the literature for the determination of deferiprone was given in Table 1. As can be seen, the current work exhibited comparable sensitivity compared with other systems.
Table 1
Comparison of analytical characteristics validated method with other reported techniques in the literature
Method
|
Sample
|
LOD
(μg mL−1)
|
Linear range
(μg mL−1)
|
Reference
|
|
Capillary electrophoresis-frontal analysis (CE/FA)
|
Aqueous solution
|
1.0
|
5.5-33.3
|
[4]
|
|
RP-HPLC
|
Pharmaceutical dosage form
|
0.21
|
60-140
|
[27]
|
|
LC-MS/MS
|
Plasma
|
0.05
|
0.1-20
|
[28]
|
|
Flame Atomic Absorption Spectroscopy(FAAS)
|
Pharmaceuticals, water and food samples
|
0.032
|
0.05–3.0
|
[25]
|
|
Spectrophotometry
|
EBC
|
0.012
|
0.05–4.0
|
[9]
|
|
Potentiometry
|
Bulk and Tablet
|
0.45
|
1.39-1391.52
|
[29]
|
|
MIL-101/agarose NCH based fluorescence assay
|
EBC
|
0.003
|
0.005-1.5
|
This work
|
3.6. Real samples analysis
To evaluate the analytical applicability and feasibility of the proposed MIL-101/agarose NCH, deferiprone determination was carried out in four EBC samples collected from patients receiving deferiprone. Table 2 presents the findings of the real sample analysis. Recovery experiments were completed by spiking to concentration of deferiprone (0.05 and 0.5 μg mL−1) to confirm the accuracy of the reported values for real sample analyses and study the no interference of other co-administered drugs. The results indicate a satisfactory recovery percentage of 94.2%–102.3%, demonstrating that this experiment is highly accurate and independent from matrix effects for determining deferiprone in biological matrices such as human EBC.
Table 2
Determination of deferiprone in real EBC samples by validated method
No.
|
Gender
|
Age (year)
|
Co-administrated drugs
|
Added (μg mL−1)
|
Found (μg mL−1)
|
Recovery (%)a
|
1
|
Female
|
66
|
Losartan, Amlodipine, Trifluoperazine
|
-
0.05
0.50
|
0.114
0.162
0.624
|
-
96.0
102.0
|
2
|
Female
|
32
|
Levetiracetam, Calcium, Vitamin D
|
-
0.05
0.50
|
0.030
0.077
0.528
|
-
94.0
99.6
|
3
|
Male
|
43
|
Sodium valproate
|
-
0.05
0.50
|
0.047
0.094
0.559
|
-
94.0
102.4
|
4
|
Male
|
61
|
Metoprolol, Duloxetine, Aspirin
|
-
0.05
0.50
|
0.077
0.126
0.587
|
-
98.0
102.0
|
a Recovery (%) = [deferiprone concentration in samples (after spiking – before spiking)/Added] × 100.