A nanocomposite based on multi-walled carbon
nanotubes grafted by molecularly imprinted
poly(methacrylic acid
–hemin) as a peroxidase-like
catalyst for biomimetic sensing of acetaminophen
Ederson dos Santos Moretti,aJuliana de F´atima Giarola,bMichele Kuceki,aMaiyara Carolyne Prete,aArnaldo C´esar Pereiraband C´esar Ricardo Teixeira Tarley*ac In the present study the synthesis of a nanocomposite based on multi-walled carbon nanotubes grafted by poly(methacrylic acid–hemin) is described. The presence of functional groups in the nanocomposite was evaluated by FT-IR, while morphological, textural and thermal stability data were evaluated by means of SEM, TEM, nitrogen adsorption–desorption assays and TGA, respectively. The material was evaluated as a biomimetic and catalytic sensor toward the electrochemical determination of acetaminophen (AC) by depositing it onto a glassy carbon electrode surface. Acetaminophen has been widely used as an analgesic and antipyretics drug and for patients with a high sensitivity to aspirin; therefore the development of analytical methods for AC determination in pharmaceutical formulations is of paramount importance for quality control. The biomimetic electrochemical sensor operation is similar to that of the biosensor based on horseradish peroxidase, where the hemin in the presence of H2O2 catalyses the oxidation of acetaminophen into N-acetyl-p-benzoquinoneimine, which in turn is reduced back to AC on the electrode at a potential of0.27 V. The variables that exert influence on the performance of the biomimetic response, including H2O2concentration (300mmol L1), pH (8.0) and concentration (0.1 mol L1) and type of buffer solution (Trizma) were investigated. Under optimized conditions the electrochemical determination of AC using multi-walled carbon nanotubes grafted by poly(methacrylic acid–hemin) immobilized on the surface of the glassy carbon electrode was carried out by square wave voltammetry (SWV), showing a limit of detection of 1.1mmol L1. The selectivity of material, ascribed to imprinted effect, was assured by comparison to a non-imprinted material (NIP). The proposed method was applied to AC determination in a pharmaceutical formulation, whose obtained values were very similar to those declared and statistically equal to the HPLC method, thus illustrating the feasibility of method for analysis of real samples.
1.
Introduction
The reliable determination of drugs in bulk and pharmaceutical formulations is of paramount importance for quality control to prevent overdose while optimizing the individual dosage regimen.1,2N-Acetyl-p-aminophenol (paracetamol or
acetamin-ophen, AC) belongs to analgesic and antipyretics drugs and has
been used extensively in the world to moderate pain and can be an alternative drug for patients with high sensitivity to aspirin. However, it has been reported that acute overdoses of AC may cause renal insufficiency due to accumulation of toxic metab-olites resulting in death of patient.3,4Although many analytical
techniques are available for the determination of AC in phar-maceutical formulation, including titrimetry,5 high
perfor-mance liquid chromatography,6 and UV-vis
spectro-photometry,7 extensive efforts have been made for the
devel-opment of electroanalytical methods, owing to their high simplicity, low-cost, sensibility and rapid analysis.8In general,
when bare carbon electrodes, such as glassy carbon or graphite paste electrodes are used, the electrooxidation of AC takes place in high overpotential, which in turn decreases the selectivity of electrochemical measurements. In addition, these electrodes can suffer from fouling by oxidation and interfering products formed and usually require periodic mechanical polishing or a
Universidade Estadual de Londrina (UEL), Departamento de Qu´ımica, Centro de Ciˆencias Exatas, Rodovia Celso Garcia Cid, PR 445, km 380, Londrina, PR 86050-482, Brazil. E-mail: tarley@uel.br; Fax: +55 43 3371 4286; Tel: +55 43 3371 4366
bDepartamento de Ciˆencias Naturais, Universidade Federal de S˜ao Jo˜ao del Rei (UFSJ), Campus Dom Bosco, Praça Dom Helv´ecio 74, F´abricas, 36301-160, S˜ao Jo˜ao del Rei, MG, Brazil
cInstituto Nacional de Ciˆencia e Tecnologia (INCT) de Bioanal´ıtica, Universidade Estadual de Campinas (UNICAMP), Instituto de Qu´ımica, Departamento de Qu´ımica Anal´ıtica, Cidade Universit´aria Zeferino Vaz, s/n, Campinas, SP 13083-970, Brazil Cite this: RSC Adv., 2016, 6, 28751
Received 24th January 2016 Accepted 9th March 2016 DOI: 10.1039/c6ra02150f www.rsc.org/advances
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activation processes, which decreases the sample throughput of analytical procedure. These drawbacks have been successfully overcome by preparation of biosensors using horseradish peroxidase as catalyst element immobilized on nanocomposite carbon nanotubes/conductor polymer,9 entrapped in graphite
paste10,11 or immobilized on core–shell ZrO@Fe
3O4
nano-particles on chitosan hybrid lm electrodeposited on the surface of an Au electrode.1In spite of biosensors offer intrinsic
selectivity toward the substrate, their successful use depends upon the properties of the native enzyme, and aer repeated measurements their catalytic activity is decreased.12 In this
sense, molecularly imprinted polymers (MIP) and biocatalyst (non-enzymatic system) based on synthetic metallic complex have proven to be interesting biomimetic materials with substantial stability over biosensors, but with similar selec-tivity.13,14Molecularly imprinted polymers are synthesized in the
presence of a molecule template, which establishes interactions with the functional monomer. Aer polymerization, the template is removed from the polymeric matrix, thereby resulting in the formation of analyte-selective binding sites.15
The usefulness of MIP as platform in electrochemical sensing depends on its adequate integration on the surface of transducer electrode and some strategies have been reported for this task. Yola et al. developed a new voltammetric sensor based on Fe@Au nanoparticles involved in 2-aminoethanethiol functionalized multi wall carbon nanotubes for the determination of cefexima in human plasma.16 The combined use of nanoparticles,
nano-carbonaceous material and molecularly imprinted polymer has been also reported. Yola et al. described the electrochemical polymerization of phenol as monomer on substrate of silver nanoparticles (AgNPs) involved in a polyoxometalate (H3PW12O40,
POM) functionalized reduced graphene oxide (rGO) deposited on the surface of glassy carbon electrode. The sensor was applied to ochratoxin A determination in wine and grape juice.17A similar
study was developed using platinum nanoparticles (PtNPs) and the sensor was applied to citrinin (CIT) determination in rye samples.18Electropolymerization of MIP on the surface of glassy
carbon electrode is another strategy for the preparation of based-MIP electrochemical sensor as reported by Gupta et al.19In this
study, a molecularly imprinted polypyrrole (PPy) was fabricated for the determination of tobramycin (TOB) in egg and milk. Molecularly imprinted technology has been very effective to generate sensitive quartz crystal microbalance (QCM). For instance, in the study reported by Gupta et al.20the development
of sensor is based on modication of gold surface of QCM chip through self-assembly monolayer formation with allyl
mercap-tane, with further polymerization of
poly(2-hydrox-yethylmethacrylate–methacryloylamidoaspartic acid) [p(HEMA– MAAsp)]lm in the presence of template kaempferol (KAE). The feasibility of sensor was evaluated by analysis of orange and apple juices for the determination of KAE. In this direction, this strategy has been successfully employed for the determination of tobra-mycin (TOB) in pharmaceuticals and foods20and lovastatin (LOV)
in red yeast rice.21The strategy of template polymerization on the
gold surface modied with ally mercaptane is also very useful for preparation of surface plasmon resonance (SPR) sensor.22,23As
observed, some strategies for the preparation of based-MIP sensor
have been reported, but a brief survey of literature reveals that few studies dedicated to the use of MIP as a electrochemical sensing device for AC determination have been noticed and one of the most common strategy involves the electrochemical synthesis of conductive MIP onto bare electrode (glassy carbon electrode and graphite electrode).24,25For instance, Teng et al. developed a layer
of conductivelm of molecularly imprinted poly(p-aminobenzene sulfonic acid) (pABSA) on the surface of glassy carbon electrode for the determination of AC.26 In a similar study, Luo et al.
designed the synthesis of a molecularly imprinted conductive polyaniline (PANI) nanoparticles using polymeric micelle as nanoreactors electrochemically deposited onto glassy carbon electrode for the determination of AC by differential pulse vol-tammetry.27 Although the developed conductive MIP presents
high selectivity toward the AC owing to the imprinting effect created during polymer synthesis; on the other hand, the elec-trooxidation peak of AC still took place in high overpotential. Thus, the incorporation of a catalyst element in the synthesis of MIP, such as metalloporphyrins as both co-monomer and cata-lytic center is an interesting way to improve the performance of MIP for electrochemical purposes, and thus, the MIP acts as an articial enzyme.28,29Furthermore, the use of conductive MIP is
limited due to the low commercial availability of conductive monomers. Therefore, the synthesis of non-conductive catalytic MIP graed on the surface of carbon nanotubes (CNT) by covalent linkages seems to be a very promising approach for obtaining MIP as an enzyme-mimicking catalyst for the electrochemical deter-mination of AC and other phenolic compounds. The use of CNT in the nanocomposite may provide high electrical conductivity to the material, ensure higher dispersibility of polymer and the nanoscale of MIP graed on the surface of CNT improves the mass transfer of analyte towards the selective binding sites, unlike the MIP synthesized by bulk method.30,31
According to aforementioned, the aim of the current study is to synthesize a catalytic nanocomposite and evaluate its use as a new electrochemical sensing platform for AC, combining the intrinsic properties of CNT, the merits of molecular imprinting and the catalytic properties of Fe(III)protoporphyrin(IX) (hemin).
The synthesized nanocomposite was based on multi-walled carbon nanotubes graed by poly(methacrylic acid–hemin) and used as an enzyme-mimicking catalyst. The material was characterized by FT-IR, TGA, SEM and TEM and the electrode performance for real analysis was checked from the electro-chemical analysis of pharmaceutical formulations.
2.
Experimental
2.1. Apparatus
Voltammetric measurements were performed at room temper-ature, with a potentiostat/galvanostat Autolab® PGSTAT-12 (Eco Chemie BV, Utrecht, The Netherlands). An electrochemical cell containing a glassy carbon electrode (3 mm) modied with nanocomposite as the working electrode, an Ag/AgCl electrode as the reference and a platinum wire as the auxiliary electrode were used. The voltammetric measurements were carried out in the absence of oxygen, by bubbling N2gas. In order to evaluate
the morphology of material, a microscope JEOL® JSM-6360 LV
equipped with dispersive energy microscopy was used. Before analysis, the material was coated with a thin layer of gold, using a Bal-Tec MED 020 equipment, in order to minimize charging under the incident electron beam. The FT-IR spectra were recorded in the 4000–400 cm1region with a
spectrophotom-eter model 8300 Shimazdu (Tokyo, Japan), using the KBr pellet conventional method. Thermal stability of materials was eval-uated from thermogravimetric analysis (TGA) using a TGA 4000 Perkin Elmer (Waltham, USA) thermobalance. For analysis,10 mg of sample was heated from 30 to 900C at a rate of 10C min1, under nitrogenow of 20 mL min1. The transmission electron microscopy images were taken using a microscope JEOL® JEM-1400 with an accelerating voltage of 120 kV. The samples were previously dispersed in ethanol by sonication for 20 min and then the suspension was dripped on copper grids and dried under vacuum. The pH measurements were per-formed using pH meter Metrohm® 827 (Herisau, Switzerland). Chromatography determination was performed on a Shimadzu liquid chromatograph equipped with a LC-20AT gradient pump, a UV-VIS diode array detector at 225.0 nm and an injectortted with a 20mL loop. Acetaminophen separation was carried out on a Shimadzu CLC-ODS (M) column (column size: 250 mm 4.6 mm i.d., particle size: 5mm) at 25C operating at aow rate of 1.0 mL min1using a methanol–phosphate buffer 0.1 mol L1 (pH¼ 6.2) as mobile phase (50 : 50, v/v).2
2.2. Reagents
Acetaminophen (AC, Sigma-Aldrich, >98%), hydroquinone (Sigma-Aldrich,$99%), catechol (Sigma-Aldrich, $98%),L-DOPA
(Sigma-Aldrich, >98%), ascorbic acid (Sigma-Aldrich, 99%), uric acid (Aldrich, >98%), methacrylic acid (MAA, Sigma-Aldrich, 99.5%), trimethylolpropane trimethacrylate (TRIM, Sigma-Aldrich, 90%), 2,20-azoisobutyronitrile (AIBN, Sigma-Aldrich, 98%), vinyltrimethoxysilane (VTMS, Sigma-Sigma-Aldrich, $97.5%), Fe(III)protoporphyrin(IX) (Sigma-Aldrich, $90%),
toluene (HPLC grade$ 99.9%), dimethyformamide (DMF, Sigma-Aldrich, $99.8%), hydroquinone (HQ, Sigma-Aldrich, $99%), Naon® (Sigma-Aldrich, 5%), sodium acetate (Sigma-Aldrich, $99%), HEPES (Sigma-Aldrich, $99.5%), sodium phosphate (Sigma-Aldrich, 96%), Trizma (Sigma-Aldrich,$98.5%), methanol (Sigma-Aldrich, 99.5%), acetic acid (Sigma-Aldrich, 99.5%), H2SO4
(Sigma-Aldrich, 95–95%), HNO3 (Sigma-Aldrich, >90%), H2O2
(Sigma-Aldrich, 29–32%) and MWCNTs, supplied by CNTs Co. Ltd. Yeonsu-Gu, Incheon, Korea 93% 10–40 nm diameter and length of 5–20 mm. All the solutions were prepared in deionized water from a water purication system Milli-Q® (Bedford, USA). 2.3. Synthesis of catalytic nanocomposite
Firstly, MWCNT was oxidized according to literature data.32300
mg of MWCNT were mixed with 40.0 mL of a HNO3/H2SO4(3 : 1,
v/v) mixture and reuxed at 65 C for 2 h. Aerwards, the
material was successively washed with deionized water (pHz 7.0) to remove acid excess, dried at 60C and stored at room temperature. Aer oxidation process, 150 mg of oxidized MWCNT were dispersed in 30.0 mL of toluene in a three-neck round-bottom ask and kept in an ultrasonic bath (40 kHz)
for 20 min. In the next step, 3.6 g of vinyltrimetoxisilane previously dissolved in 20.0 mL of toluene containing 1% (m/m) of hydroquinone was slowly added to the mixture under stir-ring. One of the three necks was sealed, the central neck was coupled to a reux condenser and the other neck was purged with nitrogen (N2) for 10 min. Aerwards, the neck was sealed
and the mixture was heated at 100C in an oil bath for 6 h. The obtained functionalized MWCNT was washed with ethanol and dried at 50 C for 6 h. For the synthesis of catalytic nano-composite, 40.0 mg of functionalized MWCNT were dispersed in 20.0 mL of toluene in aat-bottomed ask and kept in an ultrasonic bath (40 kHz) for 20 min. Then, 0.5 g of methacrylic acid (5.8 mmol) as functional monomer, 0.45 g of trimethylol-propane trimethacrylate (1.32 mmol) as cross-linking reagent, 60 mg of Fe(III)protoporphyrin(IX) (0.097 mmol), as prosthetic
group of peroxidase and 45.35 mg of AC as template molecule were added to the ask. The mixture was manually stirred, followed by addition of 80.0 mg of 2,20-azoisobutyronitrile (0.48 mmol). Nitrogen gas was purged for 5 min and then theask was sealed. The mixture was incubated for 24 h at 60C under stirring. The obtained catalytic nanocomposite was then washed with methanol/acetic acid (4 : 1, v/v) for the removal of acetaminophen, dried at 60 C for 12 h and stored in amber ask until use. The obtained material was named as MIP. The blank polymer, i.e., the non imprinted polymer (NIP) was synthesized in similar way excepted by the addition of AC. The schematic representation of synthesis of nanocomposite based on multi-walled carbon nanotubes graed by poly(methacrylic acid–hemin) is depicted in Fig. 1.
2.4. Electrochemical procedure
The nanocomposite was cast on the surface of a glassy carbon electrode by pipetting 10.0mL of sonicated suspension of 10.0 mg mL1nanocomposite dispersed in DMF followed by addi-tion of 10.0mL of 5% (m/v) Naon® and kept under heating at 45C in an oven to volatilize the solvent. The measurements of
Fig. 1 Schematic representation of synthesis of nanocomposite based on multi-walled carbon nanotubes grafted by poly(methacrylic acid– hemin).
square wave voltammetry (SWV) were carried out in a conven-tional electrochemical cell with 15.0 mL capacity containing the modied glassy carbon electrode, Ag/AgCl (KCl, 3.0 mol L1)
reference electrode and the platinum auxiliary electrode con-taining 0.1 mol L1Trizma buffer at pH 8.0 and using frequency of 10 Hz with pulse amplitude of 20 mV.
2.5. Sample preparation
Five tablets with known declared content (750 mg per tablet) were ground to a ne powder and thoroughly mixed. Then, a known mass was weighed for the sample and diluted with 5.0 mL of water and sonicated during 10 min.33The same
proce-dure was adopted to another sample containing 500 mg per tablet. Aerwards, the solution was transferred to a volumetric ask whose volume was made up to 100.0 mL and a volume of 20mL of solution was transferred into the electrochemical cell containing 0.1 mol L1Trizma buffer at pH 8.0 and the volume was made up to 10.0 mL.
3.
Results and discussion
3.1. Characterization of MWCNT and nanocomposite The FT-IR spectra of materials are depicted in Fig. 2. For the pristine MWCNT, oxidized MWCNT and MWCNT functional-ized with vinyltrimethoxysilane (Fig. 2a), the intense absorption
band observed at 3450 cm1 can be attributed to the n(O–H) stretching vibrations on the MWCNT surface and adsorbed water that forms hydrogen bonds.32,34A carbonyl band at 1630
cm1was noticed35and at 1380 cm1the low intense band is
attributed to d(O–H) related to in-plane vibration of carboxylic acid.36 The stretching of C–O from primary alcohol was
observed at 1090 cm1, while, the low intensity signals at 770 and 660 are ascribed to the Si–O stretching vibrations, which conrms the surface functionalization of MWCNT with vinyl-trimethoxysilane.37Aer polymerization with MIP and NIP, the
spectra proles were somewhat different (Fig. 2b). The broad signal in the region of 2990–2950 cm1is ascribed to symmetric
and asymmetric CH2stretching vibrations from the backbone
chain of polymer. The occurrence of a well dened band at 1726 cm1was observed, which can be assigned to stretching vibra-tion of C]O from MAA or hemin. In this case, the shoulder at 1632 cm1 can be attributed to C]C stretching from vinyl groups or overlap by OH deformation (water).38The signals at
1260 and 1160 cm1can be attributed to C–N from porphyrinic backbone of hemin or stretching of C–O from ester group and carboxylic acid.39 The signals at 1470 and 1390 cm1 can be
ascribed, respectively, to C–H from methylene and methyl groups.38The similarity of MIP and NIP spectra also conrms
the complete removal of AC from polymeric matrix of MIP using methanol/acetic acid (4 : 1, v/v) solution.
Fig. 3a and b shows the thermogravimetric curves of pristine MWCNT, oxidized MWCNT, MWCNT functionalized with
Fig. 2 FT-IR spectra for the materials. (a) MWCNT, oxidized MWCNT and functionalized MWCNT, (b) MIP and NIP.
Fig. 3 TG curves for the materials. (a) MWCNT, oxidized MWCNT and functionalized MWCNT, (b) MIP and NIP.
vinyltrimethoxysilane, MIP and NIP. As can be observed, the thermal prole of the MWCNTs are very similar to each other, with weight loss of 17% at 900C, which demonstrates that polymerization reaction of vinyltrimethoxysilane resulting in inorganic matrix was not observed. On the other hand, differ-ences in thermal proles between MIP and NIP were evident regarding the MWCNTs. Therst weight loss of 9% occurs in the range of 30–90C, which is ascribed to the volatilization of
adsorbed water. Weight loss of 25% for MIP and NIP occurs, respectively, at 390 C and 350 C, thus indicating that the former one is thermally more stable than NIP. Upon tempera-ture range of 300–470C the decomposition of segments of the
polymeric chain of methacrylic acid and cross-linking reagent trimethylolpropane trimethacrylate takes place.38 Taking into
account the temperature range varying from 200 to 470C for MIP and NIP, it can be observed a weight loss of 78%, i.e., much higher than that achieved for MWCNT. Such difference in the weight loss indicates the amount of polymer graed onto MWCNT surface of the total weight of nanocomposite.
The SEM images of pristine MWCNT, oxidized MWCNT, functionalized MWCNT, MIP and NIP are depicted in Fig. 3. The morphological characteristics of MWCNT are observed in Fig. 3a, with entangledlamentous of carbon nanotubes. The oxidation of MWCNT, as well as the silanization reaction with
vinyltrimetoxisilane, promote intense aggregation of the carbon nanotubeslaments (Fig. 4b and c). Aer graing process of polymers on the surface of MWCNT, it is possible to clearly observe an increase in the diameter of MWCNT, thus indicating that the MWCNTs are densely covered by polymers (Fig. 4d and e). From TEM images (Fig. 5) thisnding is distinctly observed, where the polymeric matrix is homogeneously distributed over the surface of MWCNT in nanoscale (10–50 nm).
The textural data, including surface area, pores volume and pores diameter of nanocomposite, play an important role in the electroanalytical performance toward the AC determination. Table 1 shows the differences in textural data between MIP and NIP, where the porosity of NIP was higher than that achieved for MIP. This result can be attributed to the greater solubility of complex template (AC)-functional monomers (MAA–hemin) in porogenic solvent during the synthesis of MIP, which reduced the solvent removal rate from polymeric matrix and, as a consequence, decreased its porosity.40The lower porosity of
MIP compared to NIP indicates in advance that differences in the electrochemical behavior of modied sensor will be attrib-uted to the imprinted cavities created in the MIP, and not to the textural data dependence.
Fig. 4 SEM images for the materials. (a) MWCNT, (b) oxidized MWCNT, (c) functionalized MWCNT, (d) MIP and (e) NIP. Magnification 30 000.
Fig. 5 TEM images for the materials. (a and b) Functionalized MWCNT, (c and d) MIP.
Table 1 Textural data of MIP and NIP nanocomposites
Nanocomposites Surface area (m2g1) Pores volume (cm3g1) Average pore diameter (nm) MIP 13.84 0.037 3.4 NIP 22.37 0.083 15.9
3.2. Electrochemical behavior of MIP and NIP nanocomposites toward the AC determination
Fig. 6a presents the cyclic voltammetric curves obtained from MIP nanocomposite electrode in 0.1 mol L1phosphate buffer solution at pH 7.0. The assays were carried out to evaluate the properties of MIP as a peroxidase-like catalyst for biomimetic sensing of AC using cathodic scan from 0.4 to0.5 V. In the absence of H2O2 and AC, it was observed a reduction peak at
0.2 V corresponding to the Fe(III)/Fe(II) redox couple of iron
present in the hemin, in similar way to the redox couple of the heme protein of horseradish peroxidase.41Aer adding H
2O2,
the cathodic peak current was increased, thus showing that hemin exhibits, as expected, catalytic properties toward the H2O2 reduction in the same way as peroxidase-based
biosen-sors. In the presence of H2O2, the addition of AC promotes
a signicant increase in the peak current, which demonstrates that the hemin in the presence of H2O2catalyses the chemical
oxidation of acetaminophen and the reaction product, i.e., N-acetyl-p-benzoquinoneimine, may in turn be electrochemically reduced on the surface of electrode at lower potential (0.27 V). In order to verify the inuence of MIP nanocomposite toward the AC determination, cyclic voltamograms were recorded using a glassy carbon electrode without any modication. Fig. 6b shows the absence of cathodic peak current in the presence of H2O2 and AC, revealing a better response for the MIP
nano-composite and its catalytic properties.
The better ability of MIP regarding NIP to recognize the AC can be observed by comparing the cyclic voltammograms recor-ded in Fig. 6a and c. As observed in Fig. 6c, the presence of hemin in the polymeric matrix of NIP makes possible to catalyze the reduction of H2O2 as previously veried for MIP (Fig. 6a).
However, only a slight increase of cathodic peak current was observed aer adding AC, which clearly proves that the stronger affinity of MIP nanocomposite toward the AC is attributed to the specic binding sites.
3.3. Effect of number of layers and Naon® concentration on the sensor preparation
A set of assays were carried out in order to dene the best conditions for the sensor preparation. Firstly, the number of layers (1 up to 3 layers) of MIP nanocomposite suspension at 10 mg L1was evaluated by pipetting 10.0mL of the suspension, followed by addition of 10.0 mL of 5% (m/v) Naon®. Cyclic voltammograms for the concentration of 50 mmol L1 AC in a 0.1 mol L1phosphate buffer solution, pH 7.0 and at 30 mV s1were recorded. The results showed that only one layer was sufficient to efficiently modify the surface of glassy carbon electrode and provide catalytic properties to the sensor. On the other hand, a larger number of layers decreases the peak current of AC most likely due to blocking of electron ow. Therefore, the casting of glassy carbon electrode was carried out with one layer. It has been reported that Naon® lm has an important role inxing the nanocomposite layer on the elec-trode surface. Hence, it was necessary to evaluate the inuence of Naon® concentration in the sensor preparation. Concen-trations of 0.5, 1.0, 1.6, 3.0 and 5.0% (m/v) were assessed. It was found that peak current for AC was very similar for all concen-trations; however, the highest stability of MIP nanocomposite was achieved by using 5% (m/v) Naon®, therefore this concentration was selected for further experiments.
3.4. Effect of scan rate on the AC electrooxidation
In order to assess the nature of electron transfer process of AC at the MIP nanocomposite, cyclic voltammograms for concentra-tion of 70 mmol L1 AC in 0.1 mol L1 phosphate buffer
Fig. 6 Cyclic voltammograms of the MIP nanocomposite electrode (a), bare glassy carbon electrode (b) and (c) NIP nanocomposite electrode in 0.1 mol L1phosphate buffer solution, pH 7.0 at 30 mV s1 in the presence of H2O2and H2O2with AC.
solution, pH 7.0 at different scan rate (20–100 mV s1) were
recorded (Fig. 7).
As veried in the Fig. 7, the slope (0.517) is very close to the theoretical expected value of 0.5, which conrms that electro-chemical reaction of AC takes place through diffusion-controlled process.2
3.5. Effect of pH and H2O2concentration
The pH of samples has an indicative role for MIP recognition containing hemin as catalytic center in a similar way to heme protein of horseradish peroxidase. Therefore, as shown in Fig. 8, the pH dependence was investigated in the range of 6.0– 8.0, to obtain the best condition for the horseradish peroxi-dase.28In the 6.0–7.5 pH range, there is no signicant difference
in the peak current. However, a higher signal was noticed at pH 8.0, so this value was chosen for subsequent work. Thisnding can be rationalized bearing in mind that the pKa value of AC is 9.5, which in turn makes easier its interaction with specic binding sites.
The H2O2 concentration ranging from 25.0–500 mmol L1
was also investigated (Fig. 9).
As shown in Fig. 9, the maximum peak current was obtained using 300.0mmol L1H2O2, and aer this value, a slight decrease
in current was observed, which is attributed to the saturation of hemin. Thus, 300.0 mmol L1 H2O2 solution was selected for
further studies.
Aer obtaining the best conditions for pH and H2O2
concentration, the effect of type of supporting electrolyte was
Fig. 7 Plot log n versus log i. Cyclic voltammograms recorded in 0.1 mol L1phosphate buffer solution, pH 7.0 in the presence of 70 mmol L1AC.
Fig. 8 Effect of pH on sensor performance. Conditions: cyclic vol-tammograms obtained from the MIP nanocomposite electrode in 0.1 mol L1phosphate buffer solution, at 30 mV s1in the presence of 10.0 mmol L1AC.
Fig. 9 Effect of H2O2concentration on sensor performance. Condi-tions: cyclic voltammograms obtained from the MIP nanocomposite electrode in 0.1 mol L1phosphate buffer solution, at pH 8.0 and 30.0 mV s1in the presence of 10.0mmol L1AC.
Table 2 Responses of MIP and NIP nanocomposite sensors to other molecules
Compound Molecular structure
Current cathodic (mA) Response percentage (%)
MIP NIP MIP NIP
Acetaminophen 1.05 0.07 100 100 Hydroquinone 0.14 0.14 13.8 200 Catechol 0.07 0.14 7 200 L-DOPA 0 0 0 0 Ascorbic acid 0 0 0 0 Uric acid 0 0 0 0
evaluated. Acetate, Hepes, phosphate and Trizma buffer solu-tions at 0.1 mol L1concentrations were evaluated as support-ing electrolytes. The assays were carried out by cyclic voltammograms obtained from the MIP nanocomposite elec-trode at pH 8.0, 30.0 mV s1, in the presence of 300.0mmol L1 H2O2and increasing concentration of AC (10.0, 30.0, 50.0 and
70.0 mmol L1). The best result in terms of sensibility (A L mmol1) was found using Trizma; therefore, the subsequent
experiments were carried out byxing this buffer solution as supporting electrolyte.
3.6. Response of MIP and NIP nanocomposite sensors to other compounds
The selectivity of MIP and NIP nanocomposite sensors was evaluated from the analysis of their response toward other compounds. Some of the compounds are structurally similar to AC and all of them present electrochemical activity. The study was carried out by cyclic voltammetry under pH 7.0 at 30.0 mV s1in the presence of 50.0mmol L1of H2O2and 10.0mmol L1
of each compound. As can be seen in Table 2, the developed electrochemical sensor shows excellent response toward the AC when compared to other electroactive molecules. Moreover, higher currents for catechol and hydroquinone were achieved for the NIP nanocomposite in detriment of MIP nanocomposite. These results conrm once again the imprinting effect created during the polymer synthesis.
3.7. Choice of electroanalytical technique andgures of merit
From previous studies, the best parameters obtained for SWV were 10 Hz, pulse amplitude of 20 mV and potential step of 4 mV, while for DPV (differential pulse voltammetry) the best values were 20 mV for pulse amplitude, 30 mV s1for scan rate and pulse time of 170 ms. Under these conditions, the analytical curves, linear range, limits of detection, determined according to IUPAC recommendation42and sensitivity summarized in Table 3
were obtained. According to achieved results, SWV has shown better gures of merit for AC determination and has been employed for determining AC in pharmaceutical formulations.
For assessing the precision of proposed method, standard solutions of 30.0 and 90.0mmol1AC were subjected to 10 intra-day measurements by SWV under optimized conditions. Relative standard deviation (RSD) values of 7.2 and 2.7% were found for respective concentrations of 30.0 and 90.0 mmol1 AC, thus demonstrating the precision of method. Regarding the stability of MIP nanocomposite sensor, it was observed that 60 measures performed in two different days may be performed using 90.0 mmol1AC without decreases of analytical performance, yielding
RSD value of 4.5%.
3.8. Analytical application of proposed method in real sample
The feasibility of method was assessed through analysis of pharmaceutical formulation. As shown in Table 4, good
Table 3 Comparison of SWV and DPV to the AC determination using the MIP nanocomposite sensora
Analytical technique Linear range (mmol1) Analytical curve LD (mmol1) S (A Lmmol1)
SWV 10.0–130.0 I (mA) ¼ 0.70 + 0.010[AC, mmol1) (r¼ 0.995) 2.7 0.010
DPV 10.0–90.0 I (mA) ¼ 1.8 + 0.028[AC, mmol1) (r¼ 0.991) 1.1 0.028
aLD¼ limit of detection, S ¼ sensitivity.
Table 4 Results of AC determination in pharmaceutical formulations by proposed method and HPLC
Sample Labeled value (mg per tablet) Sensora(mg per tablet) HPLCa E1(%) E2(%)
1 500 471.5(0.5) 472.2(5.0) 0.1 5.6
2 750 771.4(0.5) 722.9(3.5) 6.3 3.6
aResults are expressed as mean value standard deviation based on three replicates (n ¼ 3). E
1¼ sensor vs. HPLC; E2¼ sensor vs. labeled value.
Table 5 Recovery results of AC determination in pharmaceutical formulations by proposed method and HPLC
Sample Labeled value (mg per tablet) Added AC (mg per tablet) Sensora (mg per tablet) HPLCa R1(%) R2(%) 1 500 — 471.5(0.5) 472.2(5.0) — — 200 671.5(0.67) 657.1(5.8) 100 92.4 2 750 771.4(0.5) 722.9(3.5) — — 200 975.1(0.5) 916.2(4.3) 101.8 96.6
aResults are expressed as mean value standard deviation based on three replicates (n ¼ 3). R
1¼ recovery percentage by sensor; R2¼ recovery by HPLC.
agreement of achieved results with those described by manu-facturer was noticed, which indicates the absence of matrices effect. Moreover, the accuracy of proposed method was checked by comparison with HPLC data, used as a reference technique. No statistical difference was observed between proposed method (paired student t test, at the condence level of 95%, tcalc¼ 0.97 lower than ttab¼ 12.70)43and HPLC method.
The accuracy of method was also veried by addition and recovery tests. Table 5 shows that values of recovery are close to 100% for the proposed method and HPLC, thus attesting once again that components in the matrix did not affect the perfor-mance of the voltammetric sensor and the efficiency of sample treatment.
4.
Conclusions
The present study describes for therst time the development of a new electrochemical sensing platform for AC determination based on nanocomposite multi-walled carbon nanotubes graed by molecularly imprinted poly(methacrylic acid–hemin) as a peroxidase-like catalyst. From the FT-IR, TG, SEM and TEM data, it was found that the synthesis and the graing process of catalytic MIP at nanoscale level on the surface of MWCNT was successfully carried out. The use of surface molecular imprinting technique onto MWCNT for the preparation of catalytic nano-composite MIP provided a better recognition capacity for AC regarding NIP, as well as better selectivity in the presence of other phenolic compounds, thus showing to be a useful platform to obtain high selective electrochemical sensors. Regarding the analytical characteristics of the sensor, it was found similar limits of detection when compared to other reported electrochemical methods based on molecularly imprinted technology25,44,45and
biomimetic sensor.46Nevertheless, one should note that the great
highlight of proposed method relies on the synergic effect of MIP as a recognition element, the catalytic properties of hemin and conductive properties of MWCNT, thus allowing the AC deter-mination at very low potential.
Acknowledgements
The authors would like to thank the Conselho Nacional de Desenvolvimento Cient´ıco e Tecnol´ogico (CNPq) (Project No. 481669/2013-2, 305552/2013-9, 472670/2012-3), Coordenaç˜ao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) (25/ 2014), Fundaç˜ao Arauc´aria do Paran´a (163/2014), Laborat´orio de Espectroscopia da Central de Multiusu´ario da PROPPG, SANEPAR, FAPEMIG and Instituto Nacional de Ciˆencia e Tec-nologia de Bioanal´ıtica (INCT) (Project No. 573672/2008-3) for theirnancial support and fellowships.
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