Scholarly article on topic 'Simultaneous detection of ascorbic acid, dopamine, uric acid and tryptophan with Azure A-interlinked multi-walled carbon nanotube/gold nanoparticles composite modified electrode'

Simultaneous detection of ascorbic acid, dopamine, uric acid and tryptophan with Azure A-interlinked multi-walled carbon nanotube/gold nanoparticles composite modified electrode Academic research paper on "Chemical sciences"

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Arabian Journal of Chemistry
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{"Multi-walled carbon nanotubes" / "Azure A" / "Ascorbic acid" / Dopamine / "Uric acid" / Tryptophan}

Abstract of research paper on Chemical sciences, author of scientific article — Hayati Filik, Asiye Aslıhan Avan, Sevda Aydar

Abstract In this paper, multi-walled carbon nanotube/Azure A/gold nanoparticle composites (Nafion/AuNPs/AzA/MWCNTs) were prepared by binding gold nanoparticles to the surfaces of Azure A-coated carbon nanotubes. Nafion/AuNPs/AzA/MWCNTs based electrochemical sensor was fabricated for the simultaneous determination of ascorbic acid, dopamine, uric acid, and tryptophan. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the electrochemical properties of the modified electrodes. The modified electrode showed excellent electrocatalytic activity toward ascorbic acid, dopamine, uric acid, and tryptophan (pH 7.0). The experiment results showed that the linear response range for simultaneous detection of AA, DA, UA and Trp were 300–10,000μM, 0.5–50μM, 0.5–50μM and 1.0–100μM, respectively, and the detection limits were 16μM, 0.014μM, 0.028μM and 0.56μM (S/N=3). The proposed method offers promise for simple, rapid, selective and cost-effective analysis of small biomolecules. The procedure was also applied to the determination of tryptophan in spiked milk samples.

Academic research paper on topic "Simultaneous detection of ascorbic acid, dopamine, uric acid and tryptophan with Azure A-interlinked multi-walled carbon nanotube/gold nanoparticles composite modified electrode"

Arabian Journal of Chemistry (2015) xxx, xxx-xxx

King Saud University Arabian Journal of Chemistry

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Simultaneous detection of ascorbic acid, dopamine, uric acid and tryptophan with Azure A-interlinked multi-walled carbon nanotube/gold nanoparticles composite modified electrode

Hayati Filik *, Asiye Aslihan Avan, Sevda Aydar

Istanbul University, Faculty of Engineering, Department of Chemistry, 34320 Avcilar, Istanbul, Turkey Received 16 October 2014; accepted 31 January 2015

KEYWORDS

Multi-walled carbon nano-

tubes;

Azure A;

Ascorbic acid;

Dopamine;

Uric acid;

Tryptophan

Abstract In this paper, multi-walled carbon nanotube/Azure A/gold nanoparticle composites (Nafion/AuNPs/AzA/MWCNTs) were prepared by binding gold nanoparticles to the surfaces of Azure A-coated carbon nanotubes. Nafion/AuNPs/AzA/MWCNTs based electrochemical sensor was fabricated for the simultaneous determination of ascorbic acid, dopamine, uric acid, and tryp-tophan. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the electrochemical properties of the modified electrodes. The modified electrode showed excellent electrocatalytic activity toward ascorbic acid, dopamine, uric acid, and tryptophan (pH 7.0). The experiment results showed that the linear response range for simultaneous detection of AA, DA, UA and Trp were 300-10,000 iM, 0.5-50 iM, 0.5-50 iM and 1.0-100 iM, respectively, and the detection limits were 16 iM, 0.014 iM, 0.028 iM and 0.56 iM (S/N = 3). The proposed method offers promise for simple, rapid, selective and cost-effective analysis of small biomolecules. The procedure was also applied to the determination of tryptophan in spiked milk samples. © 2015 Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

* Corresponding author. Tel.: +90 212 473 70 70/17739; fax: +90 212 473 71 80.

E-mail address: filik@istanbul.edu.tr (H. Filik). Peer review under responsibility of King Saud University.

Ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp) are seen as crucial small biomolecules for physiological processes in human metabolism. It is well known that AA, DA, UA and Trp usually coexist in biological matrixes. Unnatural levels of these species will contribute to various diseases and disorders (Cooper et al., 1982; Damier et al., 1999; Mazloum-Ardakani et al., 2009; Liu et al., 2007). Tryptophan is an important amino acid owing to its essential functions in biological systems. It is a vital constituent of proteins and

http://dx.doi.org/10.1016/j.arabjc.2015.01.014

1878-5352 © 2015 Production and hosting by Elsevier B.V. on behalf of King Saud University.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

indispensable in human nutrition for establishing and maintaining a positive nitrogen balance (Fiorucci and Cavalheiro, 2002). Thus, the purpose of their concentration is significant not only for biomedical chemistry and neurochemistry but also for diagnostic and pathological research.

It is well known that AA, DA, UA and Trp usually coexist in biological matrixes. As the oxidation potentials of these species are very close, thus, the interference from each other should be expected. Thus, the subject area of the ability to selectively determine these species in a mixed solution is required. Latterly, for this design, various chemically modified electrodes have been constructed and applied for the simultaneous determination of AA, DA, UA and Trp in their mixture (Ghoreishi et al., 2014; Wang et al., 2012, 2013; Yang et al., 2011; Zhang et al., 2012; Noroozifar et al., 2011; Li et al., 2014; Kaur et al., 2013). A variety of materials have been used for the modification of electrode, such as gold nanoparticles/ over oxidized-polyimidazole composite (Wang et al., 2012), graphene hybrid tube-like structure and 3,4,9,10-perylenetetra-carboxylic acid (GS-PTCA) (Zhang et al., 2012), iron ion-doped natrolite zeolite-Multiwall carbon nanotube (Noroozifar et al., 2011), a hybrid of graphene sheets (GS) MWNTs bridged mesocellular graphene foam (MWNTs/ MGF) (Li et al., 2014) and silver nanoparticle-decorated reduced graphene oxide composite (AgNPs/rGO) (Kaur et al., 2013). Carbon nanotubes (CNTs) have drawn special research attention because of their unique properties and possible applications. To improve upon the attributes of the CNTs, low-cost and industrially feasible approaches to their modifications are always being sought by chemists and materials scientists. The recently developed chemical and electrochemical functionalization schemes that have significantly broadened the application spectrum of CNTs. Functionaliza-tion of carbon nanotubes (CNTs) has attracted considerable interest in the fields of physics, chemistry, material science and biology. Combining hybrid nanostructures of metal nano-particles (NPs) and carbon nanotubes could afford a novel scheme to prepare promising nanomaterials for the highly sensitive sensors and imaging science applications. Surface-linked Au-CNT nanocomposites may further be classified as cova-lently linked and non-covalently linked (Zhang and Olin, 2011). For non-covalent bonding, there are several ways to attach linking molecules to carbon nanotubes, including p stacking (Chen et al., 2008; Liu et al., 2003; Wang et al., 2002, 2012, Wang and Bi, 2013; Ou and Huang, 2006; Wang et al., 2007), hydrophobic forces (Richard et al., 2003; Cao et al., 2008; Xiao et al., 2008; Alexeyeva et al., 2006), and electrostatic interactions (Zhang and Olin, 2011; Jiang et al., 2003; Yao and Shiu, 2008). CNT/nanoparticle hybrid materials, in which nanoparticles are attached to CNT surfaces, have been reported to exhibit catalytic activity, enhanced electrical conductivity, and hydrogen-sensing capability, suggesting broad potential application in optoelectronics, molecular sensors and heterogeneous catalysis. Certain attributes of gold nano-particles suggest that gold-nanoparticle-functionalized CNTs may prove applicable in future fabrication of nanodevices, enabling further miniaturization of integrated circuits (Wang et al., 2006). Thionine (Zhuo et al., 2005, 2006) and N,N-bi(2-mercaptoethyl)-perylene-3,4,9,10-tetracarboxylic diimide (MEPTCDI) are also used to link carbon nanotubes and gold nanoparticles through p stacking (Wang et al., 2007; Zhou et al., 2007).

In this study, we have prepared Nafion/AuNPs/AzA/ MWCNT composites using N',N'-dimethylphenothiazin-5-ium-3,7-diamine (AzA) molecules as interlinkers for depositing metal nanoparticles on CNTs. AuNPs were non-covalently attached to MWCNTs in the presence of Azure A. Azure A/ Azure I molecules (Dezhampanah and Aghajani, 2013; Li et al., 2007) can easily interact with the MWCNTs to form a young kind of stable AzA-MWCNTs nanostructure. Chemically oxidized carbon nanotubes have many carboxyl groups, which lead to negatively charged surfaces of carbon nano-tubes. Electrostatic interactions will result between these car-boxyl groups and positively charged molecules (AzA+) (Li et al., 2007). Upon subsequent addition of negatively charged gold nanoparticles, electrostatic interaction with AzA serves to "glue" the AuNPs onto the MWCNTs (Zhou et al., 2007). The resulting Nafion/AuNPs/AzA/MWCNTs hybrid films were used for electrochemical sensing of AA, DA, UA and Trp in their quaternary mixture. Satisfactory results were also obtained from analyzing real samples using this Nafion/AuN-Ps/AzA/MWCNTs nanocomposite modified electrode. The attractive response performances of the proposed method for simultaneous detection of the four biomolecules were presented in detail.

2. Experimental

2.1. Apparatus

The voltammetric experiments were performed in an electrochemical assembly with a platinum wire as the counter electrode, a glassy carbon electrode (U = 3 mm) as working electrode and a Ag/AgCl reference electrode. Cyclic voltamme-try (CV) experiments were taken out with a Gamry Reference 600 potentiostat (Gamry, USA). All experiments were done at room temperature (25 0C). Before each experiment, the working electrode was polished with a slurry containing 0.3 im and then with 0.05 im sized aluminum oxide particles for 5 min. After each treatment, the electrode was washed and ultrasoni-cated in distilled water for 5 min to remove retained aluminum oxide particles on the electrode surface. Multiwalled carbon nanotubes (MWCNTs, u = 6-9 nm) were purchased from Sigma Aldrich Co. The pH values of the solutions were assessed by a Hanna HI 221 pH-meter using the full range of 0-14.

2.2. Reagents and materials

All chemicals used were of analytical-reagent grade, and distilled water was used throughout. Ascorbic acid, dopamine and uric acid were obtained from Sigma (St. Louis, MO, USA), and they were all used as received. The stock solutions of dopamine (1.0 x 10"2molL"*) and ascorbic acid (1.0 x 10"2 mol L"1) were made daily by dissolving dopamine hydrochloride and ascorbic acid (Merck) in ethanol and distilled water, respectively. Uric acid solution (1.0 x 10"2 -mol L"1) was prepared by dissolving the solid in a small volume of 1.0 x 10"2 mol L"1 NaOH solution and diluted to the desired concentration. The solutions were protected from light and stored at 4 0C. Before usage, all sample solutions were prepared by appropriate dilutions to the desired concentration with distilled water. Trisodium citrate (99.5%) and

HAuCl4 • 3H2O (99.999%) were purchased from Aldrich. N',N'-dimethylphenothiazin-5-ium-3,7-diamine chloride (Azure A or AzA) was purchased from Merck and it was used as received. A solution of Azure A (1 x 10~3 M) was prepared by dissolving 0.2918 g of Azure A in 25 ml ethanol and made up to 100 ml with distilled water. All potentials reported were versus the Ag/AgCl electrode (3.0 M KCl). The Nafion (5wt.% in lower aliphatic alcohols) was purchased from Aldrich. The phosphate buffer was prepared from 0.1 mol in both phosphoric acid and sodium phosphate. All the other chemicals were analytical grade, or better, and used as received. All experiments were carried out at room temperature. The bare GCE was pretreated carefully with 0.05 pm alumina slurry on a polishing cloth, rinsed thoroughly with 1:1 HNO3:H2O (v/v), and then washed with pure ethanol and distilled water, respectively. All glassware used in the colloid preparation was rigorously cleaned in a tub of freshly prepared 3:1 HCl:HNO3 and thoroughly rinsed with ultrapure water.

2.3. Analytical procedure

The experiments were conducted in phosphate buffer (0.1 M, pH 7.0) at room temperature. The electrochemical responses were measured in a conventional three-electrode system using a modified GCE (Nafion/AuNPs/AzA/MWCNTs) as working electrode, a platinum wire as counter electrode, and a Ag/AgCl (3 M KCl) electrode as reference. Differential pulse voltamme-try (DPV), CV and EIS were performed on a Gamry Electrochemical Analyzer (Bioanalytical Systems, Inc., USA). All cyclic voltammetric tests were carried out with a scan rate of 50 mV s_1 unless otherwise stated.

(1.0%) with stirring and 20 ml of HAuCl4 solution (1.0 mm) was quickly added. The solution initially develops a gray color, which switches to a lavender and finally red in 1-3 min of continuous boiling. When the solution became dark red it removed from heat. Gold-nanoparticle solutions were used within two hours of preparation. Nanoparticles used in this study bear a negative charge due to weakly adsorbed citrate ions (Li and Grennberg, 2006). In a typical preparation, 5 mg of AzA + /MWCNTs was added dropwise into 2.5 mL of as-prepared colloidal solution of AuNPs under vigorous agitation. Immediately, the pink color faded, then changed slowly into gray, indicating formation of AuNPs/AzA/ MWCNTs composites. After agitating for an additional 8 h, a black precipitate was isolated by centrifugation. The black precipitate was washed with distilled water several times. The sediment of resulted AuNPs/AzA/MWCNTs hybrid was finally suspended in distilled water (4.5 ml), then mixed with 0.5 ml of 1.0% Nafion solution and sonicated at room temperature for at least 8 h and then homogeneous suspension would be accomplished. A 5.0 iL of a homogeneous Nafion/AuNPs/ AzA/MWCNTs solution was firstly dropped on the pretreated GCE to obtain the AuNPs/AzA/MWCNTSs/GCE. Subsequently, the prepared electrode was dried in the air and then rinsed with distilled water. The modified electrodes were stored at 4 0C in the refrigerator. The suspension (Nafion/AuNPs/ AzA/MWCNTSs) was stored at 4 0C and prevented from light. This suspension was stable at least for two weeks. The schematic diagram of the adsorption process of colloid gold nano-particles is shown in the Scheme 1.

3.3. UV-vis and FT-IR spectroscopic properties

3. Results and discussion

3.1. Preparation of AzA/MWCNTs composite

Carbon nanotubes require surface activation before nanoclus-ters can be attached. The as-received MWCNTs (1.0 g) were treated with a 1:3 v/v mixture of HNO3 (65%) and H2SO4 (98%) at 50 0C for 4h with continuous ultrasonication (Li and Grennberg, 2006). The mixture was filtered and washed with distilled water until the filtrate was neutral. The treated MWCNTs were dried under air at room temperature and stored in the refrigerator. 20 mg treated MWCNTs were dispersed in 5.0 ml 1.0 x 10~4 M AzA solution and sonicated at room temperature for at least 1 h. The blue color of the AzA solution disappeared and black solution obtained. The result indicates that AzA dye was adsorbed completely on the MWCNTs surface. The mixture was washed with distilled water to get rid of the weakly adsorbed AzA dye. The fictionalized MWCNTs were stored until time of use.

3.2. Preparation of Nafion/AuNPs/AzA/MWCNTs composites

Citrate-reduced AuNPs colloids were prepared according to a modified Martin et al. method (Martin et al., 2010). Particles synthesized by citrate reduction are nearly monodisperse spheres of a size controlled by the initial reagent concentrations (Pillai and Kamat, 2004). The AuNP colloidal solution was prepared by heating 25 ml of sodium citrate solution

Prior to addition of gold nanoparticles, MWCNTSs/AzA samples were characterized by UV-Vis, and FTIR. Fig. 1 shows typical UV-Vis absorption spectra of (a) MWCNTSs/AzA solution and (b) aqueous AzA solution. Pure Azure A displayed two strong adsorbs at 285 and 605 nm (curve a, Fig. 1). The AzA/MWNTs composite also had two characteristic absorption peaks, one in the visible region around 580 nm, and the other located in the UV region around 270 nm (curve b, Fig. 1). As compared with pure AzA, the two absorption

Scheme 1 Schematic diagram of Azure A-mediated adsorption of colloidal gold nanoparticles on MWCNTs.

200 300 400 500 600 700 Wavelength (nm)

Figure 1 UV-vis spectra of Azure A (curve a), MWCNTs (curve b), AzA/MWCNTs and AuNPs/AzA/MWCNTs (curve d).

peaks exhibited a little departure, which showed an interaction between AzA and MWCNTSs. After AuNPs were adsorbed onto the AzA/MWCNTs nanocomposite film, the two peaks could also be appearing. However, a red shift for the peak at 597 nm, a blue shift for the other peak at 265 nm, and a new absorption peak at 522 nm appeared due to the characteristic peak of the AuNPs (curve c, Fig. 1), which provided evidence of the interaction between the oppositely charged AzA and AuNPs, showing that AuNPs could be preferentially adsorbed onto AzA surface. On the other hand, FT-IR spectra of MWCNTs/AzA samples (Fig. 2B) do not exhibit amide C-N stretching vibrations in the range of 2000-1000 cm-1. These observations indicate that covalent bonds have not formed between MWCNTs and AzA (Kovtyukhova and Mallouk, 2005; Wang et al., 2007), thus implying that strong interactions arise from p-p stacking between these conjugated frames.

Wavenumbers (cm1)

Figure 2 FT-IR spectra of MWCNTs(A), AzA (B) and AzA/ MWCNTs(C).

3.4. Choice of the amount of modifier

The film thickness of the porous thin film modified electrodes affects both kinetics of the electrode processes and mass transfer mechanism via diffusion through the porous film (Streeter et al., 2008; Xiao et al., 2009; Henstridge et al., 2010; Keeley et al., 2009) and thus, has a predominant role in the voltam-metric response of these electrodes toward different analytes. Cyclic voltammograms were recorded at a glassy carbon electrode modified with changing amounts of Nafion/AuNPs/ AuNPs/MWCNTs. To vary the thickness of the modifier, different volumes of Nafion/AuNPs/AzA/MWCNTs suspension in distilled water (1 mgmL-1, 1-10 iL) have been deposited on the electrode surface. The peak current of analytes clearly increased as the amount of Nafion/AuNPs/AzA/MWCNTs at a GCE from 1 to 5 iL increased. A further increase in the nation/AuNPs/AzA/MWCNTs volume decreased the current response slightly. A volume of 5 iL of the modifier suspension provides reasonable sensitivity in the voltammetric responses, and was selected as the optimum volume for modification of the electrode surface.

3.5. TEM characterization of nanocomposite materials

In solution, Azure A in protonated form (AzA+) is shown to adsorb to MWCNTs walls via p-p stacking interaction (Dezhampanah and Aghajani, 2013). The gold nanoparticles bear considerable negative charge from citrate incorporated during preparation (Santhosh et al., 2006; Sudeep and Kamat, 2005; Weitz et al., 1985), which should afford ready adsorption on the MWCNTs-AzA+ surface. The formation of AuNPs/AzA/MWCNTs was also investigated using TEM. TEM images of the expected Nafion/AuNPs/AzA/MWCNTs composite confirmed that gold nanoparticles were typically bound on MWCNTs walls, as shown in Fig. 3A. Nevertheless, when colloidal AuNPs were mixed with MWCNTs under the same condition without AzA, only few gold nanoparticles can be established on the nanotubes. The density of adsorbed gold nanoparticles along MWCNTs (i.e., without AzA+) was lower than on AzA-coated MWCNTs, as indicated in Fig. 3B.

3.6. Impedance spectroscopy

EIS was chosen to further characterize the process of the modification of GC electrode. The impedance measurements of modified electrode in 0.1 mol L-1 PBS (pH = 7.0) containing 5 mmol L-1 [Fe(CN)6]3-/4- were proceeded to investigate the conductivity and electron transfer properties of different electrodes. The Nyquist plots for all electrodes are presented in Fig. 4. It is well-known that a representative impedance spectrum is written of a semi-circle portion and a linear portion. The semicircle diameter at higher frequencies corresponds to the electron-transfer resistance (Rct), and the linear part at lower frequencies reflects the diffusion process. It can be understood that a well defined semicircle at high frequencies and a linear part at low frequencies were obtained at the bare GCE (Rct 505 ±2 X) (Fig. 4a). When Nafion/MWCNTs (Fig. 4b), Nafion/MWCNTs/AuNPs (Fig. 4c) and Nafion/MWCNTs/ AzA/AuNPs (Fig. 4d) were combined onto the electrode surface, Rct gradually decreased, attributed to the Nafion/ MWCNTs/AzA and AuNPs layers for serving as conductive

Figure 4 The Nyquist plots of the electrochemical impedance spectroscopy (EIS) at bare GCE (curve a), Nafion/MWCNTs (curve b), Nafion/AuNPs/MWCNTs (i.e., without AzA) (curve c) and Nafion/AuNPs/AzA/MWCNTs (curve d) in 5.0mmolL—1 Fe(CN)3—/4— (1:1) containing 0.1 mM PBS (pH = 7), respectively. The frequency range is from 0.01 Hz to 100 kHz.

layers and increasing the interfacial electron transfer between the electrode and the detection solution. The value of Rct was decreased from 505 ± 2 X (bare GCE) to 103 ±2 X. The value of Rct was 505 ±2 X, 148 ± 2 X, 129 ± 2 X and 103 ± 2 X for GCE, Nafion/MWCNTs, Nafion/AuNPs/MWNTs, Nafion/AuNPs/AzA/MWCNTs, respectively. The answers showed that the modified material can decrease the electron transfer resistance and improve the electron transport process.

3.7. Electrocatalytic oxidation of AA, DA, UA and Trp

CV behaviors of the Nafion/AuNPs/AzA/MWCNTs/modified GCE were investigated by adding AA, DA, UA and Trp in 0.1molL—1 PBS (pH 7.0), respectively. Fig. 5 displays the CV responses of the mixture of AA, DA, UA and Trp at the bare GCE (a), Nafion/MWCNTs (b), Nafion/AuNPs/ MWCNTs (c) and Nafion/AuNPs/AzA/MWCNTs (d), respectively. At the bare GCE, a broad oxidation peak can be

Figure 5 CVs of the bare GCE (curve a), Nafion/MWCNT (curve b), Nafion/AuNPs/MWCNTs (curve c) and Nafion/AuNPs/AzA/ MWCNT (curve d) in 0.1 M PBS (pH 7.0) containing 1.0 mM AA, 0.1 mM DA, 0.1 mM UA and 0.1 mM Trp, respectively.

watched at 0.525 V. The cause may be that the oxidation peaks of AA, DA and UA overlapped together and came into large peak. Also, an inconspicuous anodic peak at 0.887 V can be imputed to the anodic peak of Trp. On the base of the above resolutions, we can arrive at a determination that it is impossible to simultaneously observe the four small biomolecules at bare GCE. When Nafion/MWCNTs and Nafion/AuNPs/ MWCNTs were used as the working electrode, the detection sensitivity was improved significantly and effective separation of the anodic packs of AA, DA, UA and Trp was obtained, but these peak currents are lower than those of Nafion/AuN-Ps/AzA/MWCNTs. As depicted in the Fig. 5, the oxidation peak of AA, DA, UA and Trp appeared at about —19 mV, 223 mV, 359 mV and 660 mV, respectively. The separations of the oxidation peak potentials of AA-DA, DA-UA, AA-UA and UA-Trp at the surface of the modified electrode using CV and DPV were obtained at 242, 136, 378 and 301 mV, respectively, which were large enough to determine AA, DA, UA and Trp individually and simultaneously. These data imply that the favoritism of the four species was feasible. We can see that the Nafion/AuNPs/AzA/MWCNTs have higher

potential resolution, high sensitivity and suitable for simultaneous determination of AA, DA, UA and Trp.

3.8. Effect of .scan rate

The effect of scan rate (in the range of 25-300 mV s-1) on the peak currents and peak potentials at the Nafion/AuNPs/AzA/ MWCNTs in 0.1 mol L-1 phosphate buffer solution (pH 7.0) containing AA, DA, UA and Trp was investigated by CV. A good linear relationship between the peak current and the scan rate from 25 to 300 mV s-1 was obtained (figure not shown). The regression equations were Ipa (pA) = 0.045v (mVs-1) + 2.1408 for AA, Ipa (pA) = 0.2255v (mV s-1) + 207.75 for DA, Ipa (pA) = 0.6368v (mV s-1) + 37.574 for UA and Ipa (pA) = 0.2386v (mV s-1) + 42.524 for Trp, with a correlation coefficient (R) of 0.9973, 0.9925, 0.9910 and 0.9908, respectively, demonstrating the redox process of AA, DA, UA and Trp at the Nafion/AuNPs/AzA/MWCNTs/GCE was controlled by adsorption.

3.9. Effect of pH on the oxidation of AA, DA, UA and Trp

The value of human blood and urine is close to 7.0, thus 0.1 mol L-1 PBS solution with pH = 7.0 is chosen for simultaneous determination of AA, DA, UA and Trp. Also, at the pH 7.0, AA, DA, UA and Trp can be completely separated, which makes it possible to simultaneously detect them in the mixture solution. So, 0.1 mol L-1 PBS of pH 7.0 was taken as the optimum working buffer in the following field. It is clear that the sensitivity of Nafion/AuNPs/AzA/MWCNTs is higher for all the four analytes when comparing with only Nafion/ MWCNTs or Nafion/AuNPs/MWCNTs modified GCEs. Scheme 2 shows the redox reactions of all the four analytes with Nafion/AuNPs/AzA/MWCNTs composite film. In addition, the effect of the temperature of the PBS solution on the response of the modified electrode was studied in the range of 10-35 0C. Initially, the voltammetric response, increased monotonically, reaching a maximum value at approximately 25 0C, which decreased after. Thus, an operating temperature of 25 0C (room temperature) was chosen as a compromise.

3.10. Simultaneous detection of AA, DA, UA and Trp

For the simultaneous determination of AA, DA, UA, and Trp, DPV was carried out at the Nafion/AuNPs/AzA/MWCNTs modified electrode in 10 mL buffer solution (pH 7.0) (Fig. 6). DPV (step size 8 mV, pulse size 50 mV, sample period 0.3 s, pulse time 0.05 s) technique can provide a better peak resolution and current sensitivity, which is very suited for simultaneous determination of species in the admixture. The obtained optimal conditions and parameters were used for plotting the calibration curves. Fig. 6 shows the DPV responses of AA, DA, UA and Trp in a mixture in which the concentration of one substance changed, while the other three species remained constant. The slope of the linear regression line for the calibration graph of each species is nearly equal to that without the other species, indicating that they do not interfere with the detection of each other. As shown in the Fig. 6A, the peak current of AA increases linearly with the increase in AA concentration from 0.3 to 10 mM in the mixture of DA (5.0 pM), UA (30 pM), Trp (50 pM). The linear regression equation was expressed as i(pA) = 0.2773 + 2.8862 CAA (mM) and correlation coefficient of R = 0.9989. Fig. 6B illustrated the anodic peak current of DA in the mixture of AA (5.0 mM), UA (30 pM), Trp (50 pM). The dependence of the peak current on the DA concentration is shown in inset B of Fig. 6. This inset clearly shows that the plot of peak current versus DA concentration is comprised of two linear segments with different sides, corresponding to two different ranges of substrate absorption. The calibration plot of the proposed sensor is linear in two concentration ranges of 0.5-10 and 10-50 pM. The regression equation over these ranges were: i(pA) = 1.7405 + 3.5062 CDA (pM), R = 0.9996 and i(pA) = 33.503 + 0.7217 CDA (pM), R = 0.9950, respectively. Likewise, the DPV curves of UA and Trp were also recorded in Fig. 6C and D, respectively. As shown in Fig. 6C, the peak current of UA increases linearly with the increase in UA concentration from 0.5 to 50 pM in the mixture of AA (5.0 mM), DA (5.0 pM), Trp (50 pM). The linear response range was 0.550 pM and the linear regression equation was expressed as 1(pA) = 1.3532 + 1.4151 CUA (pM) and correlation coefficient

Scheme 2 Redox reactions of all the four analytes with Nafion/AuNPs/AzA/MWCNTs composite film.

Figure 6 DPVs at the Nafion/AuNPs/AzA/MWCNTs modified GCE in 0.1 M PBS (pH 7.0) (A) containing DA (5.0 iM), UA (30 iM), Trp (50 iM) and different concentrations of AA (from inner to outer): 0.3, 0.5, 0.7, 1.0, 3.0, 5.0, 7.0 and 10.0 mM; (B) containing AA (5.0 mM), UA (30 iM), Trp (50 iM) and different concentrations of DA (from inner to outer): 0.5, 0.7, 1.0, 3.0, 5.0, 7.0, 10, 20, 30, 40 and 50 iM; (C) containing AA (5.0 mM), DA (5.0 iM), Trp (50 iM) and different concentrations of UA (from inner to outer): 0.5, 1.0, 5.0, 7.0, 10, 20, 30, 40, 50 iM and (D) containing AA (5.0 mM), DA (5.0 iM), UA (30 iM) and different concentrations of Trp (from inner to outer): 1.0, 2.0, 4.0, 6.0, 8.0, 10, 20, 40, 60, 70 and 100 iM. Insets: the calibration plots of the anodic peak current response versus concentration of AA, DA, UA and Trp, respectively. (Step size 8 mV, pulse size 50 mV, sample period 0.3 s, pulse time 0.05 s).

of R = 0.9996. The calibration curve for Trp also exhibits two linear segments with regression equation as: i(iA) = 1.5736 + 0.8299 CTrp (iM) (R = 0.9966) and i(iA) = 11.72 + 0.163 CTrp (iM) (R = 0.9987) in the linear range of 1.0-10 pM and 10-100 iM, respectively. Further, the lower detection limit for AA, DA, UA and Trp were 16 iM, 0.014 iM, 0.28 iM and 0.56 M iM (S/N = 3), respectively. Moreover, the comparison of the performances of other sensors which employed different nanomaterials was also investigated. The outcomes were presented in Table 1. It could be found that the prepared electrode exhibited satisfactory detection limit and linear range for simultaneous detection of AA, DA, UA and Trp in their quaternary mixture.

3.11. Reproducibility and stability of the modified electrode

The reproducibility of the sensors (Nafion/AuNPs/AzA/ MWCNTs modified GC electrode) was evaluated by using ten equally proposed electrodes in the mixture of DA, AA, UA, and Trp. The oxidation peak potential of the four species was the same at the ten electrodes. The relative standard deviation (RSD) of peak currents was 3.0%, 4.1%, 3.9% and 3.6% for AA, DA, UA, and Trp, respectively. Similarly, the RSD values for the prepared ten equally electrodes in ten separate batches of the mixture were 4.0%, 3.2%, 3.6% and 3.8%, respectively. When the sensors were not in use, they were stored in 0.1 mol L—1 PBS (pH 7.0) at 4 0C. The stability of

Table 1 Comparison of analytical performance of Nafion/AzA/MWCNTs/AuNPs modified electrode with other nanomaterials

reported previously.

Modified electrode AA DA UA Trp References

Linear range /LOD Linear range/LOD Linear range/LOD Linear range/LOD

(lM) (lM) (lM) (lM)

AuNPs/PImox 210-1010/2 5.0-268/0.08 6.0-486/0.5 3.0-34/0.7 Wang et al. (2012)

GS-PTCA 20-420/5.60 0.4-370/0.13 4-540/0.92 0.4-140/0.06 Zhang et al. (2012)

MWCNT/FeNAZ 7.77-833/1.11 7.35-833/1.05 0.23-83.3/0.033 0.074-34.5/0.01 Noroozifar et al. (2011)

MWNTs/MGF 100-6000/18.28 0.3-100/0.06 5-1000/0.93 5-500/0.87 Li et al. (2014)

AgNPs/rGO 10-800/0.45 10-800/0.39 10-800/0.39 10-800/0.44 Kaur et al. (2013)

AzA/MWCNTs/AuNPs 300-10000/16 0.5-50/0.01 0.5-50/0.01 1-100/0.3 This work

Table 2 Determination of AA, DA, UA, and Trp in real urine samples.

Sample Analyte Detected (iM) Added (iM) Founda (iM) Recovery (%) Total value (iM)

Urine 1 AA - 500 498.7(±2.4) 99.74 -

DA - 10 10.3(±2.7) 103 -

UA 33.7 (±2.2) 15 49.1(±1.8) 100 2.02(±2.8)

Trp - 10 10.1(±2.8) 101 -

Urine 2 AA - 500 498.6(±0.8) 99.72 -

DA - 25 25.8(±1.7) 103 -

UA 17.8 (±2.2) 25 43.0(±1.2) 100.8 1.07(±2.1)

Trp - 20 20.3(±1.8) 101.5

a Average of three replicates ± standard deviation.

Table 3 Determination of tryptophan in milk by standard addition method.

Milk sample Found (iM) Added (iM) Founda (iM) Recovery (%)

Milk 1 190 ± 2.7 50 240 ± 2.7 100

Milk 2 170 ± 3.4 100 270 ± 3.4 100

Milk 3 180 ± 2.3 150 330 ± 2.3 100

a Average of three replicates ± standard deviation.

the proposed sensors was investigated over a period of 7 days and 20 days, respectively. No apparent change was observed after storage for 7 days and the response maintained 96% of the initial response. After storing for 20 days, the response also maintained 93.7% compared with the initial currents. The results implied that the constancy of the proposed sensors was acceptable.

3.12. Effect of interference

There are a variety of interference coexisting in biological samples. It was found that no interference for the detection of AA, DA, UA, and Trp was observed for the following compounds (tolerance ratio): Na + , K + , Ca2+ and Mg2+ (250), Citric acid (500), tartaric acid (800) and glucose (400). The peak current signal change was below 5.0% of all cases. The proposed Naf-ion/AuNPs/AzA/MWCNTs/GCE exhibits good selectivity, and the interference in the biological samples does not interfere with the assay.

3.13. Sample analysis

Urine and milk samples were selected as real samples for analysis by the proposed method using the standard addition method. The water samples were diluted 50 times with 0.1 M PBS (pH 7.0). In order to avoid from the interferences of the real samples matrix and to fit into the linear range of AA, DA and UA, only 1.0 mL of urine samples were added to the electrochemical cell containing 10 mL of PBS. It can be seen that AA, DA, UA, and Trp in binary mixtures could be found with the appropriate recovery in Table 2. The Nafion/ AuNPs/AzA/MWCNTs modified electrode was also used to detect tryptophan content in milk. At first, 1 mL of milk was diluted to 10-mL with PBS, and then a series of 1 x 10-3 M Trp solution was added. The concentration of tryptophan was calculated by standard addition, and results are shown in Table 3. The recovery of the spiked samples ranged between 99.7% and 103%, indicating the detection procedures are free from interferences of the milk and urine sample matrix. The

adjustment of the relative standard deviation (RSD) is below 4%, showing the possible utility of the Nafion/AuNPs/AuN-Ps/MWCNTs for the practical purpose of the four biomole-cules in real samples.

4. Conclusion

In conclusion, we have prepared Nafion/AuNPs/AzA/ MWCNTs composites using Azure A molecules as interlinkers. p-p stacking interactions between MWCNTs and Azure A, and electrostatic interactions between AzA and AuNPs nanoparticles, resulted in the formation of Nafion/AuNPs/ AzA/MWCNTs/GCE composites. The fabricated electrode exhibited excellent electrocatalytic activity toward the oxidation of AA, DA, UA, and Trp by significantly enhancing the anodic peak currents. The proposed sensor is useful and suitable for the direct simultaneous determination of AA, DA, UA, and Trp in real samples by means of the DPV. The proposed method provides a promising scheme for simultaneous detection of these species in human metabolism during early stage of diseases.

Acknowledgment

We gratefully acknowledge Istanbul University Scientific Research Fund (Project nos. BYP-20075 and UDP-26215) for financial support.

References

Alexeyeva, N., Laaksonen, T., Kontturi, K., Mirkhalaf, F., Schiffrin, D.J., Tammeveski, K., 2006. Oxygen reduction on gold nanopar-ticle/multi-walled carbon nanotubes modified glassy carbon electrodes in acid solution. Electrochem. Commun. 8, 1475-1480. Cao, W., Wei, C., Hu, J., Li, Q., 2008. Direct electrochemistry electrocatalysis of myoglobin immobilized on gold nanoparticles/ carbon nanotubes naohybrid film. Electroanalysis 20, 1925-1931. Chen, W., Lu, Z., Li, C.M., 2008. Sensitive human interleukin 5 impedimetric sensor based on polypyrrole—pyrrolepropylic acid—gold nanocomposite. Anal. Chem. 80, 8485-8492. Cooper, J R., Bloom, F.E., Roth, R.H., 1982. The Biochemical Basis

of Neuropharmacology. Oxford University Press, Oxford, UK. Damier, P., Hirsch, E.C., Agid, Y., Graybiel, A.M., 1999. The substantia nigra of the human brain. II. Patterns of loss of dopamine-containing neurons in Parkinson's disease. Brain 122, 1437-1448.

Dezhampanah, H., Aghajani, N., 2013. Study of Azure A adsorption from aqueous solution onto rice husk. Adv. Chem. Sci. 2, 51-56. Fiorucci, A.R., Cavalheiro, E.T.G., 2002. The use of carbon paste electrode in the direct voltammetric determination of tryptophan in pharmaceutical formulations. J. Pharm. Biomed. Anal. 28, 909915.

Ghoreishi, S.M., Behpour, M., Mousavi, S., Khoobi, A., Ghoreishi, F.S., 2014. Simultaneous electrochemical determination of dopa-mine, ascorbic acid and uric acid in the presence of sodium dodecyl sulphate using a multi-walled carbon nanotube modified carbon paste electrode. RSC Adv. 4, 37979-37984. Henstridge, M.C., 2010. Voltammetric selectivity conferred by the modification of electrodes using conductive porous layers or films: the oxidation of dopamine on glassy carbon electrodes modified with multiwalled carbon nanotubes. Sens. Actuators, B 145, 417427.

Jiang, K., Eitan, A., Schadler, L.S., Ajayan, P.M., Siegel, R.W., Grobert, N., Mayne, M., Reyes-Reyes, M., Terrones, H., Terrones,

M., 2003. Selective attachment of gold nanoparticles to nitrogen-doped carbon nanotubes. Nano Lett. 3, 275-277.

Kaur, B., Pandiyan, T., Satpati, B., Srivastava, R., 2013. Simultaneous and sensitive determination of ascorbic acid dopamine uric acid and tryptophan with silver nanoparticles-decorated reduced graph-ene oxide modified electrode. Colloids Surf. B 111, 97-106.

Keeley, G.P., Lyons, M.E.G., 2009. The effects of thin layer diffusion at glassy carbon electrodes modified with porous films of singlewalled carbon nanotubes. Int. J. Electrochem. Sci. 4, 794-809.

Kovtyukhova, N.I., Mallouk, T.E., 2005. Ultrathin anisotropic films assembled from individual single-walled carbon nanotubes and amine polymers. J. Phys. Chem. B 109, 2540-2545.

Li, J., Grennberg, H., 2006. Microwave-assisted covalent sidewall functionalization of multiwalled carbon nanotubes. Chem. Eur. J. 12, 3869-3875.

Li, N., Yuan, R., Chai, Y., Chen, S., An, H., Li, W., 2007. New antibody immobilization strategy based on gold nanoparticles and Azure I/multi-walled carbon nanotube composite membranes for an ampero-metric enzyme immunosensor. J. Phys. Chem. C 111, 8443-8450.

Li, H., Wang, Y., Ye, D., Luo, J., Su, B., Zhang, S., Kong, J., 2014. An electrochemical sensor for simultaneous determination of ascorbic acid dopamine uric acid and tryptophan based on MWNTs bridged mesocellular graphene foam nanocomposite. Talanta 127, 255-261.

Liu, L.Q., Wang, T.X., Li, J.X., Guo, Z., Dai, L.M., Zhang, D.Q., Zhu, D.B., 2003. Self-assembly of gold nanoparticles to carbon nanotubes using a thiol-terminated pyrene as interlinker. Chem. Phys. Lett. 367, 747-752.

Liu, A., Honma, I., Zhou, H., 2007. Simultaneous voltammetric detection of dopamine and uric acid at their physiological level in the presence of ascorbic acid using poly(acrylic acid)-multiwalled carbon-nanotube composite-covered glassy-carbon electrode. Biosens. Bioelectron. 23, 74-80.

Martin, M.N., Basham, J.L., Chando, P., Eah, S.K., 2010. Charged gold nanoparticles in non-polar solvents: 10-min synthesis and 2D self-assembly. Langmuir 26, 7410-7417.

Mazloum-Ardakani, M., Beitollahi, H., Ganjipour, B., Naeimi, H., Nejati, M., 2009. Electrochemical and catalytic investigations of dopamine and uric acid by modified carbon nanotube paste electrode. Bioelectrochemistry 75, 1-8.

Noroozifar, M., Khorasani-Motlagh, M., Akbari, R., Parizi, M.B., 2011. Simultaneous and sensitive determination of a quaternary mixture of AA DA UA and Trp using a modified GCE by iron ion-doped natrolite zeolite-multiwall carbon nanotube. Biosens. Bioelectron. 28, 56-63.

Ou, Y., Huang, M.H., 2006. High-density assembly of gold nanopar-ticles on multiwalled carbon nanotubes using 1-pyrenemethylamine as interlinker. J. Phys. Chem. B 110, 2031-2036.

Pillai, Z.S., Kamat, P.V., 2004. What factors control the size and shape of silver nanoparticles in the citrate ion reduction method. J. Phys. Chem. B 108, 945-951.

Richard, C., Balavoine, F., Schultz, P., Ebbesen, T.W., Mioskowski, C., 2003. Supramolecular self-assembly of lipid derivatives on carbon nanotubes. Science 300, 775-778.

Santhosh, P., Gopalana, A., Lee, K.P., 2006. Gold nanoparticles dispersed polyaniline grafted multiwall carbon nanotubes as newer electrocatalysts: preparation and performances for methanol oxidation. J. Catal. 238, 177-185.

Streeter, I., Wildgoose, G.G., Shao, L., Compton, R.G., 2008. Cyclic voltammetry on electrode surfaces covered with porous layers: an analysis of electron transfer kinetics at single-walled carbon nanotube modified electrodes. Sens. Actuators, B 133, 462-466.

Sudeep, P.K., Kamat, P.V., 2005. Photosensitized growth of silver nanoparticles under visible light irradiation: a mechanistic investigation. Chem. Mater. 17, 5404-5410.

Wang, Y., Bi, C., 2013. Simultaneous electrochemical determination of ascorbic acid, dopamine and uric acid using poly (tyrosine)/ functionalized multi-walled carbon nanotubes composite film modified electrode. J. Mol. Liq. 177, 26-31.

Wang, T.X., Zhang, D.Q., Xu, W., Yang, J.L., Zhu, B.D., 2002. Preparation characterization and photophysical properties of alkanethiols with pyrene units-capped gold nanoparticles: unusual fluorescence enhancement for the aged solutions of these gold nanoparticles. Langmuir 18, 1840-1848.

Wang, T., Hu, X., Dong, S., 2006. Noncovalent functionalization of multiwalled carbon nanotubes: application in hybrid nanostruc-tures. J. Phys. Chem. B 110, 6631-6636.

Wang, Z., Li, M., Zhang, Y., Yuan, J., Shen, Y., Niu, L., Ivaska, A., 2007. Thionine-interlinked multi-walled carbon nanotube/gold nanoparticle composites. Carbon 45, 2111-2115.

Wang, C., Yuan, R., Chai, Y.Q., Chen, S.H., Hu, F.X., Zhang, M.H., 2012. Simultaneous determination of ascorbic acid dopamine uric acid and tryptophan on gold nanoparticles/overoxidized-polyimi-dazole composite modified glassy carbon electrode. Anal. Chim. Acta 741, 15-20.

Weitz, D.A., Lin, M.Y., Sandroff, C.J., 1985. Colloidal aggregation revisited: new insights based on fractal structure and surface-enhanced raman scattering. Surf. Sci. 158, 147-164.

Xiao, L., Wildgoose, G.G., Compton, R.G., 2008. Sensitive electrochemical detection of arsenic (III) using gold nanoparticle modified carbon nanotubes via anodic stripping voltammetry. Anal. Chim. Acta 620, 44-49.

Xiao, L., Wildgoose, G.G., Compton, R.G., 2009. Exploring the origins of the apparent "electrocatalysis" observed at C60 film-modified electrodes. Sens. Actuators, B 138, 524-531.

Yang, S., Li, G., Yang, R., Xia, M., Qu, L., 2011. Simultaneous voltammetric detection of dopamine and uric acid in the presence

of high concentration of ascorbic acid using multi-walled carbon nanotubes with methylene blue composite film-modified electrode. J. Solid State Electrochem. 15, 1909-1918.

Yao, Y., Shiu, K., 2008. Direct electrochemistry of glucose oxidase at carbon nanotube-gold colloid modified electrode with poly(diallyl-dimethylammonium chloride) coating. Electroanalysis 20, 15421548.

Zhang, R.Y., Olin, H., 2011. Gold-carbon nanotube nanocomposites: synthesis and applications. Int. J. Biomed. Nanosci. Nanotechnol. 2, 112-135.

Zhang, W., Chai, Y.Q., Yuan, R., Chen, S.H., Han, J., Yuan, D.H., 2012. Facile synthesis of graphene hybrid tube-like structure for simultaneous detection of ascorbic acid dopamine uric acid and tryptophan. Anal. Chim. Acta 756, 7-12.

Zhou, R.J., Shi, M.M., Chen, X.Q., Wang, M., Yang, Y., Zhang, X.B., Chen, Z.H., 2007. Water-soluble and highly fluorescent hybrids of multi-walled carbon nanotubes with uniformly arranged gold nanoparticles. Nanotechnology 18, 485-603.

Zhuo, Y., Yuan, R., Chai, Y.Q., Zhang, Y., Li, X.L., Zhu, Q., 2005. An amperometric immunosensor based on immobilization of hepatitis B surface antibody on gold electrode modified gold nanoparticles and horseradish peroxidase. Anal. Chim. Acta 548, 205-210.

Zhuo, Y., Yuan, R., Chai, Y.Q., Zhang, Y., Li, X.L., Wang, N., Zhu, Q., 2006. Amperometric enzyme immunosensors based on layer-by-layer assembly of gold nanoparticles and thionine on nafion modified electrode surface for a-1-fetoprotein determinations. Sens. Actuators, B 114, 631-639.