Electrochemical Investigation of Ofloxacin Using a Functionalized Multi-Walled Carbon Nanotube Paste Electrode

Electrochemical Investigation of Ofloxacin Using a Functionalized Multi-Walled Carbon Nanotube Paste Electrode | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Electrochemical Investigation of Ofloxacin Using a Functionalized Multi-Walled Carbon Nanotube Paste Electrode Asma Gul, Muhammad Iqbal Zaman, Abdul Niaz, Amna Bibi, Madina NA, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6580404/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ofloxacin, a member of the fluoroquinolone class of antibiotics, exhibits broad-spectrum activity against both gram-positive and gram-negative bacteria by inhibiting topoisomerase enzymes. Monitoring the concentration of ofloxacin is crucial in pharmaceutical formulations, biological samples, food products, and environmental matrices. In this study, a novel, simple, selective, and sensitive voltammetric sensor was developed for the detection of ofloxacin in pharmaceutical and real-world samples. A functionalized multi-walled carbon nanotube paste electrode (MWCNTPE) was employed for the electrochemical oxidation and quantification of ofloxacin using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Measurements were carried out in 0.1 M phosphate buffer solution, with a scan rate of 0.1 V/s and a pulse period of 0.1 seconds, following an accumulation time of 60 minutes. Ofloxacin was found to adsorb irreversibly onto the electrode surface, producing a well-defined anodic peak at + 0.85 V. The sensor demonstrated high sensitivity, achieving a detection limit as low as 1×10⁻⁸ M, and exhibited excellent selectivity even in the presence of common interfering ions. Owing to its low cost, ease of preparation, and renewability, the sensor was successfully applied for the determination of ofloxacin in pharmaceutical tablets and biological samples, yielding high recovery rates. Voltammetry MWCNTPE Ofloxacin Biomedical application Acid functionalization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Ofloxacin is a member of the fluoroquinolone class of synthetic antibacterial agents [Todd and Faulds, 1991; Cheng et al., 2008], and it has demonstrated significant efficacy in treating a variety of infectious diseases caused by both Gram-positive and Gram-negative bacteria due to its broad-spectrum antimicrobial activity [Sato et al., 1982]. The molecular formula of ofloxacin is C₁₈H₂₀FN₃O₄, and its chemical structure features key functional groups: a carboxyl group (-COOH) at position 3, a keto group at position 4, and a fluorine atom at position 6 [Raj et al., 1997]. Alterations in the position of these functional groups can substantially impact the antibacterial activity of the compound [Mitscher et al., 1993]. Structurally, ofloxacin contains a tricyclic ring system, with each ring and its attached functional groups playing a critical role in disrupting or inactivating bacterial DNA [Morrissey et al., 1996]. It is well documented in literature that the Fluoroquinolone being an antibacterial works by inactivation of two enzymes in bacteria belonging to the group of DNA topoisomerases (DNA topoisomerases II and DNA topoisomerases IV) that are mainly concern with the production, repairing and replication of bacterial DNA. Topoisomerase II also called DNA gyrase has a major role to keep the chromosomes in super coiled state as well as performs the DNA repairing process. DNA topoisomerases IV is involved in the isolation of daughter chromosomes in the process of replication. If these enzymes become inactive, the bacteria will be unable to make and replicate their genetic material which results in destruction of bacteria or bacterial cell wall [Hooper 1999; Smith et al., 2001]. Ofloxacin is extensively recommend by the physician and surgeon around the globe to treat various types of pathogenic infection. These infection/diseases includes prostate gland infection (Prostatitis), skin infection (Impetigo and Cellulitis), pulmonary chest infection (T.B and Pneumonia), digestive tract infection (vomiting and diarrhea) and urinary tract infections (Cervicitis, Urethritis and Cystitis). This is because Ofloxacin have an excellent absorption capability as well as the rate of their adverse effect is very low [Francis & Adcock 2005; Christodoulou et al., 2007]. In addition, the use of Ofloxacin is not only limited to the human medication but broadly used in the treatment of veterinary diseases also [Monk and Campoli-Richards 1987; Hopkala and Kowalczuk 2000]. Ofloxacin administered both orally and intravenously and inside the body, it is metabolized to some extent while greater amount of it is excreted in urine as such within 48 hours [Todd and Faulds 1991; Zhang et al., 1998a]. The use of ofloxacin for long period of time can produce unfavorable conditions and various side effects including suppression of immune system, discomfortness of central nervous system, abdominal pain and angioneurotic edema etc. In order to avoid these complications it is very essential to use an appropriate amount of ofloxacin in drugs formulation, to check and regulate its content in the surrounding environment and find out its remains in food [Andreu and Picó et al. 2007; Martínez-Carballo et al. 2007; Stolker and Brinkman 2005]. Till date various methods have been developed for determination of ofloxacin that include HPLC, spectrophotometry, electrochemical analysis and capillary electrophoresis [Attia et al. 2012; Shervington et al., 2005; García et al., 2005, Awadallah et al., 2003; Yin et al. 2004]. A narrow range, more analysis time, specified temperature, and organic solvents of high purity and high quality make the HPLC as worst technique for routine analysis [Bae et al. 2007]. Similarly, absorption spectroscopy also suffers from a number of drawbacks including limit of detection for analyte, expensive instrumentation and vigorous handling became the reason for its failure [White 1942; Haggquist et al., 1994; Butler 1964.; Shibata et al., 1954; Merzlyak & Naqvi 2000]. Similarly majority of spectroscopic and chromatographic techniques are costly as well as involves time taking procedures. Therefore, to develop an easy, simple, sensitive, responsive and consistent method is utmost important [Heineman 1984]. In contrast to other instrumental methods, the electrochemical techniques which are based on the measurement of either current or potential in chemical reaction [Adams 1976] have large linear range, tremendous sensitivity, provide excellent accurateness with a reasonable low-cost, quick and highly responsive instrumental setup [Cheemalapati et al. 2014; Bard & Faulkner, 2001] make the electrochemical technique is one of the best tool for routine analysis [Goyal et al. 2012; Yang et al. 2008a]. In this connection attempts were made by different scientist to developed nanosensors based electrochemical methods emphasizing that carbon nano-materials possesses an excellent chemical reliability, stability, high electrical and thermal conductivity, specified large surface area which enable them suitable for a wide range of electrochemical determination when used as an electrode materials [Chen and Chatterjee 2013; Yánez-Sedeno et al., 2010; Jang et al., 2013]. Recently MWCNTs has been reported to play a vital role in electrochemical determination of various compounds such as proteins, drugs, markers of cancer as well as environmentally important anions like iodide, sulphide, nitrate, nitrite, and phosphate due to charge transfer mechanism [Nugent et al., 2001; Ensafi and Karimi-Maleh 2010; Wildgoose et al., 2006; Bibi et al., 2019]. The electrodes modified with MWCNTs have been reported to possess high sensitivity, ability to decrease the over potentials when utilized as a sensing material in electrochemical cell [Nugent et al. 2001; Ensafi and Karimi-Maleh 2010; Wildgoose et al., 2006]. Similarly the functionalization of MWCNTs with acids also creates different functional groups on the surface of MWCNTs make it more interactive with different analytes [Carabineiro et al. 2011; Alizadah et al., 2009]. In the present work functionalization of MWCNTs have been carried out with sulphuric and nitric acid, which ultimately increase the surface area, impart porosity with greater electrostatic attraction towards many chemical species especially ofloxacin [Peng et al., 2012;]. MATERIALS AND METHODS During course of our experimental work, analytical grade reagents were used without further purification. The multi-walled carbon nanotubes powder were obtained from sigma-Aldrich (Sigma-Aldrich, Germany with product code 901019) whereas standard Ofloxacin B.No:5857895, origin: England, Potency: 99.29% was obtained from Roryan Pharmaceutical Industries (Pvt) Ltd Peshawar, Pakistan. Deionized water was use throughout the experimental study, while Phosphate buffer solution (PBS) was used as a supporting electrolyte in which sodium phosphate diabasic (Na 2 HPO 4 ) obtained from Sigma-Aldrich, Germany was used. Instrumentation and Apparatus Three electrode multi-channel potentiostate systems was used to monitor the electrochemical change as a result of electron transfer. The MWCNTs paste electrode functionalized with acids were used as working electrode whereas saturated calomel and platinum wire were used as reference and counter electrode, respectively. pH meter/ion meter model (K 2000-pH Istek-Inc. Korea) was utilized for pH measurement throughout the experimental work. Reagents The stock solution of 10 − 3 M ofloxacin was prepared by dissolving 0.00361g of standard ofloxacin in 10ml of phosphate buffer, which was prepared by mixing 50 mL of 0.1 M Na 2 HPO 4 and 50 mL of 0.1M H 3 PO 3 and used as supporting electrolyte in present voltammetric study. Preparation of Paste Electrode A proper amount of powder MWCNTs were treated with a mixture of concentrated nitric and sulphuric acid having a ratio (3:1 v/v) respectively to remove the metal particles left behind during the process of synthesis and to create various functional groups on the surface of MWCNTs [Osorio et al., 2008]. The resulting mixture was stirred at room temperature (25°C) for 10 to 12 hours. The mixture was then washed with deionized water for 48 hours using dialysis membrane and the residue were then kept in oven at 120°C for 5 hours. Paste was prepared by mixing MWCNTs powdered with an appropriate amount of paraffin oil and then mix well with help of mortar and pestle for about 30 min. A disposable needle cap was filled with paste and pressed followed by insertion of copper wire for electrical connectivity. Finally the tip of the cap was cut off with sharp blade for further used as working electrode. Similarly MWCNTs paste was also functionalized with Graphene Oxide (GO) and chitosan a natural polymer of chitin for studying the electrochemical response towards ofloxacin. During the entire procedure, Pt wire was used as an auxiliary electrode while calomel electrode was as a reference electrode. Voltammetric Procedure 10 ml of the buffer solution were added to the three capped voltammetric cell and potential was adjusted in the range 0.0 to 1.5V, under which approximately 9 to 10 consecutive cyclic voltammetric anodic scans were recorded in the presence of 0.1M phosphate buffer at every new surface of MWCNTPE by using differential pulse and cyclic voltammetry techniques. For the determination of ofloxacin, 10ml of 0.1M buffer solution was taken in a cell and pH was adjusted to pH 5. The voltammetric scan was recorded for blank solution followed by addition of proper amount of standard ofloxacin in gram into the cell for electrochemical study. Finally, the potentials of the working electrode were scanned after 60 min in the form of voltammogram in anodic direction over the range of 0.0 to 1.5 V at the scan rate of 0.1V.s − 1 . All measurements were performed at room temperature (25 o C) where Pt wire was used as an auxiliary electrode while calomel electrode was as a reference electrode along with acid functionalized paste electrode. Selection of working electrode Cyclic voltammetry (CV) was studied at different electrodes such as bare glassy carbon electrode (GCE), unmodified multiwalled carbon nanotube paste electrode (MWCNTPE), graphene oxide modified paste electrode (GO-MWCNTPE) and chitosan modified paste electrode (Chi-MWCNTPE) in 1×10 − 3 M phosphate buffer containing ofloxacin. A scan rate of 0.1V.s − 1 in a potential range of 0.0 to 1.5 V was selected for elucidation of electrochemical properties of the electrode surfaces. All the experiment were run in duplicate at room temperature and constant pH5. Based on the voltammetric response, acid functionalized MWCNTPE was selected as a working electrode. Effect of pH In pH study, different samples were prepared, having 10 ml of phosphate buffer solution and pH of these samples were adjusted using standard 0.01M NaOH and 0.01M HNO 3 in the range 2–10. Cyclic voltammogram was recorded for blank and suitable amount of ofloxacin was added into the cell followed by scanning its cyclic voltammogram. The same procedure was repeated for the remaining samples, for electrochemical response in the presence of 0.1M ofloxacin. Similarly for Scan rate study, 10ml of 0.1M phosphate buffer solution was transferred into a cell and used as blank followed by adding an appropriate amount of ofloxacin. Cyclic voltammograms were recorded at different scan rates ranging from 0.005 to 10 V.s − 1 using the acid functionalized MWCNTPE was electrode. Time and calibration study of the electrode for voltammetric response of ofloxacin The accumulation time study was carried out for the optimization process of the ofloxacin on the surface of working electrode by using cyclic voltammetry. In this study a sample prepared for scan rate study was scan for cyclic voltammograms at different interval of time in the range 5 to 200 min. Similarly the calibration study was performed to check the sensitivity of electrode for detection of ofloxacin at pH 5. Both cyclic and differential pulse voltammetry (DPV) was recorded at a constant pH5 having different concentration of ofloxacin in the range 1×10 − 8 M to 1×10 − 5 M. Optimum time for accumulation of ofloxacin at the surface of the electrode from oxidation was calculated from the voltammogram. Interference study To check the selectivity of the proposed electrode, interference study was conducted under the optimized conditions using DPV technique. Different ions such as Na +, K + , Ca 2+ , Mg 2+ , Fe + 3 , NH 4+ , ascorbic acid and oxalic acid were added to solution containing ofloxacin in 0.01M phosphate buffer at background electrolyte. Voltammograms were recorded for blank and all the samples at optimized condition where 100ul of each interfering ion were introduce into the cell. Applications of the synthesized MWCNTs paste electrode The applicability and validity of MWCNTs paste electrode was evaluated for determining the concentration of ofloxacin in pharmaceutical formulations (tablets) and urine samples of the patient treated with ofloxacin or mock solution of urine using with addition of proper amount of ofloxacin. i. Determination of ofloxacin in pharmaceutical formulation. Three (03) samples were prepared by using different available brands of ofloxacin namely Tarivid 200mg, Oflobid 200mg and Albact 200mg. Each sample was prepared by dissolving 3 tablets of specific brand in 100ml of phosphate buffer followed by filtration. A suitable amount of the specified tablet sample was transferred into the voltammetric cell, and voltammogram was scanned for it. Finally an appropriate amount from standard stock solution of ofloxacin (1×10 − 3 M) was added into the cell where its voltammogram was recorded under optimized i.e., pH5 and time period of 60min. The voltamagram of the each sample was recorded as a triplicate. ii. Determination of ofloxacin in biological sample Urine samples were collected from patient treated with ofloxacin and was centrifuged at 1500 RPM for 5min. The supernatants was taken from it and filtered again. In a specific ratio, the urine and electrolyte (PBS) having total volume of 10ml, was added into the voltammetric cell as blank, and its voltammogram was recorded. Similarly, an appropriate amount from standard stock solution of ofloxacin (1×10 − 3 M) was added into the cell and voltammogram was recorded under optimized condition of time and pH. All the reading were taken as triplicate. RESULTS AND DISCUSSION Voltammetric response of ofloxacin at MWCNTs-paste electrode The electrochemical behavior of Ofloxacin was studied at GCE, MWCNTPE, GO-MWCNTPE and Chi-MWCNTPE in PBS of pH-5 containing 1×10 − 3 M of Ofloxacin at a scan rate of 0.1V.s − 1 . Cyclic voltammetric curves were recorded using different electrodes where the highest peak current was observed at the designed MWCNTPE in a potential range of 0.0 to 1.5V (Fig. 1 ). The interaction shows that MWCNTs paste electrode have the ability to increase the rate of electrons transfer between the Ofloxacin and the electrode material. It is reported that electron transfer played a vital role to facilitate an electro-chemical oxidation of Ofloxacin at MWCNTs surface due to which electro-catalytic response of the proposed electrochemical sensor enhanced [Si et al., 2018; Nugent et al., 2001]. Another reason for the extra ordinary electrochemical response is the large surface area provided by the MWCNTs paste electrode for oxidation of Ofloxacin. Contrary to that, under the similar experimental optimized conditions there was no oxidation peak appears for the Ofloxacin at GO-MWCNTPE. Similarly the oxidation peaks of Ofloxacin using bare GCE and Chi-MWCNTPE were also very low that obviously attributed to the less conductivity, as shown in Fig. 1 . Therefore MWCNTs paste electrode was selected for the rest of the studies throughout the project. The mixture of nitric acid and sulfuric acid acts as an oxidizing agent, leading to the formation of oxygenated functional groups on the surface of CNTs. The specific ratio of HNO 3 and H 2 SO 4 i.e., 3:1 keeping into account the treatment time and temperature as the influencing factors, can greatly affect the surface functionalization and the types of groups originated at the CNTs surface. It is reported that when MWCNTs treated with a mixture of nitric acid and sulfuric acid, then primarily carboxyl (-COOH) and phenolic groups are introduced onto the surface of CNTs which results in alteration of the overall surface parameters [Osorio et al., 2008; Britto et al. 1996]. Similarly oxygen-containing functional groups like hydroxyl (-OH) and carbonyl (-C = O) are also appear on the surface after acid treatment. Effect of pH The pH of an electrolyte is one of the important and key factor which determines the ability of electrode to enhance the electrochemical response of an analyte. The effect of pH on the electro-chemical behavior for the assessment of ofloxacin was investigated by using cyclic voltammetry at proposed MWCNTPE electrochemical sensors in the pH range from 2–10. As shown in the Figure.2, a gradual increase in the oxidation peak current with increase in pH from 1.0 to 5.0 was observed and a current response reached to a maximum at pH 5. Further increase in the pH from 6–10 has drastically decrease the peak current. It is well documented in the literature that during the electro-oxidation of ofloxacin an equal number of electrons and protons are discharge [Yang et al. 2008b; Zhang et al. 2013b]. From these results it was observed that maximum oxidation was take place at this pH value and therefore, was chosen as an optimum pH value for the ofloxacin investigation. It is also pertinent to mention that the oxidation peak current reach to maximum value of 67µA, where electron transfer between proposed electrode and ofloxacin was almost completed. Beyond this pH value, either electron transfer stop or reverse phenomenon is expected according to the law of mass action [Zhang et al. 2013b]. Effect of scan rate The effect of scan rate on electrochemical behavior of ofloxacin was investigated on a proposed MWCNTs-paste electrode using different scan rates ranges from 0.01–0.1 Vs − 1 . Figure.3 a reveals that different cyclic voltammograms were obtained at different scan rates and oxidation peak current increases as the scan rate increases from 0.01 to 0.1Vs − 1 .When the oxidation peak current was plotted against the scan rate, a linear relationship was found as shown in the Figure.3 b . These result demonstrates that the oxidation of ofloxacin on the surface of MWCNTs paste electrode is an irreversible as well as adsorption controlled process at low ofloxacin concentration. However, when the oxidation peaks were plotted against the square root of scan rate, it also give a linear plot as shown in Figure.3 c . These observations indicate the diffusionally controlled process at high concentration of ofloxacin. The highest oxidation peak current at 0.1 Vs − 1 scan show that mechanism of charge transfer for oxidation is greater at this condition between MWCNTs paste electrode and ofloxacin moiety. Therefore, further investigations were carried at this scan rate under the optimized condition. Influences of adsorption Accumulation Time The influence of accumulation time on the oxidation peak current of ofloxacin was also investigated in order to reveal the adsorption capability of the proposed sensor. Accumulation time is thought to be an important parameter in electrochemical study especially in cyclic voltammetry. Figure.4 show the effect of accumulation time on oxidation peak current where it is noted that the oxidation peak current rapidly increases with increasing the accumulation time ranging from 0–60 min. It is also important to note that further increase in time up to 200 min has no prominent effect on the oxidation peak current. These results indicate the saturation point where almost all vacant sites for adsorption has occupied by the analyte molecules and no further adsorption take place under the optimized experimental condition. Similarly, the functional group interactions with Ofloxacin molecules also reach the plateau condition, and hence 60 min were selected in further experimental work for accumulation time. Similarly, it is also inferred that adsorption controlled is the dominant mechanism responsible for high oxidation peak current of the ofloxacin at the surface of the MWCNTs paste electrode [Amira et al., 2023]. In cyclic voltammetry, an adsorption-controlled process, also known as a surface-controlled process, occurs when the electroactive species adsorb to the electrode surface before undergoing electron transfer. This means the rate-limiting step is the adsorption of ofloxacin, not the diffusion to the electrode surface [Amira et al., 2023]. A key characteristic of adsorption controlled processes is that the peak current is linearly proportional to the scan rate as observed in scan rate studies for ofloxacin at the surface of the paste electrode. Sensitivity of MWCNT Paste Electrode (Calibration study) The calibration curve for Ofloxacin was obtained under the optimized experimental conditions in presence of phosphate buffer solution at the MWCNT paste Electrode by using cyclic voltammetry (CV) as well as differential pulse voltammetry (DPV) to check the sensitivity of proposed sensor for the Ofloxacin. Various cyclic voltammograms were obtained by adding different concentrations of Ofloxacin ranging from 1×10 − 8 -1×10 − 5 M as shown in the Figure.5 a,b for CV and DPV respectively. The oxidation peak current increases with gradual increase in the concentrations of ofloxacin and a linear relationship was obtained between the oxidation peak current and ofloxacin concentration. Figure.5 c,d reveal that the synthesized electrode work both in lower concentration range i.e., 1×10 − 8 -5×10 − 7 M and high concentration ranges 1×10 − 6 -1×10 − 5 M with good regression coefficient (R 2 = 0.999). It is also worth mentioning that both the techniques i.e., CV and DPV work good in wide range of concentration as clear from Figure.5 e,f . The limit of detection for Ofloxacin was calculated using the relation: (LOD) = 3s/S Where small ‘s’ stands for standard deviation whereas capital ‘S’ was calculated from the slop of linear plot using straight line equation. It is obvious from result that limit of detection being 1×10 − 8 M for both CV and DPV using the functionalized MWCNTs paste electrode confirm the validity, sensitivity and selectivity of proposed sensor. According to the redox mechanism that was proposed by Kauffmann et al., [1987] the oxidation peak is due to the irreversible oxidation of the piperazine moiety of ofloxacin molecule. The comparative sensitivity of proposed sensor with other previously reported electrode by other researchers for detection of ofloxacin is given in Table 1 . Hence the proposed electrode could be an appropriate and suitable electrochemical sensor for ofloxacin determination using adsorption cyclic voltammetric techniques (CV and DPV) due to possessing high sensitivity, selectivity and greater stability over the paste electrode. The greater accumulation and adsorption is attributed to the high surface area, pore size, pore volume and availability of different surface functional groups responsible for the interaction with Ofloxacin molecules. Similar observation has been reported by [Sannia et al., 2019] for nitrite and sulphide determination. Table 1 Shows all the previously reported voltammetric oxidation methods for the determination of Ofloxacin. Electrode Technique Electrolyte/pH Potential(V) LOD(M) Reference MWNTs-CR/GE CV Phosphate buffer/6.0 + 0.9 9×10 − 9 Yang et al.,2008 MWNTs/Nafion- film /GCE CV/LSV KHP-NaOH buffer/5.5 + 1.2 1×10 − 7 Huang et al., 2008 ZnO/GR/GCE CV/DPV Phosphate buffer/5.0 + 0.92 0.33×10 − 6 Si et al., 2018 Trp-GO- CNT/GCE CV Phosphate buffer/7.0 + 0.8 1×10 − 9 Zhu et al., 2019 HMDE CV BR buffer/8.36 + 1.56 4 × 10 − 8 Gulaboskiand Jordanoski,. 2000 MWCNTPE CV/ DPV Phosphate buffer/5.0 + 0.85 1×10 − 8 Presentwork Selectivity of MWCNTs Paste Electrode (Interference study) The selectivity of the proposed MWCNTs paste electrode was investigated under optimum conditions using differential pulse voltammetry (DPV) for the detection of ofloxacin in the presence of potential interfering agents such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Fe³⁺, NH₄⁺, oxalic acid, and ascorbic acid in a phosphate buffer solution at pH 5.0. The results, as shown in Fig. 6 , indicate that these ions did not cause any significant change in the peak current of ofloxacin, demonstrating good selectivity. No noticeable interaction with either cations or anions was observed in this study. Therefore, the proposed sensor exhibits high selectivity for ofloxacin determination in the presence of common interfering ions and can be recommended for use in industrial effluent analysis as well as for qualitative determination in pharmaceutical applications. Determination of Ofloxacin in commercial tablet samples The practical applicability of the designed sensor (MWCNTsPE) was evaluated for the qualitative determination of ofloxacin in commercially available pharmaceutical tablets using the differential pulse voltammetry (DPV) technique. Three different brands of ofloxacin tablets, i.e., Tarivid 200 mg, Oflobid 200 mg, and Albact 200 mg were selected for quantitative analysis. The standard addition method was employed, where varying concentrations of a 1×10⁻³ M standard ofloxacin solution were spiked into the electrochemical cell containing the tablet solution along with phosphate buffer (pH 5.0) as the supporting electrolyte. The results, summarized in Table 2 , are the mean of three replicate measurements. It is evident from the data that the recovery of ofloxacin ranged from 97.75–103%, indicating the accuracy and reliability of the proposed sensor. These findings support the validity and potential applicability of the sensor for industrial-scale analysis and pharmaceutical quality control. Table 2 Determination of Ofloxacin in pharmaceutical samples using DPV techniques Name of tablet Tablet Amount (mg) Found Amount (mg) Recovery (%) Tarivid 200 195.5 97.75 Oflobid 200 207 103.5 Albact 200 194.1 97.05 Determination of Ofloxacin in urine sample The proposed voltammetric sensor was also successfully applied for the determination of ofloxacin in human urine samples using the standard addition method and differential pulse voltammetry (DPV) and results are presented in Table 3 . Various concentrations of a standard ofloxacin solution were spiked into the electrochemical cell containing urine samples in the presence of phosphate buffer solution (pH 5.0) as the supporting electrolyte. A high percent recovery of 102.8% was achieved, demonstrating the accuracy of the proposed method. These results indicate that the sensor is highly effective and sensitive for detecting ofloxacin in urine, without significant interference from endogenous substances. Furthermore, it is worth noting that a substantial amount of ofloxacin is excreted in urine following oral administration in patients with various diseases, which supports the relevance of urine analysis for monitoring therapeutic levels. Table 3 Determination of Ofloxacin in urine samples Added (µM) Found (µM) Recovery (%) Urine sample 300.0 308.4 102.8 Patient 300.0 378.0 126.0 CONCLUSIONS The designed electrochemical sensor based on a functionalized multi-walled carbon nanotube paste electrode (MWCNTsPE) demonstrated high sensitivity and selectivity toward the voltammetric oxidation of ofloxacin. The functionalization and high surface area of the MWCNTs significantly enhanced electron-transfer kinetics, making the electrode highly suitable for the electrochemical detection of ofloxacin. It is important to note that the large surface area and surface functional groups of MWCNTs facilitate efficient charge transfer between the electrode surface and the ofloxacin analyte. This charge transfer process is the primary mechanism behind the observed oxidation peak current, which arises after the adsorption of ofloxacin onto the electrode surface. Based on the experimental findings, it can be concluded that the oxidation process is adsorption-controlled at low concentrations of ofloxacin, transitioning to a diffusion-controlled process at higher concentrations. This dual mechanism results in a sharp and well-defined oxidation peak. The sensor was successfully applied to determine ofloxacin at very low concentrations under optimized conditions and exhibited excellent selectivity even in the presence of various potentially interfering ions. The method yielded high percent recoveries when applied to both pharmaceutical formulations and biological (urine) samples, confirming the sensor’s sensitivity and reliability. The limit of detection (LOD) was determined to be 1×10⁻⁸ M. Interference studies showed that the oxidation peak current remained unaffected by the presence of common anions and cations, underscoring the sensor's selectivity toward ofloxacin. Additionally, the sensor demonstrated a good molar absorptivity (extinction coefficient) and performed effectively across a broad concentration range from 1×10⁻⁸ to 1×10⁻⁵ M, with a strong coefficient of determination, confirming its suitability for quantitative analysis. Declarations Author Contribution Asma Gul: Writing Original Draft, Methodology, SoftwareMuhammad Iqbal Zaman: Supervision, Project Administration, Investigation, MethodologyAbdul Niaz: Supervision, Data curationAmna Bibi: Writing- Reviewing and Editing, Visualization, Conceptualization, Formal AnalysisMadina: Conceptualization, Formal AnalysisHanan E Osman: Visualization, Conceptualization, Formal Analysis ACKNOWLEDGEMENT This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Adams, R. N. (1976). Probing brain chemistry with electroanalytical techniques. Analytical Chemistry, 48(14), 1126A-1138A. Alizadeh, T., Ganjali, M. R., Norouzi, P., Zare, M., & Zeraatkar, A. (2009). A novel high selective and sensitive para-nitrophenol voltammetric sensor, based on a molecularly imprinted polymer–carbon paste electrode. Talanta, 79(5), 1197–1203. Amira, Youssef, Mohammed, Abeer, Sherif,. Voltammetric quantitative analysis of vildagliptin in bulk form and spiked human serum at a modifed electrode., J. Iranian Chemical Society (2023) 20:1503–1522 Andreu V, B. C., Picó Y (2007). Analytical strategies to determine quinolone residues in food and the environment. TrAC Trends Anal Chem. 26(6), 534–556. . Attia, M., Essawy, A. A., Youssef, A., & Mostafa, M. S. (2012). Determination of Ofloxaci using a highly selective photo probe based on the enhancement of the luminescence intensity of Eu 3+ Ofloxacin complex in pharmaceutical and serum samples. J. Fluorescence, 22(2), 557–564. Awadallah, B., Schmidt, P. C., & Wahl, M. A. (2003). Quantitation of the enantiomers of Ofloxacin by capillary electrophoresis in the parts per billion concentration range for in vitro drug absorption studies. Journal of Chromatography A, 988(1), 135–143. Bae, J. W., Kim, M. J., Jang, C. G., & Lee, S. Y. (2007). Determination of meloxicam in human plasma using a HPLC method with UV detection and its application to a pharmacokinetic study. Journal of Chromatography B, 859(1), 69–73. Bard, A. J., & Faulkner, L. R. (2001). Fundamentals and applications. Electrochemical Methods, 2(482), 580–632. Bibi, S., Zaman, M. I., Niaz, A., Rahim, A., Nawaz, M., & Arian, M. B. (2019). Voltammetric determination of nitrite by using a multiwalled carbon nanotube paste electrode modified with chitosan-functionalized silver nanoparticles. Microchimica Acta, 186(9), 1–9. Britto, P., Santhanam, K., & Ajayan, P. (1996). Carbon nanotube electrode for oxidation of dopamine. Bioelectrochemistry and Bioenergetics, 41(1), 121–125. Carabineiro, S., Thavorn-Amornsri, T., Pereira, M., & Figureueiredo, J. (2011). Adsorption of ciprofloxacin on surface-modified carbon materials. Water research, 45(15), 4583–4591. Cheemalapati, S., Devadas, B., & Chen, S. M. (2014). Novel poly-L-lysine/carboxyl-group enriched graphene oxide/modified electrode preparation, characterization and applications for the electrochemical determination of meloxicam in pharmaceutical tablets and blood serum. Analytical methods, 6(20), 8426–8434. Chen, A., & Chatterjee, S. (2013). Nanomaterials based electrochemical sensors for biomedical applications. Chemical Society Reviews, 42(12), 5425–5438. Cheng, G.W., Wu, H.L., & Huang, Y.L. (2008). Simultaneous determination of malondialdehyde and Ofloxacin in plasma using an isocratic high-performance liquid chromatography /fluorescence detection system. Analytica chimica acta, 616(2), 230–234. Christodoulou, E. A., Samanidou, V. F., and Papadoyannis, I.N. ( 2007). Development and validation of an HPLC confi rmatory method for residue analysis of ten quinolones in tissues of various food-producing animals, according to the European Union Decision 2002/657/EC. J. Sep. Sci., 30:, 2676–2686. Ensafi, A. A., & Karimi-Maleh, H. (2010). Modified multiwall carbon nanotubes paste electrode as a sensor for simultaneous determination of 6-thioguanine and folic acid using ferrocenedicarboxylic acid as a mediator. J.Electroanalytical Chemistry, 640(1–2), 75–83. Francis, P. S., & Adcock, J. L. (2005). Chemiluminescence methods for the determination of Ofloxacin. Analytica Chimica Acta, 541(1–2), 3–12. García, M. S., Albero, M. I., Sánchez-Pedreño, C., & Abuherba, M. S. (2005). Flow injection spectrophotometric determination of Ofloxacin in pharmaceuticals and urine. European J. Pharmaceutics and biopharmaceutics, 61(1–2), 87–93. Goyal, R. N., Rana, A. R. S., & Chasta, H. (2012). Electrochemical sensor for the sensitive determination of norfloxacin in human urine and pharmaceuticals. Bioelectrochemistry, 83, 46–51. Gulaboski, R., & Jordanoski, B. (2000). Square-wave voltammetry of Ofloxacin. Bulletin of the Chemists and Technologists of Macedonia, 19(2), 177–181. Hooper, D. C. (1999). Mechanisms of fluoroquinolone resistance. Drug Resist Updates. 2, 38–55. Smith, A., Pennefather, P. M., Kaye, S. B., & Hart, C. A. (2001). Fluoroquinolones. Drugs, 61(6), 747–761. Hopkala, H., & Kowalczuk, D. (2000). Application of derivative UV spectrophotometry for the determination of ciprofloxacin, norfloxacin and Ofloxacin in tablets. Acta Pol. Pharm, 57(1), 3–13. Huang, K. J., Liu, X., Xie, W. Z., & Yuan, H. X. (2008). Voltammetric behavior of Ofloxacin and its determination using a multi-walled carbon nanotubes-Nafion film coated electrode. Microchimica Acta, 162(1–2), 227–233. Jang, H. D., Kim, S. K., Chang, H., Choi, J.-W., & Huang, J. (2013). Synthesis of grapheme based noble metal composites for glucose biosensor. Materials Letters, 106, 277–280. Kauffmann, J. M., Vire, J. C., Patriarche, G. J., Nunez-Vergara, L. J., & Squella, J. A. (1987), Voltammetric oxidation of trazodone. Electrochimica acta, 32(8), 1159–1162. Martínez-Carballo E, G.-B. C., Scharf S, Gans O. (2007). Environmental monitoring study of selected veterinary antibiotics in animal manure and soils in Austria. Environ Pollut: 148(2):570–579. Merzlyak, M. N., & Naqvi, K. R. (2000). On recording the true absorption spectrum and the scattering spectrum of a turbid sample: application to cell suspensions of the cyanobacterium Anabaena variabilis. Journal of Photochemistry and Photobiology B: Biology, 58(2–3), 123–129. Mitscher, A. L., Devasthale, R. Zavod. (1993). Quinolone Antimicrobial Agents, American Society for Microbiology,2nd Edi., Washington DC, 3–53. Monk, J. P., & Campoli-Richards, D. M. (1987): a review of its antibacterial activity, pharmacokinetic properties and therapeutic use, Drugs 33 (1987) 346. Ofloxacin. Drugs, 33(4), 346–391. Morrissey, I., Hoshino, K.,Sato, K., Yoshida, A, Hayakawa, I., Bures, M. G., & Shen, L. L. (1996), Mechanism of differential activities of Ofloxacin enantiomers. Antimicrobial agents and chemotherapy, 40(8), 1775–1784. Nugent, J. M., Santhanam, K. S. V., Rubio, A. A., & Ajayan, P. M. (2001). Fast electron transfer kinetics on multiwalled carbon nanotube microbundle electrodes. Nano letters,1(2), 87–91. Osorio A, Silveira I, Bueno V, Bergmann C. H 2 SO 4 /HNO 3 /HCl-Functionalization and its effect on dispersion of carbon nanotubes in aqueous media. Applied Surface Science 2008;255:2485-9. Peng, H., Pan, B., Wu, M., Liu, R., Zhang, D., Wu, D., & Xing, B. (2012). Adsorption of Ofloxacin on carbon nanotubes: solubility, pH and cosolvent effects. J. Hazardous materials, 211, 342–348. Raj, S., Kapadia, S., & Argekar, A. (1997). Simultaneous determination of metronidazole and norfloxacin from pharmaceutical preparations by RP-HPLC. Indian drugs, 34(10), 585–589. Sato, K., Matsuura, Y., Inoue, M., Une, T., Osada, Y., Ogawa, H., & Mitsuhashi, S. (1982). In vitro and in vivo activity of DL-8280, a new oxazine derivative. Antimicrobial Agents and Chemotherapy, 22(4), 548–553. Shervington LA, A. M., Hussain B, Donnelly J. (2005). The simultaneousseparation and determination of five quinolone antibioticsusing isocratic reversed-phase HPLC: application tostabilitystudies on an Ofloxacin tablet formulation. J Pharm Biomed Anal. 39, 3–4, 769–775. Shibata, K., Benson, A. A., & Calvin, M. (1954). The absorption spectra of suspensions of living micro-organisms. Biochimica et biophysica acta, 15(4), 461–470. Si, X., Wei, Y., Wang, C., Li, L., & Ding, Y. (2018). A sensitive electrochemical sensor for Ofloxacin based on a graphene/zinc oxide composite film. Analytical methods, 10(17), 1961–1967. Skoog, Douglas. A., Donald, M. West., F, James. Holler. (1995). Fundamentals of Analytical Chemistry (7th ed.). Harcourt Brace College Publishers. ISBN 978-0-03-005938-4. Stolker AAM, B. U. (2005). Analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in food-producing animals—a review.. J Chromatogr A 1067, 1–2, 15–53. Todd, P. A., & Faulds, D. (1991). Ofloxacin a reappraisal of its antimicrobial activity, pharmacology and therapeutic use, Drugs, 42(5), 825–876. White, J. U. (1942). Long optical paths of large aperture. JOSA, 32(5), 285–288. Wildgoose, G. G., Banks, C. E., Leventis, H. C., & Compton, R. G. (2006). Chemically modified carbon nanotubes for use in electroanalysis. Microchimica Acta, 152(3–4), 187–214. Yánez-Sedeno, P., Pingarrón, J. M., Riu, J., & Rius, F. X. (2010). Electrochemical sensing based on carbon nanotubes. TrAC Trends in Analytical Chemistry, 29(9), 939–953. Yang, C., Xu, Y., Hu, C., & Hu, S. (2008a). Voltammetric Detection of Ofloxacin in Human Urine at a Congo Red Functionalized Water-Soluble Carbon Nanotube Film Electrode. Electroanalysis: An Inter. J. Devoted to Fundamental and Practical Aspects of Electroanalysis, 20(2), 144–149. Yang, C., Zhang, S., Liu, Y., & Huang, W. (2008b). Electrochemical behaviors of Ofloxacin and its voltammetric determination at carbon nanotubes film modified electrode. Frontiers of Chemistry in China, 3(3), 353–358. Yin, X. B., Kang, J., Fang, L., Yang, X., & Wang, E. (2004). Short-capillary electrophoresis with electrochemiluminescence detection using porous etched joint for fast analysis of lidocaine and Ofloxacin. Journal of Chromatography A, 1055(1–2), 223–228. Zhang, F., Gu, S., Ding, Y., Li, L., & Liu, X. (2013b). Simultaneous determination of Ofloxacin and gatifloxacin on cysteic acid modified electrode in the presence of sodium dodecyl benzene sulfonate. Bioelectrochemistry, 89, 42–49. Zhang, S., Du, Y., & Li, Y. (1998a). Altemating Current Oscillopolargraphic Titration of Ofloxacin. Chinese J. Analytical Chem, 26, 915–915. Additional Declarations No competing interests reported. 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The molecular formula of ofloxacin is C₁₈H₂₀FN₃O₄, and its chemical structure features key functional groups: a carboxyl group (-COOH) at position 3, a keto group at position 4, and a fluorine atom at position 6 [Raj et al., 1997]. Alterations in the position of these functional groups can substantially impact the antibacterial activity of the compound [Mitscher et al., 1993]. Structurally, ofloxacin contains a tricyclic ring system, with each ring and its attached functional groups playing a critical role in disrupting or inactivating bacterial DNA [Morrissey et al., 1996].\u003c/p\u003e \u003cp\u003eIt is well documented in literature that the Fluoroquinolone being an antibacterial works by inactivation of two enzymes in bacteria belonging to the group of DNA topoisomerases (DNA topoisomerases II and DNA topoisomerases IV) that are mainly concern with the production, repairing and replication of bacterial DNA. Topoisomerase II also called DNA gyrase has a major role to keep the chromosomes in super coiled state as well as performs the DNA repairing process. DNA topoisomerases IV is involved in the isolation of daughter chromosomes in the process of replication. If these enzymes become inactive, the bacteria will be unable to make and replicate their genetic material which results in destruction of bacteria or bacterial cell wall [Hooper 1999; Smith et al., 2001].\u003c/p\u003e \u003cp\u003eOfloxacin is extensively recommend by the physician and surgeon around the globe to treat various types of pathogenic infection. These infection/diseases includes prostate gland infection (Prostatitis), skin infection (Impetigo and Cellulitis), pulmonary chest infection (T.B and Pneumonia), digestive tract infection (vomiting and diarrhea) and urinary tract infections (Cervicitis, Urethritis and Cystitis). This is because Ofloxacin have an excellent absorption capability as well as the rate of their adverse effect is very low [Francis \u0026amp; Adcock 2005; Christodoulou et al., 2007]. In addition, the use of Ofloxacin is not only limited to the human medication but broadly used in the treatment of veterinary diseases also [Monk and Campoli-Richards 1987; Hopkala and Kowalczuk 2000].\u003c/p\u003e \u003cp\u003eOfloxacin administered both orally and intravenously and inside the body, it is metabolized to some extent while greater amount of it is excreted in urine as such within 48 hours [Todd and Faulds 1991; Zhang et al., 1998a]. The use of ofloxacin for long period of time can produce unfavorable conditions and various side effects including suppression of immune system, discomfortness of central nervous system, abdominal pain and angioneurotic edema etc. In order to avoid these complications it is very essential to use an appropriate amount of ofloxacin in drugs formulation, to check and regulate its content in the surrounding environment and find out its remains in food [Andreu and Pic\u0026oacute; et al. 2007; Mart\u0026iacute;nez-Carballo et al. 2007; Stolker and Brinkman 2005].\u003c/p\u003e \u003cp\u003eTill date various methods have been developed for determination of ofloxacin that include HPLC, spectrophotometry, electrochemical analysis and capillary electrophoresis [Attia et al. 2012; Shervington et al., 2005; Garc\u0026iacute;a et al., 2005, Awadallah et al., 2003; Yin et al. 2004]. A narrow range, more analysis time, specified temperature, and organic solvents of high purity and high quality make the HPLC as worst technique for routine analysis [Bae et al. 2007]. Similarly, absorption spectroscopy also suffers from a number of drawbacks including limit of detection for analyte, expensive instrumentation and vigorous handling became the reason for its failure [White 1942; Haggquist et al., 1994; Butler 1964.; Shibata et al., 1954; Merzlyak \u0026amp; Naqvi 2000]. Similarly majority of spectroscopic and chromatographic techniques are costly as well as involves time taking procedures. Therefore, to develop an easy, simple, sensitive, responsive and consistent method is utmost important [Heineman 1984]. In contrast to other instrumental methods, the electrochemical techniques which are based on the measurement of either current or potential in chemical reaction [Adams 1976] have large linear range, tremendous sensitivity, provide excellent accurateness with a reasonable low-cost, quick and highly responsive instrumental setup [Cheemalapati et al. 2014; Bard \u0026amp; Faulkner, 2001] make the electrochemical technique is one of the best tool for routine analysis [Goyal et al. 2012; Yang et al. 2008a]. In this connection attempts were made by different scientist to developed nanosensors based electrochemical methods emphasizing that carbon nano-materials possesses an excellent chemical reliability, stability, high electrical and thermal conductivity, specified large surface area which enable them suitable for a wide range of electrochemical determination when used as an electrode materials [Chen and Chatterjee 2013; Y\u0026aacute;nez-Sedeno et al., 2010; Jang et al., 2013].\u003c/p\u003e \u003cp\u003eRecently MWCNTs has been reported to play a vital role in electrochemical determination of various compounds such as proteins, drugs, markers of cancer as well as environmentally important anions like iodide, sulphide, nitrate, nitrite, and phosphate due to charge transfer mechanism [Nugent et al., 2001; Ensafi and Karimi-Maleh 2010; Wildgoose et al., 2006; Bibi et al., 2019]. The electrodes modified with MWCNTs have been reported to possess high sensitivity, ability to decrease the over potentials when utilized as a sensing material in electrochemical cell [Nugent et al. 2001; Ensafi and Karimi-Maleh 2010; Wildgoose et al., 2006]. Similarly the functionalization of MWCNTs with acids also creates different functional groups on the surface of MWCNTs make it more interactive with different analytes [Carabineiro et al. 2011; Alizadah et al., 2009]. In the present work functionalization of MWCNTs have been carried out with sulphuric and nitric acid, which ultimately increase the surface area, impart porosity with greater electrostatic attraction towards many chemical species especially ofloxacin [Peng et al., 2012;].\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eDuring course of our experimental work, analytical grade reagents were used without further purification. The multi-walled carbon nanotubes powder were obtained from sigma-Aldrich (Sigma-Aldrich, Germany with product code 901019) whereas standard Ofloxacin B.No:5857895, origin: England, Potency: 99.29% was obtained from Roryan Pharmaceutical Industries (Pvt) Ltd Peshawar, Pakistan. Deionized water was use throughout the experimental study, while Phosphate buffer solution (PBS) was used as a supporting electrolyte in which sodium phosphate diabasic (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e) obtained from Sigma-Aldrich, Germany was used.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInstrumentation and Apparatus\u003c/h2\u003e \u003cp\u003eThree electrode multi-channel potentiostate systems was used to monitor the electrochemical change as a result of electron transfer. The MWCNTs paste electrode functionalized with acids were used as working electrode whereas saturated calomel and platinum wire were used as reference and counter electrode, respectively. pH meter/ion meter model (K 2000-pH Istek-Inc. Korea) was utilized for pH measurement throughout the experimental work.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eThe stock solution of 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M ofloxacin was prepared by dissolving 0.00361g of standard ofloxacin in 10ml of phosphate buffer, which was prepared by mixing 50 mL of 0.1 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and 50 mL of 0.1M H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e3\u003c/sub\u003e and used as supporting electrolyte in present voltammetric study.\u003c/p\u003e\n\u003ch3\u003ePreparation of Paste Electrode\u003c/h3\u003e\n\u003cp\u003eA proper amount of powder MWCNTs were treated with a mixture of concentrated nitric and sulphuric acid having a ratio (3:1 v/v) respectively to remove the metal particles left behind during the process of synthesis and to create various functional groups on the surface of MWCNTs [Osorio et al., 2008]. The resulting mixture was stirred at room temperature (25\u0026deg;C) for 10 to 12 hours. The mixture was then washed with deionized water for 48 hours using dialysis membrane and the residue were then kept in oven at 120\u0026deg;C for 5 hours. Paste was prepared by mixing MWCNTs powdered with an appropriate amount of paraffin oil and then mix well with help of mortar and pestle for about 30 min. A disposable needle cap was filled with paste and pressed followed by insertion of copper wire for electrical connectivity. Finally the tip of the cap was cut off with sharp blade for further used as working electrode. Similarly MWCNTs paste was also functionalized with Graphene Oxide (GO) and chitosan a natural polymer of chitin for studying the electrochemical response towards ofloxacin. During the entire procedure, Pt wire was used as an auxiliary electrode while calomel electrode was as a reference electrode.\u003c/p\u003e\n\u003ch3\u003eVoltammetric Procedure\u003c/h3\u003e\n\u003cp\u003e10 ml of the buffer solution were added to the three capped voltammetric cell and potential was adjusted in the range 0.0 to 1.5V, under which approximately 9 to 10 consecutive cyclic voltammetric anodic scans were recorded in the presence of 0.1M phosphate buffer at every new surface of MWCNTPE by using differential pulse and cyclic voltammetry techniques.\u003c/p\u003e \u003cp\u003eFor the determination of ofloxacin, 10ml of 0.1M buffer solution was taken in a cell and pH was adjusted to pH 5. The voltammetric scan was recorded for blank solution followed by addition of proper amount of standard ofloxacin in gram into the cell for electrochemical study. Finally, the potentials of the working electrode were scanned after 60 min in the form of voltammogram in anodic direction over the range of 0.0 to 1.5 V at the scan rate of 0.1V.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. All measurements were performed at room temperature (25\u003csup\u003eo\u003c/sup\u003eC) where Pt wire was used as an auxiliary electrode while calomel electrode was as a reference electrode along with acid functionalized paste electrode.\u003c/p\u003e\n\u003ch3\u003eSelection of working electrode\u003c/h3\u003e\n\u003cp\u003eCyclic voltammetry (CV) was studied at different electrodes such as bare glassy carbon electrode (GCE), unmodified multiwalled carbon nanotube paste electrode (MWCNTPE), graphene oxide modified paste electrode (GO-MWCNTPE) and chitosan modified paste electrode (Chi-MWCNTPE) in 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM phosphate buffer containing ofloxacin. A scan rate of 0.1V.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in a potential range of 0.0 to 1.5 V was selected for elucidation of electrochemical properties of the electrode surfaces. All the experiment were run in duplicate at room temperature and constant pH5. Based on the voltammetric response, acid functionalized MWCNTPE was selected as a working electrode.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pH\u003c/h2\u003e \u003cp\u003eIn pH study, different samples were prepared, having 10 ml of phosphate buffer solution and pH of these samples were adjusted using standard 0.01M NaOH and 0.01M HNO\u003csub\u003e3\u003c/sub\u003e in the range 2\u0026ndash;10. Cyclic voltammogram was recorded for blank and suitable amount of ofloxacin was added into the cell followed by scanning its cyclic voltammogram. The same procedure was repeated for the remaining samples, for electrochemical response in the presence of 0.1M ofloxacin. Similarly for Scan rate study, 10ml of 0.1M phosphate buffer solution was transferred into a cell and used as blank followed by adding an appropriate amount of ofloxacin. Cyclic voltammograms were recorded at different scan rates ranging from 0.005 to 10 V.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using the acid functionalized MWCNTPE was electrode.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTime and calibration study of the electrode for voltammetric response of ofloxacin\u003c/h3\u003e\n\u003cp\u003eThe accumulation time study was carried out for the optimization process of the ofloxacin on the surface of working electrode by using cyclic voltammetry. In this study a sample prepared for scan rate study was scan for cyclic voltammograms at different interval of time in the range 5 to 200 min. Similarly the calibration study was performed to check the sensitivity of electrode for detection of ofloxacin at pH 5. Both cyclic and differential pulse voltammetry (DPV) was recorded at a constant pH5 having different concentration of ofloxacin in the range 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M to 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003eM. Optimum time for accumulation of ofloxacin at the surface of the electrode from oxidation was calculated from the voltammogram.\u003c/p\u003e\n\u003ch3\u003eInterference study\u003c/h3\u003e\n\u003cp\u003eTo check the selectivity of the proposed electrode, interference study was conducted under the optimized conditions using DPV technique. Different ions such as Na\u003csup\u003e+,\u003c/sup\u003e K\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e, NH\u003csup\u003e4+\u003c/sup\u003e, ascorbic acid and oxalic acid were added to solution containing ofloxacin in 0.01M phosphate buffer at background electrolyte. Voltammograms were recorded for blank and all the samples at optimized condition where 100ul of each interfering ion were introduce into the cell.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eApplications of the synthesized MWCNTs paste electrode\u003c/h2\u003e \u003cp\u003eThe applicability and validity of MWCNTs paste electrode was evaluated for determining the concentration of ofloxacin in pharmaceutical formulations (tablets) and urine samples of the patient treated with ofloxacin or mock solution of urine using with addition of proper amount of ofloxacin.\u003c/p\u003e \u003cp\u003e i. \u003cb\u003eDetermination of ofloxacin in pharmaceutical formulation.\u003c/b\u003e Three (03) samples were prepared by using different available brands of ofloxacin namely Tarivid 200mg, Oflobid 200mg and Albact 200mg. Each sample was prepared by dissolving 3 tablets of specific brand in 100ml of phosphate buffer followed by filtration. A suitable amount of the specified tablet sample was transferred into the voltammetric cell, and voltammogram was scanned for it. Finally an appropriate amount from standard stock solution of ofloxacin (1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM) was added into the cell where its voltammogram was recorded under optimized i.e., pH5 and time period of 60min. The voltamagram of the each sample was recorded as a triplicate.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eii. Determination of ofloxacin in biological sample\u003c/h2\u003e \u003cp\u003eUrine samples were collected from patient treated with ofloxacin and was centrifuged at 1500 RPM for 5min. The supernatants was taken from it and filtered again. In a specific ratio, the urine and electrolyte (PBS) having total volume of 10ml, was added into the voltammetric cell as blank, and its voltammogram was recorded. Similarly, an appropriate amount from standard stock solution of ofloxacin (1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003eM) was added into the cell and voltammogram was recorded under optimized condition of time and pH. All the reading were taken as triplicate.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVoltammetric response of ofloxacin at MWCNTs-paste electrode\u003c/h2\u003e \u003cp\u003eThe electrochemical behavior of Ofloxacin was studied at GCE, MWCNTPE, GO-MWCNTPE and Chi-MWCNTPE in PBS of pH-5 containing 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e M of Ofloxacin at a scan rate of 0.1V.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Cyclic voltammetric curves were recorded using different electrodes where the highest peak current was observed at the designed MWCNTPE in a potential range of 0.0 to 1.5V (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The interaction shows that MWCNTs paste electrode have the ability to increase the rate of electrons transfer between the Ofloxacin and the electrode material. It is reported that electron transfer played a vital role to facilitate an electro-chemical oxidation of Ofloxacin at MWCNTs surface due to which electro-catalytic response of the proposed electrochemical sensor enhanced [Si et al., 2018; Nugent et al., 2001]. Another reason for the extra ordinary electrochemical response is the large surface area provided by the MWCNTs paste electrode for oxidation of Ofloxacin. Contrary to that, under the similar experimental optimized conditions there was no oxidation peak appears for the Ofloxacin at GO-MWCNTPE. Similarly the oxidation peaks of Ofloxacin using bare GCE and Chi-MWCNTPE were also very low that obviously attributed to the less conductivity, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Therefore MWCNTs paste electrode was selected for the rest of the studies throughout the project. The mixture of nitric acid and sulfuric acid acts as an oxidizing agent, leading to the formation of oxygenated functional groups on the surface of CNTs. The specific ratio of HNO\u003csub\u003e3\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e i.e., 3:1 keeping into account the treatment time and temperature as the influencing factors, can greatly affect the surface functionalization and the types of groups originated at the CNTs surface. It is reported that when MWCNTs treated with a mixture of nitric acid and sulfuric acid, then primarily carboxyl (-COOH) and phenolic groups are introduced onto the surface of CNTs which results in alteration of the overall surface parameters [Osorio et al., 2008; Britto et al. 1996]. Similarly oxygen-containing functional groups like hydroxyl (-OH) and carbonyl (-C\u0026thinsp;=\u0026thinsp;O) are also appear on the surface after acid treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of pH\u003c/h2\u003e \u003cp\u003eThe pH of an electrolyte is one of the important and key factor which determines the ability of electrode to enhance the electrochemical response of an analyte. The effect of pH on the electro-chemical behavior for the assessment of ofloxacin was investigated by using cyclic voltammetry at proposed MWCNTPE electrochemical sensors in the pH range from 2\u0026ndash;10. As shown in the Figure.2, a gradual increase in the oxidation peak current with increase in pH from 1.0 to 5.0 was observed and a current response reached to a maximum at pH 5. Further increase in the pH from 6\u0026ndash;10 has drastically decrease the peak current. It is well documented in the literature that during the electro-oxidation of ofloxacin an equal number of electrons and protons are discharge [Yang et al. 2008b; Zhang et al. 2013b]. From these results it was observed that maximum oxidation was take place at this pH value and therefore, was chosen as an optimum pH value for the ofloxacin investigation. It is also pertinent to mention that the oxidation peak current reach to maximum value of 67\u0026micro;A, where electron transfer between proposed electrode and ofloxacin was almost completed. Beyond this pH value, either electron transfer stop or reverse phenomenon is expected according to the law of mass action [Zhang et al. 2013b].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of scan rate\u003c/h2\u003e \u003cp\u003eThe effect of scan rate on electrochemical behavior of ofloxacin was investigated on a proposed MWCNTs-paste electrode using different scan rates ranges from 0.01\u0026ndash;0.1 Vs\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Figure.3\u003csub\u003ea\u003c/sub\u003e reveals that different cyclic voltammograms were obtained at different scan rates and oxidation peak current increases as the scan rate increases from 0.01 to 0.1Vs\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.When the oxidation peak current was plotted against the scan rate, a linear relationship was found as shown in the Figure.3\u003csub\u003eb\u003c/sub\u003e. These result demonstrates that the oxidation of ofloxacin on the surface of MWCNTs paste electrode is an irreversible as well as adsorption controlled process at low ofloxacin concentration. However, when the oxidation peaks were plotted against the square root of scan rate, it also give a linear plot as shown in Figure.3\u003csub\u003ec\u003c/sub\u003e. These observations indicate the diffusionally controlled process at high concentration of ofloxacin. The highest oxidation peak current at 0.1 Vs\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e scan show that mechanism of charge transfer for oxidation is greater at this condition between MWCNTs paste electrode and ofloxacin moiety. Therefore, further investigations were carried at this scan rate under the optimized condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInfluences of adsorption Accumulation Time\u003c/h2\u003e \u003cp\u003eThe influence of accumulation time on the oxidation peak current of ofloxacin was also investigated in order to reveal the adsorption capability of the proposed sensor. Accumulation time is thought to be an important parameter in electrochemical study especially in cyclic voltammetry. Figure.4 show the effect of accumulation time on oxidation peak current where it is noted that the oxidation peak current rapidly increases with increasing the accumulation time ranging from 0\u0026ndash;60 min. It is also important to note that further increase in time up to 200 min has no prominent effect on the oxidation peak current. These results indicate the saturation point where almost all vacant sites for adsorption has occupied by the analyte molecules and no further adsorption take place under the optimized experimental condition. Similarly, the functional group interactions with Ofloxacin molecules also reach the plateau condition, and hence 60 min were selected in further experimental work for accumulation time. Similarly, it is also inferred that adsorption controlled is the dominant mechanism responsible for high oxidation peak current of the ofloxacin at the surface of the MWCNTs paste electrode [Amira et al., 2023]. In cyclic voltammetry, an adsorption-controlled process, also known as a surface-controlled process, occurs when the electroactive species adsorb to the electrode surface before undergoing electron transfer. This means the rate-limiting step is the adsorption of ofloxacin, not the diffusion to the electrode surface [Amira et al., 2023]. A key characteristic of adsorption controlled processes is that the peak current is linearly proportional to the scan rate as observed in scan rate studies for ofloxacin at the surface of the paste electrode.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSensitivity of MWCNT Paste Electrode (Calibration study)\u003c/h2\u003e \u003cp\u003eThe calibration curve for Ofloxacin was obtained under the optimized experimental conditions in presence of phosphate buffer solution at the MWCNT paste Electrode by using cyclic voltammetry (CV) as well as differential pulse voltammetry (DPV) to check the sensitivity of proposed sensor for the Ofloxacin. Various cyclic voltammograms were obtained by adding different concentrations of Ofloxacin ranging from 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e-1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M as shown in the Figure.5\u003csub\u003ea,b\u003c/sub\u003e for CV and DPV respectively. The oxidation peak current increases with gradual increase in the concentrations of ofloxacin and a linear relationship was obtained between the oxidation peak current and ofloxacin concentration. Figure.5\u003csub\u003ec,d\u003c/sub\u003e reveal that the synthesized electrode work both in lower concentration range i.e., 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e-5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M and high concentration ranges 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e-1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M with good regression coefficient (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.999). It is also worth mentioning that both the techniques i.e., CV and DPV work good in wide range of concentration as clear from Figure.5\u003csub\u003ee,f\u003c/sub\u003e. The limit of detection for Ofloxacin was calculated using the relation:\u003c/p\u003e \u003cp\u003e(LOD)\u0026thinsp;=\u0026thinsp;3s/S\u003c/p\u003e \u003cp\u003eWhere small \u0026lsquo;s\u0026rsquo; stands for standard deviation whereas capital \u0026lsquo;S\u0026rsquo; was calculated from the slop of linear plot using straight line equation.\u003c/p\u003e \u003cp\u003eIt is obvious from result that limit of detection being 1\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M for both CV and DPV using the functionalized MWCNTs paste electrode confirm the validity, sensitivity and selectivity of proposed sensor.\u003c/p\u003e \u003cp\u003eAccording to the redox mechanism that was proposed by Kauffmann et al., [1987] the oxidation peak is due to the irreversible oxidation of the piperazine moiety of ofloxacin molecule. The comparative sensitivity of proposed sensor with other previously reported electrode by other researchers for detection of ofloxacin is given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Hence the proposed electrode could be an appropriate and suitable electrochemical sensor for ofloxacin determination using adsorption cyclic voltammetric techniques (CV and DPV) due to possessing high sensitivity, selectivity and greater stability over the paste electrode. The greater accumulation and adsorption is attributed to the high surface area, pore size, pore volume and availability of different surface functional groups responsible for the interaction with Ofloxacin molecules. Similar observation has been reported by [Sannia et al., 2019] for nitrite and sulphide determination.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eShows all the previously reported voltammetric oxidation methods for the determination of Ofloxacin.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTechnique\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElectrolyte/pH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePotential(V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOD(M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWNTs-CR/GE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhosphate\u003c/p\u003e \u003cp\u003ebuffer/6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e9\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYang et al.,2008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWNTs/Nafion- film /GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV/LSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKHP-NaOH\u003c/p\u003e \u003cp\u003ebuffer/5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e1\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHuang et al., 2008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZnO/GR/GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV/DPV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhosphate\u003c/p\u003e \u003cp\u003ebuffer/5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e0.33\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSi et al., 2018\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTrp-GO- CNT/GCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhosphate\u003c/p\u003e \u003cp\u003ebuffer/7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e1\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eZhu et al., 2019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHMDE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBR buffer/8.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e4 \u0026times; 10\u0026thinsp;\u0026minus;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGulaboskiand\u003c/p\u003e \u003cp\u003eJordanoski,. 2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMWCNTPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV/ DPV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhosphate\u003c/p\u003e \u003cp\u003ebuffer/5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c5\"\u003e \u003cp\u003e1\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePresentwork\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSelectivity of MWCNTs Paste Electrode (Interference study)\u003c/h2\u003e \u003cp\u003eThe selectivity of the proposed MWCNTs paste electrode was investigated under optimum conditions using differential pulse voltammetry (DPV) for the detection of ofloxacin in the presence of potential interfering agents such as Na⁺, K⁺, Mg\u0026sup2;⁺, Ca\u0026sup2;⁺, Fe\u0026sup3;⁺, NH₄⁺, oxalic acid, and ascorbic acid in a phosphate buffer solution at pH 5.0. The results, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, indicate that these ions did not cause any significant change in the peak current of ofloxacin, demonstrating good selectivity. No noticeable interaction with either cations or anions was observed in this study. Therefore, the proposed sensor exhibits high selectivity for ofloxacin determination in the presence of common interfering ions and can be recommended for use in industrial effluent analysis as well as for qualitative determination in pharmaceutical applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Ofloxacin in commercial tablet samples\u003c/h2\u003e \u003cp\u003eThe practical applicability of the designed sensor (MWCNTsPE) was evaluated for the qualitative determination of ofloxacin in commercially available pharmaceutical tablets using the differential pulse voltammetry (DPV) technique. Three different brands of ofloxacin tablets, i.e., Tarivid 200 mg, Oflobid 200 mg, and Albact 200 mg were selected for quantitative analysis. The standard addition method was employed, where varying concentrations of a 1\u0026times;10⁻\u0026sup3; M standard ofloxacin solution were spiked into the electrochemical cell containing the tablet solution along with phosphate buffer (pH 5.0) as the supporting electrolyte. The results, summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, are the mean of three replicate measurements. It is evident from the data that the recovery of ofloxacin ranged from 97.75\u0026ndash;103%, indicating the accuracy and reliability of the proposed sensor. These findings support the validity and potential applicability of the sensor for industrial-scale analysis and pharmaceutical quality control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetermination of Ofloxacin in pharmaceutical samples using DPV techniques\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName of tablet\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTablet Amount (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFound Amount (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarivid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e195.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOflobid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e103.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbact\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e194.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Ofloxacin in urine sample\u003c/h2\u003e \u003cp\u003eThe proposed voltammetric sensor was also successfully applied for the determination of ofloxacin in human urine samples using the standard addition method and differential pulse voltammetry (DPV) and results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Various concentrations of a standard ofloxacin solution were spiked into the electrochemical cell containing urine samples in the presence of phosphate buffer solution (pH 5.0) as the supporting electrolyte. A high percent recovery of 102.8% was achieved, demonstrating the accuracy of the proposed method. These results indicate that the sensor is highly effective and sensitive for detecting ofloxacin in urine, without significant interference from endogenous substances. Furthermore, it is worth noting that a substantial amount of ofloxacin is excreted in urine following oral administration in patients with various diseases, which supports the relevance of urine analysis for monitoring therapeutic levels.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetermination of Ofloxacin in urine samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFound (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUrine sample\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e300.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e308.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e102.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e300.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e378.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e126.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe designed electrochemical sensor based on a functionalized multi-walled carbon nanotube paste electrode (MWCNTsPE) demonstrated high sensitivity and selectivity toward the voltammetric oxidation of ofloxacin. The functionalization and high surface area of the MWCNTs significantly enhanced electron-transfer kinetics, making the electrode highly suitable for the electrochemical detection of ofloxacin. It is important to note that the large surface area and surface functional groups of MWCNTs facilitate efficient charge transfer between the electrode surface and the ofloxacin analyte. This charge transfer process is the primary mechanism behind the observed oxidation peak current, which arises after the adsorption of ofloxacin onto the electrode surface. Based on the experimental findings, it can be concluded that the oxidation process is adsorption-controlled at low concentrations of ofloxacin, transitioning to a diffusion-controlled process at higher concentrations. This dual mechanism results in a sharp and well-defined oxidation peak. The sensor was successfully applied to determine ofloxacin at very low concentrations under optimized conditions and exhibited excellent selectivity even in the presence of various potentially interfering ions. The method yielded high percent recoveries when applied to both pharmaceutical formulations and biological (urine) samples, confirming the sensor\u0026rsquo;s sensitivity and reliability. The limit of detection (LOD) was determined to be 1\u0026times;10⁻⁸ M. Interference studies showed that the oxidation peak current remained unaffected by the presence of common anions and cations, underscoring the sensor's selectivity toward ofloxacin. Additionally, the sensor demonstrated a good molar absorptivity (extinction coefficient) and performed effectively across a broad concentration range from 1\u0026times;10⁻⁸ to 1\u0026times;10⁻⁵ M, with a strong coefficient of determination, confirming its suitability for quantitative analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAsma Gul: Writing Original Draft, Methodology, SoftwareMuhammad Iqbal Zaman: Supervision, Project Administration, Investigation, MethodologyAbdul Niaz: Supervision, Data curationAmna Bibi: Writing- Reviewing and Editing, Visualization, Conceptualization, Formal AnalysisMadina: Conceptualization, Formal AnalysisHanan E Osman: Visualization, Conceptualization, Formal Analysis\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENT\u003c/h2\u003e \u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdams, R. N. (1976). Probing brain chemistry with electroanalytical techniques. Analytical Chemistry, 48(14), 1126A-1138A.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlizadeh, T., Ganjali, M. R., Norouzi, P., Zare, M., \u0026amp; Zeraatkar, A. (2009). A novel high selective and sensitive para-nitrophenol voltammetric sensor, based on a molecularly imprinted polymer\u0026ndash;carbon paste electrode. Talanta, 79(5), 1197\u0026ndash;1203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmira, Youssef, Mohammed, Abeer, Sherif,. Voltammetric quantitative analysis of vildagliptin in bulk form and spiked human serum at a modifed electrode., J. Iranian Chemical Society (2023) 20:1503\u0026ndash;1522\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndreu V, B. C., Pic\u0026oacute; Y (2007). Analytical strategies to determine quinolone residues in food and the environment. TrAC Trends Anal Chem. 26(6), 534\u0026ndash;556. .\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAttia, M., Essawy, A. A., Youssef, A., \u0026amp; Mostafa, M. S. (2012). Determination of Ofloxaci using a highly selective photo probe based on the enhancement of the luminescence intensity of Eu \u003csup\u003e3+\u003c/sup\u003e Ofloxacin complex in pharmaceutical and serum samples. J. Fluorescence, 22(2), 557\u0026ndash;564.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAwadallah, B., Schmidt, P. C., \u0026amp; Wahl, M. A. (2003). Quantitation of the enantiomers of Ofloxacin by capillary electrophoresis in the parts per billion concentration range for in vitro drug absorption studies. Journal of Chromatography A, 988(1), 135\u0026ndash;143.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBae, J. W., Kim, M. J., Jang, C. G., \u0026amp; Lee, S. Y. (2007). Determination of meloxicam in human plasma using a HPLC method with UV detection and its application to a pharmacokinetic study. Journal of Chromatography B, 859(1), 69\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBard, A. J., \u0026amp; Faulkner, L. R. (2001). Fundamentals and applications. Electrochemical Methods, 2(482), 580\u0026ndash;632.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBibi, S., Zaman, M. I., Niaz, A., Rahim, A., Nawaz, M., \u0026amp; Arian, M. B. (2019). Voltammetric determination of nitrite by using a multiwalled carbon nanotube paste electrode modified with chitosan-functionalized silver nanoparticles. Microchimica Acta, 186(9), 1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBritto, P., Santhanam, K., \u0026amp; Ajayan, P. (1996). Carbon nanotube electrode for oxidation of dopamine. Bioelectrochemistry and Bioenergetics, 41(1), 121\u0026ndash;125.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarabineiro, S., Thavorn-Amornsri, T., Pereira, M., \u0026amp; Figureueiredo, J. (2011). Adsorption of ciprofloxacin on surface-modified carbon materials. Water research, 45(15), 4583\u0026ndash;4591.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheemalapati, S., Devadas, B., \u0026amp; Chen, S. M. (2014). Novel poly-L-lysine/carboxyl-group enriched graphene oxide/modified electrode preparation, characterization and applications for the electrochemical determination of meloxicam in pharmaceutical tablets and blood serum. Analytical methods, 6(20), 8426\u0026ndash;8434.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, A., \u0026amp; Chatterjee, S. (2013). Nanomaterials based electrochemical sensors for biomedical applications. Chemical Society Reviews, 42(12), 5425\u0026ndash;5438.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng, G.W., Wu, H.L., \u0026amp; Huang, Y.L. (2008). Simultaneous determination of malondialdehyde and Ofloxacin in plasma using an isocratic high-performance liquid chromatography /fluorescence detection system. Analytica chimica acta, 616(2), 230\u0026ndash;234.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChristodoulou, E. A., Samanidou, V. F., and Papadoyannis, I.N. ( 2007). Development and validation of an HPLC confi rmatory method for residue analysis of ten quinolones in tissues of various food-producing animals, according to the European Union Decision 2002/657/EC. J. Sep. Sci., 30:, 2676\u0026ndash;2686.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnsafi, A. A., \u0026amp; Karimi-Maleh, H. (2010). Modified multiwall carbon nanotubes paste electrode as a sensor for simultaneous determination of 6-thioguanine and folic acid using ferrocenedicarboxylic acid as a mediator. J.Electroanalytical Chemistry, 640(1\u0026ndash;2), 75\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrancis, P. S., \u0026amp; Adcock, J. L. (2005). Chemiluminescence methods for the determination of Ofloxacin. Analytica Chimica Acta, 541(1\u0026ndash;2), 3\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a, M. S., Albero, M. I., S\u0026aacute;nchez-Pedre\u0026ntilde;o, C., \u0026amp; Abuherba, M. S. (2005). Flow injection spectrophotometric determination of Ofloxacin in pharmaceuticals and urine. European J. Pharmaceutics and biopharmaceutics, 61(1\u0026ndash;2), 87\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoyal, R. N., Rana, A. R. S., \u0026amp; Chasta, H. (2012). Electrochemical sensor for the sensitive determination of norfloxacin in human urine and pharmaceuticals. Bioelectrochemistry, 83, 46\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGulaboski, R., \u0026amp; Jordanoski, B. (2000). Square-wave voltammetry of Ofloxacin. Bulletin of the Chemists and Technologists of Macedonia, 19(2), 177\u0026ndash;181.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHooper, D. C. (1999). Mechanisms of fluoroquinolone resistance. Drug Resist Updates. 2, 38\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith, A., Pennefather, P. M., Kaye, S. B., \u0026amp; Hart, C. A. (2001). Fluoroquinolones. Drugs, 61(6), 747\u0026ndash;761.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHopkala, H., \u0026amp; Kowalczuk, D. (2000). Application of derivative UV spectrophotometry for the determination of ciprofloxacin, norfloxacin and Ofloxacin in tablets. Acta Pol. Pharm, 57(1), 3\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, K. J., Liu, X., Xie, W. Z., \u0026amp; Yuan, H. X. (2008). Voltammetric behavior of Ofloxacin and its determination using a multi-walled carbon nanotubes-Nafion film coated electrode. Microchimica Acta, 162(1\u0026ndash;2), 227\u0026ndash;233.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJang, H. D., Kim, S. K., Chang, H., Choi, J.-W., \u0026amp; Huang, J. (2013). Synthesis of grapheme based noble metal composites for glucose biosensor. Materials Letters, 106, 277\u0026ndash;280.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKauffmann, J. M., Vire, J. C., Patriarche, G. J., Nunez-Vergara, L. J., \u0026amp; Squella, J. A. (1987), Voltammetric oxidation of trazodone. Electrochimica acta, 32(8), 1159\u0026ndash;1162.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Carballo E, G.-B. C., Scharf S, Gans O. (2007). Environmental monitoring study of selected veterinary antibiotics in animal manure and soils in Austria. Environ Pollut: 148(2):570\u0026ndash;579.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerzlyak, M. N., \u0026amp; Naqvi, K. R. (2000). On recording the true absorption spectrum and the scattering spectrum of a turbid sample: application to cell suspensions of the cyanobacterium Anabaena variabilis. Journal of Photochemistry and Photobiology B: Biology, 58(2\u0026ndash;3), 123\u0026ndash;129.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitscher, A. L., Devasthale, R. Zavod. (1993). Quinolone Antimicrobial Agents, American Society for Microbiology,2nd Edi., Washington DC, 3\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonk, J. P., \u0026amp; Campoli-Richards, D. M. (1987): a review of its antibacterial activity, pharmacokinetic properties and therapeutic use, Drugs 33 (1987) 346. Ofloxacin. Drugs, 33(4), 346\u0026ndash;391.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrissey, I., Hoshino, K.,Sato, K., Yoshida, A, Hayakawa, I., Bures, M. G., \u0026amp; Shen, L. L. (1996), Mechanism of differential activities of Ofloxacin enantiomers. Antimicrobial agents and chemotherapy, 40(8), 1775\u0026ndash;1784.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNugent, J. M., Santhanam, K. S. V., Rubio, A. A., \u0026amp; Ajayan, P. M. (2001). Fast electron transfer kinetics on multiwalled carbon nanotube microbundle electrodes. Nano letters,1(2), 87\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsorio A, Silveira I, Bueno V, Bergmann C. H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e/HNO\u003csub\u003e3\u003c/sub\u003e/HCl-Functionalization and its effect on dispersion of carbon nanotubes in aqueous media. Applied Surface Science 2008;255:2485-9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng, H., Pan, B., Wu, M., Liu, R., Zhang, D., Wu, D., \u0026amp; Xing, B. (2012). Adsorption of Ofloxacin on carbon nanotubes: solubility, pH and cosolvent effects. J. Hazardous materials, 211, 342\u0026ndash;348.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaj, S., Kapadia, S., \u0026amp; Argekar, A. (1997). Simultaneous determination of metronidazole and norfloxacin from pharmaceutical preparations by RP-HPLC. Indian drugs, 34(10), 585\u0026ndash;589.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato, K., Matsuura, Y., Inoue, M., Une, T., Osada, Y., Ogawa, H., \u0026amp; Mitsuhashi, S. (1982). In vitro and in vivo activity of DL-8280, a new oxazine derivative. Antimicrobial Agents and Chemotherapy, 22(4), 548\u0026ndash;553.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShervington LA, A. M., Hussain B, Donnelly J. (2005). The simultaneousseparation and determination of five quinolone antibioticsusing isocratic reversed-phase HPLC: application tostabilitystudies on an Ofloxacin tablet formulation. J Pharm Biomed Anal. 39, 3\u0026ndash;4, 769\u0026ndash;775.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShibata, K., Benson, A. A., \u0026amp; Calvin, M. (1954). The absorption spectra of suspensions of living micro-organisms. Biochimica et biophysica acta, 15(4), 461\u0026ndash;470.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSi, X., Wei, Y., Wang, C., Li, L., \u0026amp; Ding, Y. (2018). A sensitive electrochemical sensor for Ofloxacin based on a graphene/zinc oxide composite film. Analytical methods, 10(17), 1961\u0026ndash;1967.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkoog, Douglas. A., Donald, M. West., F, James. Holler. (1995). Fundamentals of Analytical Chemistry (7th ed.). Harcourt Brace College Publishers. ISBN 978-0-03-005938-4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStolker AAM, B. U. (2005). Analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in food-producing animals\u0026mdash;a review.. J Chromatogr A 1067, 1\u0026ndash;2, 15\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTodd, P. A., \u0026amp; Faulds, D. (1991). Ofloxacin a reappraisal of its antimicrobial activity, pharmacology and therapeutic use, Drugs, 42(5), 825\u0026ndash;876.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite, J. U. (1942). Long optical paths of large aperture. JOSA, 32(5), 285\u0026ndash;288.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWildgoose, G. G., Banks, C. E., Leventis, H. C., \u0026amp; Compton, R. G. (2006). Chemically modified carbon nanotubes for use in electroanalysis. Microchimica Acta, 152(3\u0026ndash;4), 187\u0026ndash;214.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY\u0026aacute;nez-Sedeno, P., Pingarr\u0026oacute;n, J. M., Riu, J., \u0026amp; Rius, F. X. (2010). Electrochemical sensing based on carbon nanotubes. TrAC Trends in Analytical Chemistry, 29(9), 939\u0026ndash;953.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, C., Xu, Y., Hu, C., \u0026amp; Hu, S. (2008a). Voltammetric Detection of Ofloxacin in Human Urine at a Congo Red Functionalized Water-Soluble Carbon Nanotube Film Electrode. Electroanalysis: An Inter. J. Devoted to Fundamental and Practical Aspects of Electroanalysis, 20(2), 144\u0026ndash;149.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, C., Zhang, S., Liu, Y., \u0026amp; Huang, W. (2008b). Electrochemical behaviors of Ofloxacin and its voltammetric determination at carbon nanotubes film modified electrode. Frontiers of Chemistry in China, 3(3), 353\u0026ndash;358.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin, X. B., Kang, J., Fang, L., Yang, X., \u0026amp; Wang, E. (2004). Short-capillary electrophoresis with electrochemiluminescence detection using porous etched joint for fast analysis of lidocaine and Ofloxacin. Journal of Chromatography A, 1055(1\u0026ndash;2), 223\u0026ndash;228.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, F., Gu, S., Ding, Y., Li, L., \u0026amp; Liu, X. (2013b). Simultaneous determination of Ofloxacin and gatifloxacin on cysteic acid modified electrode in the presence of sodium dodecyl benzene sulfonate. Bioelectrochemistry, 89, 42\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, S., Du, Y., \u0026amp; Li, Y. (1998a). Altemating Current Oscillopolargraphic Titration of Ofloxacin. Chinese J. Analytical Chem, 26, 915\u0026ndash;915.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Voltammetry, MWCNTPE, Ofloxacin, Biomedical application, Acid functionalization","lastPublishedDoi":"10.21203/rs.3.rs-6580404/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6580404/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOfloxacin, a member of the fluoroquinolone class of antibiotics, exhibits broad-spectrum activity against both gram-positive and gram-negative bacteria by inhibiting topoisomerase enzymes. Monitoring the concentration of ofloxacin is crucial in pharmaceutical formulations, biological samples, food products, and environmental matrices. In this study, a novel, simple, selective, and sensitive voltammetric sensor was developed for the detection of ofloxacin in pharmaceutical and real-world samples. A functionalized multi-walled carbon nanotube paste electrode (MWCNTPE) was employed for the electrochemical oxidation and quantification of ofloxacin using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Measurements were carried out in 0.1 M phosphate buffer solution, with a scan rate of 0.1 V/s and a pulse period of 0.1 seconds, following an accumulation time of 60 minutes. Ofloxacin was found to adsorb irreversibly onto the electrode surface, producing a well-defined anodic peak at +\u0026thinsp;0.85 V. The sensor demonstrated high sensitivity, achieving a detection limit as low as 1\u0026times;10⁻⁸ M, and exhibited excellent selectivity even in the presence of common interfering ions. Owing to its low cost, ease of preparation, and renewability, the sensor was successfully applied for the determination of ofloxacin in pharmaceutical tablets and biological samples, yielding high recovery rates.\u003c/p\u003e","manuscriptTitle":"Electrochemical Investigation of Ofloxacin Using a Functionalized Multi-Walled Carbon Nanotube Paste Electrode","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-27 17:52:02","doi":"10.21203/rs.3.rs-6580404/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"925377e9-d85b-455d-9789-64b871bec6d2","owner":[],"postedDate":"May 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-27T17:52:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-27 17:52:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6580404","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6580404","identity":"rs-6580404","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}