CuONPs/MWCNTs/carbon paste modified electrode for determination of tramadol: theoretical and experimental investigation

A practical technique was applied to fabricate CuO nanostructures for use as the electrocatalyst. The green synthesis of cupric oxide nanoparticles (CuO NPs) via co-precipitation is described in this paper using an aqueous extract of Origanum majorana as both reductant and stabilizer, accompanied by characterization via XRD, SEM, and FTIR. The XRD pattern revealed no impurities, whereas SEM revealed low agglomerated spherical particles. CuO nanoparticles and multi wall carbon nanotubes (MWCNTs) have been used to create a modified carbon paste electrode. Voltammetric methods were used to analyze Tramadol using CuONPs/MWCNT as a working electrode. The produced nanocomposite showed high selectivity for Tramadol analysis with peak potentials of ~ 230 mV and ~ 700 mV and Excellent linear calibration curves for Tramadol ranging from 0.08 to 500.0 µM with a correlation coefficient of 0.9997 and detection limits of 0.025. Also, the CuO NPs/MWCNT/CPE sensor shows an an appreciable sensitivity of 0.0773 μA/μM to tramadol. For the first time the B3LYP/LanL2DZ, quantum method was used to compute DFT to determine nanocomposites' connected energy and bandgap energy. Eventually, CuO NPs/CNT was shown to be effective in detecting Tramadol in actual samples, with a recovery rate ranging from 96 to 104.3%.

Tramadol is a synthetic opioid analgesic that works primarily on the central nervous system. It works through two fundamental mechanisms: agonistic binding to -opioid receptors and blocking norepinephrine and serotonin reuptake. Tramadol's pharmacokinetics, effectiveness, and safety qualities have made it a success in patients with moderate to severe chronic pain who take it three to four times a day. When compared to the usual form of Tramadol, extended-release Tramadol, a newly created modified-release tablet, would be favorable for the day-long duration and minor drug plasma variance 1,2 .
Tramadol is a substance that acts as a -agonist. [2-(dimethyl aminomethyl)-1(3-methoxyphenyl) cyclohexanol] is the chemical name. It is used to treat most forms of neuralgia, including trigeminal neuralgia, as well as moderate to severe pain. Several analytical techniques for determining Tramadol and other combination drugs have been published in the literature, including the spectrophotometric method 3,4 and spectrophotometric and spectrofluorimetric approaches [5][6][7] .
Nanotechnology is now regarded as a cutting-edge subject of research that involves the creation of nanoparticles of various sizes, shapes, and chemical structures with a wide range of possible uses 8 . For the synthesis and design of nanoparticles, many procedures have been reported, including microwave irradiation 9 , photoreduction 10 , thermal breakdown 11 and mechanical grinding 12 but these procedures are mainly costly, energy-consuming, or hazardous to humans and the environment. Environmentally friendly methods should be implemented as a result. Green synthesis refers to the development of chemical and physical techniques that are environmentally benign, economically effective, and can be scaled up for large-scale synthesis without the use of high pressure, energy, temperature, or harmful compounds. Bioreduction of metal ions employing biomolecules such as enzymes, bacteria, and plant extracts is both ecologically friendly and chemically sophisticated 13  www.nature.com/scientificreports/ Among the several green synthesis strategies, plant-mediated synthesis appears to be a promising strategy that allows for faster nanoparticle production and more stable synthesis 14 . The creation of bio-inspired nanoparticles has received much interest, as well as approaches for manipulating nanoparticle size 6,15 .
Origanum majorana is a cold-tolerant perennial plant or undershrub with pleasant pine and citrus notes. Marjoram is sometimes confused with oregano in several Middle Eastern nations, and the terms sweet marjoram and twisted marjoram are used to distinguish it from other Origanum species. It is sometimes known as pot marjoram 16 , however, this term is also applied to other Origanum cultivated species. Soups, stews, salad dressings, sauces, and herbal teas all benefit from the addition of marjoram. Sweet marjoram, also known as Origanum majorana L. (O. majorana, Lamiaceae family), is a prominent herb applied in traditional medicine for its healing qualities in gastrointestinal, ophthalmic, cardiac, and neurological problems. Significant bioactive elements of O. majorana have been identified and isolated, such as volatile compounds, terpenoids, phenolics, flavonoids, and tannins. This herb's ethnopharmacological knowledge revealed that it has antibacterial, antifungal, antiprotozoal, and antioxidant properties. The majority of the treatments are time-consuming, expensive, and necessitate the use of skilled operators and sophisticated instruments. Electrochemical determination approaches, on the other hand, are preferable for determining several biological, environmental, and pharmacological chemicals due to their quick reaction and ease of use 5,17,18 . Nevertheless, oxidizing Tramadol using traditional solid electrodes is a sluggish process that necessitates a larger over-potential. As a result, a simple and sensitive upgraded electrode for quantitative tramadol measurement is required. In contemporary voltammetry, chemically modified electrodes have become a hot topic. The intended analyte measurement becomes more specific and sensitive when these electrodes are used. Nanostructured materials have been used to change electrode surfaces to improve the sensitivity of electrochemical sensors in recent decades 19 . Nanoparticles can be utilized to modify electrodes, allowing for the detection of trace quantities of analytes by improving the sensors' sensitivity and stability 20 . Metal nanomaterials, including transition metal (Co/Ni/Cu) and their oxides 21 , have attracted a lot of attention in past years due to their various advantages of excellent electrocatalytic efficiency, long-term stability, relatively inexpensive, and ease of fabrication and construction of non-enzymatic electrochemical sensors 22 , wherein cupric oxide nanoparticles are favorable in electrocatalytic activity and electrical conductivity, making them an excellent non-enzymatic based electrochemical sensor ingredient 23,24 .
The use of carbon nanotubes in sensors and biosensors has recently received much interest. Because of their exceptional one-dimensional physical and electrical capabilities, multi-walled carbon nanotubes (MWCNTs) are extensively utilized in electroanalytical chemistry 25,26 .
We use the co-precipitation approach to make CuO NPs in this study. In addition, MWCNTs employed to modify a carbon paste electrode. The current study presents a CuONPs/MWCNTs-based tramadol electrochemical sensor that is both selective and sensitive. Finally, real-life samples of Tramadol and acetaminophen analyzed using this modified electrode. Therefore, the current study presents a CuONPs/MWCNTs-based tramadol electrochemical sensor that is both selective and sensitive.

Materials and methods
Materials. All of the compounds applied in this study were analytical grade, and they were utilized as-is, with no additional purification. Multi wall carbon nanotubes (MWCNTs) as well as cupric nitrate (Cu(NO 3 ) 2 ,5H 2 O) was acquired from Merck in Germany for this work. In addition, twofold distilled water (DW) was utilized in all of the tests. A Metrohm 797 was utilized for each electrochemical experiment. In a 10 mL one compartment electrochemical cell, SPE (DropSens; DRP-110) employed three standard electrodes: carbon (4 mm diameter) active electrodes, a graphite counter electrode, and a silver pseudo-reference electrode.
Green synthesis of CuO nanoparticles. The plant species Origanum majorana was obtained from Kerman suburb and validated by a biosystematic plant specialist. A person from herbarium center of Kerman university of medical science assisted us to collect and identify the Origanum majorana.
Origanum majorana is a cold-sensitive perennial herb or undershrub with sweet pine and citrus flavours. In some Middle Eastern countries, marjoram is synonymous with oregano, and there the names sweet marjoram and knotted marjoram are used to distinguish it from other plants of the genus Origanum. It is also called pot marjoram, although this name is also used for other cultivated species of Origanum. Marjoram has long been used as a medicinal herb. Marjoram or marjoram oil has been used to treat cancer, colds, coughs, cramps, depression, as a diuretic, ear infections, gastrointestinal problems, headaches, and paralysis, as well as arthritis, chest congestion, and muscle aches. It has also been used as an aphrodisiac, mouthwash, tea, and in poultices, tinctures, and infusions. Though not all of its historic uses are scientifically backed, the plant has verifiable medical use. For example, it contains the phenol carvacrol, which is antibacterial, antifungal and antimicrobial. Ethanol extract is cytotoxic against fibrosarcoma cell lines, ethyl acetate extract has antiproliferative properties against C6 and HeLa cells, as have Hesperetin and hydroquinone, which can be isolated from marjoram extract. Cardioprotective, hepatoprotective, antiulcerogenetic, anticholinesterase, anti-PCOS, and anti-inflammatory effects were also found in dried marjoram, marjoram tea, or in compounds extracted from marjoram. Marjoram is generally not toxic, but should not be used by pregnant or lactating womenHowever, it is always important to be cautious and consult a doctor when using medical herbs 27 . Figure 1 indicates the Origanum majorana image. To make the water based extract, O. majorana leaves were first washed with DW water to remove any attached dust particles, then chopped into very small pieces and dried in the sun. After heating 100 mL distilled water to 100 °C, 20 g dried O. majorana leaf powder was added and left to incubate for 10 min. As a result, the supplied leaf extract was allowed to cool at ambient temperature before being filtered using Whatman filter paper. 1 mmol Cu(NO3)02 was diluted in distilled water to a transparent solution to make the CuO nanoparticles. After that, a small quantity of O. majorana extract was added to the Preparation of the CuONPs/MWCNTs/carbon paste modified electrode. An Autolab potentiostat/galvanostat was used for the electrochemical experiments, and the General-Purpose Electrochemical System (GPES) software regulates the experimental settings. At 25 ± 1 °C, a standard three-electrode cell was applied. The reference, auxiliary, and working electrodes were an Ag/AgCl/KCl (3.0 M) electrode, a platinum wire, and CuONPs/MWCNTs/carbon paste. A Metrohm 710 pH meter was used to determine the pH. Buffer solutions with a pH range of 2.0-9.0 were made via orthophosphoric acid and its salts. Next, CuONPs/MWC-NTs/carbon paste was made by combining 0.01 g MWCNT with 0.95 g graphite powder and 0.04 g CuO nanoparticles in a mortar and pestle by hand. The mixture mentioned above was then combined for 20 min with 0.7 mL paraffin oil until a consistently moistened paste was achieved. After that, the paste was stuffed into the end of a glass tube (ca. 3.4 mm i.d. and 15 cm long), and a copper wire was implanted inside the carbon paste to make the electrical connection.

Characterization of CuO nanostructures.
A range of technologies used to characterize the synthesized samples, including X-ray diffraction (XRD) patterns. The FTIR alpha model of Bruker used to record Fourier transform infrared spectra. In addition, Scanning electron microscopy applied to examine the generated NPs' morphologies. The Vasco model of Nanosizer cordouan used to determine particle size and zeta potential (France). For pH measurements, a Metrohm 827 lab pH meter employed.
Statistical analysis. Student's t-tests and analysis of variances used to determine group significance. All data provided as mean ± SD. Statistical significance defined as a probability threshold of p = 0.05.
Theoretical method. The energy of adsorption (Ead) for Tramadol on MWNT calculated using DFT calculations using Guassian 03 software. DFT calculations solely carried out using 6-311 + (d) to minimize computational difficulties and the demand for immense computation potential. All of the buildings that built were geometrically optimized first. The older structures then changed using the geometrically optimized atomic locations, and algorithms run to determine the SCF energies before Ead was eventually calculated.
Ethical standards. This study was conducted following Compliance with Ethical Standards, and it did not involve human participants, animals, and potential conflicts of interest.

Results and discussion
CuO nanostructures characterization. Figure 28 . The crystallite size of CuO nanoparticles was determined to be 38.2 nm utilizing the Deby-Scherer equation 29 . The synthesized nanostructures were pure and no impurities detected. Figure 2B demonstrates the FTIR spectrum of the synthesized cupric oxide NPs. As seen, the spectrum of the CuO exists in three areas. In the first area, those peaks from 500 to 800 cm −1 exhibited a stronger absorption band related to the stretching vibrational of Cu-O vibrations, confirming the synthesis of CuO nanoparticles 30 . However, in the second area (1350 cm −1 to 1650 cm −1 ), we may observe peaks due to the presence of CO 2 in the www.nature.com/scientificreports/ air. Finally, the third area is between 2800 and 3500 cm −1 . Therefore, it could be concluded that the hydrated CuO and H 2 O in the air contribute to the peak formation. Therefore, the synthesized CuO NPs present a pure and monolithic phase according to FTIR spectra. Figure 3A, B depicts the SEM images of CuO nanoparticles. As presented in Fig. 3, the nanoparticles were uniformly sized and spherical shaped. The size of the particles estimated to be approximately 52 nm. It has been found that the biological synthesis of CuO NPs produces relatively small quasi-spherical particles of homogeneous dimension. The use of biological components in the synthesis process could describe the slight agglomeration in the as-synthesized nanoparticles. The CuO NPs synthesized from leaf extract had a spherical shape, which was consistent with previous findings 31 . Figure 4A,  www.nature.com/scientificreports/ For CuONPs/MWCNTs/carbon paste in an aqueous solution, the test results reveal anodic and cathodic peaks that are well defined and repeatable with quasi-reversible activity. The CuONPs/MWCNTs/carbon paste's longterm stability also examined over a period of three weeks. Once the reference electrode maintained at 20-22 °C, the maximum potency for tramadol oxidation stayed identical. However, the current signals decreased by b2.4 percent compared to the first response. The improved electrode's antifouling characteristics against tramadol oxidation and its oxidation metabolites examined to evaluate the CVs of the modified electrode before and after application in the corporation of tramadol. CVs obtained after cycling the potential 15 times at a scan rate of 10 mV s −1 in the presence of Tramadol. Peak potentials were constant, while currents fell via b2.4 percent. As a result, not only did the sensitivity of the analyte and its oxidation product rise at the surface of CuONPs/ MWCNTs/carbon paste, but also the fouling impact reduced as well.
Electrocatalytic oxidation of tramadol at CuONPs/MWCNTs/carbon paste. The aqueous solution's pH level influences tramadol's electrochemical behavior. As a result, pH adjustment of the solution appears to be required for tramadol electrocatalytic oxidation. By CV, the electrochemical activity of tramadol examined at the surface of CuONPs/MWCNTs/carbon paste in 0.1 M PBS at varied pH values (2.0 b pH b 9.0). Under neutral circumstances, the electrocatalytic oxidation of tramadol at the surface of CuONPs/MWCNTs/carbon paste was shown to be more favorable than in an acidic or basic media. In the CVs of CuONPs/MWCNTs/carbon paste, this manifests as a progressive increase in the anodic peak current and a parallel drop in the cathodic peak current. Thus, the optimal pH for tramadol oxidation electrocatalysis at the surface of CuONPs/MWCNTs/ carbon paste was found to be 7.0. Scheme 1 depicts the presumed mechanism for oxidation of tramadol.
To investigate the tramadol behavior and also as-produced electrode response to determine tramadol, the performance of CuONPs/MWCNTs/CPE was compared to that of MWCNTs/CPE, CuONPs/CPE, and unmodified  Effect of scan rate. The linear sweep voltammograms measurements were carried out to evaluate the association of peak current with scan rate at varied scan rates (10-400 mV/s) in the 400.0 μM tramadol-containing 0.1 M PBS (pH 7.0) on the CuONPs/MWCNTs/CPE (Fig. 6). As shown in Fig. 6, the peak currents of tramadol grow with increasing scan rates and there are good linear relationships between the peak currents (Ip) and square root of the scan rate (ν 1/2 ). The results also showed that the action is mass transfer of tramadol controlled at diffusion process.  www.nature.com/scientificreports/ ing the working electrode potential at 0.70 V (at the first potential step) and 0.40 V (at the second potential step) (Fig. 7). Using chronoamperometric studies, we determined the diffusion coefficient, D, of tramadol in buffer solution.
For an electroactive drug (tramadol in this case) with a diffusion coefficient of D, the Cottrell equation outlines the current observed for the electrochemical process underneath the mass transportation limited state.
The best fits for varied tramadol doses were found using experimental plots of I vs. t − 1/2. The slopes of the straight lines resulted plotted upon the tramadol level.The average rate of the D found to be 6.85 × 10 −6 cm 2 /s using the resultant slope and Cottrell equation.
Limit of detecting and calibrating curve. The tramadol concentration was determined using the square wave voltammetry (SWV) technique (Fig. 8). Two linear segments with slopes of 0.7441 and 0.1378 μA μM-    The stability of the response at the modified electrode. The stability of the CuONPs/MWCNTs/ CPE was examined by storing the electrode in the lab at room temperature. Then, the electrode was used for the analysis of 50 μM of tramadol from 1 to 21 days intervals in 0.1 M PBS (pH 7.0). The results showed that the electrode signal retained to 92% of its initial value after 7 days and 90% of its initial value after 21 days. these results indicated that the proposed electrochemical sensor had excellent long-term stability.
Computational method. The energy of adsorption (Ead) for Tramadol on MWNT calculated using DFT calculations using Guassian 03 software 36 . The value of Ead as calculated for tramadol adsorption on the MWNT was 5.06 × 10 -19 kcal and 4.94 × 10 -19 kcal on and inter of MWNT, respectively. However, depending on the DFT input parameters used, Ead values can vary greatly, and Ead values can also fluctuate for different poses of an adsorbent for a particular adsorptive 37 . The Ead sign is frequently used to determine whether an adsorption process is exothermic or endothermic. A negative sign in the formula for calculating Ead denotes an endothermic reaction. Thus, the DFT calculations, which agree with the experimental results, also point to the endothermic    www.nature.com/scientificreports/ character of the adsorption mechanism (to be more specific, the DFT calculations point to the endothermic feature of Tramadol adsorption on the MWNT). Figure 9 indicates different view of Tramadol on and inter of MWNT and Fig. 10 shows various view of Tramadol on and inter of MWNT.

Conclusions
The use of Origanum majorana as alkaline agent in the green production of CuO nanostructures was described in this study. One of the innovative materials employed for tramadol determination was a CuONPs/MWCNTs/ carbon paste modified electrode. CuONPs/MWCNTs nanocomposite improved tramadol oxidation selectivity and electrochemical activity. The linear calibration curve in ranges between 0.07 and 300 µM with a LOD of 0.01 µM for MO was produced using the optimal condition. Finally, the modified electrode substantially used for tramadol analysis in the real specimens. The proposed method offers a sensitive approach to detect tramadol in drug and biological formulations. Furthermore, this modified electrode may be used to identify tramadol in human plasma and urine and also drug samples.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.