3.1 Morphological analysis and characterization
Prior to its application as a fluorescent probe for Fe(III) detection, the synthesized modified GQD was subjected to characterization in order to verify its structural and optical features. Figure (2, a) displays the X-ray diffraction (XRD) patterns of graphene quantum dots (GQDs), revealing the presence of three distinct diffraction peaks. [17,18] The peak observed at an angle of 27.06 degrees (2θ) corresponds to the crystallographic plane labeled as (002) in graphene. Additionally, there are other peaks present, The angles 2θ = 31.3 and 45.1 degrees correspond to the crystallographic planes (100) and (102). The presence of three distinct diffraction peaks indicates a high level of sharpness, thereby confirming the excellent crystallinity of the produced GQDs (Fig. 2.b). Sherrer's equation was utilized to estimate the average size, denoted as L, of the nanoparticles in the processed samples. Based on Scherrer's equation, the grain size D may be estimated using the formula D = Klambda∕βcosθ. In this equation, the constant K is equal to 0.942, the x-ray wavelength λ is 0.15405 nm, the diffraction peak half-height width β is 0.247, and the diffraction angle θ is 13.4 degrees. Using these values, the calculated grain size D is 33.3 nm. However, in the case of a sample with a small particle size, the grain size determined by XRD is more than the actual size.
The morphology and structure of GQD were examined using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Despite the challenge of obtaining high-quality TEM pictures due to the poor electro-contrast between the GQDs and the carbon-coated TEM grids, it is nevertheless possible to discern nanosheets measuring 20 nm in size (Fig. 3. c). This image displays graphene quantum dots (GQDs) with a diameter of around 20 nanometers and a spherical shape.
Field-emission scanning electron microscopy (FESEM) is a sophisticated imaging method that allows for the detailed examination of the surface structure and shape of graphene quantum dots (GQDs) with high precision. When GQDs are observed using FESEM (as shown in Fig. 3. a and b), they typically appear as a cluster of small, spherical particles with sizes ranging from a few nanometers. The particles' surface morphology and texture can vary based on the synthesis and processing method. The compositions and dispersion of the element were determined using EDX-mapping (Fig. 3. d,e, and f). The field emission scanning electron microscope (FE-SEM) reveals a detailed surface morphology, displaying the variations in surface texture of the created modified GQDs indicating the distribution of elements. The chemical composition of the modified GQDs was analyzed using energy dispersive X-ray (EDX) spectroscopy, and the corresponding results are presented in Fig. 2. g. Graphene quantum dots (GQDs) exhibit a consistent circular morphology.
The Fourier-transform infrared (FT-IR) spectra of graphene quantum dots are presented in Fig. 4. a. The GQDs were functionalized using 3-(trimethoxysilyl)propyl methacrylate, resulting in broad absorption peaks at around 1376.20 cm−1 and 1550.49 cm−1. These peaks are attributed to the stretching vibrations of symmetric and asymmetric carboxyl groups, respectively. Additionally, a stretching vibration of C-OH was observed below 1350 cm-1, and broadband around 3347.19 cm−1 was attributed to the stretching vibration of hydroxyl groups. [19]
In Fig.4.b the use of luminol reveals the presence of C=O and asymmetric carboxyl group bonds at around 1571 and 1413 cm-1, respectively. Additionally, a large peak at 3500 cm-1 indicates the stretching vibration of the N-H bond in luminol. The presence of a band at approximately 1413 cm-1 can be attributed to the absorption of C–N bonds. In contrast, a band at roughly 704 cm-1 is associated with the out-of-plane bending of C-H aromatic bonds. Identifying a peak at 3100 cm-1 indicated the presence of N–H stretching vibration in the amine groups, confirming the successful integration of nitrogen atoms.
3.2 Optical properties
Figure 5 depicts the UV-Vis absorption spectra of the modified graphene quantum dots (GQDs) samples. The GQDs display a weak peak at 360 nm due to the n-π* transition in C=O groups, an identifiable band for the modified GQDs.
The fluorescence spectra in Fig.6 indicate that the highest fluorescence emission of GQDs occurred at wavelengths 465 nm while blue shifting with 428 nm maximum wavelength in the luminol-GQD was noticed due to fractionalization, respectively. The Luminol-GQD produced in its original form demonstrates a high level of fluorescence emission and possesses surface functionalization that is distinctive to nitrogen. The hydrophilicity of the GQDs, which is responsible for their high solubility in water, is a result of the presence of these functional groups. Due to the existence of these functional groups and intense emission, these Luminol-GQDs are anticipated to be an optimal choice for fluorescence sensing.
The relationship between the emission wavelength and the excitation has been investigated, and Figure 7 displays the fluorescence spectra of the Luminol-GQDs solution at different excitation wavelengths. The Figure demonstrates that the emission maximum wavelength remains constant regardless of the selected excitation wavelength. The emission of graphene quantum dots is unaffected by excitation, indicating that the size and surface state of the sp2 clusters within GQDs are uniform.
3.3 Selective detection of Fe (III)ion
Synthetic urine has been proven to exhibit a significant matrix profile when analyzed using various analytical methods. Fluorescence spectrophotometry limits the matrix effect by utilizing the selectivity of fluorescence for certain molecules that impact the analysis. Monitoring the effect of the matrix by detecting Fe(III) ions in artificial urine before analyzing real human urine was first tested. To achieve this goal, the selective sensing of Fe(III) was carried out using excitation/emission wavelengths of 314/428 nm and a pH of 7.4 in a phosphate buffer saline solution containing Luminol-GQDs using Synthetic urine as the solution media, with a concentration of 0.0819 g in 40 ml of GQD. The excitation wavelength of 314 nm was chosen as it resulted in the strongest fluorescence emission. Thus, this specific wavelength was chosen to observe and measure the impact of Fe(III) ions on fluorescence intensity. The fluorescence intensities of Luminol-GQD were measured upon introducing different concentrations of ferric ions (Fig. 8). The results indicate that Luminol-GQD has a strong ability to detect Fe (III) specifically. The process of fluorescence quenching is associated with the high attraction of Fe (III) ions to the amino group of Luminol-GQD, resulting in the formation of a stable complex. To elucidate the potential mechanism of our detecting system, an endeavor to establish a correlation between the concentration of Fe (III) ions and the luminescence intensity of Luminol-GQDs was done by employing the Stern-Volmer relationship. Fig. 9 depicts the Stern-Volmer analysis of the quenching experiment. Specifically, the ratio of initial fluorescence intensity to the fluorescence intensity after quenching (F0/F), plotted against the concentration of Fe(III). The linear relationship of the Stern-Volmer Plot over the concentration range of Fe (III) ions from 50 to 300 μM is notable. This behavior indicates that a statistic mechanism involving charge transfer likely controls the interaction between GQDs and Fe(III) ions
The regression value (R2) was found to be 0.9901 which reflects a linear relation between the quencher concentration and fluorescence intensity, the obtained R2 value indicates a linear relation between the quencher concentration and fluorescence intensity. The findings indicate that the produced Luminol-GQD can be a selective and sensitive sensor for detecting Fe(III) ions in artificial urine. The formulas for determining the limit of detection (LOD, equation 1) and limit of quantification (LOQ, equation 2) are below An abrupt decrease in photoluminescence (PL) was seen upon introducing Fe(III). The fluorescence quenching was examined at Fe(III) concentrations ranging from 1 to 300 μM. A valid linear correlation was observed, as shown in Fig. 10, demonstrating a strong linear association.
LOD = 3.3* σ /S
LOQ = 10 * σ /S
where σ is the standard deviation of the intercept and S is the slope of the linear regression plot. The LOD and LOQ were calculated to be 1.5 μM and 5.2 μM, respectively. The detection limit of Fe (III)found in this study was much lower than the reported studies in Table 1
Table 1. Comparison of Fe (III) Sensing Properties of the Luminol-GQD with Those of Recently Reported GQDs
materials
|
technique in detail
|
detection limit (μM)
|
Analysis time
|
ref
|
Luminol-GQD
|
hydrothermal method
|
1.5
|
< 3 min
|
This work
|
GQDs
|
hydrothermal/ modified hummer’s method
|
1.1
|
>24h
|
20
|
N-doped GQDs
|
ammonia through hydrothermal method
|
1
|
>24
|
21
|
GQDs
|
chemical oxidation
|
60
|
>26h
|
22
|
N-doped GQDs
|
microwave synthesis
|
100
|
>27h
|
23
|
GQDs
|
electrochemical synthesis
|
7.22
|
--
|
24
|
Graphitic GQDs
|
electrochemical synthesis
|
2
|
>96 h
|
25
|
N-doped/amino GQDs
|
chemical oxidation
|
0.5
|
>24
|
26
|
N-doped GQDs
|
hydrothermal method
|
0.5
|
>12 h
|
27
|
3.4 Analysis of Fe (III)in human urine sample
The fluorescence sensor was utilized to detect Fe(III) ions in real human urine samples, demonstrating the capabilities of the probe. Healthy volunteers provided human urine samples, which were collected following the international standard for manual urine collection. The urine liquid was either analyzed immediately or held at a temperature of -20°C until it could be analyzed. Prior to the analysis, the urine samples were spiked with varying quantities of Fe(III). In order to assess the repeatability of the suggested approach (e.g., precision), each sample was analyzed five times using the same working conditions within a single day. Fig. 11. Display the emission fluorescence of the modified graphene quantum dots (GQD) both before and after the addition of a quencher.
The implemented technique achieves a 94.75% recovery of Fe(III) ions in urine samples, with a Relative Standard Deviation (RSD) of 4.12%. This demonstrates the practicality of the suggested method for directly analyzing Fe(III) in human urine without the need for dilution or pre-treatment.