Methylene blue incorporated mesoporous silica microsphere based sensing scaffold for the selective voltammetric determination of riboflavin

Abstract

This study reports the simple, selective and sensitive voltammetric detection of riboflavin (RF) using methylene blue (MB) incorporated sulfonic acid functionalized mesoporous silica microspheres (MSM), represented as MB-SO₃H-MSM. MB-SO₃H-MSM is synthesized and characterized by spectroscopic and microscopic methods. This material is coated on a glassy carbon (GC) electrode (symbolized as GC/MB-SO₃H-MSM) to utilize it in electroanalytical applications. The electrochemical behavior of MB-SO₃H-MSM is established using the GC/MB-SO₃H-MSM electrode by cyclic voltammetry (CV) and electrochemical impedance spectroscopy techniques. The electrochemical behavior of RF at the GC/MB-SO₃H-MSM electrode is also studied by CV. Compared to bare GC and SO₃H-MSM coated GC, the GC/MB-SO₃H-MSM electrode shows favorable electron transfer kinetics as well as an enhanced and stable electrochemical response of RF. Furthermore CV and differential pulse voltammetry (DPV) are used for the quantitative determination of RF at the GC/MB-SO₃H-MSM electrode. The DPV response shows two linear calibration ranges of 10.0 nM to 15.0 μM and 15.0 to 50.0 μM. The detection limit based on the first linear calibration range is calculated as 5.0 nM with a sensitivity of 393.0 μA mM⁻¹ cm⁻². The fabricated sensing scaffold shows an excellent selectivity for RF over other soluble vitamins and interfering ions. The stability, reproducibility and determination of RF in pharmaceutical products are also demonstrated effectively.

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Introduction

Riboflavin (RF) or vitamin B₂, is a required compound for the normal metabolic activity of the human body.^1–3 The possession of two keto groups in the flavin ring of RF imparts its redox activity^4,5 which in turn supplements the energy demands of our body by mediating the conversion of vital nutrients like carbohydrates, fats and proteins into ATP.^1,2,6 Inadequacy of RF results in various physiological problems.^1,4,6 Bearing in mind, the nutritive importance of RF, daunting effects of its deficiency and prospective for constructive clinical use, a comprehensive varied practice to detect this vitamin is necessary. Several analytical techniques like HPLC,^7,8 spectrophotometry,⁹ fluorescence,¹⁰ capillary electrophoresis,¹¹ chemiluminescence,¹² have been used for RF detection. However the drawbacks of expensive instrumentation, complicated pretreatment steps, long analysis time and lower sensitivity, puts a limitation on the real time application of these techniques. In comparison to the above mentioned methods, the electrochemical methods are quite simpler, cheaper and less time consuming.^1,2,4,5 At present, several electrochemical methods using different modified electrodes have been employed to achieve this goal.^13–16 However, electrochemical determination of RF is severely interfered with by other vitamins and electroactive compounds.^6,14,17,18 Thus, there is still a necessity to explore new methods which can display selective and sensitive voltammetric detection of RF at neutral pH.

Mesoporous silica are inorganic silicates possessing hexagonal array of cylindrical pores having tunable diameter in the range of 2–12 nm. They have high surface area, large pore volume, well defined pore radius distribution and high adsorption properties.^19–22 On account of the above properties which results in structural and functional versatility, these mesoporous silica materials serve a wide range of applications in separation technology,²⁰ drug delivery,²¹ electrochemical sensors,²² heterogeneous catalysis,²³ optics,²⁴ etc. However, the chief interest of the chemist lies in to make the maximum possible utilization of material. This craving has led to the functionalization of the pores of these materials by appropriate chemical reagents which generate desirable functional groups within the cylindrical framework of the pores.^19,25–27 These functionalized mesoporous materials are endowed with mechanically stable inorganic framework, chemical reactivity of specific functional group and active sites for ion exchange.^19,25–27 All these advantageous properties of functionalized mesoporous material can be effectively brought into use to incorporate desired species into them and utilize the resulting material for various applications.^19–24 On the other hand, methylene blue (MB) is a cationic dye possessing a heterocyclic ring skeleton and extensively used as electron mediator in several reactions^28,29 due to its well-defined electrochemical properties. Various research groups have reported the incorporation of MB within different matrixes like mesoporous silica,³⁰ zeolites,³¹ zirconia–silica composite,³² sol–gel ceramic film,³³ Nafion,³⁴ graphene,³⁵ etc. and used for the fabrication of the chemically modified electrodes.

The ion-exchange capacity and porous nature of sulfonic acid functionalized silica material can strongly bind the cationic MB dye by electrostatic interaction as well as by adsorption. Therefore, the resulting hybrid MB-SO₃H-MSM material must be enriched with the properties of the mesoporous silica material as well as the electrochemical properties of the MB and can be effectively utilized for the voltammetric determination of RF. In this context, the present work reports the synthesis of MB ion-exchanged on to the sulphonic acid functionalized mesoporous silica material (MB-SO₃H-MSM) and its subsequent utilization in the electrochemical determination of RF. To the best of our knowledge, this is the first report on selective voltammetric determination of RF by MB immobilized matrix.

Experimental

Chemicals and reagents

Riboflavin (RF) and poly(vinyl alcohol) were obtained from S.D. Fine Chemicals, Mumbai, India. Tetraethoxysilane (TEOS > 98%) and 3-mercaptopropyltrimethoxysilane (MPTMS), were bought from Sigma-Aldrich (India). Cetyl trimethylammonium bromide (CTAB) was procured from Himedia (India). NH₄OH (25%), potassium ferrocyanide and potassium ferricyanide were procured from Qualigens (Mumbai, India). Phosphate buffer solutions (PBS) (0.1 M, pH 5.0 to 8.0) were prepared from KH₂PO₄ and K₂HPO₄ (Qualigens). For pH below 5.0 and above 8.0, small amount of concentrated HCl or NaOH was added to adjust the pH. Triple distilled water was used to prepare all the solutions. All other reagents were of analytical grade and used as received.

Preparation of sulfonic acid functionalized mesoporous silica microspheres

The sulfonic acid functionalized mesoporous silica microspheres (SO₃H-MSM) was synthesized according to the procedure described in literature.^19,25,36 Briefly, to an aqueous dispersion of CTAB, ethanol and ammonia were added followed by the addition of a mixture of TEOS and MPTS (80 : 20 molar ratios) under vigorous stirring condition and stirring was continued for about 2 h at room temperature. The obtained white solid product was filtered, washed thoroughly with water and ethanol, and then vacuum dried for 24 h. The removal of surfactant was done by acid extraction method.^19,25 Further the conversion of this above synthesized thiol group bearing mesoporous silica spheres (SH-MSM) to SO₃H-MSM was carried out by adding an oxidizing solution of H₂O₂ in ethanol^25,36 under stirring condition. The suspension was stirred for next 24 h and then the product was obtained by filtration followed by washing with water and ethanol. In order to protonate the sulphonate group, the obtained product (as such in wet condition) was then added to 0.1 M H₂SO₄ solution and further stirred for another 4 h. Subsequently, the final solid product was filtered, washed and vacuum dried at 60 °C for about 12 h.

Incorporation of MB into SO₃H-MSM

MB incorporated SO₃H-MSM (i.e. MB-SO₃H-MSM) was prepared by simple ion-exchange between the cationic MB and anionic SO₃H-MSM employing magnetic stirring process.^37,38 A mixture of 0.25 g of SO₃H-MSM dispersed in 50 mL of 0.5 mM aqueous solution of MB was kept under magnetic stirring for 24 h. Then the resulting blue product was filtered, washed with copious amount of water to remove any unadsorbed MB and then vacuum dried at 50 °C for 12 h. A schematic representation to illustrate the whole synthesis process of MB-SO₃H-MSM has been displayed in Fig. 1.

Fig. 1 Schematic representation of MB-SO₃H-MSM synthetic route.

Instrumentation

FT-IR spectroscopic examination of the samples was carried out using Perkin Elmer Spectrometer (spectrum two) and KBr pellet method was employed. UV-vis absorption spectrum of SO₃H-MSM and MB-SO₃H-MSM was recorded using UV-vis spectrophotometer (model 2802PC, Unico) and 1 wt% aqueous colloid of respective material was used. Powder X-ray diffraction (XRD) studies were performed on Bruker D8 Advance X-ray diffractometer using Cu-Kα radiation (λ = 0.15406 nm). Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy analysis was performed with SEM (VEGA 3 TESCAN) with EDX (Bruker, Nano Gmbh), operating at 10 kV. Entire electrochemical measurements were done using CH electrochemical workstation (Model CHI-660 C, USA). A standard three electrodes set up, where GC electrode (geometric area = 0.07 cm²) as working electrode, Pt wire as counter electrode and saturated calomel electrode (SCE) as reference electrode were used. All the electrochemical measurements were carried out at laboratory temperature and prior to each experiment N₂ gas is purged in to the supporting electrolyte for 20 min to remove dissolved oxygen.

Fabrication of modified electrodes

Glassy carbon (GC) electrode was polished with alumina on Buehler-felt pad, washed with water followed by ethanol and ultrasonicated for 5 min in water to obtain a mirror clean surface. A 2 wt% aqueous colloidal dispersion of MB-SO₃H-MSM or SO₃H-MSM was prepared with 0.01% poly(vinyl alcohol). A 10.0 μL of this aqueous colloidal dispersion was coated on the clean GC electrode which was air dried for 2 h and then employed for electrochemical studies. The resulting electrodes are represented as GC/MB-SO₃H-MSM or GC/SO₃H-MSM.

Preparation of pharmaceutical samples

A multivitamin tablet (Beplex Forte manufactured by Anglo-French drugs and industries Ltd, India, containing vitamin B₁, B₂, B₃, B₅, B₆, B₉, B₁₂, biotin and ascorbic acid) and vitamin B complex injection (Optineuron manufactured by Lupin pharmaceuticals Ltd, India, containing vitamin B₁, B₃, B₅, B₆, B₉ and B₁₂) were obtained from the local pharmaceutical store. A single tablet was grinded to fine powder using a pestle and mortar. The powder was quantitatively transferred into volumetric flask containing 10 mL of PBS (pH 7.0). The content of the volumetric flask was sonicated in ultrasonic bath for 10 min to dissolve the sample completely and then the solution was filtered. The filtered solution was further diluted with PBS to the required concentration and used for the analysis without any further treatment. Similarly, 1.0 mL of the injection solution was first mixed with 10 mL of PBS (pH 7.0) and sonicated in ultrasonic bath for 10 min to mix the sample completely. Then the solution was further diluted with PBS to obtain a solution of desired concentration and used for the analysis. The recovery analysis was performed by adding two known concentration of standard RF into the electrochemical cells containing the aliquot of pharmaceutical products.

Results and discussion

Characterization of MB-SO₃H-MSM

The incorporation of MB into the MSM material is confirmed by spectroscopy techniques (FT-IR and UV-vis), X-ray diffraction (XRD) patterns and microscopic analysis (SEM). Fig. 2A shows the FT-IR spectra of SO₃H-MSM and MB-SO₃H-MSM. The FT-IR spectrum of SO₃H-MSM (Fig. 2A(a)) shows a prominent, broad absorption band at 3435 cm⁻¹ due to Si–OH stretching vibration.³⁹ The bands at 2850 and 2929 cm⁻¹ are attributed to the symmetric and asymmetric stretching vibrations of –CH₃ group, respectively. The bending vibrations of hydrogen bonded –OH groups are related to the band observed at 1643 cm⁻¹. Asymmetric stretching mode of SO₂ vibration is observed at 1348 cm⁻¹.³⁹ The bands at 1080 and 1160 cm⁻¹ corresponds to symmetric stretching vibration of –SO₃H group and asymmetric vibration of S=O, respectively.³⁹ Si–OH vibrations can be revealed by the band at 964 cm⁻¹.⁴⁰ Except these characteristic bands of SO₃H-MSM, the FT-IR spectrum of MB-SO₃H-MSM exhibits a sharp band at 1635 cm⁻¹ which is related to the vibrations of the =N⁺ (immonium) group.⁴¹ A small shoulder at 1182 and 1598 cm⁻¹ could be assigned to the vibrations of the heterocyclic skeleton of the MB dye.⁴¹ Furthermore, the characteristic FT-IR bands of SO₃H-MSM in MB-SO₃H-MSM are found to be less intense with little shift in band position. Thus, shifting of certain peaks and emergence of new peaks at 1182 cm⁻¹ and 1598 cm⁻¹ in the FT-IR spectra of MB-SO₃H-MSM ascertains the successful incorporation of MB dye in sulfonic acid functionalized mesoporous silica material. The incorporation of MB in SO₃H-MSM material is further confirmed by UV-vis absorption study. Fig. 2B depicts the UV-vis absorption spectra of aqueous colloidal dispersion of SO₃H-MSM (spectra a) and MB-SO₃H-MSM (spectra b). Absence of any absorption peak in the spectrum of SO₃H-MSM and existence of three distinctive peaks in the spectrum of MB-SO₃H-MSM strongly supports the incorporation of MB dye in SO₃H-MSM. Two prominent peaks at 620 and 678 nm and a hump at 762 nm are observed in the spectrum of MB-SO₃H-MSM. The observed peaks correspond to dimer, monomer and protonated forms of the MB, respectively.^42,43 The low angle XRD patterns of SO₃H-MSM and MB-SO₃H-MSM in the 2θ range of 2–10° are depicted in Fig. S1 (ESI†). The low angle XRD patterns of SO₃H-MSM shows a clear peak at 2θ value of 3° which corresponds to the reflection from the d₁₀₀ plane of the sulfonic acid functionalised mesoporous silica.^25,44 Upon incorporation of MB, reduction in the peak intensity is observed which can be attributed to the reduction in the scattering of the X-rays due to the pores occupied by the dye.^45,46 Along with the low angle XRD, high angle XRD spectra (15 to 80°) of MB-SO₃H-MSM (inset of Fig. S1†) also shows a decrease in characteristic peak intensity (at 22°) of amorphous silica. The lowering down of the XRD peak intensities indicates decline in the ordered arrangement of the mesoscopic channels after the incorporation of MB.²⁵ Above XRD results also indicate that the structure of mesoporous silica is well retained even after incorporation of MB. Fig. 3 shows the SEM images and EDAX spectra of SO₃H-MSM and MB-SO₃H-MSM. The SEM image of SO₃H-MSM (Fig. 3a) shows a relatively uniform distribution of regular spherical shaped particles of an average diameter around 400 nm. Whereas the surface morphology of MB-SO₃H-MSM (Fig. 3b) does not change appreciably after the incorporation of MB on the SO₃H-MSM, however, slightly irregular thicker spheres are observed, which indicates the incorporation of MB. To confirm the presence of MB, the chemical compositions of SO₃H-MSM and MB-SO₃H-MSM were further analysed by EDAX. The microanalytical data of EDAX analysis clearly depicts the presence of elements Si, O and S in both SO₃H-MSM (Fig. 3c) and MB-SO₃H-MSM (Fig. 3d) whereas the presence of N only in MB-SO₃H-MSM assures the encapsulation of MB dye in SO₃H-MSM. The justification for encapsulation of MB dye within MSM-SO₃H is further ascertained by the corresponding elemental (S and N only) mapping image of SO₃H-MSM and MB-SO₃H-MSM shown in inset of Fig. 3c and d. The elemental mapping of SO₃H-MSM shows the presence of only S whereas that of MB-SO₃H-MSM shows both S and N which again indicates the successful incorporation of MB.

Fig. 2 FT-IR (A) and UV-vis absorption spectra (B) of SO₃H-MSM (a) and MB-SO₃H-MSM (b).

Fig. 3 SEM (a and b) and EDX spectra (c and d) of SO₃H-MSM (a and c) and MB-SO₃H-MSM (b and d). Inset of (c) and (d) shows the corresponding elemental mapping.

Electrochemical characteristics of MB-SO₃H-MSM

The electrochemical characteristics of MB-SO₃H-MSM is analysed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The CV curves of GC/SO₃H-MSM and GC/MB-SO₃H-MSM recorded in 0.1 M pH 7.0 PBS at a scan rate 20 mV s⁻¹ are depicted in Fig. 4A. No oxidation or reduction peak is observed at GC/SO₃H-MSM electrode (Fig. 4A, curve a), whereas, an evident cathodic and anodic peak appear at the GC/MB-SO₃H-MSM electrode (Fig. 4A, curve b). The observed cathodic peak at −0.26 V and the anodic peak at −0.22 V are due to the redox behaviour of the incorporated MB.^42,47 This redox peak current and peak potential of MB does not show any significant changes on continuous scan of over 25 cycles (ESI, Fig. S2†). Thus, the appearance of well-defined stable reversible redox peaks of MB at GC/MB-SO₃H-MSM electrode assures that, the cationic MB dye is strongly incorporated on the negatively charged SO₃H-MSM. The GC/MB-SO₃H-MSM electrode gave an E_1/2 value of −0.24 V with a ΔE_p value of 40 mV (20 mV s⁻¹). The ratio of I_pa/I_pc was found to be 0.56 (i.e. <1) which indicates that the dye in its reduced form is more stable within the mesoporous silica material.⁴² The CV curves of GC/MB-SO₃H-MSM electrode at different scan rates in 0.1 M pH 7.0 PBS are shown in Fig. 4B. A persistent increase in redox peak currents with the square root of scan rates (inset of Fig. 4B) is observed, which indicates a diffusion controlled electron transfer process at the GC/MB-SO₃H-MSM electrode.⁴² Furthermore, in order to examine the ease of electron transfer process at the GC/SO₃H-MSM and GC/MB-SO₃H-MSM electrodes an effective technique, electrochemical impedance spectroscopy (EIS) is utilized.^1,4 EIS is a highly sensitive technique for characterizing the surface properties of the modified electrodes. Fig. 5 shows Nyquist plots of GC/MB-SO₃H-MSM and GC/SO₃H-MSM electrodes (with and without the encapsulation of MB dyes within the SO₃H-MSM material) in 5.0 mM [Fe(CN)₆]^3−/4− (1 : 1 molar ratio) as redox probe containing 0.1 M KCl. The diameter of the semicircle obtained from the Nyquist plot corresponds to the interfacial electron transfer resistance (R_ct) of the material coated on the electrode surface. It is clearly understandable that the R_ct value of MB-SO₃H-MSM (237 Ω, curve b, Fig. 5) is less than the R_ct value of SO₃H-MSM (397 Ω, curve a, Fig. 5). This leads to the conclusion that the dye encapsulated material has enhanced conductivity as compared to the SO₃H-MSM.^1,4,48 The CV responses of 5.0 mM [Fe(CN)₆]^3−/4− (1 : 1 molar ratio) containing 0.1 M KCl are also recorded at GC/MB-SO₃H-MSM and GC/SO₃H-MSM electrodes and depicted in inset of Fig. 5. At GC/SO₃H-MSM electrode a pair of redox peaks with a peak separation (ΔE_p) of 127 mV is observed. Whereas, the GC/MB-SO₃H-MSM electrode displays an enhanced response of [Fe(CN)₆]^3−/4− redox peaks with a 88 mV of peak potential separation. This decrease of peak separation and enhanced response for [Fe(CN)₆]^3−/4− at GC/MB-SO₃H-MSM electrode is result from the higher electrical conductivity of MB-SO₃H-MSM.⁴ Thus these results (EIS and CV) collectively indicate that the incorporation of dye within sulfonic acid functionalized mesoporous silica material greatly boosts the conductivity thereby increasing the electron transfer kinetics.

Fig. 4 (A) CV curves for GC/SO₃H-MSM (a) and GC/MB-SO₃H-MSM (b) in 0.1 M pH 7.0 PBS at a scan rate of 20 mV s⁻¹. (B) CV curves for GC/MB-SO₃H-MSM at different scan rates (a → m: 10, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500 mV s⁻¹) in 0.1 M pH 7.0 PBS. Inset shows the plot of redox peak currents vs. square root of the scan rates.

Electrochemical behavior of RF at different electrodes

Fig. 6A compares the CV curves at bare GC, GC/SO₃H-MSM and GC/MB-SO₃H-MSM electrodes in absence and presence of 0.5 mM RF in 0.1 M pH 7.0 PBS at a scan rate of 20 mV s⁻¹. In the absence of RF (inset of Fig. 6A), no peak is observed at the bare GC (Fig. 6A(a′)) and GC/SO₃H-MSM (Fig. 6A(b′)) electrodes whereas the GC/MB-SO₃H-MSM (Fig. 6A(c′)) shows distinct redox peaks which can be attributed to the cathodic and anodic peak of the MB as discussed in Section 3.2 (vide supra). In the presence of RF (Fig. 6A), bare GC (Fig. 6A(a)) shows clear redox peaks at −0.49 V and −0.46 V, due to the reduction and oxidation of RF respectively. With respect to bare GC, an improved redox response for RF is observed at GC/SO₃H-MSM (Fig. 6A(b)) electrode. Slight increase in the anodic peak current and marked increase (nearly 2 folds) in cathodic peak current of RF is witnessed with a 20 mV positive shift in cathodic potential. This enhancement of the cathodic peak current with positive shift in potential at GC/SO₃H-MSM is probably due to the favorable interaction (adsorption) between the SO₃H-MSM and RF which leads to increased concentration of RF in the vicinity of the electrode surface. Further, the marked enhancement in cathodic peak current (nearly 5 folds in comparison to bare GC and 2.5 folds in comparison to GC/SO₃H-MSM electrode) with decrease of anodic peak current is observed at the GC/MB-SO₃H-MSM electrode (curve c). This large increase in cathodic current with decrease of anodic current at GC/MB-SO₃H-MSM electrode could be attributed to (i) excellent electron transport property of MB-SO₃H-MSM which promotes electron transfer of RF and (ii) synergistic effect imparted from the interaction of SO₃H-MSM and RF. In addition during the oxidative scan, in presence of RF (curve c of Fig. 6A), the observed MB peak current (at −0.22 V) is larger than the observed peak current of MB when RF is absent (curve c′ of Fig. 6A inset). This increased oxidation peak current of MB, in presence of RF may be attributed to the catalytic oxidation of reduced MB by RF, however it needs further study. In order to check the efficient reduction of RF at GC/MB-SO₃H-MSM electrode, CV measurement for the continuous incremental addition of RF is performed. Fig. 6B shows the CV curves for different concentrations of RF at GC/MB-SO₃H-MSM electrode containing 0.1 M pH 7.0 PBS. For each addition of RF the cathodic peak current at −0.49 V increases. Two linear ranges with RF concentration ranging from 0.5 to 50.0 μM and other from 50.0 to 2000 μM (inset of Fig. 6B) are obtained.

Fig. 5 Nyquist plots for 5.0 mM Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ (1 : 1 molar ratio) in 0.1 M KCl at GC/SO₃H-MSM (a) and GC/MB-SO₃H-MSM (b) electrodes. Frequency range: 100 kHz to 0.1 Hz, amplitude: 5 mV and applied potential: 0.15 V. Inset (i) shows the CV curves of GC/SO₃H-MSM (a) and GC/MB-SO₃H-MSM (b) electrodes in 5.0 mM Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ (1 : 1 molar ratio) containing 0.1 M KCl. Inset (ii) shows the bar graph for R_ct value at GC/SO₃H-MSM and GC/MB-SO₃H-MSM electrodes.

Fig. 6 (A) CV curves at bare GC (a), GC/SO₃H-MSM (b) and GC/MB-SO₃H-MSM (c) electrodes in 0.1 M pH 7.0 PBS containing 0.5 mM RF at a scan rate of 20 mV s⁻¹. Inset shows the CV curves in absence of RF at the bare GC (a′), GC/SO₃H-MSM (b′) and GC/MB-SO₃H-MSM (c′) electrodes. (B) CV curves for different additions of RF at GC/MB-SO₃H-MSM in 0.1 M pH 7.0 PBS at scan rate 20 mV s⁻¹. Inset shows the corresponding calibration curve.

Effect of scan rate and pH

Fig. 7A shows the CV curves of 0.5 mM RF in 0.1 M PBS (pH 7.0) at GC/MB-SO₃H-MSM electrode in the scan rates range 10–500 mV s⁻¹. It can be clearly seen that, the redox peak current increases progressively with the increase in scan rate. The graph of reduction peak current of RF (at −0.49 V) vs. square root of scan rate is shown in the inset of Fig. 7A. A linear plot between cathodic peak current and the square root of scan rate with a regression equation I_p (μA) = 1.02 ν^1/2 + 2.77 with R value = 0.998, suggests a diffusion controlled electrode reaction for RF at the GC/MB-SO₃H-MSM electrode.^2,4,48 Further, the impact of pH on the electrochemical reduction of RF (0.5 mM) at the GC/MB-SO₃H-MSM electrode is tested by recording CV response in solutions having different pH values ranging from 3.0–9.0 (ESI, Fig. S3†). Fig. 7B shows the plot of both peak potential and peak current against the pH values. It can be seen that when the pH is increased from 3 to 9, peak position of RF at the GC/MB-SO₃H-MSM electrode is shifted negatively and shows a linear behavior (Fig. 7B, round filled).⁴ The slope is calculated as 49.5 mV pH⁻¹, which is closer to the expected Nernstian value of 58.6 mV pH^−1 49 for the equal number of electrons and protons involved reduction of RF (eqn (1)).^50,51

Fig. 7 (A) CV curves in presence of 0.5 mM RF at GC/MB-SO₃H-MSM in 0.1 M pH 7.0 PBS at different scan rates (10–500 mV s⁻¹). Inset shows the plot between the cathodic peak current (I_pc) vs. square root of scan rate (ν^1/2). (B) Effect of solution pH on oxidation potential (solid line, round filled, blue color) and electrocatalytic peak current (solid line, square filled, black color) of 0.5 mM RF at GC/MB-SO₃H-MSM electrode.

Further, this result also shows that the electrochemical reduction current of RF is strongly influenced by the pH of the medium. As depicted in Fig. 7B (square filled), the reduction peak current of RF increases with the pH up to 7.0 and then decreases with further increase in pH. The maximum electrochemical response of RF is obtained at pH 7.0, therefore PBS with pH 7.0 is selected for the voltammetric determination of RF.

Differential pulse voltammetric determination of RF

Differential pulse voltammetry (DPV) measurements are carried out for the sensitive determination of RF in the concentration range of 10.0 nM to 50.0 μM at GC/MB-SO₃H-MSM electrode and shown in Fig. 8A. A well-defined cathodic peak at −0.42 V is observed and on increasing RF concentration the peak current increases with a slight shift in peak potential. Plot of peak current versus RF concentration shows (Fig. 8B) two linear ranges, viz. 10.0 nM to 15.0 μM and 15.0 to 50.0 μM with correlation coefficients of 0.988 and 0.997 respectively. Dual linear range in the determination of RF, may be due to the adsorption of the reduction products of RF at the electrode surface causing fouling of the surface thereby diminishing the electron transfer process at high concentrations of RF.²² Inset of Fig. 8B shows the enlarged view of first linear range from which the detection limit (based on 3S of blank) and sensitivity of the modified electrode towards RF determination is calculated to be 5.0 nM and 393.0 μA mM⁻¹ cm⁻², respectively. Wide linear range, low detection limit and reduction potential at the GC/MB-SO₃H-MSM electrode for RF determination are very much comparable or superior to other reported electrodes for RF determination (Table 1).^{1–6,13–15,50,51}

Fig. 8 (A) DPV curves for the addition of RF at the GC/MB-SO₃H-MSM electrode in N₂ saturated 0.1 M pH 7.0 PBS (0.01–50.0 μM RF). Inset shows the enlarged DPV curves corresponding to the addition from 0.01–1.0 μM. (B) shows the calibration plot corresponding to the addition from 0.01–50.0 μM of RF. Inset shows the calibration plot for addition from 0.01–15.0 μM.

Table 1 Comparison of analytical parameters of RF determination at GC/MB-SO₃H-MSM electrode with some other reported modified electrodes

Method^a	Sensing platform^b	pH, medium^c	Linear range (μM)	Detection limit (nM)	Reference
a Method: DPV = differential pulse voltammetry, CV = cyclic voltammetry, ASDPV = anodic stripping differential pulse voltammetry, SWV = square wave voltammetry, SWAdSV = square-wave adsorptive stripping voltammetry.b Sensing platform: OLA–NiO/MWCNTs GC = oleylamine nickel oxide multiwalled carbon nanotubes, rMoS2–graphene/A32/Au = reduced molybdenum disulfide graphene 32-mer homoadenine ssDNA oligonucleotides modified gold electrode, nano-Cr–SnO2 modified GC = nano chromium tin oxide modified glassy carbon electrode, CILE = carbon ionic liquid electrode, PEDOT/ZrO2NPs/GCE = poly(3,4-ethylenedioxythiophene)/zirconia nanoparticles modified glassy carbon electrode, ds-DNA-modified PGE = double stranded deoxyribonucleic acid modified pencil graphite electrode, sparked-BiSPEs = sparked bismuth oxide screen-printed electrode, BiFE = bismuth film electrode, α-Fe2O3/MWCNT/AuNPs modified GC electrode = α iron oxide multiwalled carbon nanotube gold nanoparticle modified glassy carbon electrode, GC/MB-SO3H-MSM = glassy carbon electrode modified with methylene blue incorporated sulfonic acid functionalized mesoporous silica material.c Medium: PBS = phosphate buffer solution.
DPV	OLA–NiO/MWCNTs GC	7.0, PBS	0.009–55.9	1	1
DPV	rMoS2–graphene/A32/Au electrode		0.025–2.25	20	2
DPV	Nano-Cr–SnO2 modified GC electrode	5.0, PBS	0.2–100	107	4
DPV	CILE	2.0, Na2SO4	0.008–0.11	0.1	5
			0.11–1.0
DPV	PEDOT/ZrO2NPs/GCE	7.0, PBS	0.05–300	12	6
CV	Co^2+-Y/CPE	5.0, KNO3	1.7–34.0	710	13
DPV	ds-DNA-modified PGE	7.0, PBS	0.5–7.0	360	14
SWV	Sparked-BiSPEs	4.5, acetate buffer	0.001–0.1	0.7	15
SWAdSV	BiFE	4.0, acetate buffer	0.3–0.8	100	50
			1.0–9.0
SWV	α-Fe2O3/MWCNT/AuNPs modified GC electrode	7.0, PBS	0.3–60.0	6	51
DPV	GC/MB-SO3H-MSM	7.0, PBS	0.01–50	5	This work

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Selectivity, stability and reproducibility

The selectivity and anti-interference benefits of the sensing scaffold, GC/MB-SO₃H-MSM are examined through DPV. The DPV responses of 10.0 μM of RF in presence of 50 fold excess concentration of possible interference (ascorbic acid, Fe₂SO₄, MgCl₂, glucose, uric acid, glycine NaCl, alanine, vitamin B₁, vitamin B₃, vitamin B₆ and vitamin B₉) are obtained in N₂-saturated 0.1 M PBS (pH 7.0). Fig. 9 shows the relative value of current at the GC/MB-SO₃H-MSM sensing scaffold containing 10 μM RF in 0.1 M pH 7.0 PBS in the presence of 50 times excess of above mentioned interferences. It is clearly depictable that the response to the interferences is less than ±10% thereby indicating excellent tolerance behavior and selectivity of GC/MB-SO₃H-MSM electrode for the determination of RF over common interferences. The operational stability and antifouling properties of the GC/MB-SO₃H-MSM electrode for RF reduction is checked by studying the change in current response after running 25 continuous cycles in 0.5 mM RF (ESI, Fig. S4†). After running 25 continuous cycles a loss of approximately 4% in the current signal was noticed and retaining 96% of the initial current response thereby indicating efficient operational stability and antifouling properties of the GC/MB-SO₃H-MSM electrode for RF reduction. The storage stability of the GC/MB-SO₃H-MSM electrode (when stored in air and 0.1 M pH 7.0 PBS separately) is checked by taking the responses of the electrode in presence of 0.5 mM RF. After storage, the electrode is reused on 15^th, 30^th and 45^th days respectively. The storage stability responses are depicted in ESI, Fig. S5† which clearly shows that the GC/MB-SO₃H-MSM sensing scaffold displays good storage stability in both air as well as in 0.1 M PBS. A slightly more stable response is observed when the electrode is store in air as compared in PBS. This may be attributed due to the slow leaching of MB dye in PBS, which results in lower current response. The reproducibility of the RF response is examined by taking ten individual GC/MB-SO₃H-MSM electrodes prepared by same procedure (ESI, Fig. S6†). The average current response for 0.5 mM RF at these ten freshly prepared GC/MB-SO₃H-MSM electrodes is calculated to be −9.39 μA with a relative standard deviation (RSD) of 1.76%, signifying an effective reproducibility of the fabricated electrode. The reproducibility of the measurement at various concentration of RF is also performed by taking three parallel measurements with three independent GC/MB-SO₃H-MSM electrodes. The calibration curve shown in Fig. 8 (vide supra) correspond the average of the three parallel measurements along with the corresponding error bars (±1S, where S denote the standard deviation). The obtained linearity and fitting error of the calibration curve signifies the excellent reproducibility of the measurements. Therefore, the GC/MB-SO₃H-MSM electrode possesses excellent selectivity, good stability and acceptable reproducibility for the determination of RF.

Fig. 9 Relative current at the GC/MB-SO₃H-MSM electrode for the determination of 10.0 μM RF in the presence of 50 fold excess of possible interferents.

Determination of RF in pharmaceutical products

The practical application of the fabricated sensing scaffold towards the RF determination is evaluated by determining the concentration of RF in various pharmaceutical products (multivitamin tablets and vitamin B complex injection). The determination of RF in pharmaceutical products is carried out by DPV technique. Based on the DPV response of pharmaceutical products and the constructed calibration curve (Fig. 8B), the amount of RF present in pharmaceutical products are estimated. The estimated amount of RF by this proposed modified electrode is in fine agreement with the RF amount present in pharmaceutical products, indicating the negligible interference of other vitamins and interfering ions. Further to evaluate the performance of the proposed electrode, two different known amount of standard RF are spiked into the pharmaceutical samples and analyzed again. The obtained results and recoveries are summarized in Table 2. It can be seen that the obtained recoveries for the different samples are satisfactory along with the acceptable RSD value. These results also signify the good selectivity of proposed electrode over other vitamins and severe matrix of the samples. Thus, on the basis of the above results, it is expected that the GC/MB-SO₃H-MSM sensing scaffold can be successfully used for RF detection in pharmaceutical products (Fig. 9).

Table 2 Determination of RF (addition/recovery) in pharmaceutical formulations using GC/MB-SO₃H-MSM by DPV

Sample	Amount of RF from the sample (μg)	Amount of standard RF added (μg)	Total amount of RF present in mixture (μg)	Amount of RF found in mixture (μg) (average of 3 experiments)	Recovery (%)	RSD (%) (n = 3)
Multivitamin tablet	49.8	0	49.8	50.9	102.2	0.70
		18.8	68.6	69.4	101.2	1.86
		28.2	78.0	79.4	101.8	0.65
Vitamin B complex injection	16.7	0	16.7	16.5	98.8	1.37
		9.4	26.1	26.0	99.6	3.82
		18.8	35.5	35.3	99.4	1.34

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Conclusions

MB incorporated on to SO₃H-MSM (MB-SO₃H-MSM) is synthesized and characterized using FT-IR, UV-vis absorption, SEM, XRD and EDAX analysis. The electrochemical behaviour of GC/MB-SO₃H-MSM electrode is studied by CV and EIS. The GC/MB-SO₃H-MSM electrode is utilized for the electrochemical determination of RF and it exhibits enhanced reduction current response for RF determination in comparison to GC/SO₃H-MSM and bare GC electrodes. Synergistic effect of MB with SO₃H-MSM results in high voltammetric response for RF. The sensing scaffold displayed a wide linear calibration range (10.0 nM to 50 μM), low detection limit (5.0 nM), high sensitivity (393 μA mM⁻¹ cm⁻²) with good selectivity, stability and reproducibility. Practicality of the proposed sensor is tested in various pharmaceutical products and the obtained results favour the potential applicability of the proposed sensor for RF determination in real samples.

Acknowledgements

Financial support from CSIR (project number: 01(2708)/13/EMR-II), UGC (project number: F 42-271/2013 (SR)) and DST, New Delhi is gratefully acknowledged. RG acknowledges UGC for senior research fellowship. We are grateful to Dr S. A. John for SEM facilities.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra12336h

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