hyaluronic acid-multiwalled carbon nanotubes modified electrode for determination of tryptamine

Biosensors and Bioelectronics 31 (2012) 277–283

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Electrochemical sensor based on molecularly imprinted film at polypyrrole-sulfonated graphene/hyaluronic acid-multiwalled carbon nanotubes modified electrode for determination of tryptamine Xianrong Xing a , Su Liu b , Jinghua Yu a , Wenjing Lian a , Jiadong Huang c,∗ a

College of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China College of Resources and Environment, University of Jinan, Jinan 250022, PR China c College of Medicine and Life Sciences, University of Jinan, Jinan 250022, PR China b

a r t i c l e

i n f o

Article history: Received 19 August 2011 Received in revised form 16 October 2011 Accepted 18 October 2011 Available online 24 October 2011 Keywords: Molecularly imprinted polymers Electrochemical sensor Polypyrrole-sulfonated graphene composites film Hyaluronic acid-multiwalled carbon nanotubes bionanocomposites Tryptamine detection

a b s t r a c t An imprinted electrochemical sensor based on polypyrrole-sulfonated graphene (PPy-SG)/hyaluronic acid-multiwalled carbon nanotubes (HA-MWCNTs) for sensitive detection of tryptamine was presented. Molecularly imprinted polymers (MIPs) were synthesized by electropolymerization using tryptamine as the template, and para-aminobenzoic acid (pABA) as the monomer. The surface feature of the modified electrode was characterized by cyclic voltammetry (CV). The proposed sensor was tested by chronoamperometry. Several important parameters controlling the performance of the molecularly imprinted sensor were investigated and optimized. The results showed that the PPy-SG composites films showed improved conductivity and electrochemical performances. HA-MWCNTs bionanocomposites could enhance the current response evidently. The good selectivity of the sensor allowed three discriminations of tryptamine from interferents, which include tyramine, dopamine and tryptophan. Under the optimal conditions, a linear ranging from 9.0 × 10−8 mol L−1 to 7.0 × 10−5 mol L−1 for the detection of tryptamine was observed with the detection limit of 7.4 × 10−8 mol L−1 (S/N = 3). This imprinted electrochemical sensor was successfully employed to detect tryptamine in real samples. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Biogenic amines are nitrogenous low molecular weight organic compounds, which have been derived from enzymatic decarboxylation of some free amino acids and proteins (Huang et al., 2009; Pastore et al., 2005). They exist in a wide variety of foods especially in protein-rich foods such as fish and fish products, meat, dairy products, beverages and other fermented foods (Saaid et al., 2009). Biogenic amines in low concentrations are essential for many physiological functions. But at high concentrations, they can cause some deleterious effects, such as nausea, hot flushes, cold sweat, palpitations, headaches, rash, high or low blood pressure and death in severe cases (Proestos et al., 2008; Tang et al., 2011). Tryptamine is one of the biogenic amines, which is synthesized from the decarboxylation of the tryptophan (Tatsumi and Ueda, 2011). It is found in trace amounts in the brains of mammals and is believed to play a role as a neuromodulator or neurotransmitter (Jones, 1982). The consumption of foods containing high concentrations of tryptamine may cause high blood pressure.

∗ Corresponding author. Tel.: +86 531 89736122; fax: +86 531 82769122. E-mail address: chm [email protected] (J. Huang). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.10.032

Various methods have been developed for the analysis of tryptamine such as high performance liquid chromatography (Innocente et al., 2007), ion chromatography (Favaro et al., 2007), micellar electrokinetic capillary chromatography (Wang et al., 2003), capillary zone electrophoresis (Kvasniˇcka and Voldˇrich, 2006), high performance liquid chromatography/tandem mass spectrometry (Gosetti et al., 2007), and immunoassays (Skerritt et al., 2000). Although these methods can offer good selectivity and detection limit, they often are cost intensive, time consuming, qualified personnel and complex pre-treatment steps. Accordingly, it still remains a great challenge to develop a suitable analytical technique to detect tryptamine. Recently, molecular imprinting technology has become a wellestablished analytical tool, which has been widely used for the preparation of polymeric materials that have the ability to specifically bind species (Aghaei et al., 2010; Ma et al., 2011). Molecular imprinting involves positioning functional monomers around the target molecules (template) by covalent interaction or noncovalent interaction, followed by polymerization (Fang et al., 2009). After removing the template, molecularly imprinted polymers (MIPs) were obtained. MIPs possess surface cavities complementary to the template. In addition, MIPs possess several advantages over their biological recognition element, including stability,

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easiness of preparation, selective recognition material for the template. As a new class of materials possessing high selectivity and affinity for the target molecule, MIPs have been widely applied in sensor development (Zhang et al., 2010a). Due to their excellent sensitivity, rapid response, simplicity, low cost and in vivo detection, electrochemical sensors (e.g. voltammetric, potentiometric, conductometric and capacitance) have obtained wide applications in medical, biological and environmental analysis (Sun et al., 2009; Zhang et al., 2010b). In order to enhance the sensitivity and selectivity of the electrochemical sensor, a variety of materials have been employed to modify electrode, such as conducting polymer, carbon nanotubes (CNTs), and biomaterials. Graphene, as a new form of carbon, is a two-dimensional sheet of carbon atoms bonded through sp2 hybridization. Owing to their novel properties such as large specific surface area, high thermal and mechanical properties, high electrical conductivity, graphene is an ideal material for the preparation of electrochemical sensors and biosensors (Bo et al., 2011; Shan et al., 2010). Moreover, graphene-based composite materials have received increased attention. Hong et al. reported a uric acid electrochemical sensor based on the composite of gold nanoparticles and 1-pyrene butyric acid functionalized graphene modified glassy carbon electrodes exhibited rapid response and high sensitivity. The composite showed strong electrocatalytic activity and high electrochemical stability (Hong et al., 2010). Al-Mashat et al. reported on the synthesis of a graphene/polyaniline nanocomposite and its application in the development of a hydrogen gas sensor. Because of the high surface-to-volume ratio of the incorporated graphene nanosheets, the developed sensor has higher sensitivity toward hydrogen gas than that of only graphene and polyaniline nanofibers based gas sensors (Al-Mashat et al., 2010). Due to its biocompatibility, hydrophilicity and ease of processing into useful structures, hyaluronic acid (HA) is of high interest for various applications. HA is a major extracellular matrix component that consists of glucuronic acid and N-acetylglucosamine as a repeating disaccharide unit and is a nonsulfated glycosaminoglycan. Filip et al. investigated a sensing platform for a mediatorless electrochemical detection of NADH based on a biocompatible nanocomposite consisting of single-walled carbon nanotubes (CNTs) dispersed in a HA. The NADH sensor exhibits a good longterm operational stability (95% of the original sensitivity after 22 h of continuous operation) (Lee and Schmidt, 2010; Filip et al., 2011). In this article, we proposed a novel imprinted electrochemical sensor based on polypyrrole-sulfonated graphene (PPySG)/hyaluronic acid-multiwalled carbon nanotubes (HA-MWCNTs) for sensitive detection of tryptamine. Polypyrrole (PPy), one of the most important conducting polymers, were used to enhance the current response of the proposed electrode in the experiment. However, PPy is usually mechanically weak and insulating in its neutral state, in order to improve PPy mechanical, electrical, or electrochemical properties, the PPy/SG composite was prepared. Owing to large specific surface area, high thermal and mechanical properties, high electrical conductivity, the PPy/SG composite film grown at 0.7 V is porous, conductive and electroactive (Liu et al., 2010). Therefore, PPy/SG composite can be used as a modified electrode material in the experiment. Own to high aspect ratio, ultra-light weight, high mechanical strength, high electrical conductivity, high thermal conductivity and high surface-to-volume area, carboxylated MWCNTs were also used to enhance the current response of the proposed electrode (Filip et al., 2011). Electropolymerization was employed for the preparation of MIPs film. Tryptamine was employed as the template molecule, and para-aminobenzoic acid (pABA) as the functional monomer. It has been shown that pABA can be easily electropolymerized on various substrate materials and form films with good chemical and mechanical stability

(Liu et al., 2011). So pABA was chosen as the functional monomer for electropolymerization. The electrochemical performance of the developed MIPs sensor was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The selectivity, reproducibility, and stability of developed sensor were also tested. Moreover, it was employed to detect tryptamine in cheese and lactobacillus beverage samples. 2. Materials and methods 2.1. Reagents and materials Tryptamine and graphite were purchased from Jingchun Co., Ltd. (Shanghai, China). Hyaluronic acid (HA from Streptococcus equi), multiwalled carbon nanotubes (MWCNTs) and pyrrole were obtained from Sigma–Aldrich Company (USA). Sulfuric acid, nitric acid, hydrazine, dodecylbenzene sulfonic acid and sodium borohydride were purchased from Beijing Chemical Technology Co., Ltd. (China). Cheese and lactobacillus beverage samples were obtained from a local market. All chemicals were analytical grade. Deionized water was used throughout this study. 2.2. Equipments Cyclic voltammetry (CV) and chronoamperometry experiments were carried out with a model VersaSTAT 3 electrochemical workstation (Princeton Applied Research, USA). Electrochemical impedance spectroscopy (EIS) experiments were performed on IM6ex (Germany). The impedance spectra were recorded upon the application of the bias potentials in the frequency range of 100 mHz–10 kHz, using an ac voltage of 5 mV amplitude. Electrochemical measurements were performed using a three-electrode system consisting of a KCl saturated Ag/AgCl reference electrode, a platinum wire auxiliary electrode and a modified glassy carbon working electrode. Solution pH was measured on FE 20 MettlerToledo pH meter (Switzerland). All Fourier transform infrared (FTIR) spectroscopic measurements were performed on a Bruker Vertex 70 spectrometer (Germany). Atomic force microscopy (AFM) was carried out with AFM (Nanoscope@ IV, USA). Scanning electron microscope (SEM) images were obtained using field emission SEM (ZEISS, Germany). The fluorescence imagings were performed on FLS920 (England). 2.3. Preparation of sulfonated graphene (SG) First of all, graphite oxide (GO) was prepared by a modified Hummers method (Hummers and Offeman, 1958). Graphite power was oxidized using the sulfuric acid solution contained NaNO3 and KMnO4 , and then H2 O2 was slowly added to the mixture. When no gas evolved, excess deionized water was added in the mixture. The reaction mixture was filtered and washed with HCl aqueous solution and deionized water. The filtrate was dissolved in deionized water and removed the impurities by dialysis. Finally, the filtrate was centrifugated for 30 min to remove residual graphite. The spectrum of graphite oxide was shown in Fig. S1(a). It illustrated the presence of C–O (at 1060 cm−1 ), C–O–C (at 1250 cm−1 ), and C O (at 1720 cm−1 ) (see Fig. S1(a) in supplement). Then GO was obtained. SG was prepared from graphite oxide according to Si method (Si and Samulski, 2008): (1) prereduction of graphene oxide with sodium borohydride at 80 ◦ C for 1 h to remove the majority of the oxygen functionality; (2) sulfonation with the aryl diazonium salt of sulfanilic acid in an ice bath for 2 h; and (3) postreduction with hydrazine (100 ◦ C for 24 h) to remove any remaining oxygen functionality. As shown in Fig. S1(b), the absence of the peaks at 1250 cm−1 , and 1060 cm−1 indicates the epoxide groups have been removed. The characteristic peaks of –SO3 H groups (1190 cm−1 )

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was found. The results clearly indicated that sulfonated graphene was successfully prepared (see Fig. S1(b) in supplement). The thickness of used graphene was measured by atomic force microscopy (AFM) to be 1.35 nm (see Fig. S2 in supplement).

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2.4. Glassy carbon electrode (GCE) pretreatment Bare GCE was polished using 0.05 ␮m alumina slurry on microcloth pads and rinsed thoroughly with deionized water. The GCE was activated by polarization at +1.6 V for 60 s and at −1.6 V for the same period before use. Prior to surface modification, the GCE was scanned by cyclic voltammetry (CV) from −0.2 to +0.6 V in K3 [Fe(CN)6 ] solution until repeating cyclic voltammograms appeared. Then, the electrodes were washed with deionized water and dried before use.

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2.5. Sensor fabrication Firstly, the PPy-SG was modified on the surface of the electrode by the electropolymerization. A controlled amount of SG (1.5 mg mL−1 ) was added into 0.01 mol L−1 dodecylbenzene sulfonic acid aqueous solution and sonicated for 15 min. Then pyrrole monomer (0.05 mol L−1 ) was dissolved in this emulsion solution under ultrasonic stirring for 15 min. All of the electropolymerizations were performed at a constant potential of 0.7 V for 500 s (Liu et al., 2010). The PPy-SG/GCE electrodes were allowed to dry. Secondly, the carboxylation MWCNTs were performed as described previously (Qin et al., 2010). A 50 mg MWCNTs were dissolved in a 40 mL mixture of concentrated sulfuric acid and nitric acid (3:1, v/v) for ultrasonic agitation for 4 h. The mixture was washed with deionized water by centrifugation (10,000 rpm) until the pH of the resulting solution became neutral. As shown in Fig. S3, after MWCNTs were carboxylated, the characteristic peaks at 1720 cm−1 was found due to the stretching vibrations of C O of –COOH. It demonstrated that the –COOH has been bonded to MWCNTs (see Fig. S3 in supplement). Thus, carboxylated MWCNTs was successfully prepared. The functionalized MWCNTs were dried in an oven at 110 ◦ C. Such prepared carbon nanotube carboxylate was confirmed to be MWCNTs-COOH. Dispersions of MWCNTs-COOH were prepared from 1 mg of MWCNTs-COOH in 1 mL of 0.1% HA. Then HA-MWCNTs bionanocomposites were obtained. The PPySG/GCE was modified by casting of 20 ␮L HA-MWCNTs solution, and allowed to dry. Finally, poly(pABA) with imprinting of tryptamine-modified HA-MWCNTs/PPy-SG/GCE was prepared by electropolymerization for 10 scans in the range from −0.6 to 1.0 V at a scan rate of 100 mV s−1 from a HCl solution (0.01 mol L−1 ), which contained 20 mmol L−1 pABA and 1 mmol L−1 tryptamine and was purged with nitrogen for 10 min before use. The electrodes were allowed to dry overnight. As a control, non-imprinted polymers (NIPs) were fabricated following the same procedure, but in the absence of the template. 2.6. Electroanalytical measurements Electrochemical measurements to characterize the MIPs films were carried out in 5.0 mmol L−1 K3 [Fe(CN)6 ] solution containing 0.2 mol L−1 KCl at room temperature (25 ◦ C). CV measurements were performed over a potential range from −0.2 to +0.6 V at a scan rate of 50 mV s−1 . Chronoamperometry was performed and all sensing potentials were set at 0.4 V. The equilibrium time of each curve was set at 800 s. All experiments except the characterization of the MIPs modified electrode were carried out using chronoamperometry.

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Potential/V Fig. 1. CV of the bare GCE (a), PPy-SG/GCE (b), HA-MWCNTs/PPy-SG/GCE (c), tryptamine-MIPs/HA-MWCNTs/PPy-SG/GCE (d) and removed tryptamine (e) in aqueous solution consisting of 5 mmol L−1 of K3 [Fe(CN)6 ] and 0.2 mol L−1 of KCl. The voltage range: −0.2 to 0.6 V; scan rate: 50 mV s−1 .

3. Results and discussion 3.1. Characterization 3.1.1. Characterization of electrochemical behavior of the modified electrodes Cyclic voltammetry (CV) is the effective and convenient tool to monitor the electrons transmission procedure of the modified electrode (Zhang et al., 2010a,b). The electrochemical behavior of the stepwise fabrication process was studied in 5.0 mmol L−1 K3 [Fe(CN)6 ] solution containing 0.2 mol L−1 KCl. K3 [Fe(CN)6 ] was served as an electrochemical probe. The results were shown in Fig. 1. As shown in Fig. 1a, a couple of typical redox peaks of K3 [Fe(CN)6 ] appeared at bare GCE. When the surface was covered with the PPy-SG composites layer, an increment of the redox peak current in the curve of the electrode was observed in Fig. 1b. It may be that the incorporation of highly conductive SG sheets (conductivity) into PPy greatly improved the conductivity of the composite. We made a further experiment to confirm this. It can be seen that the PPy-SG composites showed larger redox peak compared to PPy or SG from Fig. 2(a). Moreover, the chronoamperometric response of the electrode during the processes of electropolymerization of PPy film and PPy/SG composite film at a constant potential of +0.7 V was used to confirm this. In both curves, the currents response increased immediately, mainly due to doublelayer capacitance charges. As a result, the currents response of i–t curve recorded in the PPy/SG system is higher than that recorded in the PPy system. This is mainly due to that a thin compact film of PPy was deposited on the surface of GCE, which increased the electrical resistance of the working electrode. However, SG sheets were co-polymerized into PPy matrix to form a porous film, which increased the conductivity of the composite film and the electrode surface area for electrochemical polymerization (Liu et al., 2010) (see Fig. S4 in supplement). Coupled with the introduction of the HA-MWCNTs, an obvious increment of the redox peak current was observed in Fig. 1c. It is well known that MWCNTs had the special properties involving stable physical and chemical character, small dimensional size, and excellent catalytic. Furthermore, HA was used as a dispersing agent to solubilise MWCNTs owing to its film-forming ability, biocompatibility, nontoxicity and adhesion. From Fig. 2(b), HA-MWCNTs bionanocomposites showed more

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Fig. 2. (a) CV of the bare GCE (a), SG/GCE (b), PPy/GCE (c), PPy-SG/GCE (d); (b) CV of the bare GCE (a), PPy-SG/GCE (b), MWCNTs/PPy-SG/GCE (c), HA/PPy-SG/GCE (d), HA-MWCNTs/PPy-SG/GCE (e) in aqueous solution consisting of 5 mmol L−1 of K3 [Fe(CN)6 ] and 0.2 mol L−1 of KCl. The voltage range: −0.2 to 0.6 V; scan rate: 50 mV s−1 .

reversible electrochemistry, higher short-term stability and higher selectivity. HA was electrochemically inactive matrix with a predominant role as a dispersing agent for MWCNTs (Filip et al., 2011). When the imprinted film was electrosynthesized on the surface of HA-MWCNTs/PPy-SG/GCE, the peak current was not observed (Fig. 1d). It may be that the K3 [Fe(CN)6 ] could not pass through the layer of polymer to arrive at the surface of electrode. As shown in Fig. 1e, after the template removal, the redox current of Fe(CN)6 3− increased. It can be ascribed that removal of the template, the formation of recognition sites or binding cavity made electronic transmission possible and K3 [Fe(CN)6 ] could pass through the cavity and reach the surface of the electrode more easily. In EIS, the semicircle diameter equals the electron-transfer resistance (Ret ). Fig. S5 shows the EIS of the modified electrode at different modification stages (see Fig. S5 in supplement). It was observed that the EIS of the bare GCE displayed a small semicircle at high frequencies, suggesting very low Ret to redox probe [Fe(CN)6]3−/4− in Fig. S5(a). After the bare electrode was modified with PPy-SG nanocomposite film, the resistance for the redox probe decreased (Fig. S5(b)), implying that PPy-SG is excellent electric conducting material and could accelerate the electron transfer. Subsequently, when HA-MWCNTs nanoparticles were loaded on the surface of PPy-SG/GCE, the EIS showed a large decrease in diameter, indicating that the coating of HA-MWCNTs nanoparticles enhanced the electric conductivity in Fig. S5(c). As shown in Fig. S5(d), after the imprinted film was electrodeposited onto the surface of HA-MWCNTs/PPy-SG/GCE, the EIS exhibited a large increase in diameter, suggesting that the imprinted film formed an additional barrier and further prevented the redox probe to the electrode surface. These results were accordant with CV assays as described in above detail. 3.1.2. Characterization of the removal of template SEM images and fluorescence imaging are used to confirm the removal of tryptamine. As observed in Fig. S6(a), when the imprinting film was immobilized on the HA-MWCNTs/PPy-SG/GCE, the electrodeposited imprinted film was distributed regularly throughout the electrode surface. Therefore, tryptamine-MIPs/HAMWCNTs/PPy-SG/GCE was successfully fabricated. After being washed with PBS, tryptamine was removed from the molecularly imprinted polymer. As illustrated in Fig. S6(b), tryptamine imprinted pores were revealed on the surface (see Fig. S6 in supplement). Meanwhile, the fluorescence experiment was carried out in the elution solution. The elution solution was found at 354 nm

with emission spectra (Fig. S7). This result was consistent with the result of the standard tryptamine solution. It confirmed that the tryptamine was removed.

3.2. Optimization of experimental parameters 3.2.1. Optimization of scan cycles The thickness of polymer membranes could easily be adjusted by controlling the number of cycles during the electropolymerization process. As shown in Fig. 3(a), the current response reached maximum with 10 cycles, and then decreased with further increasing of cycle number. In general, if the imprinted polymer membranes are too thick, template molecules situated at the central area of the polymer membranes cannot completely be removed from polymer matrix (Xie et al., 2010). Thus, 10 cycles could obtain an optimal membrane thickness to provide the highest sensitivity to tryptamine.

3.2.2. Optimal ratio of template and monomer The amount of the imprinted sites of MIPs was affected by the ratio of template and monomer. So the ratio of template to monomer was studied in our investigations. The corresponding results were shown in Fig. 3(b). It was observed that the largest current response was obtained when template–monomer ratio was 1:20. While the monomer–template ratio was higher than 20:1, the current response became decreasing, the possible reason was that the amount of the template molecules decreased, the formation of recognition sites or binding cavity in the MIPs films decreased. The optimization ratio of template to monomer could make the proposed sensor obtain the optimum sensing performance. Therefore, the optimal monomer–template ratio was 20:1.

3.2.3. Optimal loading of SG and carboxylated MWCNTs The effect of the loading of SG and carboxylated MWCNTs on the response of sensor were studied. As shown in Fig. S8, with the increase of loading of SG, the current response became increasing. It was observed that the largest current response was obtained when the loading of SG was 1.5 mg mL−1 . As shown in Fig. S9, the largest current response was obtained when the loading of carboxylated MWCNTs was 1 mg mL−1 . The possible reason was that MWCNTs was found agglutination and poor dispersibility when the loading of carboxylated MWCNTs was higher than 1 mg mL−1 .

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Fig. 3. (a) Effect of the different cycles of the electropolymerization of MIPs: a, 5; b, 10; c, 15; d, 20; e, 25; f, 30; (b) Effect of the monomer–templates ratio on the response of the sensor to tryptamine: a, 10:1; b, 15:1; c, 20:1, d, 25:1, e, 30:1.

3.2.4. Optimization of pH The effect of K3 [Fe(CN)6 ] solution with different pH values from 4.0 to 9.0 on the current response performance of the MIPs/HA-MWCNTs/PPy-SG/GCE and NIPs/HA-MWCNTs/PPySG/GCE electrode toward tryptamine was studied by using the CV. The results revealed a maximum response was observed at about pH 7.0, indicating that when the solution pH is 7.0, it can be facilitated the interaction between tryptamine and the imprinted film. Meanwhile, the response current of the NIPs/HA-MWCNTs/PPy-SG/GCE electrode was lower than that of the MIPs/HA-MWCNTs/PPy-SG/GCE electrode, which suggested that binding sites at the imprinted film existed and tryptamine reached easily the surface of the electrode by imprinted cavities which contain functional group matching with the template, while the absence of the imprinted cavities on the tryptamine electrode resulted in the reduction of the peak current. 3.2.5. Optimization of temperature The effect of K3 [Fe(CN)6 ] solution with different temperatures on the current response performance of the MIPs/HAMWCNTs/PPy-SG/GCE and NIPs/HA-MWCNTs/PPy-SG/GCE electrode toward tryptamine was studied by using the CV. The largest response was observed at 25 ◦ C.

of the proposed sensor. The results were shown in Fig. 5. Noticeably, the current response of the imprinted sensor was stronger than that of the non-imprinted sensor, and the response toward tryptamine was stronger than that toward structural analogs. This might attribute that the MIPs sensor provided a thin imprinted polymer layer on the sensor surface. This layer had functional groups and selective cavities that specifically interacted with the template molecular tryptamine. The results indicated that the proposed sensor exhibited good selectivity toward tryptamine. 3.5. Reproducibility, repeatability and stability To investigate the reproducibility of the proposed technique, under the same conditions, three sensors fabricated independently were examined. The results indicated that the imprinted sensor showed excellent reproducibility. The relative standard deviation (RSD) was 4.8% (see Table S1 in supplement). To investigate the repeatability of the MIPs sensor, the experiments were performed in 5 × 10−6 mol L−1 tryptamine using the same sensor. Five electrodes were prepared by using the same modification method. Then, the five electrodes were used to determine the same tryptamine solution (5 × 10−6 mol L−1 ). The calculated RSD were about 5.0% (n = 5) (see Table S2 in supplement). The results showed that the proposed sensor had good

3.3. Calibration curve of the sensor 500

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To investigate the affinity of the MIPs electrode and NIPs electrode to tryptamine, amperometric measurement of MIPs and NIPs electrodes were carried out. Under the optimized conditions, the determination of tryptamine at different concentrations was performed. The results of the amperometric detection were shown in Fig. 4. The current response was linear to tryptamine concentration varying from 9.0 × 10−8 mol L−1 to 7.0 × 10−5 mol L−1 . The detection limit was 7.4 × 10−8 mol L−1 (r = 0.992). Compared with other method (Proestos et al., 2008), the linear range and limit of detection obtained from the developed sensor were better.

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Selective recognition of the template molecule was an important merit for a MIPs sensor. In order to check the selectivity of tryptamine imprinted sensor, some species were chosen as interference based on structural similarity and interaction types. The MIPs electrode and NIPs electrode were used to study the selectivity

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Fig. 4. Amperometric curves of sensor in tryptamine solution at different concentrations: a, MIPs; b, NIPs.

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MIP NIP

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In this study, tryptamine molecule was successfully imprinted on the PPy-SG/HA-MWCNTs/GCE. The electropolymerization was employed as the preparation of MIPs film. The results showed that the PPy-SG composite films showed improved conductivity and electrochemical performance. HA had been used as a dispersing agent to solubilise MWCNTs. HA-MWCNTs bionanocomposites could enhance the current response evidently. Under optimization of the effective parameters, the proposed electrochemical sensor showed high selectivity, sensitivity and speed, appropriate for tryptamine detection, including in real sample analysis.

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Interferents Fig. 5. Amperometric measurement of tryptamine and structural analogues on imprinted electrode: a, tryptamine; b, tyramine; c, dopamine; d, tryptophan.

repeatability. We also performed the regeneration experiment of developed MIPs sensor. The regeneration procedure was as follows: in the experiment, after the detection, the electrodes were washed by 0.1 mol L−1 PBS (pH 5.0) for 5 min to extract the templates. When the redox peak currents of these electrodes were the same as Fig. 1e, the regeneration procedure was finished. It revealed that binding of tryptamine to the “cavity” was reversible. The electrodes were allowed to dry. Then the electrodes were used to detect tryptamine solution again. Afterwards, the electrodes were washed again and the next detection was processed likewise. The experimental results demonstrated the MIPs sensor could be regenerated very well. The long-term stability of the sensor was also important. The stability of the sensor has been examined by monitoring the current response for 5 × 10−6 mol L−1 tryptamine at regular intervals (2 day) for a period of two weeks. After two weeks, the sensor retained 87% of current response. It was suggested that the proposed sensor possessed acceptable storage stability.

3.6. Application To evaluate the applicability of the proposed sensor, the concentration of tryptamine in cheese and lactobacillus beverage samples were determined applying the standard addition method. The analytical results were shown in Table 1. The recovery was in the range of 91.5–103%, indicating that the imprinted sensor might be preliminarily applied for the determination of tryptamine in the real sample. The comparative data suggested superiority of the present sensor over some earlier reported methods (Lavizzari et al., 2006) (see Table S3 in supplement).

Table 1 Determinations of tryptamine in cheese and lactobacillus beverage samples. Sample

Added (mol L−1 )

Found (mol L−1 )

Recovery (%)

Cheese

1 × 10−7 3 × 10−7 5 × 10−7

9.51 × 10−6 2.76 × 10−7 5.06 × 10−7

95.1 92 101.2

Lactobacillus beverage

1 × 10−7 3 × 10−7 5 × 10−7

9.15 × 10−6 3.09 × 10−7 4.86 × 10−7

91.5 103 97.2

This work was supported by the National Natural Science Foundation of the People’s Republic of China (No. 31171700, 30972056 and 30911140278), the Foundation for Outstanding Young Scientist in Shandong Province (No. BS2009NY001), the Scientific and Technological Development Plan in Shandong Province (No. 2010GNC10960), the Natural Science Foundation of Shandong Province (No. ZR2010DQ025) and the Shandong Province Higher Educational Science and Technology Program (No. J10LB14). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2011.10.032. References Aghaei, A., Hosseini, M.R.M., Najafi, M., 2010. Electrochim. Acta 55, 1503–1508. Al-Mashat, L., Shin, K., Kalantar-zadeh, K., Plessis, J.D., Han, S.H., Kojima, R.W., Kaner, R.B., Li, D., Gou, X.L., Ippolito, S.J., Wlodarski, W., 2010. J. Phys. Chem. C 114, 16168–16173. Bo, Y., Yang, H.Y., Hu, Y., Yao, T.M., Huang, S.S., 2011. Electrochim. Acta 56, 2676–2681. Fang, C., Yi, C.L., Wang, Y., Cao, Y.H., Liu, X.Y., 2009. Biosens. Bioelectron. 24, 3164–3169. Favaro, G., Pastore, P., Saccani, G., Cavalli, S., 2007. Food Chem. 105, 1652–1658. ˇ coviˇcová, J., Tomˇcík, P., Gemeiner, P., Tkac, J., 2011. Talanta 84, 355–361. Filip, J., Sefˇ Gosetti, F., Mazzucco, E., Gianotti, V., Polati, S., Gennaro, M.C., 2007. J. Chromatogr. A 1149, 151–157. Hong, W.J., Bai, H., Xu, Y.X., Yao, Z.Y., Gu, Z.Z., Shi, G.Q., 2010. J. Phys. Chem. C 114, 1822–1826. Huang, K.J., Wei, C.Y., Liu, W.L., Xie, W.Z., Zhang, J.F., Wang, W., 2009. J. Chromatogr. A 1216, 6636–6641. Hummers, W.S., Offeman, R.E., 1958. J. Am. Chem. Soc. 80, 1339. Innocente, N., Biasutti, M., Padovese, M., Moret, S., 2007. Food Chem. 101, 1285–1289. Jones, R.S.G., 1982. Prog. Neurobiol. 19, 117–139. Kvasniˇcka, F., Voldˇrich, M., 2006. J. Chromatogr. A 1103, 145–149. Lavizzari, T., Veciana-Nogués, M.T., Bover-Cid, S., Mariné-Font, A., Vidal-Carou, M.C., 2006. J. Chromatogr. A 1129, 67–72. Lee, J.Y., Schmidt, C.E., 2010. Acta Biomater. 6, 4396–4404. Liu, A.R., Li, C., Bai, H., Shi, G.Q., 2010. J. Phys. Chem. C 114, 22783–22789. Liu, Y.T., Deng, J., Xiao, X.L., Ding, L., Yuan, Y.L., Li, H., Li, X.T., Yan, X.N., Wang, L.L., 2011. Electrochim. Acta 56, 4595–4602. Ma, J., Yuan, L.H., Ding, M.J., Wang, S., Ren, F., Zhang, J., Du, S.H., Li, F., Zhou, X.M., 2011. Biosens. Bioelectron. 26, 2791–2795. Pastore, P., Favaro, G., Badocco, D., Tapparo, A., Cavalli, S., Saccani, G., 2005. J. Chromatogr. A 1098, 111–115. Proestos, C., Loukatos, P., Komaitis, M., 2008. Food Chem. 106, 1218–1224. Qin, X., Wang, H.C., Wang, X.S., Miao, Z.Y., Chen, L.L., Zhao, W., Shan, M.M., Chen, Q., 2010. Sens. Actuators, B 147, 593–598. Saaid, M., Saada, B., Ali, A.S.M., Saleh, M.I., Basheer, C., Lee, H.K., 2009. J. Chromatogr. A 1216, 5165–5170. Si, Y.C., Samulski, E.T., 2008. Nano Lett. 8, 1679–1682. Shan, C.S., Yang, H.F., Han, D.X., Zhang, Q.X., Ivaska, A., Niu, L., 2010. Biosens. Bioelectron. 25, 1070–1074. Skerritt, J.H., Guihot, S.L., McDonald, S.E., Culvenor, R.A., 2000. J. Agric. Food Chem. 48, 27–32. Sun, D., Zhang, Y., Wang, F.R., Wu, K.B., Chen, J.W., Zhou, Y.K., 2009. Sens. Actuators, B 141, 641–645.

X. Xing et al. / Biosensors and Bioelectronics 31 (2012) 277–283 Tang, T., Qian, K., Shi, T.Y., Wang, F., Li, J.Q., Cao, Y.S., Hu, Q.B., 2011. Food Control 22, 1203–1208. Tatsumi, H., Ueda, T., 2011. J. Electroanal. Chem. 655, 180–183. Wang, Q.J., Yu, H., Li, H., Ding, F., He, P.G., Fang, Y.Z., 2003. Food Chem. 83, 311–317.

283

Xie, C.G., Gao, S., Guo, Q.B., Xu, K., 2010. Mikrochim Acta 169, 145–152. Zhang, J., Wang, Y.Q., Lv, R.H., Xu, L., 2010a. Electrochim. Acta 55, 4039–4044. Zhang, Z.H., Hu, Y.F., Zhang, H.B., Luo, L.J., Yao, S.Y., 2010b. J. Electroanal. Chem. 644, 7–12.