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Sensors and Actuators B 251 (2017) 65–73

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo ur nal home page: www.elsev ier .com/ locate /snb

new strategy for NADH sensing using ionic liquid crystals-carbonanotubes/nano-magnetite composite platform

ada F. Atta ∗, Soha A. Abdel Gawad, Ekram H. El-Ads, Asmaa R.M. El-Gohary,hmed Galal

hemistry Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

r t i c l e i n f o

rticle history:eceived 10 March 2017eceived in revised form 26 April 2017ccepted 6 May 2017vailable online 8 May 2017

a b s t r a c t

Glassy carbon (GC) electrode is modified by stepwise manual casting of successive layers of ionicliquid crystals (ILC)/carbon nanotubes (CNTs) and magnetite nanoparticles. The composite [GC/(ILC-CNTs)/Fe3O4)] is used as a successful NADH biosensor. Molecular interaction of NADH is improved throughthe conductive and dense structure of ILC/CNTs. Magnetite nanoparticles act as electron-exchange “anten-nas” resulting in enhanced charge transfer at the interface, selective and sensitive oxidation of NADH.

eywords:ADH biosensorarbon nanotubes

onic liquid crystalsano-magnetitemperometry

The type of ionic liquid used is key factor for sensor performance. The figures of merit for the proposedNADH sensor are: linear dynamic range of 5–700 �mol L−1, a sensitivity of 0.0102 �A �mol−1 L, a detec-tion limit of 34.6 nmol L−1 and a limit of quantification of 0.115 �mol L−1. The sensor showed a stableamperometric response and anti-interfering ability in presence of ascorbic acid, tryptophan, ibuprofenand morphine.

© 2017 Elsevier B.V. All rights reserved.

. Introduction

Carbon nanotubes (CNTs) are highly conjugated systems andave unique properties including electronic, mechanical and chem-

cal characteristics [1]. CNTs have been applied extensively inlectrochemical sensors construction [1–5]. Their surfaces adsorp-ion and penetration ability are important characteristics forensor fabrication. Consequently, CNTs are conveniently applica-le for enzyme and redox systems immobilization because of theirydrophobic and biocompatible nature [1–3]. CNTs have goodffinity towards organic molecules to form non-covalent struc-ures; some examples include DNA, proteins and �-cyclodextrins6–8]. The increase in the surface area of CNTs modified electrodes attributed to the fact that the surface of CNTs is consid-red formed of nano-electrode arrays. Therefore, CNTs have beensed in constructing biosensors for the determination of someompounds such as cysteine, ascorbic acid, neurotransmitters, tra-adol, nucleic acids and NADH [1–5].

CNTs can be functionalized through chemical or physical trans-

ormations which facilitate the rate of electron transfer of redoxrocesses taking place at their surfaces [9–16]. Ionic liquid crys-als (ILCs) functionalization allows their role as host to CNTs guest

∗ Corresponding author.E-mail address: anada@sci.cu.edu.eg (N.F. Atta).

ttp://dx.doi.org/10.1016/j.snb.2017.05.026925-4005/© 2017 Elsevier B.V. All rights reserved.

resulting in ordered structure at the interface [9,16,17]. Further-more, it was reported that the interesting dispersion of CNTs intoionic liquids “ILs” did not change the symmetrical conjugated �-system structure of CNTs [18–21]. At the same time, non-covalent�-� interactions take place between the aromatic moieties of ILsand CNTs [9,16,18–21]. This leads to a decrease in the electric resis-tance while maintaining high ionic conductivity.

On the other hand, ILCs and ILs possess distinct properties[23–25]. The most important among those is the presence of ionicunits in the mesophase that is unique for ILCs. Different drugs andcompounds were determined successfully using ILCs in differentcomposite electrodes [26–28].

Iron oxide nanoparticles “Fe3O4” are widely employed as sur-face modifiers in sensors. The catalytic activity of Fe3O4 is crucialfor the direct charge transfer at the electrolyte/electrode inter-face [3–5,29–32]. CNTs-Fe3O4 nanoparticles have been used for thedetermination of pesticides [3], dopamine [4] and glucose [5], etc.

Dihydro-nicotinamide adenine dinucleotide (NADH) and its oxi-dized form (NAD+) are important coenzymes and contribute toelectron transfer reactions in living cells [33–36]. NADH is widelyused in medicine for the treatment of critical diseases such asAlzheimer and Parkinson. Therefore, the determination of NADH

is of prime importance for biotechnological and pharmaceuticalindustries. Several techniques have been used for the determina-tion of NADH including HPLC [35], capillary electrophoresis [36],flow injection [37,38], chemiluminescence [39], colorimetry [40]

66 N.F. Atta et al. / Sensors and Actuators B 251 (2017) 65–73

Scheme 1. Schematic representation of the proposed electrode, GC/(ILC-CNTs)/Fe3O4 used for the amperometric sensing of NADH.

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Fig. 1. (A) Cyclic voltammograms of 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4recorded at different working electrodes; bare GC, GC/CNTs, GC/CNTs/Fe3O4,

A mixture of CNTs and ILC was dissolved in 1 mL DMF and thenultra-sonicated for one hour to obtain homogeneous suspension.The amount of ILC (from 0 to 3 mg) in the suspension with 1 mg

nd electrochemistry [41]. Electrochemical methods are advan-ageous because they are simple, rapid, less costly and relativelyensitive. Some challenges still face electrochemical methods forADH determination such as high overpotential of oxidation, low

electivity and surface fouling by oxidative products [34]. CNTsave been applied in different modified electrodes for NADH oxida-ion such as: fluphenazine on CNTs [1], polymerized phenothiazineyes on CNTs [2], CNTs-ferrocene dicarboxylic acid [42], activatediclosamide on CNTs [43], carbon nanofibers modified glassy car-on [44] and Pd-nanoparicles-CNTs nanohybrid [45].

In the present work, we introduce a new strategy to modify thelassy carbon electrode with ILC-CNTs mixture and Fe3O4 nanopar-icles. The proposed sensor, GC/(ILC-CNTs)/Fe3O4, combines theharacteristic features of its individual components toward NADHxidation. CNTs offer several unique properties such as relativelyigh conductivity, biocompatibility and high apparent active sur-

ace area for electrochemical oxidation of NADH [1–5]. ILC canenetrate the spacing between the nanotubes resulting in the

ormation of a matrix with enhanced ionic and electronic con-uctivities [17,19–22]. The assembled Fe3O4 nanoparticles overILC-CNTs) matrix result in increased number of active sites avail-ble for the electrochemical reaction (Scheme 1).

. Experimental

.1. Chemicals and reagents

All chemicals were used as received without furtherurification. Multi-walled carbon nanotubes (MWCNTs)length = 0.1–10 �m, >90%), iron (III) oxide magnetite (Fe3O4)diameter < 50 nm), 1-butyl-1-methylpiperidinium hexafluo-ophosphate ionic liquid crystal (ILC), 1-butyl-4-methylpyridiniumetrafluoroborate (IL1) and 1-n-hexyl-3-methylimidazoliumetrafluoroborate (IL2) ionic liquids were obtained from Sigma-ldrich. �-nicotinamide adenine dinucleotide (NADH), ascorbiccid (AA), morphine (MO), tryptophan (Try), dimethylformamideDMF), sodium sulfate, sodium chloride, sodium nitrate, sodiumihydrogen phosphate and disodium hydrogen phosphateere supplied by Aldrich Chem. Co. Phosphate buffer solu-

ion (PBS, 0.1 mol L−1) was used as the supporting electrolyte1 mol L−1 K2HPO4 and 1 mol L−1 KH2PO4). The pH was adjustedsing 0.1 mol L−1 H3PO4 and 0.1 mol L−1 KOH. All solutions were

repared from analytical grade chemicals and distilled water.

GC/(ILC-CNTs) and GC/(ILC-CNTs)/Fe3O4 electrodes, scan rate 50 mV s−1. (B) Cyclicvoltammograms of 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4 recorded at GC/(ILC-CNTs)/Fe3O4 and GC/(ILC-CNTs-Fe3O4) electrodes, scan rate 50 mV s−1.

2.2. Electrochemical cell and equipment

The working electrode was a glassy carbon electrode (GC)(� = 3 mm) from BASi (USA), the reference electrode was Ag/AgCl(4 mol L−1 KCl saturated with AgCl) from BASi (USA) and 6.0 cm(� = 0.5 mm) Pt wire from Alfa Aesar (USA) was used as the aux-iliary electrode. The GC electrode was polished using an alumina(2 �m)–water slurry until no visual scratches were observed.The electrochemical characterizations were performed using aBAS epsilon-electrochemical analyzer (Bioanalytical systems, USA)(with ±5 mV error). All experiments were performed at 25 ± 0.2 ◦C.For the amperometric experiments the following parameters wereused: the applied potential = 225 mV, the time limit = 20 s and thesample interval = 1 s.

The microstructure of the samples was investigated usingQuanta FEG 250 instrument. A glassy carbon sheet of large surfacearea was used to prepare the samples required for the SEM analysis.

2.3. Construction of the proposed sensor

CNTs prepared in DMF has been optimized and the suspension con-

N.F. Atta et al. / Sensors and Actuators B 251 (2017) 65–73 67

Table 1Values of anodic peak current (Ipa/�A) and potential (Epa/mV) of 1 mmol L−1

NADH/0.1 mol L−1 PBS/pH 7.4 at scan rate 50 mVs−1 recorded at different workingelectrodes; bare GC, GC/CNTs, GC/CNTs/Fe3O4, GC/(ILC-CNTs), GC/(ILC-CNTs)/Fe3O4,GC/(ILC-CNTs-Fe3O4), GC/(IL1-CNTs)/Fe3O4 and GC/(IL2-CNTs)/Fe3O4.

Electrode Ipa (�A) Epa (mV)

Bare GC 4.00 675GC/CNTs 13.85 257GC/CNTs/Fe3O4 33.85 245GC/(ILC-CNTs) 42.73 210GC/(ILC-CNTs)/Fe3O4 54.44 223

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Fig. 2. Cyclic voltammograms of 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4 recordedat GC/(ILC-CNTs)/Fe3O4, GC/(IL1-CNTs)/Fe3O4 and GC/(IL2-CNTs)/Fe3O4, scan rate50 mV s−1.

Fig. 3. Cyclic voltammograms of 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4 recordedat GC/(ILC-CNTs)/Fe3O4 at different scan rate values from 20 to 400 mV/s. Inset: Plotof the anodic peak current of NADH as a function of the square root of scan rate.

GC/(ILC-CNTs-Fe3O4) 44.44 254GC/(IL1-CNTs)/Fe3O4 41.82 282GC/(IL2-CNTs)/Fe3O4 24.44 242

aining 2 mg ILC and 1 mg CNTs in 1 mL DMF shows the maximumurrent response compared to other ratios (Figure not shown). Thenliquot of 10 �L of ILC-CNTs mixture was casted on the polishedurface of GC. The electrode was left to dry and this electrode is rep-esented as GC/(ILC-CNTs). The amount of Fe3O4 (from 0 to 3.5 mg)n the suspension containing 1 mL DMF has been optimized and the

aximum current response was obtained in the suspension con-aining 2.5 mg Fe3O4 in 1 mL DMF (Figure not shown). Then 10 �L ofe3O4 suspension was casted on the GC/(ILC-CNTs) electrode andeft to dry. This electrode is represented as GC/(ILC-CNTs)/Fe3O4.he proposed electrode, GC/(ILC-CNTs)/Fe3O4, was then ready forlectrochemical tests. The most common solvent used for effectiveNTs dispersion is DMF. Ethanol as solvent for example resulted inggregation of CNTs.

Other design of the sensor was also investigated to select theest arrangement with the highest current response. It involveshe preparation of a mixture of 1 mg CNT, 2 mg ILC and 2.5 mg ofe3O4 in 1 mL DMF and sonicated for one hour. Then, 10 �L of thisixture was casted on the GC and the electrode is represented asC/(ILC-CNTs-Fe3O4).

. Results and discussion

.1. Electrochemistry of NADH at different modified electrodes

The electrocatalytic oxidation of NADH at different modifiedlectrodes has been investigated using cyclic voltammetry. Fig. 1(A)hows the electrochemistry of 1 mmol L−1 NADH/0.1 mol L−1

BS/pH 7.4 at different modified electrodes. Table 1 summarizeshe anodic peak currents and the anodic peak potentials of NADHt the studied electrodes.

The mechanism of NADH oxidation involves two electrons andne proton through an electrochemical-chemical-electrochemicalECE) pathway. The oxidation mechanism is summarized aseported earlier in the following steps [34,46,47]:

NADH → NADH•+ + e−

(Irreversible oxidation step, electrochemical) (1)

ADH•+ → NAD• + H+(Deprotonation step, chemical) (2)

AD• → NAD+ + e−(Oxidation step, electrochemical) (3)

The overall equation is represented as:

ADH → NAD+ + H+ + 2e− (4)

The electrochemical oxidation of NADH at previously studied

lectrodes exhibits an irreversible behavior. From the obtainedesults we found that the anodic peak current of NADH increasesy 3.46, 8.46, 10.68 and 13.61 folds at GC/CNTs, GC/CNTs/Fe3O4,C/(ILC-CNTs) and GC/(ILC-CNTs)/Fe3O4, respectively compared to

GC. In addition, the anodic peak potential of NADH at GC/(ILC-CNTs)/Fe3O4 shifts negatively by 0.452 V compared to bare GC.Synergistic interactions between the individual components of theproposed sensor result in both thermodynamic and kinetic favor-ing as manifested in a decrease in overpotential and increase incurrent signal. A second design for the proposed sensor was inves-tigated in order to select the better arrangement with the highestcurrent response. Fig. 1(B) shows the electrochemical response of1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4 at GC/(ILC-CNTs)/Fe3O4and GC/(ILC-CNTs-Fe3O4). The results indicated that mixing ILC,CNTs and Fe3O4 nanoparticles leads to lower current responsetowards NADH oxidation compared to the first method (Table 1).This may be attributed to less availability of Fe3O4 nanoparticlessites in the second case. Therefore, the proposed sensor formed bythe systematic casting of Fe3O4 nanoparticles over the conductivecomposite of ILC-CNTs is more favored than the mixing of the threecomponents approach.

68 N.F. Atta et al. / Sensors and Actuators B 251 (2017) 65–73

Fig. 4. (A) Cyclic voltammetric response of GC/(ILC-CNTs)/Fe3O4 electrode in1 mmol L−1 NADH/0.1 mol L−1 PBS of different pH values from 3.7 to 11. Inset: plotof the anodic peak potential of NADH versus the pH values. (B) Cyclic voltammet-ric response of the GC/(ILC-CNTs)/Fe3O4 electrode in 1 mmol L−1 NADH in differenttypes of supporting electrolytes. All electrolytes were 0.1 mol L−1 in concentra-t0

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the anodic peak potential shifts to more positive values. The cur-

ion in distilled water. Buffer (I) is 5 mmol L−1 Na2HPO4, 5 mmol L−1 NaH2PO4 and.1 mol L−1 NaCl.

.2. Effect of type of ionic liquid

The dispersion of CNTs in ILs leads to enhanced ionic/electroniconductivity of the composite. It is therefore important to examinehe effect of changing the type of ILs in (ILs-CNTs) composite on theensor response. Fig. 2 shows the electrochemistry of 1 mmol L−1

ADH/0.1 mol L−1 PBS/pH 7.4 at GC/(ILC-CNTs)/Fe3O4, GC/(IL1-NTs)/Fe3O4 and GC/(IL2-CNTs)/Fe3O4. The obtained results provedhat higher current response and less positive oxidation poten-ial are achieved in case of ILC (Table 1). This is attributed to theharacteristic features of ILC compared to the other ILs. ILC showshe features of ILs and LCs including enhanced anisotropic ioniconductivity, high polarizability, highly ordered films with the aidf its solid state structure and spontaneous molecular orientation

27,48]. These features facilitate the formation of highly conductive

atrix with dense structure. This matrix becomes more tangible forhe immobilization of Fe3O4 nanoparticles in organized manner.

Fig. 5. (A) SEM of GC/CNTs, (B) GC/(ILC-CNTs), (C) GC/(ILC-CNTs)/Fe3O4.

3.3. Effect of scan rate

The effect of applying different scan rates on the electrochemicalresponse of 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4 at GC/(ILC-CNTs)/Fe3O4 is investigated (Fig. 3). Upon increasing the scan ratefrom 20 mV/s to 400 mV/s, the anodic peak current increases and

rent increases linearly with the square root of the scan rate as anindication of diffusion-controlled process at the proposed sensor.The shift in the oxidation potential with the scan rate is an indica-

N.F. Atta et al. / Sensors and Actuators B 251 (2017) 65–73 69

Table 2Effect of the electrolyte type on the peak potentials (Epa/ mV) and anodic peak currents (Ipa/�A) of 1 mmol L−1 NADH observed at GC/(ILC-CNTs)/Fe3O4 electrode, scan rate50 mVs−1.

Supporting electrolytea pH Epa (mV) Ipa (�A)

NaNO3 6 240 39.09NaCl 5.8 254 42.22Buffer Ib 6.6 221 43.33Na2SO4 10.4 250 46.67PBS 7.4 223 54.44

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a All electrolytes were 0.1 mol L−1 in concentration in distilled water.b Buffer (I) is 5 mmol L−1 Na2HPO4, 5 mmol L−1 NaH2PO4 and 0.1 mol L−1 NaCl.

ion of the system irreversibility [34]. A linear relation is obtainedetween the anodic peak current of NADH and the square root ofhe scan rate between 20 and 400 mVs−1 (inset of Fig. 3).

.4. Effect of solution pH and type of supporting electrolyte

The effect of pH of the supporting electrolyte is an importantarameter affecting the electrochemical performance of NADH sen-or. Fig. 4A shows the CVs of 1 mmol L−1 NADH/0.1 mol L−1 PBS ofifferent pH values (3.7–11) at GC/(ILC-CNTs)/Fe3O4. Changing theH of PBS affects the anodic peak current and potential of NADH.his is attributed to protonation/deprotonation that affects thelectron transfer process. The current response shows distinct vari-tion in the pH range studied. The pH that dominates the humanody, namely the blood, is around 7.4. Therefore, it will be advan-ageous to test the performance of the proposed sensor in bufferolution of pH 7.4.

The linear relation between the anodic peak potentials and pH3.7–7.4) (Inset of Fig. 4A) is fitted to the following equation:

pa(V) = 0.416–0.026 pH (5)

The correlation coefficient is: r2 = 0.994. The slope of this relations (−0.026 V/pH) suggesting that the oxidation of NADH involves

ig. 6. Amperomteric response of GC/(ILC-CNTs)/Fe3O4 with successive additions of NADnset (1): Amperomteric response of GC/(ILC-CNTs)/Fe3O4 with successive additions of Nnset (2): Calibration curve for NADH for concentrations from 5 to 700 �mol L−1 on GC/(IL

two electrons and one proton through an ECE mechanism as men-tioned before [34,46,47].

In addition, the effect of the type of supporting electrolyteused in the preparation of NADH solution on the electrochemicalresponse of NADH at GC/(ILC-CNTs)/Fe3O4 is studied. Fig. 4B showsthe CVs of 1 mmol L−1 NADH prepared in 0.1 mol L−1 concentrationof different supporting electrolytes namely; PBS, NaNO3, Na2SO4,NaCl and buffer I (consists of 5 mmol L−1 Na2HPO4, 5 mmol L−1

NaH2PO4 and 0.1 mol L−1 NaCl). The pH of each supporting elec-trolyte, the anodic peak current and oxidation potential values ofNADH are summarized in Table 2. The anodic peak potential ofNADH is nearly the same in all the studied supporting electrolytes.While the anodic peak current values are highly affected by thesupporting electrolyte type and the highest current response isobtained in case of PBS. This may be attributed to the differencein the kinetic effects upon varying the supporting electrolyte. Theanions of the supporting electrolyte affect the conductivity level ofthe proposed surface as a function of the charge/size ratio [49].

3.5. Morphology of the different modified electrodes

The morphology of the different modified electrodes is investi-gated using scanning electron microscopy. Fig. 5(A–C) shows theSEM of GC/CNTs, GC/(ILC-CNTs) and GC/(ILC-CNTs)/Fe3O4, respec-

H from 5 �mol L−1 to 700 �mol L−1).ADH from 5 �mol L−1 to 50 �mol L−1).C-CNTs)/Fe3O4 electrode. 0.1 mol L−1 PBS/pH 7.4 and applied potential 225 mV.

70 N.F. Atta et al. / Sensors and Actuators B 251 (2017) 65–73

Table 3Comparison of figures of merits for GC/(ILC-CNTs)/Fe3O4 with different modified electrodes mentioned in literature for NADH determination.

Electrode Linear dynamic range (�mol L−1) Sensitivity (�A/�mol L−1) Detection limit (nmol L−1)

GC modified with CNTs and fluphenazine [1] 15–84 40 × 10−3 (�A �mol−1 L cm−2) 5 × 103

poly(3,4-ethylenedioxythiophene) modified glassy carbonelectrode [34]

5–45 0.026 3.8 × 103

1-butyl-3-methylimidazolium tetrafluoroborate ionicliquid and CNTs with chitosan modified GC [18]

0.2–26.0 0.0844 60

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GC modified with CNTs and activated niclosamide [43] 10–280

Carbon nanofibers modified GC electrode [44] 30–2140

GC/(ILC-CNTs)/Fe3O4 [This work] 5–700

ively. A network structure of concentric and overlapped nanotubesre observed in Fig. 5A acting as supporting nano-electrode arraysor further incorporation of nanoparticles or biomolecules [1–5].ig. 5B shows the SEM of (ILC-CNTs) composite in which the ILCenetrates the spacing between the CNTs. Supplement (1) showsEM pictures of cross-section of the surface of a layer containingLC-CNTs. A denser surface is observed due to the combination ofLC and CNTs. This conductive matrix is receptive for the orderedomogenous assembling of Fe3O4 nanoparticles as shown in Fig. 5Cesulting in larger active surface area and higher current signal.ig. 5C shows homogenous immobilization of Fe3O4 nanoparticles.he average diameter of Fe3O4 nanoparticles calculated from SEM

s about 73.12 nm.

.6. Amperometric response of NADH oxidation

The sensitivity of GC/(ILC-CNTs)/Fe3O4 toward NADH determi-ation is examined using amperometric technique. Fig. 6 showshe amperometric steps recorded upon successive additions of

ADH in the concentration range of 5–700 �mol L−1 at an appliedotential of 225 mV. Inset (1) shows the amperometric response

n the concentration range of 5–50 �mol L−1. The amperometricurrent signals increase linearly upon increasing NADH concentra-

ig. 7. CV for 1 mmol L−1 NADH and 1 mmol L−1 MO mixture prepared in 0.1 mol L−1 PBSADH mixture prepared in 0.1 mol L−1 PBS/pH 7.4 at GC/(ILC-CNTs)/Fe3O4. Inset (2) CV fot GC/(ILC-CNTs)/Fe3O4.

2.4 × 10 3 × 10257 × 10−6 11 × 103

0.0102 34.6

tion. The linear relation between the amperometric current andNADH concentration is shown in inset (2) in the linear range of5–700 �mol L−1. The linear regression equation is fitted to the fol-lowing equation:

Ip(�A) = 0.0102C(�mol L−1) + (0.0326) (6)

The figures of merit are: correlation coefficient is 0.987, sensi-tivity is 0.0102 �A �mol−1 L, detection limit is 34.6 nmol L−1 andquantification limit is 0.115 �mol L−1.

The detection limit (DL) and quantification limit (QL) were cal-culated from the following Eqs. (7) and (8), respectively:

DL = 3(s/b) (7)

QL = 10(s/b) (8)

Where “s” is the standard deviation and “b” is the slope of thecalibration curve.

The proposed NADH amperometric sensor, GC/(ILC-CNTs)/Fe3O4, shows wider linear range, good sensitivity andlower detection limit compared to other NADH sensors reportedin the literature (Table 3).

/pH 7.4 at GC/(ILC-CNTs)/Fe3O4. Inset (1): CV for 1 mmol L−1 AA and 0.1 mmol L−1

r 1 mmol L−1 NADH and 1 mmol L−1 Try mixture prepared in 0.1 mol L−1 PBS/pH 7.4

N.F. Atta et al. / Sensors and Actuators B 251 (2017) 65–73 71

Fig. 8. (A) Chronoamperogram of GC/(ILC-CNTs)/Fe3O4 at the oxidation potential of NADH (225 mV) in 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4. The inset: the relationbetween the current values of 1st and 15th cycles at GC/CNTs, GC/(ILC-CNTs) and GC/(ILC-CNTs)/Fe3O4 electrodes recorded in 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4. (B)R H/0.1c nset (G l L−1

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epeated cycles stability up to 10 cycles for GC/(ILC-CNTs)/Fe3O4 in 1 mmol L−1 NADycles for bare GC in 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH 7.4, scan rate 50 mV/s. IC/(ILC-CNTs)/Fe3O4 and bare GC toward the oxidation of 1 mmol L−1 NADH/0.1 mo

.7. Simultaneous determination of NADH in presence ofommon interferences

It is necessary to examine the anti-interference performance ofhe proposed sensor towards NADH determination in the presencef common interferences. One of the popular interferents that exist

n human fluids is ascorbic acid (AA) because of its similar oxidation

otential compared to NADH. It is therefore important to study theetermination of NADH in presence of AA [2,27,34,41,48]. In addi-ion, morphine (MO) is one of the severe pain relief drugs [50] andt may interfere with NADH determination in patients under MO

mol L−1 PBS/pH 7.4, scan rate 50 mV/s. Inset (1): Repeated cycles stability up to 102): The relation between the normalized current% versus the number of cycles forPBS/pH 7.4 upon repeated cycles up to 10 cycles.

treatment. Tryptophan (Try) is an essential amino acid to humansand animals because of its physiological roles. It is an essentialcomponent in human nutrition for preserving a positive nitrogenbalance [42]. Therefore, the simultaneous determination of NADHin presence of AA, MO or Try is very crucial from the clinical point ofview. Fig. 7 shows the CV of 1 mmol L−1 NADH and 1 mmol L−1 MOprepared in 0.1 mol L−1 PBS/pH 7.4 at GC/(ILC-CNTs)/Fe3O4. For

all binary mixtures studied: NADH/MO (Fig. 7), AA/NADH (Fig. 7,Inset 1) and NADH/Try (Fig. 7, Inset 2), two well-resolved oxidationpeaks are obtained using the proposed sensor. Peak potentials areobtained at 0.264 V/0.394 V, −0.03 V/0.257 V, 0.276 V/0.707 V with

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eak separations of 0.130 V, 0.260 V and 0.431 V, respectively. It isoticed that the oxidation peak potential for AA at this sensor elec-rode appears at relatively lower value than normally expected atconventional” electrode surfaces. This could be attributed to either

facile electron transfer or the change of ascorbate redox transi-ion mechanism in the environment of ionic liquid. Furthermore,he cyclic voltammograms of pure interferents (AA, Try or MO) onhe top of the CV’s of the mixtures with NADH were shown in theupplement figures (2, 3 and 4), respectively.

The relative decrease in the oxidation peak current values cor-esponding to NADH is due to its competitive adsorption with

O, AA or Try. The oxidation products arising from AA, the firstnalyte oxidized (at relatively lower potential); compete at thelectrode interface with the “next” oxidized analyte (NADH). It ismportant to mention however, that the NADH peak is still wellesolved. Another important factor is the background elevation thats observed upon determining a binary mixture.

It is also recommended to determine NADH in presence ofbuprofen (IBP) [51]. IBP is an antipyretic and analgesic off-the-helf drug. The CV of the mixture (Figure not shown) depicted twoesolved peaks at 0.258 V and 1.349 V for NADH and IBP oxidationsith peak separation of 1.091 V.

.8. Stability of the proposed sensor toward NADH determination

The stability of the proposed sensor is an important factorffecting its performance. GC/(ILC-CNTs)/Fe3O4 shows a sta-le current response upon amperometric studies in 1 mmol L−1

ADH/0.1 mol L−1 PBS/pH 7.4 at the oxidation potential (0.225 V)Fig. 8A). In addition, repeated CV cycles stability (up to 15 cycles)ere compared between GC/CNTs, GC/(ILC-CNTs) and GC/(ILC-

NTs)/Fe3O4 electrodes in 1 mmol L−1 NADH/0.1 mol L−1 PBS/pH.4. The relation between the current of the 1st and 15th cyclesas drawn for the three different electrodes (Inset of Fig. 8A). The

urrent of the 1st and 15th cycles in case of GC/(ILC-CNTs)/Fe3O4as still much higher compared to other electrodes GC/CNTs andC/(ILC-CNTs), respectively.

Furthermore, we have studied the response of GC/(ILC-NTs)/Fe3O4 and bare GC toward the oxidation of 1 mmol L−1

ADH/0.1 mol L−1 PBS/pH 7.4 upon repeated CV cycles up to 10ycles (Fig. 8B and inset 1). Inset (2) shows the response in termsf normalized current% versus the number of cycles up to 10 cyclesor GC/(ILC-CNTs)/Fe3O4 and bare GC.

The anodic peak current of NADH at GC/(ILC-CNTs)/Fe3O4ecreases by 19.8% and 8.3% from cycle 1 to cycle 2 and from cycle

to cycle 10, respectively. While the anodic peak current of NADHt bare GC decreases by 33.5% and 27.3% from cycle 1 to cycle 2nd from cycle 3 to cycle 10, respectively. The data display relativetability for the proposed sensor compared to GC.

In addition, NADH was determined at the surface of GC/(ILC-NTs)/Fe3O4 for 4 different runs with very small relative standardeviation of 1.95%. Also, the reproducibility of the sensor prepara-ion has been examined by recording the independent response ofhree similarly prepared electrodes of GC/(ILC-CNTs)/Fe3O4 (Sup-lement Fig. 5). Relative standard deviation of 1.18% is obtainedhowing good reproducibility of sensor preparation with stableesponse.

Also, the intra-day precision was estimated by the analysis of theame concentration in a single assay run three times with relativetandard deviation of 2.23%. The inter-day precision was estimated

y the analysis of the same concentration in three separate assayuns three times with relative standard deviation of 2.10%. Theseesults demonstrated that good reproducibility, excellent precisionnd stable response were obtained at the modified electrode.

[

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4. Conclusion

In the present study, the proposed sensor GC/(ILC-CNTs)/Fe3O4is successfully employed for amperometric determination of NADH.The unique characteristics of each component of the composite filmelectrode acted synergistically to enhance the current responsefor NADH oxidation. A relatively wide linear dynamic range forthe calibration curve, good sensitivity, relatively lower detectionand quantification limits were realized. It is possible to determineNADH distinctly in binary mixtures of common interferences fromAA, MO, Try and IBP.

Acknowledgment

The authors would like to acknowledge the financial supportfrom Cairo University through the Vice President Office for ResearchFunds.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.snb.2017.05.026.

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Biographies

Nada F. Atta earned her PhD from the University of Cincinnati; she is currentlyProfessor of Physical Chemistry at the Chemistry Department, Faculty of Science,Cairo University. The research interests of Professor Atta are the area of modi-fied electrodes for sensing and electrocatalysis applications, corrosion protectionusing advanced materials and nano-structured materials including graphene, car-bon nanotubes and nano-mixed oxides.

Soha A. Abdel Gawad earned her PhD in Physical Chemistry from Cairo University,Faculty of Science. She is currently Professor at the same department. ProfessorAbdel Gawad research interest is in electrochemistry and corrosion in molten salts.

Ekram H. El-Ads earned her Master and PhD degrees from Cairo University, Fac-ulty of Science where she is currently Lecturer of Physical Chemistry. Her researchinterests are in the fields of nanotechnology, conducting polymers, self-assemblymonolayers, nanoparticles, surfactant, inorganic mixed metal oxides, catalysis, sen-sors and biosensors.

Asmaa R.M. El-Gohary is a Master Degree candidate at the Department of Chem-istry, Faculty of Science, Cairo University. She earned her BSc Degree in chemistrywith distinction from the same University. Ms. El-Gohary research interests is inthe area of developing new materials for sensitive determination of biologically andmedically important molecules.

Ahmed Galal earned his PhD from the University of Cincinnati. He worked as Deanof the Faculty of Science at Cairo University where he is now Professor of Chemistry

and Materials in the Department of Chemistry. His research interests are in theareas of electrochemical sensors, nano-materials, conducting polymers, corrosionand passivity of metals and alloys and environmental Chemistry. Other researcharea includes developing new electrochemical treatment methods for cleaning theenvironment from potential pollutants.