Rosa Damascena mediated ZnO-Red Ochre nanocomposite for the electrochemical determination of 5-Fluorouracil

2.1 Materials, reagents, and solutions

The names of all the chemicals used in this research, along with the chemical formula, the name of the manufacturer, and their purity, are listed in Table 1. Graphite powder is amorphous, and its particle size is less than 50 µm. Phosphate buffer solutions, PBS, were prepared by mixing 0.1 M solutions of phosphate salts, and its pH was adjusted in the desired pH using 0.1 M NaOH or H₃PO₄ solutions. Ampoules containing 250 mg of 5-FU per 5 ml were bought from a drugstore in Isfahan, Iran.

2.2 Apparatus

The XRD pattern was obtained by PANalytical X’pert under Cu-K radiation, λ = 1.541 A˚, 2θ = 20–80, to analyze the RO nanoclay and determine the crystal structure and the size of RDZRONCs. The morphology of RDZRONCs was studied using a scanning electron microscope Leo 1430 Vp (Carl Zeiss). Also, the functional groups of RDZRONCs were characterized by Shimadzu 8900 FT-IR spectroscopy. The micron proton system used in this study was purchased in 2000 by the Atomic Energy Organization of Iran from Oxford Microbial Company and installed on the nuclear and nuclear research center's vendor accelerator. The launch of this system was carried out in late 2004. The proton beam required for elemental analysis is generated by the 3 MeV accelerator and nuclear physics unit. This bar has a dimension of 2 × 2 mm² for daily analysis. However, to create a micron beam, by an object diaphragm and a diaphragm, the diameter of the beam is reduced to less than 9 mm. Then the plate is converted by three triangular magnetic poles of less than 10 µm in diameter. The micron beam is swept by two oscillators in two directions, y and ×, and the sample's surface is in a maximum dimension of 2.5 × 2.5 mm. (The broom level in this research is 0.5 × 5 mm square). Therefore, in this way, the two-dimensional distribution of the elements in the sample can be measured by the micro-pixel technique. To determine the resolution of the measurement using the micro-pixel technique, the proton beam is projected onto a metal net, for example, of the iron contained in the test chamber. Also, the degree of separation of sample measurement is known as the distance between the vertical and horizontal lines in the net (about 120 µm). In this experiment, a 1.5-meV proton beam with a pulsed flow of about 10–20 pA was used. The proton beam diameter in this test is abou9t 10 µm. Autolab device manufactured by Company Chemie-Eco Netherlands, model PGSTAT320N, was used for electrochemical measurements consist of the CV, DPV, CC, CA, and EIS. For potentiostat tests, three-electrode cells containing working (CPE, modified or unmodified), reference (saturated Ag/AgCl electrode), and auxiliary (platinum wire electrode) are commonly used in this research. The voltammograms, chronoamprograms, chronocolograms, and Nyquist curves were recorded using NOVA 1.8 software. Then the required parameters were obtained by using this software and discussed. Nyquist curves were obtained for 0.1 M KCl solution containing 1 mM Fe²⁺/Fe³⁺ redox after 15 min of equilibrium, in the frequency range of 10 kHz-0.1 Hz with an amplitude of 0.01 V. Metrohome pH meter model 827 was used to prepare the buffers.

2.3 Preparation of the RD extract

RD flowers were collected from Qamsar, Kashan, Iran, and after washing with re-distilled water, it was dried and turned into powder. 5 g of dried and grounded RD flowers was added to 100 ml of re-distilled water in a 250 ml Erlenmeyer flask and sonicated at 70 °C for 20 min. The resulting solution was filtered with Whatman No. 1 filter paper and was used to green synthesis RDZRONCs.

2.4 Preparation of RO nanoclay

RO was collected from the Persian Gulf of Iran. For the green synthesis of RO nanoclay, 100 ml of ethanol 70 % v/v was added to 0.5 g of purified RO and sonicated for 20 min. In the next step, 20 ml of RD extract was added to the supernatant solution and subjected to 50 kHz ultrasonic for one hour. Finally, the obtained precipitate was calcined at 600 °C.

2.5 Green synthesis RDZRONCs

For the green synthesis of RDZRONCs, 0.1 g of RO nanoclay was dispersed in 10 ml of ethanol and then added to 40 ml of 10 mM zinc acetate dihydrate solution. To this solution was added 10 ml of RD extract as a reducing and capping agent and kept at 60 °C on a magnetic plate for about 24 h under continuous stirring. The resulting precipitate was centrifuged at 5000 rpm for 10 min and washed with re-distilled water to remove impurities. The green synthesized RDZRONCs were dried at 80 °C and calcinated at 600 °C for 3 h.

2.6 Preparation of the RDZRONCs/CPE

To make the unmodified CPE, weigh 0.5 g of graphite powder, then grind it well in a porcelain mortar for 20 to 30 min, then add about 7 to 8 drops of paraffin to the ground powder and mix it to be uniform. The homogenized paste was then pressed into a glass tube with a cross-section of 1.34 mm. For the preparation RDZRONCs/CPE, all the steps are similar to those mentioned for making the unmodified CPE, except that the values of 0.002, 0.004, 0.006, and 0.008 g of the RDZRONCs were separately added to 0.5 g of graphite powder. In addition, a thin copper wire, inserted through the end of the glass tube into the paste, was used to make an electrical connection between Autolab and the drug solution in electrochemical measurements.

2.7 Prepare real samples

To prepare the serum solution, place the sample in a centrifuge to separate the plasma portion, then 1 ml of it is transferred with a micropipette into a 10 ml volumetric flask and brought to volume with deionized water. Then, 1 ml of the resulting solution was transferred by micropipette into the cell containing 5 ml of PBS with specified pH, and the voltammogram was obtained. After collecting the urine sample from a healthy volunteer, it was stored at 4 °C. In order to prepare the sample, 5 ml of the sample was centrifuged for 5 min at a speed of 5000 rpm. Then it was diluted twice with phosphate buffer (pH = 8). To minimize the effect of the matrix, the standard addition technique was used for the serum and urine samples. In this technique, certain values of 5-FU standard solution were spiked into the sample solution so that the resulting current of voltammograms is in the linear range of the proposed technique.

3.1 Characterization of RO nanoclay

3.1.1

3.1.1 Micro-PIXE of RO nanoclay

Fig. 1 (a) shows the distribution of phases in RO nanoclay obtained from Micro-PIXE analysis. As a result, hematite (Fe₂O₃), quartz (SiO₂), and alumina (Al₂O₃), with a relative percentage of 43 %, 31.6 %, and 10.9 %, respectively, form the main phases of RO nanoclay. The remaining phases included magnesium 4.5 %, calcium 7.5 %, potassium 2 %, and trace amounts of 0.2 % sulfur, 0.2 % chloride, 0.3 % titanium, 14.4 % manganese, chromium 014 / 0 % on 016 % and sodium is 0.076 %. Fig. 1 (b) shows the Micro-PIXE elemental maps of iron, calcium, aluminum, potassium, magnesium, sulfur, silicon, and chlorine. As can be seen, the sample has a uniform and completely homogeneous distribution. Also, in an area where there is a cavity or space associated with one element, it is uniformly and homogeneously filled with other elements.

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Fig. 1

Distribution of phases in RO nanoclay (a), and elemental maps (b) obtained from Micro-Pixe analysis.

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3.1.2

3.1.2 XRD of RO nanoclay

X-ray powder diffraction (XRD) analysis was used to evaluate the main mineral phase of synthesized RO nanoclay. As shown in the XRD pattern, Fig. 2 (a), hematite (Fe₂O₃) and quartz (SiO₂) were the major phases of RO nanoclay. The characteristic peak at 2θ = 33.31° corresponds to the (1 0 4) miller index (reference code: 01–087-1166), confirming the presence of the hematite phase in RO nanoclay. Also, the characteristic peak at 2θ = 26.81° corresponds to the (0 1 1) miller index (reference code: 01–078-1252), confirming the quartz phase. In addition, the average size of RO nanoclay was obtained using the Scherrer equation $(L=\frac{\dot{K}\lambda }{\beta \mathrm{C}\mathrm{o}\mathrm{s}\theta })$ of 39.90 nm.

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Fig. 2

XRD pattern of RO nanoclay(a), XRD pattern of the RDZRONCs (b). and FT-IR spectrum of the RDZRONCs (c).

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3.2 Characterization of green synthesized nanocomposite RDZRONCs

3.2.3

3.2.3 FE-SEM and TEM aanalysis of RDZRONCs

The FE-SEM image of green synthesized RDZRONCs is shown in Fig. 3 (a). The RDZRONCs had a mostly hexagonal crystal structure with an average diameter equal to 50 nm. Also, energy scattering spectroscopy (EDX) was used to investigate the elemental composition of RDZRONCs. In the EDX spectrum (Fig. 3 (c)), the Fe, Si, Zn, and O elements were seen as indicators.

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Fig. 3

FE-SEM image (a and b), elemental composition from EDX (b), and TEM images (d and f).

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In order to compare the size, morphology and uniformity of distribution of RDZRONCs nanoparticles, their TEM image was prepared. Electron microscope model LEO912-AB manufactured by LEO company was used to determine the shape and size of RDZRONCs nanoparticles. For Transmission Electron Microscope (TEM) analysis, first the grid surface was covered with a thin layer of Form Var and a drop of the sample was placed on it. After drying the sample, its image was prepared. As can be seen in Fig. 3 (d,f), the particles were mainly spherical and their diameter was in the range of 40–50 nm. Also, in some areas, they are piled up or scattered together.

3.3 Study of the electrode surface

CV and EIS techniques were used to evaluate the RDZRONCs/CPE surface. To compare the active surface area of the CPE and the RDZRONCs/CPE, the actual surface area of each electrode was obtained using the Randles Sevik equation:

I_pa, n, A, D, ν, C₀ are the peak anode current, number of electrons transferred, electrode surface area, diffusion coefficient, scan rate, and concentration of K₃Fe(CN)₆, respectively. For this purpose, CVs were obtained at different scan rates for each electrode in 0.1 M KCl solution containing 1 mM K₃Fe(CN)₆ at different scan rates. Then, using the slope of the I_pa-ν^1/2 curve and considering that the values of n and D for K₃Fe(CN)₆ are equal to 1 and 7.6 × 10^-6, respectively, the surface area for the CPE is equal to 0.032, and for RDZRONCs/CPE equal to 0.072. Therefore, the surface area of RDZRONCs/CPE is 2.25 times the surface area of the CPE, that it is due to the large specific surface area of the RDZRONCs.

EIS is one of the most reliable techniques for examining the electrode surface based on the resistance to electron transfer at the surface. For this purpose, the Nyquist curves for the CPE, RO nanoclay/CPE, and the RDZRONCs/CPE for 1 mM Fe²⁺/Fe³⁺ redox in 0.1 M KCl solution were obtained (Fig. 4, left side). In the Nyquist curve, the value of Z_re, on the x-axis, represents the resistance to electron transfer, R_ct, at the electrode surface. Higher R_ct indicates slower electron transfer resulting in less current in voltammetric techniques, lower R_ct, indicates faster electron transfer, therefore, more current is observed in voltammetric techniques. Based on Fig. 4 and using the equivalent circuit (Pavel et al., 2005) to fit the Nyquist curve data (insets of Fig. 4), the R_ct value for the CPE was 7.02 kΩ, for the RO nanoclay/CPE 3.64 kΩ and for the RDZRONCs/CPE was 1.54 kΩ. These results indicate that the electrical conductivity of the green synthesized RDZRONCs is very good and facilitate electron transfer at the electrode surface.

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Fig. 4

Left: Nyquist diagram CPE (a), RO nanoclay/CPE (b), and RDZRONCs/CPE (c) and inset is equivalent circuit fitted with impedance data, at the solution containing 1 mM of Fe²⁺/Fe³⁺ redox and 0.1 M KCl, right: CVs for CPE, RO nanoclay/CPE and RDZRONCs/CPE, (a-c) in buffer solution, (d-f) buffer solution with 5-FU. Conditions: 100 µM 5-FU, 0.1 M PBS at pH 8.

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3.4 Electrochemical response of 5-FU on the electrode surface by CV technique

Cyclic voltammograms of the CPE, RO nanoclay/CPE, and RDZRONCs/CPE in the absence and presence of the drug were evaluated to assess the electrocatalytic properties of RDZRONCs/CPE (Fig. 4, right side). A comparison of the CVs of curve (c) and curve (d) in Fig. 4 shows that the electrochemical response of 5-FU at the CPE surface (curve (d)) appeared at a potential of 1.11 V with I_pa = 0.271 μA, at the RO nanoclay/CPE surface (curve (e)) appeared at a potential of 1.08 V with I_pa = 0.894 μA. In contrast, for RDZRONCs/CPE (curve (f)) the 5-FU electrochemical signal is observed at a potential of 1.03 V with I_pa = 1.282 μA. Based on these results, the peak potential of 5-FU in the RO nanoclay/CPE and RDZRONCs/CPE shifts 30 and 80 mV to a more negative potential than the CPE, respectively. As a result, the RO nanoclay and RDZRONCs have catalytic properties for 5-FU oxidation. On the other hand, the peak current oxidation of 5-FU for the RO nanoclay/CPE and RDZRONCs/CPE are 3.29 and 4.73 times that of the CPE due to the increased effective surface area of the electrode in the presence of RO nanoclay and green synthesized RDZRONCs. Therefore, RDZRONCs increase the measurement sensitivity of 5-FU due to their high electrical conductivity and catalytic properties.

3.5 Optimization of chemical and instrumental parameters

Chemical and instrumental parameters such as the amount of modifier in the electrode fabrication, the type of electrolyte (buffer), the pH value of the solution, and instrumental parameters in the DPV technique, were optimized to obtain the maximum current.

3.5.1

3.5.1 Optimization of the amount of RDZRONCs

Based on the above discussion, RDZRONCs can enhance the current responses of 5-FU. The amount of RDZRONCs was impressive on I_p in DPV. Fig. 5 shows the effect of change in the amount of RDZRONCs on the enhancement of the voltammogram peak height of 5-FU. As shown, From 0.001 to 0.002 g of RDZRONCs 3 µA increase in peak oxidation current was observed, and from 0.002 to 0.003 g of RDZRONCs, the current increased slightly. However, from 0.003 to 0.004 g of modifier, 2 µA increase in peak oxidation current is visible. With an increase of more than 0.004 g of the modifier, the current has also decreased. Therefore, the electrode with 0.004 g of RDZRONCs was used for subsequent studies.

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Fig. 5

Effect of the amount of RDZRONCs on the peak current of 100 µM 5-FU.

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3.5.2

3.5.2 Effect of pH

The pH of the supporting electrolyte is an essential parameter for the electro-oxidation of 5-FU on the RDZRONCs/CPE. Several buffers, such as ammonia, acetate, Britton Robinson, and phosphate, were used as the supporting electrolyte in the determination of the 5-FU solution using the DPV technique. A higher oxidation peak current was obtained for PBS. Thus, PBS was used as the supporting electrolyte in the continuing experiments. The effect of pH in the range of 4–10, I-pH diagram, and E-pH diagram for the PBS buffer as a suitable buffer was shown in Fig. 6 (a), Fig. 6 (b), and Fig. 6 (c), respectively. Based on Fig. 6 (a) and Fig. 6 (b), the maximum peak current of 5-FU is obtained at pH = 8.0. The maximum peak current depends on the form of protonation and deprotonation of 5-FU species in the electrochemical cell (Bukkitgar and Shetti, 2016; Hadi et al., 2018). In other words, in acidic pH, the protonated form of 5-FU is dominant, and in basic pH, the deprotonated form of 5-FU is dominant (Bukkitgar et al., 2015). The peak current is dependent on the structure of the analyte. Fig. 6 (c) shows that the oxidation potential of 5-FU tends to have negative potentials as the pH increases, and there is a linear relationship between E_p and pH, following the equation below.

(2)

$$E_{\mathit{pa}}\left(V\right)=1.5938-0.0582pH,R2=0.9871$$

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Fig. 6

DPVs obtained by RDZRONCs/CPE in 0.1 M phosphate buffer with pHs of 4 to 10 containing 5 µM 5-FU (a), the effect of pH on the peak current of 5-FU (b), and the effect of pH on the peak oxidation potential of 5-FU (c).

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The above result displays the participation of proton in the electrochemical reactions of 5-FU. According to the proposed mechanism for fluorouracil oxidation and the interventions of H⁺ ions, changing the pH changes the oxidation of the species so that by increasing the concentration of H⁺ ions and decreasing the pH, the species oxidation reaction takes place at more positive potentials. Given the slope of equation (2) for 5-FU, −58 mv/pH, it can be concluded that the ratio m/n is approximately 1. Therefore, the number of protons and electrons exchanged in the electrochemical reaction of 5-FU are equal to each other and are consistent with the results reported in the literature (Bukkitgar and Shetti, 2016; Hadi et al., 2018).

3.6 Investigation of the effect of potential scan rate on the electrochemical behavior of 5-FU

By examining the influence of potential scan rate, ν, on cyclic voltammograms, it is possible to investigate the penetration or absorption of the reaction of 5-FU on the surface of the RDZRONCs/CPE. According to the Randles-Sevik equation, equation (1), linearity relationship of I_p-ν^1/2 indicates that the 5-FU oxidation process at RDZRONCs/CPE is under mass transfer control and the fluorouracil for oxidation penetrate to the electrodes. The Tafel equation is used for obtaining the α (electron transfer coefficient) parameter, based on the data of the I-E curves and then using the slope of the curve of the logI_p,-Efrom the following.

According to Fig. 7 (c), the slope of the Tafel line is equal to 6.3245. By putting the values of R = 8.314 j mol⁻¹ K⁻¹, T = 298 K, and F = 96485C mol⁻¹ and considering that the n for 5-FU is 2 (Bukkitgar and Shetti, 2016; Hadi et al., 2018), the value of α for 5-FU is 0.81. On the other hand, the results of Fig. 7 (a) show that with increasing scan rate, the peak potential value shifts to more positive values. The observation of this behavior can be attributed to the irreversibility of the oxidation process (Hadi et al., 2018; State et al., 2022). The relationship between log Ip and log υ is linear, and the slope of the resulting line is 0.62 (Fig. 7 (d)). If the value of the slope is close to 1, it indicates that the process is controlled by adsorption, and if the slope is close to 0.5, the process is controlled by diffusion (Laviron, 1979). Therefore, it was again concluded that the electrochemical process of 5-FU at RDZRONCs/CPE is diffusion controlled.

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Fig. 7

CVs obtained for 100 µM 5-FU in 0.1 M PBS at pH = 0.8 on the surface of RDZRONCs/CPE with scan rates of 1.0 to 50.0 mV s⁻¹ (a), changes in the electrocatalytic current relative to the ν^1/2 (b), and Tofel diagram obtained from the current–potential curve recorded at the 30 mV s⁻¹ scan rate (c), dependence of log I on logν (d), relationship between E_p and logν (e), relationship between E_p and ν (f).

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Pursuant to Laviron's theory (Ganduh et al., 2021), for an irreversible electrode process, the relationship between scan rate and peak potential, E_p, is expressed through equation (4).

In equation (4), E⁰, and k⁰ are formal potential (V), and the heterogeneous catalytic rate constant (s⁻¹), respectively. Fig. 7 (e) shows that the relationship between E_p and log υ is linear. The value of nα is obtained using the slope of the resulting line equal to 1.69. The value of α can be calculated using equation (5).

In equation (5), E_p/2 is the potential at which the current is half the peak value. Considering that the value of E_p-E_p/2 is equal to 0.058 V, the value of α is equal to 0.82. Considering that the E_p-E_p/2 value is 0.058 V, the α value is equals 0.82. The value of α obtained from equations (4) and (5) is agrees with the value obtained from equation (3). Finally, by determining the value of α, the value of n was obtained 2.05≈2. The k⁰ value can be calculated from the intercept of the resulting line from the log ν-E_p graph (Fig. 7 (e). The value of E⁰ is obtained by using the graph obtained from E_p in relation to ν (Fig. 7 (f)) by extrapolating the resulting line to the vertical axis, ν = 0. Consequently, E⁰ and K⁰ were obtained as 0.995 and 2.2 × 10⁴ s⁻¹, respectively.

3.7 Reaction mechanism

Since in section 3–6, the number of electrons was equal to 2, and in section 3.5.2, the number of protons and electrons were equal in the electrochemical reaction of 5-FU, therefore, the possible electrochemical reaction for 5-FU can be justified by Fig. 8. According to Fig. 8, first the π electron in the 5-FU ring is removed and component 1 is produced. But component 1 is very unstable and is quickly attacked by OH– ions and turns into component 2. Finally, by removing a proton and an electron, the final product is created. The oxidation of 5-FU is facilitated by increasing the electron density in the π bond of C⚌C. The presence of the electron-withdrawing group F in the fifth position of 5-FU molecule causes a decrease in the electron density due to its electron-withdrawing property, but on the other hand, the mesomeric effect of the F atom can increase the electron density. Considering that the mesomeric effect of the halogen atom plays a more important role, as a result, the presence of the F atom in the 5-FU molecule facilitates the oxidation of this molecule (Wang et al., 2012; Jasim et al., 2022; Mahdi et al., 2021; Mahdi et al., 2020; Aljeboree et al., 2020; Aljeboree et al., 2021).

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Fig. 8

Possible mechanism of 5-FU oxidation reaction.

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3.8 Investigation of oxidation of 5-FU by CA and CC methodes

To obtain the diffusion coefficient D for 5-FU, its oxidation by the RDZRONCs/CPE by CA technique has been studied. First, 5-FU chronoamperograms were obtained in different concentrations, and from them, Ip diagrams were drawn relative to t^−1/2. Then, by plotting the slope of the graph curve I- t^−1/2 in terms of different concentrations of 5- FU and using Cottrell’s equation, equation (6), D was calculated.

In this equation, D is the diffusion coefficient (cm².s⁻¹), C is the concentration of 5-FU (mol.cm⁻³), A is the surface area of the electrode (cm²), and n is the number of electrons participating in the reaction. According to Fig. 9 (a), Fig. 9 (b), and equation (6), D by CA technique was obtained 3.8 × 10^-5 cm².s⁻¹. So, first, chronocoulogram diagrams were drawn for different concentrations of 5-FU. Then Q-t1/2 graphs were drawn from the resulting chronocoulogram graphs. Bby plotting the slope of the lines obtained from the Q-t^1/2 graphs relative to the concentration, the value of D was obtained using equation (7).

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Fig. 9

Chronoamperograms obtained from solutions with concentrations of 10, 20, 30, 40, 60, 70, 80 and 100 μM 5-FU in 0.1 M PBS solution with pH = 8.0 (a), diagram I-t^−1/2 for different concentrations of 5-FU,(inset of a) Slope of I-t^−1/2diagram in terms of 5-FU concentration (b), chronocoulograms obtained from solutions with concentrations of 10, 20, 30, 40, 60, 70, 80 and 100 μM 5-FU in 0.1 M PBS solution with pH = 8.0 (c), and diagram Q-t^1/2 for different concentrations of 5-FU,(inset of c) Slope of Q-t^1/2 diagram in terms of 5-FU concentration (d).

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According to equation (7), D was obtained 3.8 × 10^-5 cm².s⁻¹ by the CC technique, which is in good agreement with the value obtained by the CA technique.

3.9 Calibration curve and detection limit

Because the differential pulse voltammetry technique has a higher current sensitivity than cyclic voltammetry, this technique is used to estimate the detection limit. After optimizing the test conditions and obtaining the most appropriate electrochemical response for the RDZRONCs/CPE to 5-FU using DPV obtained for different concentrations of 5-FU, the calibration curve was drawn (Fig. 10). Fig. 10 (a)-(p) shows that the variations of the measured current are linear in terms of 5-FU concentration in the range of 0.05 to 140 μM. After drawing the calibration curve and obtaining the line equation, it is possible to obtain the detection limit of the technique with the equation DL = 3S_b/m (Killedar et al., 2021; Shetti et al., 2020). In this equation, S_b and m are the standard deviations of the blank and the slope of the line of the calibration curve, respectively. The detection limit was calculated to be 0.0016 µM. Table 2 compares the detection limit and the linear range obtained from this technique with the values reported in some of the articles. By comparing the results of this technique with the previously reported techniques, it can be seen that the detection limit of this technique is better than some reports, and in some cases, the linear range of the proposed technique is wider.

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Fig. 10

DPVs in 0.1 M PBS with RDZRONCs/CPE at pH = 0.8, with concentrations of 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120 and 140 μM 5-FU and inset; current changes measured in terms of 5-FU concentration.

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3.10 Stability, repeatability, and reproducibility of RDZRONCs/CPE

Voltamograms of solution 10 µM of 5-FU were obtained with RDZRONCs/CPE for 50 consecutive days. The results showed that after this period, the response current of the electrode changes to 98.2 % of its initial value. Therefore, the desired electrode has good relative stability. To evaluate the accuracy of the technique, DPVs for 10 µM of 5-FU under optimal conditions were recorded 15 times. The RSD of the resulting currents was 1.4 %. The results indicate that the proposed technique for measuring 5-FU has good accuracy. To evaluate the reproducibility of the technique, 5 electrodes with the optimal amount of RDZRONCs were prepared, and then DPVs of 10 μM 5-FU were recorded under optimal conditions for each of the electrodes. The RSD value was 2.7 %.

3.11 Investigate the effect of interferences

In this study, the effect of some interfering species, which are often present in biological systems and can interfere with the measurement, has been studied. The study is that if the current from the presence of the interfering species is less than 5 % different from the peak current of the drug in the absence of the interferences, such interference will not interfere with the measurement of the drug. However, if this ratio is more than 5 %, it will interfere with the measurement of the drug. Table 3 shows the interference effects of various species on the determination of 5-FU. To investigate the interferences in the measurement of 5-FU, a 10 μM solution of 5-FU in 0.1 M PBS with pH = 8.0 was used under optimal conditions. The results show that most of the studied species will not interfere with measuring 5-FU. Therefore, it can be concluded that the electrode modified with RDZRONCs has good selectivity, and this electrode can be easily used to measure 5-FU in real samples.

3.12 Measurement of 5-FU in real samples by standard addition technique

To evaluate the efficiency of the technique, after drawing the standard curve, the amount of 5-FU in the 0.250 mg/0.5 ml fluorouracil ampoule was measured by the standard addition technique. For this purpose, 10 ml of a solution with a concentration of 30 μM 5-FU in PBS buffer with pH = 8.0 was prepared, DPV was recorded, and its current was measured. Then, specific volumes of the standard solution of 5-FU were added to the drug solution each time, and DPVs were recorded. After extracting the results of these voltammograms, the standard addition curve was drawn. The measurements were repeated 4 times, and the confidence limits for the 97 % confidence level were calculated. The results are given in Table 4. The effect of physiological tissue on real samples was also evaluated with the standard addition technique. The results of these studies are also presented in Table 4. According Table 4, the complex composition of real samples has little effect on 5-FU recovery.