A surface-modified graphene – carbon-based composite sensor for the voltammetric assessment of pyridoxine in food and pharmaceutical samples

Abstract

A novel poly (glutamic acid)-modified graphene carbon paste sensor (PGMGCPS) was developed by the electro-polymerization technique for the analysis of Pyridoxine (PYX). Scanning electron microscopy (SEM), X-ray diffraction (XRD), Energy dispersive X-ray (EDX) and Electrochemical impedance spectroscopy (EIS) were employed to gain a deep insight into the surface modification and elemental analysis of the unmodified graphene carbon paste sensor (UGCPS) and PGMGCPS. The study of the impact of scan rate on the PYX analysis at the PGMGCPS surface revealed that the oxidation process is diffusion-controlled and at an optimum pH of 6.0 of PBS (phosphate buffer solution, 0.2 M) , the sensor demonstrated a detection limit (DL) of 0.09 × 10⁻⁷ M and 0.35 × 10⁻⁶ M through CV and DPV methods, respectively. A probe into the crucial attributes like repeatability, reproducibility, stability and concurrent analysing capacity signified the designed sensor as an efficient electrochemical tool to analyse PYX in food and pharmaceutical samples.

KEYWORDS:

• Pyridoxine
• cyclic voltammetry
• differential pulse voltammetry
• carbon paste electrode (CPE)
• graphene paste electrode (GPE)
• glutamic acid (GA)

1. Introduction

A healthy lifestyle emphasize the importance of a balanced diet which provides the necessary vitamins, nutrients and minerals for the maintenance of cells, tissues and other organs of the human body [1]. Vitamins are organic micronutrients with various biological roles in human nutritional and metabolic processes [2]. PYX, commonly known as Vitamin B₆, is one form of water-soluble Vitamin B which is converted into an active form called Pyridoxal 5′ – phosphate [3]. This active form has a role of a crucial cofactor in several enzymes-induced physiological processes like biosynthesis of sphingolipids, gluconeogenesis, neurotransmitters and metabolism of various amino acids [4]. PYX has various medical applications such as in the treatment of anaemia, pregnancy sickness and radiation sickness and for various pathological disorders [5,6]. PYX is an essential vitamin which is vital for the brain and immune system functioning, transportation of oxygen around the body, neurotransmitter production and assists in other physiological processes as well. PYX is supplied through both processed and unprocessed foods which are fortified to improve people’s health [7]. The growing interest has given rise to the development of many methods for the quantitative and qualitative analysis and detection of the PYX in various food and pharmaceutical samples [8]. The deficiency of PYX causes central nervous system-related serious diseases such as Parkinson’s and Schizophrenia [9].

The rising requirement and production of both known and novel derivatives with pharmaceutical action involves the call for developing and implementing simplistic and reliable methods for the analysis and detection of the naturally present and manufactured PYX derivatives. Spectrophotometric methods [10], HPLC (high-performance liquid chromatography) [11,12], RP-HPLC (reversed-phase high-performance liquid chromatography) [13–15], the chemiluminescent method [16], liquid chromatography [17], potentiometric methods [18] have been employed to determine PYX in various food and pharmaceutical samples. However, these techniques are expensive and laborious and hence cannot be applied for the determination and analysis of all compounds [19]. Among the extensively used analytical techniques, the electrochemical methods are almost certainly the most rapid, the most modest and those exhibit greatest performance [20–24]. The voltammetric methods like CV and DPV are very adaptable and reliable as they provide ample experimental details and insights into both the thermodynamic and kinetic aspects of various chemical systems [25–27]. In addition, these techniques are simple and sensitive and provide great results within a shorter analysis time than conventional methods in the detection of various biological and pharmaceutical samples [28,29].

Large surface area, high chemical and thermal stability, superior conductivity, high mechanical strength and biocompatibility of carbonaceous materials are attributed to their widespread applications in electro-analytical techniques [30]. The morphological structure of carbonaceous materials can be altered and elements contained in its chemical structure can be modified [31,32]. This can be achieved by chemical methods and the modification results in an amorphous carbon material with enhanced surface area with larger pores [33–36]. Graphene, an allotrope of carbon with a two-dimensional nanostructure network, possesses a large surface area with exceptional thermal conductivity [37,38]. These qualities contribute to the enhanced sensitivity of the sensors comprising graphene as electrode material, resulting in superior catalytic activity and improved kinetics of the electrochemical process [39,40].

This novel approach towards the effective determination and analysis of PYX involves the electro-polymerization of GA on the graphene-carbon composite electrode surface. The outcome of the various assessments proves the designed electrode to be affordable, efficient, reliable and practically sensitive towards PYX detection.

2. Materials and methods

2.1. Reagents and their procurement

PYX (99.9% pure) and glutamic acid (99% pure) were bought from Molychem, Mumbai, India. Graphite powder (94% pure), disodium hydrogen phosphate (purity of 99.5%,), monosodium dihydrogen phosphate (99% pure) and Silicone oil (90% pure) were obtained from Nice Chemicals, Cochin, India. Graphene nanoplatelets (6–8 nm thick, 5 µm wide) were procured from the Tokyo chemical industry, Tokyo, Japan. All the reagents and chemicals utilized in the analysis were of analytical reagent grade. The necessary solutions were freshly made by dissolving a calculated amount of chemicals in double distilled water (DDW). DDW required for the analysis was obtained from a VITSIL-VBSD/VBDD purification chamber.

2.2. Real sample preparation

PYX tablets bought from the local medical store were crushed and dissolved in DDW. Whatman filter paper was used to filter the solution and the filtrate obtained was centrifuged. The clear supernatant solution was used for the analysis.

Potatoes procured from the local market were peeled and then ground separately to extract the juice. The juice obtained was diluted with DDW and Whatman filter paper was used to filter the solution and the filtrate obtained was centrifuged. The clear supernatant solution was used for the analysis.

2.3. Instrumentation

Voltammetric measurements were recorded in a computer-equipped electrochemical analyzer, CHI-6038E (USA). This is configured with three electrodes, a calomel electrode (reference electrode), a platinum electrode (counter electrode) and a working electrode (PGMGCPS/ UGCPS). The surface characterizations of the sensors were performed using SEM, EDX and XRD instruments at the Research Laboratory, Vijnana Bhavan, University of Mysore.

2.4. Sensor fabrication

The preferred sensors were developed using the conventional and established approach.

UGCPS: The composite was prepared by hand mixing the graphene nanoplatelets and graphite powder. Silicone oil is added to the composite mixture as a binder and blended well in an agate mortar for around 15 min to obtain a well-mixed paste. This paste was meticulously pressed into the 3 mm groove of the Teflon tube and rubbed on a soft tissue paper to achieve an even surface. Electrical connectivity is established by the insertion of a copper wire through the Teflon tube.

PGMGCPS: The UGCPS was subjected to electro-polymerization with 1 mM GA in supporting electrolyte (pH 5.5, 0.2 M PBS) at 0.1 V/s scan rate by the CV method in the potential gap of −0.1 to 1.5 V for 20 segments (10 cycles), as depicted in Figure 1(a). The developed PGMGCPS surface was rinsed carefully with DDW to remove the unabsorbed GA if any. Before this, the number of cycles was optimized by recording CV for 5, 10, 15 and 20 cycles. The number of polymerization cycles highly influences the electrochemical conduct of PYX at the developed electrode surface. The result portrayed that the electro-polymerization for 10 cycles yielded the highest anodic peak current for PYX in pH 6.0 (Figure 1(b)). During the process, the monomer GA progressively forms a poly (GA) layer on UGCPS transforming it into a highly conductive polymer-based sensor (as depicted in Scheme 1). This conductive polymer layer shows a strong covalent and hydrophobic interaction with the UGCPS surface [41]. The formation of a poly (GA) layer on the amalgamated nano-structured graphene and the graphite powder yields a sensor which is highly porous with amplified electro-active sites and an enlarged electro-active surface area. These attributes of the proposed PGMGCPS contribute to excellent and impressive electrocatalytic activities.

Figure 1. CV response for (a) electro polymerization of GA on UGCPS at −0.2 to 1.5 V potential window (b) number of cycles versus peak current.

Scheme 1. Pictorial representation of poly (GA) layer on the PGMGCPS surface.

Bare carbon paste sensor (BCPS): BCPS was prepared by blending graphite powder and silicone oil in a ratio of 70:30 for 15 min in an agate mortar. The obtained homogeneous paste was filled into a Teflon tube, as mentioned in the above procedure (fabrication of UGCPS). The resulting sensor was used to compare the effectiveness of the sensor material.

3. Results and discussions

3.1. Influence of electrode material on oxidation peak current of PYX

The electrocatalytic performance of PYX was investigated at BCPS and UGCPS surfaces to compare the sensing capacity of the sensor material. Figure 2 symbolizes the CV recorded for 1.0 mM PYX in 0.2 M PBS at a scan rate of 0.1 V/s at BCPS (curve “a” with an oxidation peak current of 19.69 µA) and UGCPS (curve “b” with an oxidation peak current of 24.46 µA) surfaces. The UGCPS, a composite sensor material made of graphene nanoplatelets and graphite powder, exhibits higher current sensitivity than the BCPS made of graphite powder alone. This comparative analysis prompted us to select the composite as the sensor material and electro-polymerization of UGCPS further increased the efficacy and sensitivity for the analysis of PYX (discussed in Section 3.3).

Figure 2. CV output for PYX at BCPS (curve a) and UGCPS (curve b) at a scan rate of 0.1 V/s.

3.2. Morphological study of the electrode surface and active surface area calculation

SEM, XRD, EIS, EDX and CV methods were applied to investigate the morphology of the electrode surfaces and elemental composition and to calculate the active surface area of UGCPS and PGMGCPS.

SEM analysis: Figure 3(a) depicts the external structure of bare carbon paste, Figure 3(b,c) represents the SEM images which reveal the difference in the surface morphology of UGCPS and PGMGCPS, respectively. The unmodified sensor shows an even and thick film of carbon on the surface and the modified sensor surface displays the deposition of dense spongy-like structures. The variation in the surface morphology accounts for the proliferated surface area and the enhanced electro-catalytic performance.

Figure 3. SEM images of (a) carbon paste, (b) graphene-carbon paste composite (c) PGMGCPS.

XRD analysis: Figure 4 depicts the XRD images of the working electrode material which provides a profound insight into the crystal structure of the components. The analysis was performed at 5° to 80° and the UGCPS revealed peaks at 2θ = 26.6, 26.9, 32.05 and 54.64 at high intensity. These intense peaks symbolize the crystalline structure of graphite and graphene composite. Contrarily, the PGMGCPS showed diffraction patterns at 2θ = 26.5 and 32.08 at lower intensity. It is evident from the images that the diffraction patterns are asymmetric due to the scattering of the polymer matrix with slightly amorphous structures [42,43].

Figure 4. XRD images of UGCPS and PGMGCPS.

EDX analysis: The elemental composition of the working electrodes was analysed by the EDX technique. The outcome revealed that the UGCPS comprises carbon (C), oxygen (O) and silicon (Si), as represented in Figure 5(a). In Figure 5(b), the composition of PGMGCPS portrays the presence of an additional element Nitrogen (N) which is purely due to the poly (GA) layer. Hence, the analysis authenticates the successful electro-polymerization in attaining the desired PGMGCPS sensor.

Figure 5. EDX images of (a) UGCPS and (b) PGMGCPS.

EIS: EIS, an efficient method, was utilized to examine the electro-catalytic behaviour and conductivity of the working electrodes. The investigation was carried out at optimum conditions with K₄[Fe(CN)₆] (1.0 mM) in KCl (0.1 M, supporting electrolyte). The Nyquist plot was derived from the values obtained and an equivalent electric circuit, comprising salient parameters such as R_s (solution resistance), R_ct (charge transfer resistance), CPE (constant phase element) and W (Warburg impedance), was fitted according to the Nyquist plot (Figure 6(b)). The Nyquist plot displays the larger semicircle with a higher R_ct value (220 Ω) for UGCPS and the comparatively smaller semicircle with a lower R_ct value (165 Ω) for PGMGCPS and these data authenticate that the effective activation of the UGCPS surface via electro-polymerization (with GA) was successfully achieved. This modification elevated the electro-catalytic performance and conductive nature of PGMGCPS.

Figure 6. (a) CV output of 1 mM K₄ [Fe (CN)₆], curve a for UGCPS and curve b for PGMGCPS in 0.1 M KCl, (b) Nyquist plots, curve a for UGCPS and curve b for PGMGCPS with an equivalent electric circuit.

CV was recorded for 1.0 mM K₄[Fe (CN)₆] in 0.1 M KCl as a supporting electrolyte at the surface of UGCPS and PGMGCPS at a potential range of −0.2 V to 0.6 V (at 0.1 V/s scan rate). Figure 6(a) shows the current sensitivity for UGCPS (curve a) and PGMGCPS (curve b). The modified sensor exhibits a fine peak with high current sensitivity compared to the unmodified sensor. The electro-active surface area (A) of UGCPS and PGMGCPS was evaluated by employing the Randles-Sevcik equation, (1) $\begin{array}{r}{I}_{\text{pa}}=2.69\times {10}^5{n}^{3/2}A{D}^{1/2}C{\nu }^{1/2}\end{array}$(1) where “I_pa” is the peak current at the anode (µA), “n” represents the number of electrons, “D” is the diffusion co-efficient (7.6 × 10⁻⁶ cm²/s), C is the concentration of PYX (mM) and “ν” denotes the scan rate (V/s). The active surface area was 0.0112 cm² for UGCPS and 0.1027 cm² for PGMGCPS. The higher active surface area for the electro-chemically modified sensor confirms the GA polymer layer formation on UGCPS elevating the sensing power and resulting in superior electro-catalytic activity.

3.3. Electro-catalytic action of the sensors towards PYX

The electro-catalytic activities of UGCPS and PGMGCPS towards the oxidation of PYX were assessed using the CV technique. CV measurements were made for both the sensors in the presence of PYX in 0.2 M PBS (pH of 6.0) at 0.1 V/s scan rate and in the absence of PYX as well. Figure 7 shows that the PGMGCPS (curve a) generates a highly sensitive and elevated anodic peak current than that of the UGCPS (curve c) for PYX. PGMGCPS surface does not disclose any electrochemical behaviour for only PBS (without PYX, curve b). According to the obtained CV data, the greater electrochemical response of PYX with a faster rate of electron transfer at the PGMGCPS surface is due to the electrochemical modification which enhanced the active surface area resulting in high conductivity.

Figure 7. CV depiction of PYX in 0.2 M PBS (pH 6.0) at UGCPS (curve c) and at PGMGCPS (curve a), only PBS at PGMGCPS (curve b) at a scan rate of 0.1 V/s.

3.4. Influence of accumulation time

The accumulation time of PYX on the PGMGCPS surface greatly affects the electro-chemical activity and this was studied at optimal conditions. Figure 8(a,b) portrays the anodic peak current of 1.0 mM PYX versus the accumulation time which indicates the upliftment of the current sensitivity from 0 s (s) to 20 s and then the sensitivity decreases with time. The accumulation time of 20 s displays a very definite anodic peak with high current due to the saturation of PYX at the 20th second on the surface of the fabricated sensor. Further analysis of PYX at PGMGCPS was conducted by considering the accumulation time of 20 s.

Figure 8. (a) CV output of PYX (1.0 mM in PBS of pH 6.0) at PGMGCPS at varied time intervals (b) plot of anodic peak current versus accumulation time.

3.5. Study of the effect of pH on peak current

The pH of the PBS has a great influence on the electro-catalytic activities of the target molecules at the surface of the sensor. This was examined by recording the CVs for 0.1 mM PYX in PBS (0.2 M) in various pH solutions ranging from 5.5 to –8.0 (Figure 9(a)). This variation is due to the difference in the electrostatic attraction and repulsion between the PYX and the surface of the proposed sensor. Figure 9(c) depicts the E_pa versus pH graph, from which it is evident that E_pa values decline from 0.8199 to 0.5935 V as the pH is varied from 5.5 to –8.0. The linear regression equation (LRE) is represented as (2) $\begin{array}{r}{E}_{\text{pa}}\left(\text{V}\right)=1.2505\text{--}0.0825\text{pH}\left(\text{V}/\text{pH}\right),\left(R^2=0.9942\right)\end{array}$(2)

Figure 9. (a) CV for 1.0 mM PYX at PGMGCPS surface at different pH, (b) plot of I_pa versus pH, (c) E_pa versus pH graph.

The slope value of 0.0825 in the above equation is approximately closer to the theoretically existent slope value and this indicates that the oxidation of PYX on the surface of PGMGCPS involves an equal number of electrons and protons. The augmentation of the oxidation peak current is observed for pH 6.0 and at lower and higher pH the peak current degrades. Hence, pH 6.0 is considered the optimal pH for the analysis of PYX at the PGMGCPS surface.

3.6. Influence of potential sweep rate on anodic peak current

CVs recorded for various scan rates altered from 0.025 – 0.325 V/s for 1.0 mM PYX in pH 6.0 at the surface of PGMGCPS as depicted in Figure 10(a). The current peak at the anode (I_pa) enhances with the ascending potential sweep rates and the peak potential (E_pa) shifts towards the positive values. Figure 10(b) represents the linearity of I_pa with the square root of the scan rate and LRE can be represented as (3) $\begin{array}{r}{I}_{\text{pa}}\left(\mu \mathrm{A}\right)=9.56+1.86{\nu }^{1/2}\left(\text{V}/\text{s}\right),R^2=0.9967\end{array}$(3)

Figure 10. CV output of (a) anodic peak current versus peak potential at varying scan rate from 0.025 to 0.325 V/s (b) plot of I_pa versus ν^1/2 (c) plot of log I_pa versus log ν (d) graph of E_pa versus log ν.

The LRE for log I_pa versus log ν graph (Figure 10(c)) can be written as (4) $\begin{array}{r}\text{log }{I}_{\text{pa}}\left(\mu \mathrm{A}\right)=3.68+0.62\text{log}\nu \left(\text{V}/\text{s}\right),R^2=0.9988\end{array}$(4)

The R² value of 0.9967 (≈1) in Equation (3) and slope value of 0.62 (approximately close to 0.5) in Equation (4) reveals that the electron transfer process is diffusion-controlled kinetics. Figure 10(d) portrays the graph of E_pa versus log ν and LRE can be expressed as below (5) $\begin{array}{r}{E}_{\text{pa}}\left(\text{V}\right)=0.8458+0.1075\text{log}\nu \left(\text{V}/\text{s}\right),R^2=0.9793\end{array}$(5)

The slope obtained from Equation (5) was applied in Laviron’s equation [44] to evaluate the number of electrons involved in the electrochemical oxidation of PYX. (6) $\begin{array}{r}{E}_{\text{pa}}=E^0+\left[\frac{2.303\text{RT}}{\alpha \text{nF}}\right]\log \left[\frac{\text{RT}{k}^0}{\alpha \text{nF}}\right]+\left[\frac{2.303\text{RT}}{\alpha \text{nF}}\right]\log \nu \end{array}$(6) where “n” represents the electron count involved in the oxidation of PYX, “E⁰” denotes the standard redox potential, “α” denotes the charge transfer co-efficient, k⁰ is the heterogeneous rate constant (/s), “R” is the universal gas constant (8.314 J/mol/K), “T” is the absolute temperature (298 K), “E_pa^” is the peak potential, “ν” is the scan rate and “F” is the Faraday’s constant (96,485 C/mol). Upon substitution, the number of electrons “n” was 1.99 ≈ 2 and the possible electrochemical oxidation of PYX has been depicted in Scheme 2.

Scheme 2. Oxidation of PYX on the PGMGCPS surface.

Furthermore, the surface coverage area (Γ) of PGMGCPS was calculated using the below equation (7) $\begin{array}{r}Q=\text{nFA}\mathrm{\Gamma }\end{array}$(7) where “Q” is the charge availed from the area under the peak, “n” denotes the number of electrons, “F” represents Faraday’s constant and “A” is the electro-active surface area. The corresponding values were substituted in Equation (7) and the obtained surface coverage area was 10.12 ÅM cm⁻².

3.7. Limit of detection, limit of quantification and sensitivity

At optimal conditions, DL and QL of PYX of the modified and developed sensor were evaluated with DPV and CV methods. CV and DPV were recorded by increasing the concentration of PYX in 0.2 M PBS (pH 6.0), as represented in Figure 11(a,c), respectively. The linear relationship between varied concentrations and I_pa of PYX was studied and has been depicted in Figure 11(b,d). The LRE can be expressed as below (8) $\begin{array}{rl}{I}_{\text{pa}}\left(\text{A}\right)& =2.96\times {10}^{-5}+0.0127\left[\text{PYX}\right]\\ & \left(\text{M}\right)\left(R^2=0.9964\right)\text{by}\text{DPV}\end{array}$(8) (9) $\begin{array}{rl}{I}_{\text{pa}}\left(\text{A}\right)& =2.35\times {10}^{-5}+0.3458\left[\text{PYX}\right]\\ & \left(\text{M}\right)\left(R^2=0.9966\right)\text{by}\text{CV}\end{array}$(9)

Figure 11. Peak current versus peak potential for various concentrations of PYX ranging from 0.1 to 1.0 µM (a) CV response, (c) DPV response, (b) & (d) corresponding calibration curves.

The DL and QL values were calculated by the equations, DL = 3 S/N and QL = 10 S/N, where, “S” is the standard deviation of the blank and “N” is the slope of I_pa versus [PYX]. The DL and QL were 0.35 and 1.17 µM by DPV and 0.009 and 0.03 µM, respectively by CV. A comparison of the developed sensor with a few earlier reported works for PYX detection is tabulated in Table 1. From the available information, it can be concluded that the fabricated sensor in the current work exhibits an improved electro-catalytic response for PYX assessment by the DPV and CV method with a lower DL.

Table 1. Techniques, electrodes and DLs of reported works.

Technique/ Buffer	Electrode/Modifier	Linear range	Detection Limit	Reference
DPV/PBS	GCE/MWCNT	5 × 10^−7–1 × 10^−4 mol L^−1	2 × 10^−7 M	45
DPV/BRBS	diamond electrode doped with boron (unmodified)	7–47 µM	3.76 µM	46
CV/KCl	GCE/chromium(III) hexacyanoferrate(II)	1.33 × 10^−6–1.32 × 10^−5 mol L^−1	3.46 × 10^−7 mol L^−1	47
LSV/KCl	CPE/vanadyl(IV) salen complex	4.5 × 10^−4–3.3 × 10^−3 mol L^−1	3.7 × 10^−5 mol L^−1	48
CV/Acetate buffer	CPE/copper(II) hexacyanoferrate(III)	1.2 × 10^−6–6.9 × 10^−4 mol L^−1	4.1 × 10^−7 mol L^−1	49
DPV/PBS	CPE/iron oxide nanoparticle	8.88 µM to 1000.0 µM	9.06 µM	50
DPV/Acetate buffer	pencil graphite electrode (unmodified)	5 × 10^−6–10^−3 M	2.8 µM	51
DPV/PBS	carbon ceramic electrode/ MWCNT	2.5 × 10^−7–10 × 10^−5 M	95 nM	52
DPV/PBS	screen-printed graphite electrode/zinc ferrite nano-particles	0.8–585.0 µM	0.17 µM	53
SWV/PBS	polycrystalline gold electrode/copper nano-particles	0.3–2.7 µM	0.087 µM	54
DPV/BRBS	GCE/Ethylenediamine	0.02–0.40 µg/mL	7.00 ng/mL	55
DPV/Tris buffer	CPE/crown ethers	0.4 µg cm^−3	0.6–100 µg cm^−3	56
SWV/PBS	CPE/ZnO/CuO nanosheets, ionic liquids	8.0 × 10^−7 M	1.0 × 10^−6 to 1.2 × 10^−3 M	57
DPV/PBS	GCE/gold nanoparticles and MWCNT	0.53 µg mL^–1	1.59–102.74 µg mL^–1	58
CV/DPV /PBS	PGMGCPS	0.1 µM to 1.0 µM	0.009 µM/ 0.35 µM	proposed work

Notes: PBS – phosphate buffer solution; BRBS – Britton-Robinson buffer solution; MWCNT – multi-walled carbon nanotube; GCE – glassy carbon electrode; CPE – carbon paste electrode.

3.8. Concurrent analysis of PYX and Rutin (RTN)

DPV technique was employed to assess the selectivity of the PGMGCPS in the detection of PYX even in the presence of other molecules. The selectivity of both UGCPS and PGMGCPS was inspected and compared towards the oxidation of 1 mM PYX in the presence of 1 mM RTN. Both PYX and RTN vitamins are present in various fruits and vegetables. These molecules readily undergo electrochemical oxidation in the same potential window and hence exhibit excellent electrochemical activity when analysed simultaneously. It is evident from Figure 12(a) that UGCPS displays less sensitive anodic peaks for PYX and RTN. Contrarily, PGMGCPS reveals well-defined anodic oxidation peaks with relatively elevated current for both PYX and RTN. The DPV data demonstrates that the electro-catalytic performance of PGMGCPS in PYX analysis in the presence and absence of RTN is similar, thus validating the efficiency of PGMGCPS in selectively detecting PYX even in the presence of similar chemical species. Furthermore, the concentration of RTN varied from 0.7 µM to 1.0 µM (keeping the PYX concentration constant). The analysis (as depicted in Figure 12(b)) resulted in increased oxidation peak current as the RTN concentration was increased, thus authenticating the potentiality of the proposed sensor in PYX detection.

Figure 12. DPV depicting (a) simultaneous detection of PYX and RTN at the UGCPS and PGMGCPS surface in 0.2 PBS and (b) concentration variation of RTN with constant PYX.

3.9. Impact of interfering ions and vitamins

The impact of the interfering metal ions and other vitamins on the oxidation peak potential was examined. The investigation was carried out for 1 mM PYX in PBS (0.2 M, pH 6.0) at 0.1 V/s scan rate at the PGMGCPS surface. The outcome revealed that the peak potential values were affected and varied to some extent but did not exceed the ±5% limit which has been represented in Figure 13. This indicates that the presence of interfering molecules will not have an effect on PYX detection at the PGMGCPS surface.

Figure 13. CV response depicting the variation of potential versus interfering species.

3.10. Food and pharmaceutical sample analysis

For validating the applicability of PGMGCPS in real samples, the CV technique was applied for the detection of PYX in pharmaceutical and food samples. The PGMGCPS produced recoveries of approximately 97% (in food samples) and 95% to 97% (in pharmaceutical samples) through the spike addition method and the same has been represented in Table 2. These data reveal that PGMGCPS is an apt tool for PYX analysis in food and pharmaceutical samples as well, with an excellent recovery rate.

Table 2. Recovery rate of real samples.

Sample	Added [µM]	Found [µM]	Recovery [%]
PYX tablets	0.2	0.196	97.76
	0.3	0.287	95.65
	0.4	0.380	95.08
	0.5	0.476	95.25
Potato sample	0.2	0.194	97.11
	0.3	0.291	97.00
	0.4	0.399	97.46
	0.5	0.488	97.57
	0.6	0.583	97.13

3.11. Reproducibility, repeatability and stability of PGMGCPS

The reproducibility of the PGMGCPS was assessed by documenting CV with the same PYX in 0.2 M PBS with five developed sensors. A relative standard deviation (RSD) of 4.88% was exhibited by PGMGCPS, signifying superior reproducibility. Additionally, the repeatability of the projected sensor was assessed by recording CV with five different PYX in 0.2 M PBS with the same PGMGCPS. The sensor provided a relative standard deviation (RSD) of 4.60%, suggesting higher repeatability. The proposed sensor’s stability in PYX detection was done by recording CV for 30 cycles. The results reveal that 98.58% of the initial peak current was restored, hence validating the appreciable stability of the sensor.

4. Conclusion

The present work constitutes the development of a highly sensitive, selective, reliable, electrochemically polymerized sensor for the analysis of PYX. The proposed PGMGCPS in this work exhibits an enhanced electro-catalytic behaviour compared to the UGCPS in the PYX analysis employing CV and DPV techniques. The EIS study enabled the evaluation of the electro-active surface area to be 0.0112 cm² for UGCPS and 0.1027 cm² for PGMGCPS. The surface coverage area (Γ) was 10.12 ÅM cm⁻² and the augmentation in the electro-active surface area substantiates the increased electrochemical performance of PGMGCPS. An insight into the impact of pH and scan rate discloses the diffusion-controlled kinetics involving two electrons and two protons transfer in the oxidation of PYX in 0.2 M PBS. Furthermore, a low DL of 0.09 × 10⁻⁷ M and 0.35 × 10⁻⁶ M was obtained for PGMGCPS through CV and DPV methods, respectively, with good linearity. The developed sensor shows high reproducibility (RSD = 4.88%), repeatability (RSD = 4.60%) and stability (with 98.58% of the initial current restoration) making it an efficient device for the analysis of PYX in pharmaceutical and food samples with a convincing rate of recovery.

Ethical issues

No ethical issues for this work.

Acknowledgements

J.G. Manjunatha gratefully acknowledges the financial support from VGST, Bangalore under the Research project. No. KFIST(L2)/GRD-1020/2021-22/430. Sameh M. Osman gratefully acknowledges the financial support from Researchers Supporting Project Number (RSP2024R405), King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

J.G. Manjunatha gratefully acknowledges the financial support from VGST, Bangalore under the Research project. No. K-FIST (L2)/GRD-1020/2021-22/430. Sameh M. Osman gratefully acknowledges the financial support from Researchers Supporting Project Number (RSP2023R405), King Saud University, Riyadh, Saudi Arabia.