A Fluorescence System Composed of Nitrogen-doped Graphene Quantum Dots and Gold Nanoparticles Coated With Phenylalanine for Selective and Sensitive Quantication of Piroxicam in Biological Samples

In this study, a sensitive uorimetric method is proposed for the determination of piroxicam using nitrogen graphene quantum dots (N-GQDs) and gold nanoparticles coated with phenylalanine. The uorescence emission of N-GQDs at 440 nm decreases with the increase of gold nanoparticles coated with phenylalanine. However, the addition of piroxicam causes the release of gold nanoparticles from the surface of quantum dots followed by the retrieval of the uorescence emission of N-GQDs. Under the optimum conditions, the calibration graph was linear in the concentration range of 2.0-35.0 nmol L -1 for piroxicam with a limit of detection of 0.11 nmol L -1 . The developed method was successfully applied for the determination of piroxicam in urine and serum samples.


Introduction
Piroxicam (PX) is a non-steroidal anti-in ammatory drug (NSAID) that plays an important role in reducing pain in a variety of arthritis and other post-operative conditions [1]. Although, this drug is vastly employed in human and veterinary medicine, unfortunately, some common side effects including headache, gastrointestinal disorders, dizziness, palpitations, skin rashes, and tinnitus have been observed in the patients consuming this drug [2,3]. The increased request for its production increased its release as an important biological pollutant into the e uent of pharmaceutical industries. Thus, the design of a sensitive and selective method for quanti cation of the low amount of this compound in environmental samples as well as human blood and urine is necessary. Several analytical methods include spectrophotometry [4], liquid chromatography [5], spectro uorometry [6], and capillary electrophoresis [7] were applied for the determination of piroxicam. Among the mentioned method, uorescence techniques with the unique properties of high sensitivity, simple operation, and stability have attracted researchers in analytical and biological elds [8,9]. Fluorescence resonance energy transfer (FRET), introduced as a technology in uorescence methods in which energy can be transferred from uorophore as a donor to an acceptor via dipole-dipole coupling process [10,11]. This technique has been successfully utilized for the detection of different biological molecules [12][13][14] and ions [15,16], but there is no report on the use of this method for the determination of piroxicam. In the design of a FRET system, the selection of proper donor-acceptor pairs is important for the achievement of good e ciency.
Graphene quantum dots (GQDs) as a new kind of zero-dimensional carbon nanomaterial with oxygencontaining functional groups and sizes smaller than 100 nm, has been employed in different elds including catalysis, biosensors, biological labeling, and bioimaging [17][18][19][20][21][22]. GQDs have distinct advantages of excellent biocompatibility, good water solubility, low toxicity, strong photoluminescence, and large surface area. Thus, in the design of uorescent nanoprobes, GQDs are a promising alternative to the conventional quantum dots of heavy metals and carbon dots [23,24]. The numerous oxygencontaining hydroxyl and carboxyl groups, give the GQDs good capability of modi cation with functional groups making them favorable as electron acceptors and electron donors.
Gold nanoparticles (Au NPs) with the great advantages of the excellent surface plasma resonance, obvious color change, and high extinction coe cient have been used in colorimetric and uorimetric sensors [22,25]. The fabrication of uorescence sensors based on gold nanoparticles and graphene quantum dots provides a sensitive and selective nanoprobe for the quanti cation of biomolecules and drugs [22,25]. The operation of these uorescence sensors is based on the capability of Au NPs in quenching the uorescence intensity of graphene quantum dots through the FRET mechanism [26].
Herein, blue emission nitrogen-doped graphene quantum dots were chosen as a donor, and gold nanoparticles coated with phenylalanine as an acceptor in the design of a uorescent nanoprobe for the sensitive and selective FRET determination of piroxicam. The uorescence intensity of N-GQDs was signi cantly quenched (off-status) in the presence of Au NPs coated with phenylalanine due to the uorescence resonance energy transfer (FRET). However, the addition of the piroxicam resulted in the hydrogen bond formation between the functional groups of gold nanoparticles and drug followed by the release of Au NPs from the surface of quantum dots, and retrieval of uorescent intensity of N-GQDs.
All materials were applied with the highest purity. A 50.0 µg mL −1 stock solution of piroxicam was prepared by diluting 2.5 mg of piroxicam to 50 mL in a volumetric ask with double distilled water and stored in the refrigerator. Solution of phenylalanine (25.0 mmol L −1 ) was prepared by dissolving proper amounts of it in double-distilled water. Tetrachloroauric acid (25.0 mmol L −1 ) solution for synthesis of gold nanoparticles was also prepared and stored in the refrigerator.

Preparation of N-GQDs
N-GQDs were prepared by the reported study [26]. The citric acid (1.26 g) and urea (1.08 g) were added into a backer containing 30.0 mL of double-distilled water and stirred until a clear solution was obtained. Then, the solution was transferred into a Te on-lined stainless autoclave and heated at 170°C for 5 hours. The product was centrifuged for 10.0 minutes at 3500 rpm for removing the larger particles and stored in the refrigerator.

Gold nanoparticles coated with phenylalanine preparation
For the synthesis of the gold nanoparticles coated with phenylalanine, 300.0 µL of phenylalanine solution as a reduction and modi cation reagent with the concentration of 25.0 mmol L −1 was added to a 100 mL beaker containing 15.0 mL of water, under stirring. The mixture was heated to the boiling point and while heated, 1.0 mL of tetrachloroauric acid solution (1.0 mmol L −1 ) was added to it. After a few minutes, the solution turned pinkish-red, and when the reaction has completed, the colloidal solution was rapidly cooled by placing the vessel and its content into an ice water bath. The prepared nanoparticles were stored in sealed dark containers in the refrigerator for further experiments.

Sample preparation
Serum and urine samples were provided by a healthy volunteer who in the past three months did not consume any drugs. The urine sample was prepared by centrifugation at 5000 rpm for 15 minutes and passing the supernatant through a 0.45 µm lter. Then, its piroxicam content was determined according to the given procedure.
For the preparation of the serum sample, 2.0 mL of acetonitrile was added to the 4.0 mL of the human serum sample, the mixture was centrifuged at 10000 rpm for 10 minutes, the supernatant was transferred to a 25.0 mL volumetric ask and diluted to the mark with distilled water. Finally, its piroxicam content was measured by the developed procedure.
To prepare a sample of piroxicam capsules, the contents of 10 capsules of piroxicam (10 mg/capsules) were mixed and ground well. Then, a precise amount of it, equivalent to one-tenth of the weight of one capsule (0.1652 g) was put into a beaker, some water was added to it, and after sonicating for 10 minutes, was passed through a 0.45 µm Millipore lter. Then, the ltrate was diluted properly with double distilled water to get a solution with the drug concentration within the linear range of the calibration graph. The amount of piroxicam was determined according to the given procedure.

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 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 λ max 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 λ max is shifted toward the red wavelength. But, further increase in time has no noticeable effect on the λ max or intensity of absorption, and the uorescence 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 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 rst 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 uorescence was recorded. The blank solution was prepared with the same ingredient except for the drug and its uorescence under the same conditions was recorded. As shown in Fig. 3a, the addition of phenylalanine causes a decrease in the uorescence 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 uorescence 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 uorescence 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 rst 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 uorescence intensity of N-GQDs due to the resonance energy transfer mechanism. However, the addition of piroxicam to this mixture has no signi cant effect on the uorescence intensity, indicating that piroxicam can not directly bond to the Au NPs.
The fourth experiment was done as the rst 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 uorescence 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 selfabsorption and uorescence 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 uorescence 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 con rms 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 in uence 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 pKa s values of 1.86 and 5.46. So, when the pH of a solution is greater than its pKa 2, 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 xed 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 xed 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 in uence 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 xed 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 in uence 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 Ca 2+ , 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 coe cient of determination of 0.9949 (where y is the analytical signal (F-F 0 )/F 0 and x is the piroxicam concentration (nmol L −1 ). The detection and quanti cation 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 con rmed 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% con dence level is in good agreement with the value claimed (10.0 mg/tablet). 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.

Conclusion
In this study, N-GQDs were synthesized and an optical sensor was designed for the determination of piroxicam based on the off-on uorescence intensity of N-GQDs using Au NPs coated with phenylalanine. The developed method was successfully applied to the measurement of piroxicam in the complex matrices of human urine and serum samples. The structural properties of N-GQDs were investigated by various spectroscopic methods. The developed method possesses the advantages of proper detection limit without the need for separation and preconcentration step, good selectivity, and simplicity.

Declarations Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Competing Interests