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An Amperometric Immunosensor Based on an Ionic Liquid and Single-Walled Carbon Nanotube Composite Electrode for Detection of Tetrodotoxin in Pufferfish Yun Zhang,† Yuxia Fan,† Jian Wu,§ Xichang Wang,† and Yuan Liu*,† †

College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, People’s Republic of China College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, People’s Republic of China

§

ABSTRACT: An amperometric immunosensor based on a composite electrode of single-walled carbon nanotubes and ionic liquid n-octylpyridinum afluorophosphate (SWCNT−ILE) was developed for the determination of tetrodotoxin (TTX). Compared with the glassy carbon electrode (GCE), the electrode combined advantages of carbon nanotubes and ionic liquid, which exhibited the excellent antifouling ability of p-nitrophenol (PNP) so that it remarkably improved the stability of the pnitrophenyl phosphate-based sensor. Combining the enzyme-linked immune sorbent assay (ELISA) by alkaline phosphatase (AP) and magnetic particles immobilized with antigens, a real-time assay of tetrodotoxin was developed by amperometric immunosensors. Under the optimium condition, the developed sensor demonstrated a linear range of tetrodotoxin from 2 to 45 ng/mL with a low detection limit of 5 ng/mL. Furthermore, the amperometric immunosensor was applied to determine TTX in real samples and could be used as an effective and sensitive sensor for direct detection of tetrodotoxin within 20 min. KEYWORDS: single-walled carbon nanotubes, ionic liquid n-octylpyridinum afluorophosphate (OPFP), alkaline phosphatase (AP), p-nitrophenol (PNP), magnetic particles, tetrodotoxin (TTX)

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INTRODUCTION Ionic liquids (ILs), widely known as the green electrolyte, are a kind of promising material adopted in the field of electrochemistry on account of their excellent physicochemical properties and good biocompatibility.1 In addition, ILs are typically composed of organic cations with a variety of anions and exhibit unique properties:2−5 (1) ILs contain specific low melting point (below 100 °C);2 (2) ILs are used as electrolytes, characterized by high ionic conductivity, nonflammability, and chemical stability;3 and (3) ILs are considered as good solvents for organic and inorganic chemicals and have easily tuned physical and chemical properties through the control of functionality of the cationic or anionic structures.4,5 In recent years, our group has developed an application of ILs in electrochemical analysis due to their high ionic conductivity and catalytic activity. Moreover, an ionic liquid modified electrode with improved electrochemical performance has been explored.6,7 Combined with carbon materials, ILs were employed to form conductive composites for the preparation of various electrodes, such as IL−graphite,8 IL− carbon nanofibers,9 and IL−graphene.10 In our previous work, OPFP was introduced for the fabrication of carbon paste electrode,11 which showed high-performance electrode properties with various excellent features such as resistivity toward biomolecule fouling and high rates of electron transfer. Due to the high stability and electrochemical conductivity, OPFP was considered a good component in fabricating electrodes. Carbon nanotubes (CNTs) have been widely used in electrochemical analysis due to their unique properties such as large active surface area, good mechanical resistance, and high electronic conductivity.12 The successful use of CNTs for electroanalytical applications is probably due to the ability of © 2016 American Chemical Society

the nanomaterial to promote electron transfer in electrochemical reactions. The most important significance of the electrocatalytic effect of CNTs has been attributed to the activity of edge-plane-like graphite sites at the ends of CNTs.13 Therefore, CNTs could be used as robust and advanced carbon electrode material for the design of sensing platforms. Nowadays, single-walled carbon nanotubes (SWCNT) and multiple-walled carbon nanotubes (MWCNT) were used broadly and commonly in the field of CNTs. In our study, there were three reasons for us to choose a SWCNT rather than a MWCNT. First, SWCNTs have better performance and electrical conductivity in electrochemical analysis.14 Second, SWCNTs possess unique and outstanding mechanical, optical, thermal, and biological functional properties. 15 Third, SWCNTs have been more extensively demonstrated in various areas, such as composite materials, nanoelectronics, optics, and biomedicine.16−18 Magnetic particles are attractive due to their small volume and various functionalities. Integration of magnetic particles into immunoassays and other bioanalytical methodologies is a valuable approach to allow efficient target capture, enrichment, and convenient separation. In addition, large signal amplification can be achieved by preconcentration of the target and attachment of numerous enzyme labels to magnetic particles. In view of the above characteristics of magnetic particles, they have been extensively used for fabricating electrochemical19 and colorimetric biosensors.20 Therefore, the application of Received: Revised: Accepted: Published: 6888

May 30, 2016 July 28, 2016 August 10, 2016 August 10, 2016 DOI: 10.1021/acs.jafc.6b02426 J. Agric. Food Chem. 2016, 64, 6888−6894

Article

Journal of Agricultural and Food Chemistry

buffer solution (pH 8.5) was used as the electrolyte solution. Mixtures of 0.15 M PBST and 10 mM PBS buffer (pH 7.4) containing 2% BSA were prepared in advance. All aqueous solutions were prepared using ultrapure water (Millipore, 18.2 MΩ·cm). Apparatus. All of the electrochemical measurements were performed on a CHI 440 electrochemical workstation (CH Instruments, USA). The electrochemical cell was assembled with a conventional three-electrode system: a saturated Ag/AgCl reference electrode, a Pt wire auxiliary electrode, and the prepared working electrode (SWCNT−ILE). Voltammetric experiments were carried out in a one-compartment 10 mL cell. A magnetic stirrer was used to stir the test solution during the amperometric analysis. A SpectraMax M5 multimode microplate reader (Molecular Devices, USA) was employed to allow the measurement of the absorbance from each well of a 96-well microtiter plate. The scanning electron microscopy (SEM) experiment was carried out on a Philips XL-30 ESEM. Transmission electron micrographs were obtained with a JEM-1200EX (JEOL, Japan). All of the measurements were carried out at 25 ± 1 °C. The synthesis of the magnetic particles was performed on a MagneSphere Technology Magnetic Separation Stand (Promega Biotech Co., Ltd., Madison, WI, USA). Preparation of Electrode. The homemade SWCNT−ILE was prepared according to the following steps, which were similar to our previous work.35 First, 0.9 g of OPFP and 0.1 g of single-walled nanotubes were mixed manually and fully by pestle and mortar for 20 min. Then, the electrode was prepared firmly by packing a part of the mixed paste into the electrode cavity (1.8 mm diameter) of a glass sleeve using a spatula and heated in an oven with a temperature higher than the melting point of OPFP (melting point = 65 °C) at 90 °C for 3 min. Finally, electrode contact was established via a copper wire backward. The new surface of the working electrode was obtained by smoothing the electrode onto the weighing paper. For comparison, a glassy carbon electrode (GCE) was prepared. Prior to the determination, GCE was hand-polished with alumina powder (Al2O3, size = 0.5−5 μm) until a mirror finish was obtained and then sonicated in ethanol and pure water, respectively, for 5 min to further eliminate particles absorbed on the electrode surface. Finally, the electrode was dried in N2 atmosphere for later use. Sample Preparation. Considering the toxicity of the terodotoxin, the samples were always prepared carefully. After the samples had been collected from a local market, they were cut into small pieces and fully homogenized with regard to the representative portion (skin, internal organs, or ovaries). Five grams of homogenized samples was added into a 50 mL polystyrene centrifuge tube. The mixture was oscillated for 3 min, bathed in water for 20 min, and cooled to room temperature. After the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was adjusted to pH 6.5−7.4 with 1 M sodium hydroxide and was oscillated for 30 s. Blank samples were prepared from nontoxic pufferfish according to the same procedure. Extract liquid was saved at 4 °C for later use. Preparation of Magnetic Particles. Magnetic particles were prepared using the hydrothermal synthesis method. In brief, a solution of FeCl3 was prepared by adding 1.35 g of FeCl3·6H2O into a clear solution of 40 mL of ethylene glycol, followed by the addition of 3.6 g of sodium acetate and 1 g of PEG 20000. The mixture was stirred vigorously for 60 min and then sealed in a 50 mL autoclave. The autoclave was maintained at 200 °C for 8 h and then cooled to room temperature. The black products were washed three times with ethanol and deionized water alternately and finally dissolved in 40 mL of ethanol. The resulting magnetic microspheres were then characterized by transmission electron microscopy. Immobilization of BSA−TTX on Magnetic Particles. The synthesized magnetic particles were dissolved with 25 mM MES (pH 6) to a concentration of 50 mg/mL, and then 50 μL of NHS (50 mg/ mL) and 50 μL of EDC (50 mg/mL) were added into the tube and incubated at room temperature for 30 min with slow tilt rotation. The prepared magnetic particles were washed twice with 300 μL of MES, and the supernatant was removed by magnetic separation. A volume of 150 μL of BSA−TTX (100 ng/mL) was added to the activated magnetic particles. The mixture was vortexed and incubated at room

magnetic particles has been applied with enzyme immunoassay21−23 over the past two decades. Tetrodotoxin (TTX) is one of the most potent lowmolecular-weight marine neurotoxins in various marine organisms.24 It has been regarded as a typical sodium channel inhibitor for the small molecular and paralysis toxin, which is one of the most toxic nonprotein substances in nature.25,26 Upon binding, this toxin interrupts the passive influx of sodium ions and can result in numbness, tingling, and respiratory paralysis. TTX is fatal to human health because of its high toxicity and accumulation in seafood. The mortal dose of TTX is 2 mg for humans.27 Moreover, TTX poisoning is most prevalent in Asian countries, but multiple deaths occur annually due to TTX poisoning in Japan28 and the United States.29 Therefore, it is necessary to develop analytical methods to determine TTX to support food safety. Up to now, numerous methods such as bioassays in mouse,30 surface plasmaon resonance biosensors,31 high-performance liquid chromatography (HPLC) assays,32 and liquid chromatography−mass spectrometry (LC-MS) assays33,34 have been reported for the determination of TTX. However, these methods showed inherent limitations including low sensitivity, high cost, and time consumption. Therefore, the development of a sensitive and simple method for the rapid evaluation of TTX is highly urgent. In this work, an amperometric immunosensor based on a novel carbon composite electrode consisting of ionic liquid noctylpyridinum hexafluorophosphate (OPFP) and single-walled carbon nanotubes was developed. This composite electrode brought new capabilities for electrochemical analysis by combining the advantages of ionic liquid and single-walled carbon nanotubes. Furthermore, combined with ELISA and magnetic particles immobilized with antigens, the amperometric immunosensor based on the composite electrode was applied to determine TTX. Experimental results showed that the proposed immunosensor could be used as an effective and sensitive sensor for direct detection of TTX within 20 min. To our knowledge, this is the first time an amperometric immunosensor has been used to determine TTX in real samples.

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MATERIALS AND METHODS

Reagents. All of the reagents and chemicals were of analytical reagent grade and used without any further purification. Ionic liquid noctylpyridinum hexafluorophosphate (OPFP) was purchased from Shanghai Chengjie Co. Ltd. (Shanghai, China). Single-walled carbon nanotubes (SWCNT, purity > 95%, diameter = 1−2 nm, length = 5− 30 μm) was obtained from Nanjing Xianfeng Nanomaterials and Technology Co. Ltd. (Nanjing, China). Casein, p-nitrophenyl phosphate (PNPP), p-nitrophenol (PNP), tris(hydroxymethyl)aminomethane (Tris), alkaline phosphate (AP), and AP-conjugated goat anti-mouse IgG antibody (AP−anti-mouse IgG), 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride (EDC), MES monohydrate (MES), and N-hydroxysuccinimide (NHS) were purchased from Sigma (St. Louis, MO, USA). Tetrodotoxin (TTX), BSA−TTX, and anti-TTX were purchased from Jianlun Biology and Technology Co. Ltd. (Guangzhou, China). Sodium carbonate, sodium bicarbonate, monopotassium phosphate, sodium acetate, sodium hydrogen phosphate, sodium chloride, potassium chloride, ferric chloride hexahydrate, ethanol, polystyrene, and sodium hydroxide were obtained from Sinopham Chemical Reagent Co. Ltd. (Shanghai, China). Polyethylene glycol (PEG 20000) was purchased from Biosharp (Heifei, China). Tween-20 was obtained from Shanghai Jimei Medical Biology and Technology Co. Ltd. (Shanghai, China). BSA was obtained from Sangon (Shanghai, China). A 0.5 M Tris 6889

DOI: 10.1021/acs.jafc.6b02426 J. Agric. Food Chem. 2016, 64, 6888−6894

Article

Journal of Agricultural and Food Chemistry temperature for at least 30 min with slow tilt rotation and then treated with PBST twice to remove the spare BSA−TTX. The coated magnetic particles were blocked by adding 10 mM PBS buffer (pH 7.4) containing 2% BSA and incubated for 2 h with slight stirring at room temperature. The magnetic particles were resuspended in 10 mM PBS buffer (pH 7.4) and stored at 4 °C for further study. Preparation of Electrochemical Immnoassay. The electrochemical detection of sample analysis was performed after indirect immunoassay. One hundred microliters of TTX and 100 μL of antiTTXA were added to a volume of 30 μL of synthetic magnetic particles coated with BSA−TTX in a centrifuge tube (2 mL) and incubated at 37 °C for 1 h. The tube was then washed three times with PBST. AP-conjugated goat anti-mouse IgG antibody was added and incubated at 37 °C for 30 min. After the tube had been washed three times with PBST, Tris buffer was added to the tube for 5 min. Finally, the solution in the tube was transferred to the electrochemical workstation for detection using a CHI 440 electrochemical workstation (CH Instruments, USA), adding substrate PNPP solution (0.2 M) at 100 μL per hole to the prepared tubes under electrochemical detection.

Figure 2. SEM image of the surface of SWCNT−ILE composite electrode.

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as high stability, electrochemical reactivity, and low background current. Figure 3A shows the cyclic voltammograms of PNPP and PNP at SWCNT−ILE. As shown in Figure 3A (curve a), there

RESULTS AND DISCUSSION Characteristics of SWCNT−ILE. Figure 1 showed the typical cyclic voltammograms of the GCE and SWCNT−ILE in

Figure 1. Cyclic voltammograms for 1.0 mmol L−1 [Fe(CN)6]3‑/4‑ solution at the GCE (curve a, solid line) and SWCNT−ILE (curve b, dash line) between −0.8 and 0.3 V at 10 mV s−1.

the presence of 10 mM [Fe (CN)6]3−/4−. It was clear that the electron transfer rate was slower on the commercial GCE (Figure 1, curve a) which could be ascribed to the lack of conductive binder in GCE. The SWCNT−ILE (Figure 1, curve b) exhibited better redox peaks with a small peak potential separation of 80 mV, suggesting that the dramatic increase in the electron transfer rate was ascribed to the presence of the conductive binder OPFP. The result showed that the combination of single-walled carbon nanotubes and ionic liquid could bring a sensitive electrode into the fabrication of electrochemical sensors. Figure 2 shows the SEM image of the surface of SWCNT− ILE composite electrodes. The composite electrode exhibited uniform surface topography and unique structure, indicating that the solid binder OPFP could fill well into the space among carbon nanotubes.36 Moreover, after being heated over the melting point of OPFP and cooled to room temperature, the electrode exhibited strong stability. This characteristic could be ascribed to the good adherence of OPFP with carbon nanotubes. In addition, the composite electrode had advantages of simple operation and low cost in the electrochemical detection. Due to the viscosity and high conductivity of OPFP, this novel electrode exhibited highly attractive properties, such

Figure 3. Cyclic voltammograms of 3 mmol L−1 PNPP (panel A, curve a), 0.15 mmol L−1 PNP and 3 mmol L−1 PNPP (panel A, curve b), and 0.15 mmol L−1 PNP (panel B) at the SWCNT−ILE between 0 and 1.25 V at 10 mV s−1.

was no redox peak under the detection in the presence of PNPP, whereas a redox peak was investigated at 0.75 V in the presence of PNPP and PNP in Figure 3A (curve b). In addition, as shown in Figure 3B, there was a redox peak investigated at 0.75 V in the presence of PNP, which suggested that only the PNP had redox reaction during the detection. The 6890

DOI: 10.1021/acs.jafc.6b02426 J. Agric. Food Chem. 2016, 64, 6888−6894

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Journal of Agricultural and Food Chemistry

decline to the stable baseline, whereas the current at SWCNT− ILE (Figure 5B) rapidly increased first and then remained stable until the end. This phenomenon showed that the surface of GCE would be passive due to the existence of PNP during electrochemical analysis, which led to the electrode being useless for detection. However, there were plentiful sites on the surface of carbon nanotubes that could have better conductivity. Ionic liquid could generate a layer of protection membrane on the surface of the composite electrode. Therefore, SWCNT−ILE was superior in resisting fouling and more stable during detection caused by the above characteristics. As shown in Figure 6, the enzymatic reaction of alkaline phosphate presented a well-defined tendency. With the increase

results indicated that there was an irreversible oxidation reaction on the SWCNT−ILE. At the same time, the existence of PNPP had no effects on the determination of PNP. As shown in Figure 4, by adding the PNP at intervals of 120 s, the

Figure 4. Amperometric response of 0.15 mmol L−1 PNP using SWCNT−ILE. Applied potential = +0.75 V; scan rate = 10 mV s−1.

current at SWCNT−ILE led to a stable rise during the detection time, suggesting that SWCNT−ILE had good electrochemical signals of PNP. Figure 5 shows the amperometric responses of PNP at GCE and SWCNT−ILE in 0.5 M Tris (pH 8.5). After the addition of PNP at 200 s, the current at GCE (Figure 5A) rushed to

Figure 6. Amperometric responses of SWCNT−ILE in 0.1 mol L−1 Tris solution and 3 mmol L−1 PNPP for additions of AP: (a) 0 U L−1; (b) 12.5 U L−1; (c) 1250 U L−1. Applied potential = +0.75 V; scan rate = 10 mV s−1.

of concentration of alkaline phosphate, current also increased gradually under the reaction time. This illustrated that the intensity of the current signal in the process of enzymatic reaction was related to the concentration of alkaline phosphate under moderate concentration of substrate, which supported the detection in immune reactions. The concentrations of antigens (BSA−TTX) and magnetic particles were optimized, indicating that the best immobilization proportion was 30 μg of BSA−TTX per 1 μL of magnetic particles. In addition, several different dilutions of BSA−TTX stock solution were measured while the active sites on magnetic particles were saturated. In this part, magnetic particles are covalently linked with antigens BSA−TTX. To make the magnetic particles work more effectively, the optimal concentration of antigen BSA−TTX was optimized with a spectrometric method. As shown in Figure 7, the result showed that there was a rising tendency when the concentration of BSA−TTX was 100 ng/ mL. Therefore, the optimized concentration of BSA−TTXs immobilized to the magnetic particles we chose was 100 ng/ mL, where BSA−TTX could compete with the analytes. The optimized incubation time for immune reaction was 30 min. Application of Amperometric Immunoassay in Real Samples. As shown in Figure 8, the electrochemical immunoassay of tetrodotoxin presented a well-defined calibration curve, which was correlated with the concentration of standard samples of TTX in the range of 2−45 ng/mL. Figure 9 shows the detection of tetrodotoxin in standard

Figure 5. Amperometric responses of 0.15 mmol L−1 PNP at GCE (A) and SWCNT−ILE (B). Applied potential = +0.75 V; scan rate = 10 mV s−1. 6891

DOI: 10.1021/acs.jafc.6b02426 J. Agric. Food Chem. 2016, 64, 6888−6894

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Journal of Agricultural and Food Chemistry

Figure 7. Curve of the optimization of BSA−TTX immobilized on magnetic particles using the spectrometric method.

Figure 9. Equation of calibration curve of different concentrations of TTX in the standard samples by electrochemical method (A) and spectrometric method (B). The concentrations of TTX were 0, 2, 5, 15, and 45 ng/mL.

Figure 8. Amperometric responses of different concentrations of TTX in standard samples: (a) 0 ng mL−1; (b) 2 ng mL−1; (c) 5 ng mL−1; (d) 15 ng mL−1; (e) 45 ng mL−1.

Table 1. Determination of Tetrodotoxin (TTX) in Real Samplesa

samples using the amperometric method and the spectrometric method, respectively. As Figure 9A shows, the current was correlated with the concentration of tetrodotoxin in the amperometric method. As Figure 9B shows, the absorbance under 450 nm was correlated with the concentration of tetrodotoxin in the spectrometric method. To evaluate the practicality of the assay for TTX in real samples, detections of TTX by an electrochemical method with amperometric immunosensors and by a spectrometric method under the absorbance of 450 nm were carried out. As shown in Table 1, the results showed the content of tetrodotoxin in real samples. The contents of the tetrodotoxin in real samples were relatively lower because most pufferfishes were farmed on the market. Furthermore, blank samples of tetrodotoxin were added to standard samples for spiking studies to verify the accuracy of the developed method. The recovery ratios are given in Table 2. The amprometric immunoassay was compared with the spectrometric method for the determination of TTX in real samples, indicating its feasibility in use. The results showed that the amperometric immunoassay produced satisfactory results with an average recovery of 98.16%. Additionally, the amperometric immunoassay had advantages of smaller volume, faster responses, and lower cost. The reported method provided accuracy for the quantitative determination of TTX in real samples with a simple operation within 20 min.

TTX concentration (ng mL−1) sample

electrochemical method

1 2 3 4 5

6.51 3.72 6.74 8.36 7.92

± ± ± ± ±

spectrometric method

0.03 0.02 0.02 0.03 0.02

6.42 3.52 6.51 8.12 7.78

± ± ± ± ±

0.02 0.03 0.02 0.02 0.03

a

All samples were analyzed using the standard addition method (n = 3).

Table 2. Recovery of Tetrodotoxin in Blank Samples Using Amprometric Immunosensorsa spike (ng mL−1) 0 2 5 15 45

recoveryb (%) 94.86 95.30 101.65 92.49 106.50

± ± ± ± ±

0.07 0.06 0.09 0.08 0.1

RSD (%) 10.47 9.03 12.37 11.68 13.03

a

All samples were analyzed using the standard addition method (n = 3). bFound value was obtained by multiplying the equivalent volume of blank samples and real sample.

An amperometric immunosensor based on a composite electrode of single-walled carbon nanotubes and ionic liquid 6892

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OPFP with the combination of magnetic particles and immunoassay was developed for the determination of TTX in real samples. Combined with the SWCNT−ILE, the proposed amperometric immunosensor presents a good performance for detection of tetrodotoxin in real pufferfish, exhibiting advantages of simple operation, rapid detection, and low cost. Such a simple, fast, sensitive, and real-time method could be applied widely for the determination of the toxin in marine organisms.

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AUTHOR INFORMATION

Corresponding Author

*(Y.L.) Phone: (86-21) 6190-0380. Fax: (86-21) 6190-0365. Email: [email protected]. Funding

This work was supported by the Natural Science Foundation of China (No. 31571918). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.6b02426 J. Agric. Food Chem. 2016, 64, 6888−6894

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DOI: 10.1021/acs.jafc.6b02426 J. Agric. Food Chem. 2016, 64, 6888−6894