Abstract
A sensitive and selective voltammetric sensing method has been developed for simultaneous determination of dopamine (DA) and uric acid (UA) using ferrocene carboxylic acid modified halloysite nanotubes (Fc/HNT) on the surface of glassy carbon electrode (GCE). It was found that beneath optimum experimental condition, the electrocatalytic oxidation of DA and UA occurred at +0.08 V and +0.22 V at Fc/HNT/GCE. These oxidation peak potentials are less positive and ensuing peak current values are almost thrice than unmodified GCE. A well-resolved peak potential window (∼150 mV) for oxidation of DA and UA were observed in the modified electrode system. Differential pulse voltammetry (DPV) method was employed for electrochemical detection of DA and UA using Fc/HNT/GCE at trace level. The calibration curves for DA and UA were obtained within the dynamic linear range of 6.0 × 10−7 to 60 × 10−5 M (R2 = 0.9959) and 1.3 × 10−6 to 100 × 10−5 M (R2 = 0.9925), respectively with the detection limits of 0.02 and 0.03 μM by using DPV method. An enhanced stability with good reproducibility was obtained from amperometry studies under hydrodynamic condition. The amperometric method was applied for detection of DA and UA present in pharmaceutical products and urine samples.
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Ferrocene and its derivatives are used in the field of fuel additives,1 redox-mediators,2 organic synthesis,3 medicinal functions4 and materials engineering.5 It is widely used as redox probe because it undergoes a single electron reversible redox reaction and excellent mediator for the development of biosensors species like D-penicillamine,6 uric acid,7 dopamine,8 glutathione9 and hydrochlorothiazide.10 The surface modification of ferrocene can be accomplished by various self-assembly of ferrocene-terminated thiols, covalent immobilization using diazonium salt, electrostatic stabilization using charged polymer electrolytes and forming layer by layer assembly of polyallyamine or chitosan tethered ferrocene molecules.11–14
The DA and UA coexisted in extracellular fluids and serum in mammals, which plays a vital role in human metabolism; specifically, it controls the central nervous system.15 DA is a neurotransmitter that plays vital role in functioning of central nervous, cardiovascular and hormonal systems, whose abnormal level causes schizophrenia, attention deficit hyperactivity disorder (ADHD), restless legs syndrome (RLS) and Parkinson's disease.16 On the other hand, UA is a primary final product of metabolism of purine nucleotides, a defect in this metabolism causes hyperuricemia, gout and pneumonia.17 Several analytical methods have been reported for the determination of DA and UA which include spectrophotometry,18 chemiluminescence,19,20 high-performance liquid chromatography (HPLC),21,22 fluorometry23,24 and capillary electrophoresis (CE).25,26 However, the above methods could not explain the importance of selectivity and sensitivity to the determination of DA and UA. Therefore, the electrochemical methods are more accessible for DA and UA sensing and quantification due to their low price, untroubled process and high sensitivity. Though, the electrochemical detection of DA and UA are hampered by the close oxidation potential of alternative species, usually at high concentration of biological fluids. In the past, this issue was overcome by using electrodes modified with a self-assembled monolayer. However, the chemically modified electrodes (CMEs) were proved to have a very low detection limit because of their large potential window and high electro-catalytic activity. For example, pristine graphene,27 chitosan protected graphene,28 copper nanoparticles incorporated polypyrrole film,29 silver nanoparticles incorporated zeolite30 and graphene oxide templated polyaniline31 modified electrodes were shown to be used for the assay of DA and UA. Dursun et al., established an electrochemically deposited PtNPs on multiwalled carbon nanotube (MWCNT) for the concurrent determination of DA, UA and ascorbic acid (AA).32 Lin et al., demonstrated the use of PtNPs deposited polydopamine coating on MWCNT for simultaneous detection of DA and UA.33 Beitollahi et al., proved a simultaneous determination of DA and UA using a carbon paste electrode (CPE) modified CdTe quantum dots.34
Halloysite [Al2Si2O5(OH)4.2H2O with 1:1 (Al : Si ratio) layer] (Scheme 1) is naturally occurring alumina silicate with hallow nanotubular structure.35 It consists of gibbsite octahedral sheet (Al−OH) groups on internal surface and siloxane groups (Si−O−Si) on external surface. The halloysite nanotube (HNT) length varies from 1 to 15 μm and their diameter ranges from 10 to 150 nm.36 It has been widely used as electrocatalyst,37 adsorbents,38,39 electronic components,40 pharmaceutics for drug delivery,41,42 nanoreactors,43 nanocomposites44 and additives in polymers matrix.45–47 Recent reports shows that selective modification/functionalization of silica and alumina parts with silanes48 and phosphonic acid49 to aid in the drug delivery, where drug was loaded within the pore of HNT. A selective modification of silica and alumina in HNT is an interesting characteristic for targeted drug delivery50 and for selective removal of environmental pollutants.51 Sohn et al.,52 reported on the aggregation and stabilization of carboxylic acid functionalized HNT. Later, it was demonstrated that HNT can be used as a template to prepare porous carbon nanotube with narrow pore size through surface modification by hydrothermal carbonization process followed by chemical etching of silica component. Such kind of nanostructure hollow tube shows excellent supercapacitor ability.53
Scheme 1. Schematic illustration of crystalline structure of HNT.
In the present investigation, we tend to explain the development of immobilized redox active molecules like Fc within the HNT surface and its study of electrocatalytic behavior on electrochemical oxidation of biologically important molecules such as DA and UA. DPV method was exploited for the selective and sensitive detection of DA and UA at Fc/HNT/GCE as a result of its high current sensitivity and better peak resolution with well-resolved peak shape without any overlapping of oxidation peak current values. The amperometric method was applied for detection of DA and UA present in pharmaceutical products and urine samples. The electrode stability and interference of other biologically significant molecules were also tested.
Experimental
Materials
Halloysite nanotubes, ferrocene carboxylic acid, dopamine hydrochloride and uric acid were purchased from Sigma Aldrich, Bangalore. Potassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4) and potassium chloride (KCl) were received from SRL Pvt. Ltd., India. Acetone was purchased from Ranboxy Laboratories, India. Ethanol (99.9%) was bought from Vijaya Scientific Pvt. Ltd., Chennai, India. Nitric acid was purchased from Spectrum Chemicals, Bangalore. All other chemicals obtained from commercial sources without any further purification processes. Double distilled (DD) water was used for the preparation of all stock solutions.
Buffer solution with a pH of 7.0 was prepared by mixing of 0.1 M KCl, 0.1 M KH2PO4 and 0.1 M K2HPO4 in 250 ml standard flask using DD water. The pH of the solution was checked using Elico-pH meter at room temperature (RT). The stock solution of DA (0.1 M) and UA (0.1 M) were prepared freshly with DD water and stored in a dark room at 5°C. Nitrogen gas was purged 10 min for the removal of oxygen in buffer solution before doing all electrochemical studies.
Preparation of Fc/HNT nanocomposite
To prepare Fc/HNT nanocomposite, 50 mg of halloysite was dispersed in 5 mL of ethanolic solution of ferrocene carboxylic acid (5 mg) and kept for 2 h at ultrasound bath (Fisher Scientific FB15051 ultrasonic bath cleaner, 37 kHz). Finally, Fc/HNT nanocomposite was centrifuged at REMI micro centrifuge (Model No. RM-12) with 2000 rpm. Then, the nanocomposite washed with ethanol for thrice to remove unbounded Fc from surface modified HNT and allowed to dry in a desiccator at RT.
Preparation of Fc/HNT modified electrode
GCE was polished before performing each experiment by alumina (0.5 micron powder) paste and rinsed exhaustively with DD water and then washed with 1:1 v/v ratio of nitric acid and acetone. Finally, the GCE surface washed with DD water at ultrasonic bath for 5 min and then dried at RT. About, 2 mg of Fc/HNT was dispersed in 1 mL of ethanol and then sonicated for a two minutes to obtain a homogenous dispersion of Fc/HNT. The colloid suspension (5 μL) was dropped on the surface of GCE and allowed to dry at RT. The schematic diagram shows the electrocatalytic oxidation of DA and UA corresponding to Fc/HNT modified GCE (Scheme 2).
Scheme 2. Schematic diagram shows the electrocatalytic oxidation of DA and UA corresponding to Fc/HNT/GCE.
Instruments
FT-IR spectra were recorded by a Perkin-Elmer 180 with a resolution of 4 cm−1 for 32 scans over a wave number range of 4000–400 cm−1. Morphological and structural investigations were carried out using field emission scanning electron microscopy (FE-SEM, SU6600, Hitachi, Japan). The elemental composition was carried out by energy dispersive X-ray spectrometer (EDAX, 8121-H, Japan). Thermal stability was analyzed by thermogravimetric analysis (TGA) using a TGA Q5000, Thermal Analyzer (TA) instruments at a heating rate of 20°/min, from room temperature up to 800°C, under nitrogen atmosphere. Electrochemical experiments were carried out using Gamry, USA model 330 including PV220 software and a CHI 660A electrochemical instrument, USA. The working and counter (auxiliary) electrodes were glassy carbon electrode (GCE) (of 3 mm diameter) and platinum wire (0.5 mm diameter, BAS Instruments, USA) respectively. An Ag/AgCl electrode was used as a reference electrode (Ag/AgCl/KClsat). Bioanalytical system (BAS, USA) polishing kit was used to polish the GCE surface.
Electrochemical impedance spectroscopy (EIS) measurements were performed using the CHI-660A electrochemical instrument. The preparation of electrolyte using 0.1 M KCl containing 0.5 mM [Fe(CN)6]3–/4– redox probe. All solutions were purged with high purity nitrogen gas about 10 min before performing all electrochemical experiments.
Results and Discussion
Ferrocene carboxylic acid have been widely used for numerous electrocatalytic oxidation studies because of its water solubility and used in a solution based electrocatalytic reactions. The use of ferrocene based CPE by anchoring of polymer backbone and functionalization on the surface of ordered mesoporous carbon, porous silica substrate and zeolite microporous structure are certain examples for electrochemical studies.54,55 In this study, we demonstrate the use of Fc through surface modification based on self-assembling behavior of carboxylic acid functional group.56 Recently, Roof et al. reported incorporation of TiO2 nanoparticles and Fc in CPE for simultaneous determination of glutathione and tryptophan.57 In line with this, we have shown that surface modification of HNT using Fc; which act as a facile electron transfer for the electrochemical oxidation of certain biomolecules. A self-assembled monolayer of Fc was uniformly distributed within the surface of phyllosilicate tubular nanoclay. The surface modification of HNT with innumerable functional groups, including carboxylic acid was established from the recent study.58 While treating halloysite nanoclay with an ethanolic solution of Fc, the carboxylate ion forms a strong electrostatic interaction which was confirmed by FT-IR, TGA and cyclic voltammetry studies. In addition, a stable voltammetric redox behavior was noted, meanwhile continuous cyclic voltammetry studies without a loss of its redox behavior in phosphate buffer medium shows a robust integrity of monolayer assembly.
Preparation and characterization of Fc/HNT nanocomposite
FT-IR spectra of Fc, HNT and Fc/HNT are shown in Fig. 1. From the spectrum of HNT, the absorption peak at 3712 cm−1, 3616 cm−1 and 3481 cm−1 are attributed to the stretching of inner surface and inner hydroxyl groups.59 The peaks at 1022 cm−1, 787 cm−1, 748 cm−1 and 669 cm−1; it is observed that the first two bands are assigned for symmetric stretching and in-plane vibration of Si-O groups; and last two bands are ascribed to the perpendicular stretching of Si-O groups (Fig. 1b). Deformation vibration of –OH inner hydroxyl groups (Al2OH), Al-O-Si and Si-O-Si were exhibited at 914 cm−1, 549 cm−1 and 454 cm−1 respectively. In Fig. 1a, a strong band at 1645 cm−1 is attributed to stretching vibration of carbonyl group of Fc.60 The modified Fc/HNT, a carboxylic acid (-COOH) group was shifted at 1660 cm−1 (Fig. 1c), which confirms that the carbonyl group attaches to the HNT surface.
Figure 1. FT-IR spectra of a) Fc, b) HNT and c) Fc/HNT.
The surface morphology of HNT nanoclay was investigated by FESEM image. As seen from Fig. 2A, the nanotubular structure of HNT with diameters in the range of 20–120 nm was found to be open-ended. The average size of the HNT nanoclay was estimated to be 20 nm. The elemental composition of nanoclay was confirmed by energy dispersive X-ray analysis (EDAX) as shown in Fig. 2B. The major constituents for HNTs were Al, Si and O. Thermal stability and surface modification of HNT and Fc/HNT were confirmed by TGA analysis as shown in Fig. 2C. From HNT, the major weight loss attributed at 470°C to 568°C which correspond to dehydroxylation of structural alumina group.61 In Fc/HNT, the primary weight loss arise at 30°C to 120°C, which may correspond to the loss of adsorbed water molecules;46 second weight loss arrive at 260°C to 360°C, may be due to structural decomposition62 and third weight loss appear at 420°C to 570°C, correspond to dehydroxylation process of aluminosilicates.44 The final weight loss for Fc/HNT sample at 590°C to 700°C may be due to the loss of Fc nanoparticles on HNT that concludes the existence of Fc nanoparticles are immobilized on HNT surface.
Figure 2. (A) FE-SEM image of HNT, (B) EDAX spectrum of HNT and (C) TGA curves of a) HNT and b) Fc/HNT
Cyclic voltammetry and EIS behavior of Fc/HNT/GCE
Figure 3A shows the cyclic voltammograms of a) bare GCE, b) HNT and Fc/HNT modified GCE in the presence of 0.1 M KCl containing phosphate buffer solution (pH 7.0) at a scan rate of 50 mV/s. As can be seen, a bare GCE and HNT/GCE exhibits a flat and drab voltammetric response, whereas Fc/HNT/GCE shows a well resolved redox peak and its anodic and cathodic peak potentials were found to be +0.108 V and +0.049 V (vs. Ag/AgClsat), respectively. The separation of anodic peak current is larger than that of cathodic peak current value and its potential peak separation (ΔEp = Epa – Epc) was found to be 59 mV at 50 mV/s and redox peak current ratio is equal close to one (Ipa/Ipc ≈ 1) which indicates that the electrochemical reaction is reversible one. Electrochemical stability and reversibility of Fc/HNT/GCE were done by a repetitive potential sweep at a scan rate of 50 mV/s. Figure 3B shows that the peak current does not change during the continuous potential cycles which imply that the monolayer assembly of ferrocene carboxylic acid is stable enough for electrochemical studies.
Figure 3. (A) Cyclic voltammograms of a) bare GCE, b) HNT/GCE and c) Fc/HNT/GCE in 0.1 M KCl containing PBS (pH 7.0). Scan rate: 50 mV/s. (B) Cyclic voltammograms of Fc/HNT/GCE in 0.1 M KCl containing PBS (pH 7.0) at continuous cycle (0–100 cycles). (C) Cyclic voltammograms of Fc/HNT/GCE in 0.1 M KCl containing PBS (pH 7.0) at various scan rates a) 25 b) 50 c) 100 d) 200 e) 300 f) 400 and g) 500 mV/s. (D) Linear plot of anodic and cathodic peak current vs. υ from (C).
With increase of potential scan rate of the surface modified Fc/HNT/GCE, the redox peak current values are also increasing and the anodic and cathodic peak potential (Epa and Epc) separations does not change63 significantly from the scan rates of 25–500 mV/s (Fig. 3C). The observed peak current (Ipa and Ipc) has a linear relationship with scan rate (υ), which indicates that the modified GCE is adsorption controlled process i.e. Fc molecules are strongly immobilized on the HNT surface (Fig. 3D). The electron transfer behavior of surface-modified electrode process was further confirmed by electrochemical impedance spectroscopy (EIS) by employing electrochemical probe to study charge transfer across the electrode solution interface. The semicircular portion of Nyquist plot at higher frequencies corresponds to electron-transfer limited process and its diameter is equal to electron transfer resistance (Rct), which reflects electron transfer kinetics of redox probe on electrode interface.64 Meanwhile, the linear part of Nyquist plot at lower frequencies corresponds to diffusion process.65 Figure 4 shows Nyquist plots for a) bare GCE, b) HNT and c) Fc/HNT modified GCE in the presence of 0.5 mM Fe(CN)64−/3− in 0.1 M KCl as a supporting electrolyte. The bare GCE exhibits a linearity that represents the characteristics of a diffusion limited process. Modified GCE contain semicircular portions, suggesting that the behavior is electron-transfer limited process. HNT/GCE shows a higher interfacial resistance (332Ω), indicating small interface impedance as compared to bare GCE. The interfacial resistance (494Ω) of Fc/HNT/GCE has higher than that of HNT/GCE, when a resistance is introduced into the electrode/solution system, leading to a lower rate of electron transfer of K3[Fe(CN)6]3−/4−. This result implies that Fc/HNT successfully immobilized on GCE surface. From cyclic voltammetry and electrochemical impedance spectroscopy studies, the electrochemical behavior of Fc/HNT/GCE is purely surface confined redox process.
Figure 4. Nyquist plots of a) bare, b) HNT and c) Fc/HNT/GCE in 0.5 mM K3[Fe(CN)6]3−/4− containing 0.1 M KCl as the supporting electrolyte. AC amplitude: 5 mV; frequency range: 0.01 Hz to 100 kHz. Inset is the Randles circuit.
Electrocatalytic oxidation of DA and UA at Fc/HNT/GCE
Electrocatalytic behavior of glassy carbon electrode modified Fc/HNT was used toward the oxidation peak current value of DA.66 Figure 5 shows the cyclic voltammetric behavior of DA on bare GCE (absence and presence) and Fc/HNT/GCE in presence of 0.1 M KCl containing PBS (pH 7.0) at a scan rate of 50 mV/s. In bare GCE, DA exhibits a poor anodic peak current with an oxidative peak potential of +0.025 V (vs. Ag/AgClsat) whereas, the oxidative peak potential of Fc/HNT/GCE is shifted toward less positive side (less than 9 mV) and the observed peak current value is four times superior than bare GCE. The redox current increases with each addition of 0.1 M DA solution and its linear ranges from 3 × 10−4 to 20 × 10−4 M as shown in Fig. 6A. Cyclic voltammogram were recorded in DA at various scan rates in the ranges of 20–100 mV/s that increases redox peak current. The linearity of anodic peak current (Ipa = 6.281x – 7.755, R2 = 0.9972) is possessed by diffusion controlled electron transfer process (Fig. 6B).
Figure 5. Cyclic voltammograms of a) and b) bare GCE (absence and presence of DA) and c) Fc/HNT/GCE in the presence of 50 μl in 0.1 M of DA at scan rate 50 mV/s in 0.1 M KCl containing PBS (pH 7.0).
Figure 6. (A) Cyclic voltammograms of Fc/HNT/GCE in 0.1 M KCl containing PBS (pH 7.0) for different concentrations (0.3 to 2.0 mM) of DA at a scan rate 50 mV/s. Inset: Plot of electrocatalytic peak current vs. DA concentration. (B) Cyclic voltammograms of DA at various scan rates a) 20 b) 40 c) 60 d) 80 and e) 100 mV/s. Inset: Plot of electrocatalytic peak current vs. υ1/2.
A similar trend was observed in case of electrochemical oxidation of UA67 using Fc/HNT/GCE. Cyclic voltammetry behavior of UA on bare GCE (absence and presence) and Fc/HNT/GCE in the presence of 0.1 M KCl containing PBS (pH 7.0) are shown in Fig. 7. In bare GCE, UA shows a small oxidation peak current and peak potential at +0.27 V (vs. Ag/AgClsat) however the modified Fc/HNT/GCE in the presence of UA exhibits a sharp, well-defined oxidation peak current and potential at +0.23 V (vs. Ag/AgClsat). Further, linear peak current values are noted, while increasing the concentration of UA from 3 × 10−4 M to 26 × 10−4 M which exhibits the better electrocatalytic behavior of Fc/HNT/GCE as shown in Fig. 8A. The influence of potential scan rate of electrochemical oxidation of UA was investigated at pH 7.0. The observed peak current values were found to be directly proportional to square root of sweep rate range of 20–100 mV/s, which implies that electrocatalytic oxidation of UA is diffusion controlled electron transfer process (Fig. 8B).
Figure 7. Cyclic voltammograms of a) and b) bare GCE (absence and presence of UA) and c) Fc/HNT/GCE in the presence of 50 μl in 0.1 M of UA at scan rate 50 mV/s in 0.1 M KCl containing PBS (pH 7.0).
Figure 8. (A) Cyclic voltammograms of Fc/HNT/GCE in 0.1 M KCl containing PBS (pH 7.0) for different concentrations (0.33 to 2.64 mM) of UA at a scan rate 50 mV/s. Inset: Plot of electrocatalytic peak current vs. UA concentration. (B) Cyclic voltammograms of UA at various scan rates a) 20 b) 40 c) 60 d) 80 e) 100 f) 120 and g) 140 mV/s. Inset: Plot of electrocatalytic peak current vs. υ1/2.
Effect of pH on oxidation of DA and UA at Fc/HNT/GCE
In general, the electrochemical activity of biologically important molecules redox behavior is dependent upon pH of the medium.68 The electrochemical responses of both DA and UA was studied in 0.1 M KCl containing PBS with different pH range (pH 1.0 to 11.0) at Fc/HNT/GCE by cyclic voltammetric method (Figures 9A and 9B). A shift in oxidation peak potential for both DA and UA was observed, while increasing the pH of medium, indicating that redox behavior of DA and UA at Fc/HNT/GCE are pH dependent reaction. The observed anodic peak current values are higher for both system at pH 7.0 and then decreases gradually with increasing the pH medium. Figure 9C shows variation of Ipa versus variation of pH in DA and UA, it can be clearly observed that the peak potential and current were closely related to pH value (pH 7.0) of supporting electrolyte. The linear regression equation of DA and UA were Epa (V) = 0.7235−0.0526 pH (R2 = 0.9975) and Epa (V) = 0.3561−0.0489 pH (R2 = 0.9977) as shown in Figure 9D. The slope of 52.6 mV and 48.9 mV per pH unit, which may close to the Nernstian value (0.059 V/pH) for a two protons/two electrons reaction.69,70 This indicates the electrocatalytic oxidation of both DA and UA at Fc/HNT modified GCE implied as two protons and two electrons redox process.
Figure 9. Effect of pH on (A) Cyclic voltammograms of 0.1 M of DA and UA at Fc/HNT/GCE in 0.1 M KCl containing PBS with various pH values (pH 1, 3, 5, 7, 9 and 11) at scan rate of 50 mV/s. (B) Variation of anodic peak current vs. pH and (C) Plot of E/V vs. pH.
From the above observation, the reaction scheme would probably via the following mechanistic steps. In the first step, the zero valent ferrocene moiety can be oxidized into ferrocenium ion (Fc+) followed by an electron transfer reaction. Then, Fc+ catalysis the oxidation of DA or UA leads to form a dopaminequinone and allantoin. The electron settling between surface confined Fc and Fc+ enhanced peak current of DA and UA. Due to the reversible changes in electronic state of surface confined mediator, which facilitates the overall redox reactions71 as shown in Fig. 10.
Figure 10. Mechanism of the electrode reaction of DA and UA on Fc/HNT/GCE.
Determination of rate constants on electrocatalytic oxidation reactions
The chronoamperometry method was employed to determine diffusion coefficient and catalytic reaction rate constant of DA and UA oxidation process at Fc/HNT modified GCE. Figures 11A and 12A shows the current-time relationships of Fc/HNT/GCE obtained by setting the working electrode potentials of +0.034 V and +0.22 V (vs. Ag/AgCl/KClsat) for DA and UA, respectively at different concentration ranges in 0.1 M KCl containing PBS (pH 7.0). In order to calculate the diffusion coefficient (D) of DA and UA, the experimental plots of I versus t−1/2 were drawn using comparison graphs of a) to d) that results in straight lines (Figs. 11B and 12B). According to the Cottrell equation,72
![Equation ([1])](https://content.cld.iop.org/journals/1945-7111/163/10/B543/revision1/d0001.gif)
where 'n' is number of electrons, 'F' is Faraday constant (C/mol), A is electrode area (cm2), c is the bulk concentration of an analytes (mol/cm3) and D is the diffusion coefficient (cm2/s). The diffusion coefficient (D) value can be obtained from the slopes of the linear plot (I vs. t−1/2) for DA and UA. The average value of DA and UA were found to be 3.3 × 10−6 cm2/s and 2.4 × 10−6 cm2/s respectively, which agrees equitably with last pervious literatures.73,74 Thus, this result shows that the better electrocatalytic oxidation of DA and UA occurs at surface modified Fc/HNT/GCE.
Figure 11. (A) Chronoamperograms obtained at Fc/HNT/GCE in the absence and presence (0.05, 0.1, 0.15 and 0.2 mM) of DA in 0.1 M KCl containing PBS (pH 7.0) at potential step of 34 mV vs. Ag/AgCl/KClsat electrode, respectively. (B) Cottrell plots drawn using data obtained from chronoamperograms b), c), d) and e) of A). (C) Plot of Ic/IL versus t1/2. Data was obtained from the chronoamperograms of a) and d).
Figure 12. (A) Chronoamperograms obtained at Fc/HNT/GCE in the absence and presence of (0.05, 0.1, 0.15 and 0.2 mM) of UA in 0.1 M KCl containing PBS (pH 7.0) at potential step of 220 mV vs. Ag/AgCl/KClsat electrode, respectively. (B) Cottrell plots drawn using data obtained from chronoamperograms b), c), d) and e) of A).
In addition, chronoamperometry is also employed to evaluate the catalytic rate constant of DA at Fc//HNT/GCE (Fig. 11C). The catalytic reaction rate constant was determined according to the method described in Ref. 75:
![Equation ([2])](https://content.cld.iop.org/journals/1945-7111/163/10/B543/revision1/d0002.gif)
where IC and IL is the catalytic current and limiting current of Fc/HNT/GCE in the presence and absence of DA and γ = khCbt (Cb is the bulk concentration of DA (mol/cm3)) is the argument of error function. In all cases where γ exceeds 2, the error function is almost to 1 and the above equation can be written as:
![Equation ([3])](https://content.cld.iop.org/journals/1945-7111/163/10/B543/revision1/d0003.gif)
where kh and t are the catalytic rate constant (cm3/mol/s) and time elapsed (s) respectively. Equation 3 can be used to calculate the rate constant of catalytic process kh. The kh value was found to be 1.9 × 104 cm3 mol−1 s−1 for oxidation of DA, which is close to the reported value.76 This value illuminates the sharp feature of the catalytic peak observed for the oxidation of DA at the surface of Fc/HNT/GCE. This value was derived from the slope of a plot of IC/IL vs. t1/2 for oxidation of 0.2 mM DA.
Further, the heterogeneous rate constant (ks) for the oxidation of UA at Fc/HNT/GCE was calculated by Velasco equation:77
![Equation ([4])](https://content.cld.iop.org/journals/1945-7111/163/10/B543/revision1/d0004.gif)
where Do is apparent diffusion coefficient (cm2/s), Ep is oxidation peak potential, Ep/2 is half-wave oxidation peak potential and υ is scan rate (mV/s). The Do value was determined based on Cottrell slope obtained from chronoamperometry technique. The estimated ks value for oxidation of UA at Fc/HNT/GCE was found to be 1.47 × 10−3 cm/s. The value of ks illuminates the sharp feature of the catalytic peak observed for the catalytic oxidation of UA at the surface of Fc/HNT/GCE. The observed ks value is nearly close to the previously reported results.78
Simultaneous determination of DA and UA at Fc/HNT/GCE
The simultaneous determination of DA and UA mixtures was performed at Fc/HNT/GCE by DPV method, which eliminates the residual charging current and the pure Faraday current value only measured. DPV was employed for simultaneous determination of DA and UA at Fc/HNT/GCE in 0.1 M KCl containing PBS (pH 7.0). Electrochemical determination of DPV shows the concentration of one species incessantly with other species kept constant. Anodic peak current of DA increased linearly by addition of DA in the presence of 50 μL of 1 mM UA kept constant. On the other hand, oxidative peak current of UA increased linearly by the addition of known amount UA in the presence of 20 μL of 1 mM DA kept constant under optimized pH value. As the concentration of DA and UA increased simultaneously, the peak potential of UA or DA remained constant (Figs. 13A and 13B). The oxidation peak current of DA and UA at surface of Fc/HNT/GCE was proportional to concentration of added substrates in linear ranges from 0.6 × 10−6 M to 60 × 10−5 M and 1.3 × 10−6 M to 100 × 10−5 M and correlation coefficient is 0.9959 and 0.9925 respectively. The detection limits (3σ/slope, σ is standard deviation) of DA and UA were found to be 0.2 × 10−7 M and 0.3 × 10−7 M respectively. Comparison of Fc/HNT/GCE and other modified electrodes reported in pervious literatures are shown in Table I. The analytical parameter shows that Fc/HNT/GCE exhibits an electrocatalytic behavior for the independent determination of DA and UA. This result shows that Fc and Fc+ inside the clay nanotubes act as an electron transfer mediator that indicates the electrocatalytic oxidation of DA and UA.
Figure 13. (A) Differential pluse voltammograms of DA at Fc/HNT/ GCE in 0.1 M KCl containing PBS (pH 7.0) in the presence of 50 μM UA. The DA concentrations are 0.66, 1.3, 2.0, 2.6, 3.3, 4.0, 4.6, 5.3, 6.0 μM (from a-i). (B) UA at Fc/HNT/GCE in 0.1 M KCl containing PBS (pH 7.0) in the presence of 20 μM DA. The UA concentrations are 1.3, 2.6, 4.0, 5.3, 6.6, 8.0, 9.3, 10.6 μM (from a-h). (C) Simultaneous determination of DA and UA using Fc/HNT/GCE in presence of 0.1 M KCl containing PBS (pH 7.0). Concentrations of the two compounds: DA (0.1 × 10−7 M to 60 x 10−5 M) and UA (0.1 × 10−7 M to 60 × 10−5 M). Scan rate: 20 mV/s, Pulse width: 20 mV, Pulse Amplitude: 25 mV.
Table I. Comparison table of the different electrodes.
| Conc. range (μM) | R2 | Detection Limit (μM) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| aElectrode | Method | DA | UA | pH | DA | UA | DA | UA | Ref. |
| GNP/Ch/GCE | DPV | 0.2–80 | 1.2–100 | 7.0 | 0.9982 | 0.9994 | 0.12 | 0.60 | 79 |
| PAA-MWNTs/GCE | LSV | 0.04–3 | 0.3–10 | 7.0 | - | - | 0.02 | 0.10 | 80 |
| EBNBH/CNPE | DPV | 0.1–900 | 20–600 | 7.0 | 0.9981 | 0.9989 | 0.08 | 15.0 | 81 |
| PANI-AuNP | DPV | 7–148 | 29–720 | 7.0 | - | - | 3.0 | 20.0 | 82 |
| LaPO4/CPE | DPV | 0.4–11 | 2.7–24 | 7.0 | 0.9995 | 0.9991 | 0.13 | 0.9 | 83 |
| Au-CA-MWNT | DPV | 0.2–100 | 1–100 | 5.0 | - | - | 0.02 | 0.1 | 84 |
| PPGE | DPV | 0.15–15 | 0.3–150 | 5.0 | 0.9991 | 0.9988 | 0.03 | 0.12 | 85 |
| MCMS/Thionine/GCE | DPV | 2–30 | 9–100 | 5.0 | 0.9995 | 0.9991 | 0.50 | 2.30 | 86 |
| MWCNT @ PDOP@PtNP/GCE | DPV | 0.25–20 | 0.3–13 | 6.0 | 0.9990 | 0.9990 | 0.08 | 0.12 | 31 |
| PAM/rGO/GCE | CV | 0.3–50 | 1–50 | 7.0 | 0.9967 | 0.9984 | 0.1 | 0.5 | 87 |
| Fc/HNT/GCE | DPV | 0.6–600 | 1.3–100 | 7 | 0.9959 | 0.9925 | 0.02 | 0.03 | This work |
aGNP – Gold Nanoparticles GCE – Glassy Carbon Electrode PAA – Poly (Acrylic acid), DPV – Differential Pulse Voltammetry MWNTs – Multiwalled Nanotubes CNPE – Carbon Nanotube Paste Electrode MCMS – Magnetic Chitosan Microsphere LSV – Linear Sweep Voltammerty PPGE – Pretreated Pencil Graphite Electrode CPE – Carbon Paste Electrode PDOP @ PtNP – Polydopamine @ Platinum Nanoparticles PAM/rGO – Polyacrylamide/reduced Graphene oxide
The excellent electrocataytic activity with well resolved peak separation provides a sensitive for simultaneous determination of DA and UA using Fc/HNT/GCE in PBS (pH 7.0). In the presence of both analytes, two well resolved independent anodic peaks were observed for DA and UA, peak separation between these two analytes was found to be 150 mV. The peak potential for an individual analyte does not affects in presence of other electroactive species, which is essential for the independent determination of each analytes. Simultaneous electrochemical response of DA and UA still increased linearly with increase in their concentrations and their linear ranges from 1.0 × 10−6 M to 60 × 10−5 M and 10 × 10−5 M to 60 × 10−5 M with the detection limit of 1.1 μM and 2.0 μM, respectively (Fig. 13C). These results demonstrate that simultaneous determination of DA and UA at Fc/HNT/GCE achieved optimal electrocatalytic activity, lower detection limit, wider linear range, higher selectivity and sensitivity.
Amperometry detection of DA and UA at Fc/HNT/GCE
Amperometry method can be easily measure the current response for the each addition of DA and UA with respective time under stirring condition. The typical steady-state catalytic current-time response of Fc/HNT/GCE under constant stirring for step-wise injection of 20 μM of 1 mM DA (50 s) and 30 μM of 1 mM UA (30 s) into 0.1 M KCl containing PBS (pH 7.0) at applied potential of +0.034 V and +0.22 V (vs. Ag/AgCl). Figures 14A and 14C clearly show that oxidation peak current increases by increasing the concentration of DA and UA. Amperometric response, increased linearly in range from 0.04 × 10−7 M to 4.4 × 10−6 M and 0.08×10−7 M to 6.4 × 10−6 M for DA and UA, respectively. Linear calibration was obtained, with a coefficient of DA and UA are 0.9977 and 0.9976 respectively, which demonstrates the better relationship between oxidation current and concentration. Limit of detection was calculated in the graph of DA and UA were found to be 12 nM and 23 nM based on signal-to-noise ratio (S/N = 3) respectively (Figs. 14B and 14D).
Figure 14. (A) Amperometric response of Fc/HNT/GCE at an applied potential 34 mV to subsequent addition of different concentrations from of 1 mM DA in the presence of 0.1 M KCl containing PBS (pH 7.0). (B) Calibration curve of peak current vs. concentration of DA. (C) Amperometric response of Fc/HNT/GCE at an applied potential 220 mV to subsequent addition of different concentrations from of 0.1 mM UA in the presence of 0.1 M KCl containing PBS (pH 7.0). (D) Calibration curve of peak current vs. concentration of UA.
The reproducibility of the developed sensor was evaluated by using amperometric method. Five, different modified electrodes were constructed and their peak current response to 1 μM concentration of DA and UA were investigated. The relative standard deviation (RSD) was found to be 2.8% and 2.3% for DA and UA respectively (Fig. 15), confirming that the Fc/HNT/GCE was highly reproducible. The influence of interference species present in the reaction medium was also investigated at Fc/HNT/GCE along with DA and UA by amperometrric method. Suppression of peak current values of DA and UA was investigated at Fc/HNT/GCE by various possible interfering substances like ascorbic acid (10 times), citric acid, cysteine, tyrosine, tartaric acid, caffeine, glucose, sucrose, NaCl, Ca2+, Mg2+, Zn2+, aspartic acid, epinephrine (EP), and L- dopa as shown in Fig. 16. These substances did not interfere with the concurrent determination of DA and UA peak current up to a minimum of 100 fold excess.
Figure 15. Linear current response of DA and UA (1 μM) at different freshly prepared Fc/HNT/GCE (n = 5) in 0.1 M KCl containing PBS (pH 7.0).
Figure 16. (A) Amperometric response of several interefering compounds at Fc/HNT/GCE in presence of 0.1 μM of DA and UA via 0.1 M KCl containing PBS (pH 7.0).
Real sample analysis
Determination of DA in dopamine hydrochloride injection
The Fc/HNT modified electrode for analysis in practical samples was tested. Amperometric method was used in this experiment for the analyses of real sample of DA injection in pharmaceutical products. The injection solution (standard concentration of DA in 10 mg/mL, 1 mL per injection) was diluted in 50 mL standard flask with DD water and 100 μL was pipetted out into each series of 10 mL volumetric flasks and made up with 0.1 M PBS (pH 7.0). An aliquot of 15 mL of this solution was placed in the electrochemical cell for the determination of DA using above amperometric method. The analytical results are listed in Table II. These results were satisfactory and acceptable, showing that the proposed method could be effectively used for the determination of DA in commercial sources.
Table II. Determination of DA and UA mixture in dopamine injection (n = 5).
| DA | UA | |||||||
|---|---|---|---|---|---|---|---|---|
| DA injection | UA Added | Found | Recovery | R. S. D. | Found | Recovery | R. S. D. | |
| Samples | (μM) | (μM) | (μM) | (%) | (%) | (μM) | (%) | (%) |
| 1 | 1 | 0 | 0.98 | 98 | 2.22 | - | - | - |
| 2 | 1 | 10 | 0.97 | 97 | 2.43 | 9.83 | 98.3 | 3.3 |
| 3 | 2.5 | 0 | 2.46 | 98.4 | 2.34 | - | - | - |
| 4 | 2.5 | 25 | 2.43 | 97.2 | 2.38 | 24.2 | 96.8 | 2.56 |
| 5 | 5.0 | 0 | 5.21 | 104.2 | 1.78 | - | - | - |
| 6 | 5.0 | 50 | 5.12 | 102.4 | 1.90 | 50.56 | 101.1 | 1.76 |
| 7 | 7.5 | 0 | 7.52 | 100.2 | - | - | - | - |
| 8 | 7.5 | 75 | 7.49 | 99.8 | 2.95 | 74.37 | 991 | 3.7 |
| 9 | 10 | 0 | 9.91 | 99.1 | - | - | - | - |
| 10 | 10.0 | 100 | 9.94 | 99.4 | 2.72 | 100.4 | 100.4 | 1.98 |
Determination of UA in human urine samples
In using the amperometric method for determination of UA in human urine samples were also investigated. Before measurement, all urine samples were diluted 200 times with PBS (pH 7.0) in order to fit into the linear range of real samples. 1 mL of the urine sample was added to the electrochemical cell containing 15 mL of PBS medium and the certain amount of DA was spiked using the standard addition method. The binary mixtures of DA and UA were obtained between 91.4% and 102.1% as shown in Table III. The recoveries indicate the accuracy and repeatability of the proposed method.
Table III. Determination of DA and UA in urine samples (n = 5).
| Samples | Analyte | Detected (μM) | Added (μM) | Found (μM) | R.S.D (%) | aRecovery (%) |
|---|---|---|---|---|---|---|
| Urine 1 | DA | - | 5 | 4.57 | 2.3 | 91.4 |
| UA | 10.68 | 5 | 16.02 | 102.1 | ||
| Urine 2 | DA | - | 10 | 10.3 | 1.1 | 103 |
| UA | 15.87 | 10 | 25.67 | 99.2 | ||
| Urine 3 | DA | - | 20 | 19.89 | 1.3 | 99.4 |
| UA | 9.09 | 20 | 28.45 | 97.8 |
Conclusions
A novel electrochemical sensor for simultaneous detection of DA and UA were fabricated based on Fc/HNT modified GCE. Analysis by EIS revealed a lowering of charge transfer resistance by many fold due to the modification of the electrode. The modified electrode exhibited a strong electrocatalytic activity toward the oxidation of DA and UA, respectively. The influence of many interfering substances was tested for peak current response and found that the present system is free from any momentous interference due to presence of redox active molecules like AA, EP, aspartic acid, L-dopa or glucose. Moreover, modified electrode could be used for analysis in real samples. Futhermore, the preparation of modified electrode reveals a simple and easier than all previously reported studies. Thus, the most important issues for the direct estimation of DA and UA in pharmaceutical products and urine samples has been proposed. Mechanisms are proposed to elucidate the variation in response owing to pH, high response to AA and reduction of signal due to interfering substances. This Fc/HNT/GCE was found to be a potentially valuable tool for designing efficient and extremely selective electrochemical sensor design.
Acknowledgment
The authors (Dr. K. P and S. M) thank National Centre for Nanoscience and Nanotechnology, University of Madras for providing instrumental facilities.