Fabrication of MWCNTs and Ru Doped TiO₂ Nanoparticles Composite Carbon Sensor for Biomedical Application

The synthesized RuTiO₂ nanoparticles size and crystal structure were analyzed by powder XRD analysis (Phillips PW1729, Cu kα), SEM (JEOL JSM-6360), Energy-dispersive X-ray spectroscopy (EDX) and TEM Philips (CM200) followed by electrode morphology study by AFM (Model: Nano Surf AG - easy scan; Make: Nano Surf, Switzerland). An electrochemical analyzer (CHI Company, D630, USA) is utilized to carry out voltammetric measurements. The analyzer was incorporated by three electrode system, the primary functioning sensor as RuTiO₂/MWCNTs composite based carbon paste electrode (RuTiO₂/MWCNTs-CPE), an auxiliary sensor as a platinum wire, and reference sensor as an Ag/AgCl (3.0 M KCl) correspondingly. Both the bare and new fabricated CPE sensing surfaces were redeveloped via polishing with the use of filter paper and then water wash was followed, prior to each measurement. By utilizing pH meter (Elico Ltd., India), the pH measurements were performed.

CLZ, MWCNTs and all other chemicals procured from Sigma Aldrich and were used as devoid of any additional purification. In this whole experiment double distilled water was used. Methanol is used to prepare the analyte stock solution of CLZ (0.1 mM) and stored at low temperature. Supporting electrolyte solution with different pH varying from 3.0 to 11.2 were prepared with a 0.2 M concentration by adding the appropriate amount of sodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄) and trisodium phosphate (Na₃PO₄) which is commonly known as phosphate buffer saline (PBS).^44,45

Ruthenium doped TiO₂ nanoparticles were prepared as by the literature.^16,43,46 Liquid impregnation method was inherited to prepare the ruthenium (Ru) doped TiO₂ nanoparticles (anatase). Primarily an exact quantity of (0.8%) RuCl₃.3H₂O (SRL, India) was weighed and then dissolved in 100 ml of HCl (0.2 M) solution. The procedure was followed by the addition of 1.0 g TiO₂ nanoparticles (Anatase). The resultant slurry was stirred carefully for 3 hours and kept for 1 whole day until it settles. The slurry was dried and ground in a mortar to get a fine powder. The nanoparticles were calcined at 400°C, which is below the phase transformation temperature of TiO₂.

Before modification, CPE was prepared by the homogeneous blending of the powder form of graphite with paraffin oil in 7:3 ratios. The resultant paste was firmly packed in a hollow polytetrafluoroethylene tube (PTFE), and the surface was smoothened. The surface activation was carried at pH 5.0, by cycling it in the potential range 0.0–1.2 V with scan rate 50 mVs⁻¹. The paste was carefully removed prior packing a new paste, after every measurement. For the fabrication of RuTiO₂-CPE and MWCNTs-CPE, RuTiO₂ nanoparticles and MWCNTs were finely homogenized with carbon paste and filled in the cavity of PTFE tube. The RuTiO₂/MWCNTs composite electrode was prepared by adding both RuTiO₂ nanoparticles and MWCNTs to the CPE matrix in an appropriate amount.

By utilizing a mortar and pastel, the CLZ tablets, i.e., clozapex (25 mg CLZ per tablet) were finely ground. The related weight with the stock solution was dissolved and diluted up to 100 ml with pH 5.0. Proper dissolution was attained by sonication for ten minutes. The precision of the proposed technique was tested by recovery examines. By adopting standard addition method, the evaluation of studied excipients meddling with clinical dosage forms and accurateness of the current technique were analyzed.

From healthy volunteers, urine samples were collected. At room temperature (25 ± 0.1°C) the collected samples were centrifuged (4383 G) for 5 minutes. CLZ fortified urine sample (1 mM) was prepared by treating 1 mL untreated urine of a healthy individual with 1 ml CLZ solution (0.1 mM). The appropriate volume of analyte fortified urine sample was diluted with phosphate buffer of pH 5.0. Square wave voltammograms were recorded, and the amounts of CLZ in spiked human urine samples were calculated using the calibration graph.

To calculate the active surface vicinity of the new fabricated electrode, Randles - Sevcik equation was utilized as usual. From the calculation, we found 0.042 cm² area for CPE while modified CPE shows four times higher area than the bare. KCl (0.1 M) was taken as supporting electrolyte, the test solution was K₃Fe (CN)₆ (1.0 mM) and diffusion coefficient (D₀) of 7.6 × 10⁻⁶ cm² s⁻¹, to acquire the result. CV technique was adopted for the determination at different sweep rates with a temperature of 298 K.⁴⁷

In electrochemical study and analysis, the morphological features of the sensing base, modifiers structure place a fundamental role. Some muddled assembly were observed in SEM image, which indicative of vast surface area (Figure 1A). While in TEM, the non-uniform cylindrical crystalline structures were dispersed (Figure 1B). The dark tiny spots scattered were assumed to be particles of ruthenium resting on TiO₂ nanoparticles with the breadth of 10–15 nm and 30–35 nm of length roughly. From the EDX examination, noticed that the synthesized nanomaterials largely consisted titanium and oxygen with a slight quantity of Ru (Figure 1C). The XRD pattern of the Ru-TiO₂ particles in Figure 1D shows as a function of reaction temperature. From the X-ray analysis, synthesized Ru-TiO₂ particles reveal the existence of anatase phases in the reaction temperature.

Zoom In Zoom Out Reset image size

The tailored electrodes sensing base characterization was done by using AFM study. The AFM images of all the sensors utilized in current work (CPE, MWCNTs/CPE, Ru-TiO₂/CPE, and Ru-TiO₂/MWCNTs-CPE) were given in Figure 2. The morphological features of analyzed sensors were recorded in Table I.

Zoom In Zoom Out Reset image size

Enhancement of oxidation peak can be achieved by the study of preconcentration time impact, which helps in the transport of analyte molecules from solution to sensing base. Thus the study was carried out in a range of 0–120 s, and at 80 s the maximum oxidation peak was notified (Figure 3). This effect indicates saturated adsorption on the modified electrode was achieved at 80 s. Hence the same accumulation time was inherited for further studies.

Zoom In Zoom Out Reset image size

The modifier amount is also responsible for the different electrochemical behavior. So, its study stands vital in studying the voltammetric behavior of analyte and electrocatalytic activity of the electrode. The fabrication of modified CPE is already described in experimental section. Voltammograms were recorded while varying the weight of both the nanoparticles was added during the sensor fabrication and the fluctuations in the peak current as well as in the peak potential were noted. Finally, from the study, we come to know that the usage of 3.0 mg of RuTiO₂ nanoparticles and 1.8 mg of MWCNTs was found be optimum to fabricate modified CPE. Beyond this amount, saturation occurs, which may be the enlarged measure of modifier onto the sensing base resist the easy electron transfer, and as a result, the peak current of CLZ was declined after addition of modifier.

In Figure 4, CVs of CPE, RuTiO₂-CPE, MWCNTs-CPE, and RuTiO₂/MWCNTs-CPE, in the presence and absence of 0.1 mM CLZ at 100 mVs⁻¹ in pH 5.0 were evidenced. Without the analyte, no redox peaks were detected at all the electrodes, but there is an increase in the background current, indicative of the surface area enlargement. From this, it can be proved that RuTiO₂/MWCNT-CPE is electrochemically dormant in the preferred range of potential. In the active presence of 0.1 mM CLZ, a reversible behavior through moderately weak peak currents occurred for bare CPE, at an anodic potential (E_pa) = 0.549 V, the cathodic potential (E_pc) = 0.498 V, with peak current 3.283 μA and 0.290 μA respectively. While at RuTiO₂ /MWCNTs-CPE, an accentuated and well-defined redox peaks with (E_pa) = 0.522 V, (E_pc) = 0.471 V, with peak current 14.19 μA and 0.619 μA correspondingly (Figures 4A and 4B). So it is palpable that both the modifiers task is to increase the peak currents by accelerating the electron transfer kinetics, which makes them as an efficient modulator in CLZ electrochemical redox reaction. The current enhanced electrochemical behavior CLZ is due to the energy difference between the two modifiers, and the great surface characteristics. In addition, there might be some interaction between the composite material and CLZ electrostatically, which can cause the high loading efficiency of CLZ on modified CPE.

Zoom In Zoom Out Reset image size

The electrode reaction, composite material (RuTiO₂/MWCNTs) catalytic behavior, and analyte response may influence by the variation in pH of the solution. Therefore, the electrochemical response of 0.1 mM CLZ was studied by CV technique, over the pH range of 3.0–11.2 in 0.2 M PBS (Figure 5). As the pH of PBS solution was increased, there was a less positive shifting of peak potentials, symptomatic of the participation of protons in the reaction and easy oxidation.⁴⁸ At pH 5.0, highest peak current was noticed for CLZ detection (Figure 5B). From the study, we plotted E_p (peak potential) versus pH of PBS. The redox peaks were increased gradually from pH 3.0 to 5.0, later on, decreased conversely till 11.2 pH. This can be due to the less number of protons, which can facilitate the process at high pH, and thus may diminish the density of peaks. Therefore, for further analytical studies pH, 5.0 was chosen. From the plot E_p versus pH (Figure 5A), acquired linear equation is as follows; E_p = −0.041 pH + 0.7247; R₂ = 0.978. The slope value indicates that in the CLZ oxidation protons and electrons were involved in equal number.^49,50

Zoom In Zoom Out Reset image size

The impact of scan rate study plays an important stand in the understanding of reaction mechanism and the process of sensor involved. Thus, the voltammograms of CLZ were recorded on the RuTiO₂/MWCNTs composite CPE surface at varying scan rates in pH 5.0 by CV (Figure 6). From the study, it was evidenced that the anodic peak current (I_p) was linearly increased with sweep rate (υ) enlargement (Figure 6A). The primary results of this study put emphasis on the linear correlation between the square root of sweep rate (υ^1/2) and the peak current, which demonstrates that the process may be a typical diffusion-controlled one: I_p (μA) = 52.45 υ^1/2 – 3.8012; R² = 0.967. Further, the slope of log I_p of anodic peak current vs. log υ plot results with a straight line and it's a slope was found to be 0.604 (Figure 6B), which proves that the present electrochemical reaction is a diffusion controlled process. The slope value is closest to the theoretical value 0.5 for a diffusion-controlled process^51,52 corresponding to the following equation: log I_p (μA) = 0.604 log υ + 1.697; R² = 0.988. In addition a good linear relation was observed between E_p and log ν (Figure 6C) with the regression equation: E_p (V) = 0.009 log υ + 0.5209; R² = 0.8067. Scan rate and Ep relationship for a process involved by the sensor can be stated by Laviron's theory.⁵³

Zoom In Zoom Out Reset image size

Where α is the transfer coefficient, k⁰ is the standard heterogeneous rate constant of the reaction; 'n' stands for a number of electrons transferred, 'υ' is the sweep rate and E⁰ stands for the formal redox potential. Other symbols have their usual meaning. According to Bard and Faulkner,⁵⁴ α can be calculated as,

Where E_p/2 is the potential, where the current is at half the peak value. So, from this we got the value of α to be 0.56. k⁰ was calculated to be 2.256 × 10³ s⁻¹, from the intercept of E_p versus log υ. Further, the number of the electron (n) transferred in the electro-oxidation of CLZ was calculated to be 2.

At pH 5.0, CLZ shows a sharp anodic peak on the surface of RuTiO₂/MWCNTs composite CPE. From the complete study, the participation of protons and electrons involved in the oxidation process of CLZ was determined, and it was found to be two. Therefore, the following scheme was anticipated as a probable electro-oxidation mechanism of CLZ on the modified sensing surface. The metabolites of CLZ⁺ cleavage and degradation of the piperazine ring assumed as the products P resulted in the electrochemical reaction (Scheme 2).

Scheme 2. Possible electrode reaction mechanism of Clozapine (CLZ).

Figure 7 displays the square wave voltammograms of CLZ at RuTiO₂/MWCNTs composite CPE over the concentration range of 0.01 μM to 0.25 μM in pH 5.0. To avoid the sizeable capacitive current and to get more improved resolution and sensitivity, SWV technique was inherited in this work.

Zoom In Zoom Out Reset image size

The linearity was observed in the range of 0.01 μM to 0.07 μM, with an equation: I_p (μA) = 194.12 C (μM)+1.149; R²  = 0.984. From the calculation, 0.057 nM of LOD and 0.19 nM of LOQ was found by using following equations 3 Sb/M and 10 Sb/M (S = blank standard deviation, M = slope) respectively.⁵⁵ The current research suggests highly sensitive method with low detection and quantification limit for CLZ compared to sensor performances of the earlier reported publications (Table II). The reason behind this is probably allied with the sensing surface effects, modifier behavior, high electrocatalytic effects of RuTiO₂/MWCNTs-CPE, recommending that the tailored electrode stood promising for lowest amount detection of CLZ.

Study of excipients scrutinized to investigate the behavior of CLZ in the presence of some general biological metabolites, which frequently coexist with CLZ in clinical as well as in real samples. For these investigations, the interfering species were added at various concentrations (a hundred fold) higher than the concentration of the analyte. Usually, our body environment contains these natural metabolites in high concentration compared to the concentration of the analyte. Therefore, here 0.1 mM CLZ was analyzed with some available and essential excipients (10 mM). The addition of filler materials (lactose, sucrose, glucose, and dextrose), organic species (ascorbic acid, citric acid, uric acid and tartaric acid) and adhesive, such as gum acacia, caused no significant effect on the SWV response for CLZ. The result indicates that the potential of the drug changed slightly but not exceed ± 5%, which suggests that CLZ reactions at the sensing base, does not affect the existence of any metabolites tested (Figure 8). Hence, the fabricated sensor can be capably utilized for CLZ detection.

Zoom In Zoom Out Reset image size

The samples for the analysis were prepared as discussed in Fabrication of the electrode subsection. The SWV method was inherited for the determination. The analytical results obtained are presented in Table III. The results explicitly specify that CLZ can be reliably assayed from its drug using the proposed sensor.

The samples for the analysis was prepared as discussed in the experimental subsection. By utilizing the calibration graph, recovery studies were carried out. For this, known amounts of pure CLZ were mixed with limited amounts of pre-analyzed formulation, and the mixtures were analyzed. The total amount of the drug was then determined, and the amount of added drug was calculated by difference. The values of recovery and RSD (Table IV) were observed to be acceptable.

Repeatability studies were carried out with the analyte (0.1 mM), to evaluate the efficiency of the tailored sensors steadiness. Fig. 9 depicts the SWV responses of CLZ oxidation in 0.2 M PBS containing 0.1 mM CLZ for 20 successive measurements. It can be seen that a small difference was observed in the oxidation current of the CLZ for the 20 measurements with the RSD of 1.76%. In addition, the oxidation peak current was retained about 92% from its initial current. This result endorsed that the developed sensor has the excellent stability and acceptable repeatability for the CLZ detection.

Zoom In Zoom Out Reset image size

In addition, the developed CLZ sensor was scrutinized for the consecutive weeks to assess the storage ability. It was showed the retention peak current about 91.6% after 4 weeks for the 20 measurements, which indicates that the developed RuTiO₂/MWCNTs-CPE sensor has excellent storage stability. Thus obtained results signify that the fabricated sensor is highly stable and reproducible in both the preparation procedure and determinations.