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imultaneous determination of ascorbic acid, dopamine, uric acid andolic acid based on activated graphene/MWCNT nanocompositeoaded Au nanoclusters
del A. Abdelwahaba,∗, Yoon-Bo Shimb,∗
Department of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, EgyptDepartment of Chemistry and Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735, Republic of Korea
r t i c l e i n f o
rticle history:eceived 20 April 2015eceived in revised form 27 June 2015ccepted 3 July 2015vailable online 4 July 2015
eywords:ctivated grapheneu nanoclustersopamine
a b s t r a c t
An electrochemical sensor for the simultaneous determination of ascorbic acid (AA), dopamine (DA),uric acid (UA) and folic acid (FA) using gold nanoclusters (AuNCs)/activated graphene (AGR)/MWCNTnanocomposite was fabricated. The AGR/MWCNT nanocomposite was prepared via the electrochemicalreduction of GR/MWCNT while AuNCs were formed onto the AGR/MWCNT film through the electrode-position of Au. The AuNCs/AGR/MWCNT nanocomposite was characterized using different techniques,such as scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS), X-rayphotoelectron spectroscopy (XPS) and cyclic voltammetry (CV). The nanocomposite sensor exhibitedsharp and obvious peaks for the oxidation of AA, DA, UA and FA as compared to a bare electrode.The AuNCs/AGR/MWCNT probe displays an efficient electron mediating behavior with well separated
scorbic acidric acidolic acid
oxidation peak potentials between AA to DA, DA to UA and UA to FA were 0.21, 0.14 and 0.28 V, respec-tively. The linear calibration curves of AA, DA, UA and FA were observed from 10–150 �M, 1.0–210 �M,5.0–100 �M and 10–170 �M with detection limits of 0.27 ± 0.1 �M, 0.08 ± 0.02 �M, 0.10 ± 0.03 �M and0.09 ± 0.01 �M, respectively. In addition, the analytical application of the proposed sensor was success-fully conducted for the determination of these species in human urine real samples.
Dopamine (DA) is a neurotransmitter that plays an impor-ant role in central nervous system, serving as an antecedent ofdrenaline and noradrenaline and used to help maintain hormonalalance as well as emotion control [1]. Deficiency of DA in humanody may result in some serious neurological disorders, such asarkinson’s disease [2]. Ascorbic acid (AA) is an essential vitaminn the diet of humans and is present in mammalian brain along
ith various neurotransmitter amines. It has been used in therevention and treatment of the common cold, mental illnesses,
nfertility, cancers and AIDS [3]. Uric acid (UA) is the primarynal product of purine metabolism in human body. Abnormal
evels of UA lead to some of diseases, including gout, Lesch–Nyhanyndrome and hyperuricemia [4]. Folic acid (FA) is a water solubleitamin B which produced by plants and microorganisms. It can
promote the formation of red blood cells, and is believed to bea blood tonic, initially identified as an anti-anemia and growthfactor. A lack of FA gives rise to gigantocytic anemia, associatedwith leukopenia, devolution of mentality and psychosis [5]. SinceAA, DA, UA and FA are coexisting in physiological fluids, such ashuman urine and blood serum, it is important to monitor theirconcentrations with more sensitive and selective sensor.
Due to the redox behavior of these compounds, electrochemi-cal methods appear to be suitable for the analytical determinationswith high sensitivity, simple operation, low cost and fast response[6,7]. However, a significant obstacle to monitor these species con-centrations is the overlapping of their anodic peak potentials atcovenantal electrodes resulting in poor selectivity [8]. Thus, thesimultaneous determination of AA, DA, UA and FA is essentialto investigate their physiological functions. Various modificationshave been applied on the simultaneous determination of two orthree of these species, such as assembled monolayers, polymer
films, metal and metal oxide nanoparticles [9–14].
Graphene (GR) has received considerable interest of recentresearch for electrode modifications and biosensor applications.Due to its high electrical conductivity, chemical stability and
iocompatibility [15], GR has been used for various applications,uch as fuel cells, capacitors and sensors [16–18]. Of these, GRxide and hetero atoms-doped GR composites could be suggesteds potential candidates because of its abundant oxygen groups thatromote functionalization for potential active and modificationf electrode [19–26]. On the other hand, the electrochemicalctivation of GCE shows significant enhancement in its physicalnd electrochemical properties through increasing the electrodeurface active area which hence increases the electrical conductiv-ty by generation of active sites in the electrode surface [27]. Thectivated GCE has been used for simultaneous determination of AA,A and UA [28,29]. In this context, it is expected that the activationf graphene (AGR) composite MWCNT could introduce more activeites in the nanocomposite structure and hence facilities the elec-ron transfer process between target analyte and electrode surface.
In the present study, a new method for the simultaneousetermination of AA, DA, UA and FA was carried out using god nano-lusters (AuNCs) functionalized AGR/MWCNT nanocomposite. AGRnd MWCNT play an important role in the sensor fabrication dueo their unique electrochemical conductivity and excellent biolog-cal activity. Since the AGR/MWCNT nanocomposite can provide
biological environment similar to that of native system. Hence,arget molecules could maintain their biological and electrochem-cal activity when detected with the nanocomposite. While, AuNCsave been used not only to improve the sensor sensitivity byroviding a large active surface area but also possess a good stabil-
ty. In addition, the electrochemical activation of GR/MWCNT filmhowed an effective result in the present sensor. Since this pro-ess introduces active sites in the AGR/MWCNT surface through theormation of carboxylate groups. Thus, the AGR/MWCNT nanocom-osite becomes more conductive which may therefore act asemplates for high electrocatalytic activity of the analyets. Due tohe coexisting of AA, DA, UA and FA in physiological fluids, it ismportant to monitor their concentrations for diagnosing diseasesnalysis. So far, there have been no reports revealing the simulta-eous determination of four species AA, DA, UA and FA. The sensorrovided a good performance for the simultaneous determinationf AA, DA, UA and FA by not only greatly enhanced their currentesponses, but also resolved the overlapping peak potentials asell as decreased the overpotentials. In addition, the nanocompos-
te sensor was successfully applied for the determination of thesepecies in human urine real samples.
. Materials and methods
.1. Materials
Graphite powder, ascorbic acid (AA), dopamine (DA), uric acidUA), folic acid (FA), HAuCl4, sodium dihydrogen phosphate, diso-ium hydrogen phosphate and dimethylformamide (DMF) wereurchased from Sigma and Aldrich (USA). MWCNT with averageiameter of about 12 nm and 99% purity was purchased from Iljinanotech (South Korea). All other chemicals were of extra purenalytical grade and used without further purification. All solutionsere prepared with doubly distilled water obtained from a Milli-Qater purifying system (18 M� cm).
.2. Instruments
Cyclic voltammograms (CVs), square wave voltammogramsSWVs) were recorded using a Potentiostat/Galvanostat, Kosentech
odel PT-1 (Busan, South Korea). Scanning electron microscopySEM) images were obtained using a Cambridge Stereoscan 240.-ray photoelectron spectroscopy (XPS) experiments were per-
ormed using a VG scientific ESCA lab 250 XPS spectrometer
d Actuators B 221 (2015) 659–665
coupled with a monochromated Al K� source having chargecompensation. Electrochemical impedance spectroscopy (EIS) wasrecorded with an EG&G PAR 273A Potentiostat/Galvanostat anda lock-in amplifier (PAR EG&G, Model 5210) linked to a per-sonal computer. The frequency was scanned from 100 kHz to10 Hz at the open circuit voltage, acquiring five points per decade.The amplitude of sinusoidal voltage of 10 mV was used. TheAuNCs/AGR/MWCNT/GCE, GR/MWCNT/GCE and bare GCE, with anelectrode area of 0.07 cm2, were used as working electrodes. Ref-erence electrode was Ag/AgCl and counter electrode was platinumwire.
The preparation of AuNCs/AGR/MWCNT nanocomposite wasperformed as follows: Firstly, GR was synthesized according toprevious method [30]. Then, a 1.0 mg/mL of GR/MWCNT nanocom-posite was prepared by dispersing a 10 mg of GR with 10 mg ofMWCNT in a 10 mL of DMF solution and sonicated for 30 min at25 ◦C. After polishing GCE with 0.05 �m alumina slurries on a pol-ishing cloth to a mirror finish it was rinsed with second distilledwater. Thereafter, 10 �L of GR/MWCNT nancomposite was dropcaste onto GCE surface and dried in air. Then, the activation ofGR/MWCNT film was obtained by cycling the potential from +0.6to −1.8 V for ten cycles in pH 7.0 PBS to produce AGR/MWCNTfilm. Then AuNCs were formed onto the AGR/MWCNT film viaan electrochemical deposition of AuNCs by scanning the poten-tial from +0.2 to −1.0 V at scan rate of 50 mV s−1 for 15 cyclesin a 0.5 M H2SO4 solution containing 0.25 mM HAuCl4 [31]. Then,the AuNCs/AGR/MWCNT electrode was rinsed thoroughly withdoubly distilled water and stored at room temperature until use(Scheme 1).
3. Results and discussion
3.1. Preparation of the AuNCs/AGR/MWCNT nanocomposite
The AGR/MWCNT film was obtained through the electrochem-ical activation of GR/MWCNT by scanning the potential from +0.6to −1.8 V for ten cycles in 0.1 M PBS (pH 7.0) (Fig. 1A). As shown,well-defined reduction and oxidation peaks were observed dur-ing the first scan at −1.53 and +0.35 V which might be due to thereduction of C O groups and the formation of COOH groups in theAGR/MWCNT, respectively. These redox peaks currents increasedas the number of cycles increased, indicating the electrochemicalreduction of GR/MWCNT takes place on the electrode surface toproduce the AGR/MWCNT nanocomposite film. The AGR/MWCNTfilm was further investigated by cyclic voltammetry. Fig. 1B (dottedline) shows the CV recorded for AGR/MWCNT modified electrodein a 0.1 M H2SO4 solution during the potential scanning from 0to +1.4 V at a scan rate of 0.1 V/s. As can be seen, well-definedredox peaks were observed at 0.38 and 0.26 V, due to the elec-tron transition between the quinone and hydroquinone groupsof the AGR/MWCNT [32], while these peaks were not observedduring the CV recorded for the GR/MWCNT film (Fig. 1B, dashedline). In addition, the deposited AuNCs onto the AGR/MWCNTwas also investigated in a 0.1 M H2SO4 solution as shown inFig. 1B (solid line). A sharp and obvious reduction peak of Au wasobserved at +0.85 V, clearly indicating the successful formation ofthe AuNCs/AGR/MWCNT electrode.
3.2. Characterization of the AuNCs/AGR/MWCNT nanocomposite
To investigate the morphology of the AuNCs/AGR/MWCNTnanocomposite, Fig. 2A showed the SEM images of stepwiseof the sensor fabrication: (i) GR/GC, (ii) AGR/MWCNT/GC,
A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 221 (2015) 659–665 661
cation
(AsthTo5anFrAGtH
F(i
Scheme 1. Schematic representation of the fabri
iii) AuNCs/AGR/MWCNT/GC and (iv) enlarged image ofuNCs/AGR/MWCNT/GC. As can be seen, the image of GR/GChows a smooth surface while GR covered wholly and uniformlyhe GC surface. The morphology of AGR/MWCNT/GC shows aomogeneous nanocomposite structure of AGR and MWCNT.he image of AuNCs/AGR/MWCNT/GC displays the attachmentf AuNCs onto the AGR/MWCNT layer with average diameter of0 nm which might be due to that the growth of AuNCs occurredround the deposited Au. In addition, the AuNCs/AGR/MWCNTanocomposite sensor was further characterized using XPS.ig. 2 showed XPS spectra of (B) C1s, (C) O1s and (D) Au4f peaksecorded for (i) GR/MWCNT/GC, (ii) AGR/MWCNT/GC and (iii)
uNCs/AGR/MWCNT/GC. The spectrum of C1s and O1s peaks forR/MWCNT/GC were appeared at 284.3 and 533.0 eV, respec-
ively which is corresponding to the C O bonds (Fig. 2Bi, 2Ci).owever, after the electrochemical activation of GR/MWCNT,
ig. 1. (A) CVs in consecutive potential scans of GR/MWCNT/GCE in a 0.1 M PBS (pH 7.0) at 1dotted line) and AuNCs/AGR/MWCNT/GCE (solid line) in 0.1 M H2SO4 solution. (C) CVs rn a 5.0 mM K3[Fe(CN)6] solutions.
of AuNCs/AGR/MWCNT nanocomposite sensor.
the intensity of these peaks decreased indicating the formationof AGR/MWCNT (Fig. 2Bii, 2Cii). A spectrum obtained for theAuNCs/AGR/MWCNT/GC electrode showed two peaks at 83.5 and87.3 eV, which are related to Au4f7 and Au4f5 orbitals, respec-tively (Fig. 2Diii). SEM and XPS results confirmed the successfulfabrication of AuNCs/AGR/MWCNT nanocomposite.
To investigate the conductivity of the modified electrode, EISstudies were carried out. Fig. 2E shows the Nyquist plots recordedfor bare and AuNCs/AGR/MWCNT nanocomposite electrodes in a5.0 mM [Fe(CN)6]3−/4− solution. A Randle circuit was employedto analyze the obtained impedance results (inset of Fig. 2E).Where, Rs is the solution resistance, Rp1, Rp2 are the polarization
resistances, W is the Warburg element, and CPE1, CPE2 are theconstant phase elements. The charge transfer resistances Rp1 andRp2 of the AuNPs/AGR/MWCNT electrode were much smaller thanboth AGR/MWCNT and bare electrodes. This indicated that, the
00 mV s−1 of scan rate. (B) CVs of GR/MWCNT/GCE (dashed line), AGR/MWCNT/GCEecorded for the AuNCs/AGR/MWCNT (solid line) and bare (dashed line) electrodes
662 A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 221 (2015) 659–665
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ig. 2. (A) SEM images for (i) GR/GC, (ii) GR/MWCNT/GC, (iii) AuNCs/AGR/MWCNC) and Au4f (D) peaks recorded for (i) GR/MWCNT/GC, (ii) AGR/MWCNT/GC anuNCs/AGR/MWCNT electrodes in a 5.0 mM [Fe(CN)6]3−/4− solution.
uNPs/AGR/MWCNT nanocomposite layer improved the conduc-ivity of the modified electrode by facilitating the rate of chargeransfer process.
.3. Electrochemical behavior of the AuNCs/AGR/MWCNTanocomposite
Voltammetric properties of the AuNCs/AGR/MWCNT nanocom-osite were characterized using K3[Fe(CN)6] as a benchmark redoxeaction for modified electrode. Fig. 1C shows the CVs recorded for.0 mM K3[Fe(CN)6] with the AuNCs/AGR/MWCNT (solid line) andare (dashed line) electrodes. As shown, an obvious increase in theedox peak currents and a narrow peak potential difference werebserved in the CV recorded for AuNCs/AGR/MWCNT as comparedith a bare electrode, indicating a fast electron transfer process.
he active surface area of the AuNCs/AGR/MWCNT electrode wasalculated using the Randles–Sevcik equation [33]:
p = 2.69 × 105 AD1/2n3/2 �1/2C
here n is the number of electrons transferred, A is the
ctive surface area, D is the diffusion coefficient of K3[Fe(CN)6]6.7 × 10−6 cm2/s) [34], C is the concentration of analyte (mol/cm3),nd � is the scan rate (V/s). The large active surface area obtained forhe AuNCs/AGR/MWCNT electrode (0.453 cm2) as compared with
and (iv) enlarged image of AuNCs/AGR/MWCNT/GC. XPS spectra of C1s (B), O1sAuNCs/AGR/MWCNT/GC. (C) Nyquist plots of (i) bare (ii) AGR/MWCNT and (iii)
a bare electrode (0.048 cm2), indicating the higher electrochemicalactivity of the modified electrode.
3.4. Electrochemical oxidation of AA, DA, UA and FA with theAuNCs/AGR/MWCNT nanocomposite
The electrocatalytic response of the AuNCs/AGR/MWCNT elec-trode toward the oxidation of AA, DA, UA and FA was investigated.Fig. 3 shows the CVs recorded for (A) 1.0 mM AA, (B) 100 �M DA, (C)100 �M UA and (D) 100 �M FA in 0.1 M PBS (pH 7.0) at 100 mV s−1
of scan rate with the AuNCs/AGR/MWCNT (solid lines) and bare(dashed lines) electrodes. As shown, the anodic peak currents ofAA, DA, UA and FA at the AuNCs/AGR/MWCNT nanocomposite weremuch higher with negative shift in their peak potentials as com-pared to a bare electrode, indicating greatly promoted the chargetransfer reactions of AA, DA, UA and FA with the nanocompositefilm.
In order to investigate the reliability of the proposed sensor forsimultaneous determination of AA, DA, UA and FA, square wavevoltammetry (SWV) appears to be a very sensitive and high res-
olution method for determining trace amount of analytes. Fig. 3Eshows the SWVs of a mixture solution of AA, DA, UA and FA atbare and AuNCs/AGR/MWCNT electrodes. At a bare electrode, over-lapped and small broad peaks were observed for the oxidation
A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 221 (2015) 659–665 663
F A, (C)( id line3
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has a long-time stability as well as good reproducibility andcan be used over one month without significant change in itsresponse.
Table 1Comparison of the AuNCs/AGR/MWCNT nanocomposite results with recentlyreported methods.
ig. 3. CVs recorded in 0.1 M PBS (pH 7.0) containing (A) 1.0 mM AA, (B) 100 �M Dsolid lines) electrodes. (E) SWV of bare (dashed line) and AuNCs/AGR/MWCNT (sol0 �M FA.
f these species. While, the AuNCs/AGR/MWCNT nanocompositexhibited four well-defined and remarkable oxidation peaks withreatly enhanced currents at 0.04, 0.25, 0.39 and 0.67 V for AA, DA,A and FA, respectively. The separation peak potentials betweenA to DA, DA to UA and UA to FA were 0.21, 0.14 and 0.28 V,espectively. The high sensitivity and selectivity obtained usinghe proposed sensor might be attributed to the hybrid nanomateri-ls of AuNCs/AGR/MWCNT integrating the advantage properties ofhe nanocomposite by increasing the activity of electrode surface,hich may therefore significantly enhanced the electron transferrocess.
.5. Sensitivity of the AuNCs/AGR/MWCNT nanocompositelectrode
To evaluate the performance of the AuNCs/AGR/MWCNT elec-rode for simultaneous determination of AA, DA, UA and FA, SWVsf the additions of various concentrations of these species wereecorded in 0.1 M PBS (pH 7.0). As shown in Fig. 4A, the oxida-ion peak currents simultaneously increased with the increasingoncentrations of AA, DA, UA and FA in the solution and their oxi-ation peak potentials remained steady. The linear calibration plotsere obtained for AA, DA, UA and FA in the range of 10–150 �M,
.0–210 �M, 5.0–100 �M and 10–170 �M, with detection limits of
.27 ± 0.1 �M, 0.08 ± 0.02 �M, 0.10 ± 0.03 �M and 0.09 ± 0.01 �M,espectively. The lowest detection limits obtained with the presentensor as compared to others reported methods (Table 1), indicat-ng that the AuNCs/AGR/MWCNT electrode could provide a goodlatform for the effective analytical detection.
.6. Stability, selectivity and reproducibility of theanocomposite sensor
In order to examine the stability of the AuNCs/AGR/MWCNT
lectrode, it was examined over a period of one month dur-ng which the peak currents intensity of AA, DA, UA and FAecayed by about 4.5%, 3.7%, 3.9% and 5.2%, respectively. This
ndicated that the AuNCs/AGR/MWCNT sensor has a long-time
100 �M UA and (D) 100 �M FA for a bare (dashed lines) and AuNCs/AGR/MWCNT) electrodes in 0.1 M PBS (pH 7.0) containing 30 �M AA, 40 �M DA, 20 �M UA and
stability. To study the selectivity of the nanocomposite sensor,the effects of possible interfering species, such as glucose, cys-teine, glutathione, glutamic acid, alanine and glycine with thedetection of AA, DA, UA and FA was investigated. No change inthe current response was observed for AA, DA, UA and FA inthe presence of 0.1 M of these interferences indicating that thepresent sensor is selective to the determination of these specieseven in the presence of high concentrations of common phys-iological interferences (figure not shown). The reproducibilityof the proposed sensor was also investigated with four elec-trodes prepared under the same condition. The relative standarddeviations (R.S.D.) of these electrodes were 2.8%, 1.8%, 2.5% and2.9% for the current responses of AA, DA, UA and FA, respec-tively. These results suggested that the AuNCs/AGR/MWCNT sensor
d Iron oxide/reduced graphene oxide/glassy carbon electrode.e Gold/reduced graphene oxide.f Graphene/poly-cyclodextrine/multi wall carbon nanotube/glassy carbon elec-
664 A.A. Abdelwahab, Y.-B. Shim / Sensors and Actuators B 221 (2015) 659–665
Fig. 4. (A) SWVs of AuNCs/AGR/MWCNT/GCE in 0.1 M PBS (pH 7.0) containing different concentrations of AA, DA, UA and FA. (B) Enlarged SWVs from a to e additions. (C)T
3
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he linear calibration plots of AA, DA, UA and FA.
.7. Analytical applications
To evaluate the validity and reliability of the proposed sensor inractical applications, the analytical determination of AA, DA, UAnd FA in two human urine real samples were performed using thetandard addition method. The human urine samples were diluted0 times using 0.1 M PBS (pH 7.0). SWVs were recorded after 200 �Lf human urine sample was added into 10 mL PBS (pH 7.0). Inrder to ascertain the correctness of the results, certain amountsf AA, DA, UA and FA were added into the above mentioned dilutedample and were then detected. The analytical results are sum-arized in Table 2. The recovery of the spiked samples ranged
etween 97% and 109%, indicating the successful application of the
uNCs/AGR/MWCNT nanocomposite for the determination of AA,A, UA and FA in real samples.
able 2nalytical results of AA, DA, UA and FA determination in human urine samples usinguNCs/AGR/MWCNT nanocomposite sensor (n = 5).
A sensitive sensor for the simultaneous determination of AA, DA,UA and FA was developed using the AuNCs modified AGR/MWCNTnanocomposite. The sensor combines the high electrical conduc-tivity and biocompatibility of AGR/MWCNT with the excellentcatalytic activity of AuNCs which was used not only to enhancethe sensor sensitivity but also provide a high stability of the sen-sor. The AuNCs/AGR/MWCNT sensor exhibited a good platform fornot only greatly enhanced the current responses of the oxidation ofAA, DA, UA and FA, but also resolved the overlapping peak poten-tials as well as decreased the overpotentials of these species. Thenanocomposite probe showed satisfactory results when applied forthe determination of these substances in human urine real samples.
Acknowledgement
This work was supported by the National Research Foundationof Korea (NRF) a grant funded by the Korea government (MSIP) (No:20100029128).
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Biographies
Adel A. Abdelwahab received his Ph.D. from Department of Chemistry at PusanNational University, South Korea, in 2010. He is working as an Assistant Professorat Department of Chemistry, Al-Azhar University. His current research interests arethe development of electrochemical sensors and biosensors, modified electrodes,study of electron transfer reaction of enzymes and proteins, and characterization ofconducting polymers and their applications.
Yoon-Bo Shim received his Ph.D. from Department of Chemistry at Pusan NationalUniversity, South Korea, in 1985. He is working as a Professor at Department ofChemistry and a director at Institute of BioPhysio Sensor Technology (IBST), Pusan
(protein, DNA, enzyme, etc.)/chemical-sensors, electroanalytical method of tracebiological, organic species with modified electrodes, electron transfer of organiccompounds and proteins on the biomembranes, and characterization of conductingpolymers and their applications.
