Characterization of N-GQDs
The characterization of N-GQDs synthesized by the one-step hydrothermal method was done by FT-IR, UV-Vis spectra, x-ray diffraction (XRD), and transmission electron microscopy (TEM).
The FT-IR spectrum of N-GQDs is shown in Figure 1a. The broad absorption band appearing at the 3315 cm−1 corresponds to the O-H and N-H stretching vibration, respectively, and the band observed at the 1090 cm−1 corresponds to the C-O stretching vibrations. The bands that appeared at 2372 and 1636, 1576, and 1388 cm−1 are related to C-H, NH-CO, C-C, and C-N stretching vibrations, respectively. Thus, the FT-IR spectrum of N-GQDs indicates its successful synthesis.
The absorption spectrum of the synthesized N-GQDs (Fig. 2b) exhibits a strong absorption band with a maximum at 235 nm related to the π-π* transitions of aromatic C=C bond (sp2), and another strong absorption band with a maximum at 342 nm due to the n-π* transition of C=O bond. The appearance of the absorption maximum in the blue region of the spectrum is an indication of the small size of the synthesized N-GQDS.
TEM image of synthesized N-GQDs (Fig. 1c), shows the formation of spherical particles with uniform distribution. According to Fig. 1c, the dimensions of N-GQDs are in the range of 3.3-5.3 nm with an average diameter of 4.3 nm.
The XRD pattern of the N-GQDs is provided in Figure 1d. In this pattern, a broad diffraction peak (002) with a maximum at 2θ of about 24.3° is observed which is related to the graphite structure and proves the formation of these quantum dots.
The role of amino acids in the preparation of gold nanoparticles
Alpha-amino acids are compounds that differ in the type of group in their alpha position. In this study, the amino acid phenylalanine was used as a coating and reducing agent to prepare Au NPs. Thus, in the preparation of gold nanoparticles, some amino acids act as a reducing agent and the excess amino acid binds to the surface of the gold nanoparticles (Fig. 2). The gold atoms acting as Lewis acid coordinate with the unbonded electron pair of the amino group of phenylalanine. Functionalization of Au NPs with amino acids resulted in the stabilization and dispersion of the nanoparticles in water. The presence of amino acids on the surface of Au NPs with functional groups of O-C=O and N-H is useful for binding it to other molecules.
UV-Vis spectrum of Au NPs coated with phenylalanine as a function of times
One of the methods of recognition of Au NPs is the study of its UV-Vis spectrum. Gold nanoparticles have an absorption band in the range of 500 to 600 nm with a λmax that depends on the size of the nanoparticles. To investigate the formation of gold nanoparticles, the absorption of the sample was recorded at different times interval from the start of synthesis (Fig. 1S). As it is observed, when the time is increased up to 20 minutes, the absorption intensity is increased and the λmax is shifted toward the red wavelength. But, further increase in time has no noticeable effect on the λmax or intensity of absorption, and the fluorescence intensity becomes constant at the wavelength of 552 nm. For the explanation of this observation, the gold nanoparticles can be assumed as clusters made of gold atoms. Then, according to the bond theory, the greater the number of gold atoms connected, the smaller the distance between HUMO and LUMO in the clusters, and the less energy required for the electron transfer between these two levels, resulting in a shift in maximum absorption toward the longer wavelength. Thus, the Au NPs coated with phenylalanine have a λmax of absorption at 522 nm with a synthesis time of 20 minutes.
Characterization of Au NPs coated with phenylalanine
To confirm the successful binding of phenylalanine on the surface of the Au NPs, FT-IR analysis was performed. A comparison of the spectra of phenylalanine, Au NPs, and Au NPs coated with phenylalanine (Fig. 2S) revealed that the spectrum of Au NPs coated with phenylalanine has an absorption band at 3354 cm−1 related to the OH group on the surface of Au NPs. Furthermore, the absorption band at 1646, 1544, and 1211 cm−1 are related to the C=O group, the aromatic C=C of the phenylalanine benzene ring, and the stretching vibration of C-O and C-N of the phenylalanine, respectively. The above results indicate the successful binding of phenylalanine to the surface of Au NPs.
The TEM image of gold nanoparticles coated with phenylalanine (Fig. 2S) shows that these nanoparticles are spherical with an average diameter of 37.8 ± 0.5 nm.
Optical N-GQDs sensor for piroxicam determination
To evaluate how the system works and present a suitable mechanism, various experiments were done. In the first experiment, 500.0 µL of N-GQDs, 300.0 µL of phenylalanine (25.0 mmol L−1), 200.0 µL of phosphate buffer (pH = 7.0), and 500 µL of piroxicam solution with the concentration of 11.0 nmol L−1 was transferred into a quartz cell and after 10.0 minutes its fluorescence was recorded. The blank solution was prepared with the same ingredient except for the drug and its fluorescence under the same conditions was recorded. As shown in Fig. 3a, the addition of phenylalanine causes a decrease in the fluorescence intensity of N-GQDs at 440 nm due to the formation of hydrogen bonds between the amino group of the amino acid and the acidic and amide groups of N-GQDs. The addition of piroxicam to this system resulted in a further decrease in the fluorescence intensity due to the hydrogen bond formation between the drug and phenylalanine.
The second experiment was done as the previous one but instead of phenylalanine solution, 300.0 µL distilled water was used. The results (Fig. 3b) revealed that upon addition of the drug, the fluorescence intensity of N-GQDs slightly is reduced due to the hydrogen bond formation between the drug and hydroxyl and amide groups on the N-GQDs surface.
The third experiment was performed as the first one but 300.0 µL of Au NPs was used instead of phenylalanine solution. The result of this investigation (Fig. 3c) shows that the addition of Au NPs resulted in a decrease in the fluorescence intensity of N-GQDs due to the resonance energy transfer mechanism. However, the addition of piroxicam to this mixture has no significant effect on the fluorescence intensity, indicating that piroxicam can not directly bond to the Au NPs.
The fourth experiment was done as the first one but the phenylalanine solution was replaced with a 300.0 µL solution of Au NPs coated with phenylalanine. As shown in Fig. 3d, in the absence of the drug the fluorescence intensity of the N-GQDs is decreased. This observation can be explained based on the Forster resonance energy transfer mechanism that the excited electrons in N-GQDs are adsorbed by the Au NPs coated with phenylalanine, and the resulted cavity in the ground state is replaced with the resonance electron of the compound. The result of these electron transitions and resonance is self-absorption and fluorescence quenching. The addition of piroxicam to this mixture causes the release of the Au NPs from the N-GQDs surface, followed by the retrieve of the fluorescence intensity. The release of gold nanoparticles from the N-GQDs surface can be due to the formation of the hydrogen bonds between phenylalanine and the drug. This experiment confirms the important role of the Au NPs coated with phenylalanine in the designed method for the determination of piroxicam. The possible mechanism of interaction of N-GQDs with Au NPs coated with phenylalanine and piroxicam is shown in Fig. 4.
Optimization of experimental conditions
The influence of pH on the analytical signal was investigated by changing the pH within the range of 4.0-9.0 using the phosphate buffer. As shown in Fig. 5a, the analytical signal is maximized at the pH of 7.0 that was chosen as the optimal pH for further studies. Piroxicam is a diprotic amphoteric drug with pKas values of 1.86 and 5.46. So, when the pH of a solution is greater than its pKa2, the anionic form of piroxicam predominate in the solution. Thus, at the pH of 7.0, the drug is mainly in its anionic form and the electron transfer takes place from the piroxicam to the electropositive Au NPs containing empty d orbital and the analytical signal is maximized. However, at higher pH, the electron transfer to the empty d orbital is completed resulting in the decrease in analytical signal.
The effect of N-GQDs volume in the range of 100.0-700.0 µL on the analytical signal was considered. It was observed that (Fig. 5b) in the presence of a fixed amount of piroxicam, the analytical signal increases by increasing the volume of N-GQDs up to 500 µL and then decreases. The decrease in the signal at a higher N-GQDs volume can be due to the increase in the ratio of N-GQDs to the fixed concentration of piroxicam and Au NPs coated with phenylalanine. Thus, the volume of 500.0 µL was used as the optimal volume of N-GQDs.
The influence of the Au NPs coated with phenylalanine volume in the range of 100.0- 600.0 µL on the analytical signal was examined. The results of this study (Fig. 5c) indicate that the analytical signal increases up to the 300.0 µL of nanoparticles and then decreases. The decrease in analytical signal in a volume greater than 300.0 µL can be attributed to the increase of volume of Au NPs coated with phenylalanine in comparison to the fixed concentration of the drug, in which case the extra gold nanoparticles adsorb to the surface of N-GQDs and reduce the analytical signal. Therefore, a volume of 300.0 µL was selected as the optimal volume.
The contact time between the drug and N-GQDs in the presence of Au NPs coated with phenylalanine was also optimized by varying the contact time within the range of 2-25 minutes. An increase in contact time up to 10 minutes, increased the analytical signal and then remained constant. In other words, for complete interaction of drug with phenylalanine and release of the Au NPs from the surface of N-GQDs, a contact time of 10.0 minutes is required.
Selectivity studies
To evaluate the proposed method selectivity for measuring piroxicam, the influence of some drugs in the same category with piroxicam such as naproxen, aspirin, and ibuprofen, as well as other drugs (lamotrigine, carbamazepine, tetracycline, and diclofenac) and compounds (glycine, ascorbic acid, histidine, uric acid, and glucose) and metal ions including Ca2+, Na+, and K+ on the analytical signal was examined. An error of less than 5% was considered at the random experimental error. For this purpose, the standard solution of each of these materials with a concentration of 110.0 nmol L−1 was measured separately by the designed method. Based on the results of this study (Fig. 3S), the effect of most of these species with a concentration of 10 times of the piroxicam on the analytical signal is negligible and the analytical signal of aspirin, ibuprofen, and naproxen (at a concentration of 110.0 nmol L−1) is about half of the piroxicam analytical signal with the concentration of 11.0 nmol L−1. Thus, the designed method has good selectivity for the measurement of piroxicam.
Analytical features
At the optimized conditions, the analytical performance of the developed method for the determination of piroxicam was investigated. The signal was linearly increased with increasing the piroxicam concentration within the range of 2.0 to 35.0 nmol L−1 (Fig. 6) with the equation of y = 0.0048x + 0.0169 and coefficient of determination of 0.9949 (where y is the analytical signal (F-F0)/F0 and x is the piroxicam concentration (nmol L−1). The detection and quantification limits were found to be 0.11 and 0.36 nmol L−1, respectively. The intra and inter-day precisions were determined by six independent replicate measurements of piroxicam at 11.0 nmol L−1 and were 2.2 and 4.5%, respectively.
Real sample analysis
The capability of the developed method for measurement of the piroxicam in plasma and urine samples was evaluated. For investigation of the reliability of the method, each sample was spiked at two concentration levels of piroxicam and the recoveries were of the added analyte was calculated. The results (Table 1) exhibited that the recoveries of all spiked samples are in the range of 94.5-101.8%, indicating the accuracy of the developed method for the quantitative piroxicam measurements in these samples types. The reliability of the method was also confirmed by analyzing the commercial capsules of piroxicam. The mean amount of piroxicam was determined to be 9.7 ± 0.2 mg/capsule which at 95% confidence level is in good agreement with the value claimed (10.0 mg/tablet).
Table 1
Determination of piroxicam in real samples
Sample
|
Added (nmol L−1)
|
Found* (nmol L−1)
|
Recovery (%)
|
Plasma
|
-
|
N.D
|
-
|
|
9.0
|
8.6 ± 0.3
|
95.6
|
|
11.0
|
11.2 ± 0.5
|
101.8
|
Urine
|
-
|
N.D
|
-
|
|
9.0
|
8.8 ± 0.4
|
97.8
|
|
11.0
|
10.4 ± 0.3
|
94.5
|
* Mean and standard deviation of three independent analysis. |
Comparison of the analytical performance of the method with other reported methods
The analytical performance of the developed method was compared with some other reported methods for the measurement of piroxicam. The results of this study (Table 2) indicate that the limit of detection of the designed method is lower than most of the compared methods and has a reproducibility better or comparable with them. In addition, the determination of piroxicam with this designed optical sensor is simple and fast.
Table 2
Comparison of the developed method with other reported methods for determination of piroxicam
Method
|
Linear range
(nmol L−1)
|
Detection limit
(nmol L−1)
|
RSD
(%)
|
Real sample
|
Reference
|
LC
|
3.0-18000
|
0.011
|
-
|
Serum
|
[27]
|
CV
|
0.5-100.0
|
0.11
|
3.11
|
Serum
|
[28]
|
CV-SWV
|
8.7× 102-2.6 × 104
|
1 × 102
|
3.8
|
River water
|
[29]
|
PV
|
0.5 × 103-5.0 × 104
|
50.0
|
2.22
|
Serum
|
[30]
|
DPV
|
100.0-7.0 × 104
|
40.0
|
4.2
|
Serum
|
[31]
|
PL
|
3.01 × 103-6.04 × 104
|
1.47 ×103
|
4.1
|
Drug
|
[32]
|
LC-MS-MS
|
11.0-56.0
|
2.0
|
3.0
|
Serum
|
[33]
|
SFC-MS-MS
|
11.0-11.25 × 103
|
-
|
6.6
|
Serum
|
[34]
|
PL
|
2.0-35.0
|
0.11
|
2.2
|
Urine-Serum
|
Present study
|
LC: Liquid chromatography, CV: Cyclic voltammetry, SWV: Square wave voltammetry, PV: Pulse voltammetry, DPV: Differential pulse voltammetry, LC-MS-MS: Liquid chromatography-tandem mass spectrometry, PL: Photoluminescence, SFC-MS-MS: Supercritical fluid chromatography-tandem mass spectrometry. |