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Sensitive voltammetric responses and mechanistic insights into the determination of residue levels of endosulfan in fresh foodstuffs and raw natural w...
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An Electrochemical Immunosensor Based on Chemical Assembly of Vertically Aligned Carbon Nanotubes on Carbon Substrates for Direct Detection of the Pesticide Endosulfan in Environmental Water Guozhen Liu,*,† Shuo Wang,‡ Jingquan Liu,§ and Dandan Song† †

Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, P. R. China ‡ Tianjin Key Laboratory of Food Nutrition and Safety, Faculty of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China § Laboratory of Fiber Materials and Modern Textile, School of Chemical and Environmental Engineering, Qingdao University, Qingdao 266071, Shandong, P. R. China ABSTRACT: A glassy carbon substrate was covalently modified with a mixed layer of 4-aminophenyl and phenyl via in situ electrografting of their aryldiazonium salts in acidic solutions. Single-walled carbon nanotubes (SWNTs) were covalently and vertically anchored on the electrode surface via the formation of amide bonds from the reaction between the amines located on the modified substrate and the carboxylic groups at the ends of the nanotubes. Ferrocenedimethylamine (FDMA) was subsequently attached to the ends of SWNTs through amide bonding followed by the attachment of an epitope, i.e., endosulfan hapten to which an antibody would bind. Association or dissociation of the antibody with the sensing interface causes a modulation of the ferrocene electrochemistry. Antibody-complexed electrodes were exposed to samples containing spiked endosulfan (unbound target analyte) in environment water and interrogated using the square wave voltammetry (SWV) technique. The modified sensing surfaces were characterized by atomic force microscopy, XPS, and electrochemistry. The fabricated electrochemical immunosensor can be successfully used for the detection of endosulfan over the range of 0.01−20 ppb by a displacement assay. The lowest detection limit of this immunosensor is 0.01 ppb endosulfan in 50 mM phosphate buffer at pH 7.0.

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simplicity, cost-effectiveness, and high sample throughput.8 Moreover, immunoassays are field-portable and can be performed on a simple and inexpensive instrument. Since Langone and Van Vunakis pioneered the work for the detection of cyclodienes by a radioimmunoassay,9 several immunoassays capable of detecting endosulfan have been described in the literature.10−13 However, those methods were of insufficient sensitivity for the analysis of water samples and can give false results due to the coexistence of the nontoxic metabolite, endosulfan diol.14 Electrochemical immunoassays, in particular, are attractive for the detection of endosulfan because of the high specificity resulting from the introduction of the endosulfan monoclonal antibody to the sensing interface. Montoya and co-workers have developed an enzyme-linked immunosorbent assay based on monoclonal antibodies for the detection of organochlorine pesticides.15 The authors claim that endosulfan can be determined in a competitive assay from 2 to 50 nM with a detection limit of 1 nM (2.46 ppb). The

ndosulfan is a broad-spectrum pesticide widely used in agriculture to control insects and mites.1−3 It is also used extensively in public health applications in developing countries.4 Development of an immunobiosensor represents a challenging target, as Chinese Water Quality Guidelines set levels for endosulfan in drinking water of less than 0.01 ppb. Several studies have shown that endosulfan may exist in field water samples somewhat longer than in pure water by binding to sediments and soil particles. For assessment of the possible chronic health and environmental effects of long-term exposure to pesticides, extended monitoring of ground, surface, and drinking water, as well as analytical techniques with sufficiently low levels of detection, are essential. Endosulfan is generally analyzed by instrumental methods, such as gas chromatography with electron capture detection,5 gas chromatography/mass spectrometry,6 or high-performance liquid chromatography.7 Each of these methods needs extraction, cleanup, and concentration of the sample. This is labor-intensive, timeconsuming, and expensive, making it the rate-limiting step in environmental studies. Therefore, there is a need for a rapid, simple, and cost-effective method of analysis for endosulfan. Immunochemical techniques such as the immunoassay have lately gained a position as alternative and/or complementary methods for the analysis of agrochemicals because of their © 2012 American Chemical Society

Received: October 18, 2011 Accepted: March 26, 2012 Published: March 26, 2012 3921

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sensitivity has been greatly improved, but it still cannot meet the requirement for monitoring endosulfan in water samples. Previously, a label-free immunosensor using oligo(phenylethynylene) molecular wires (MWs) for electron transfer and poly(ethylene glycol) (PEG) for resisting unspecific protein adsorption was developed for the detection of small molecule free biotin by a displacement assay.16 In this design, MWs serve dual purposes in being rigid, thereby allowing access to the biotin by the binding antibodies without hindrance from the surface, and being an efficient conduit for electron transfer,17 which is necessary as the ferrocene is located approximately 20 Å above the electrode surface. These attractive advantages of MWs, in that they are rigid and give well-defined molecular architectures and efficient electron transfer, were also observed by other researchers.18−21 The MWs, however, suffer the disadvantages of being either unstable in air, difficult to synthesize in large quantities, or both. Thus, it is crucial to find an alternative to these molecular wires, especially for the application of immunosensors for real analytes. Vertical alignment of single-walled carbon nanotubes (SWNTs) on surfaces provides new materials with interesting properties for future applications in electronic, optoelectronic, and sensing devices.22−24 Robust forests of SWNTs have been successfully achieved via chemical assembly on carbon surfaces that were first modified with amine-terminated aryldiazonium salt layers.25 Highly ordered covalent anchoring of carbon nanotubes through carbon−carbon bonds to electrode surfaces modified with aryldiazonium salts has also been reported.26 Both structures show excellent stability over a wide potential range and are resistant to degradation from sonication in acids, bases, and organic solvents. An immunosensor based on SWNT-modified glassy carbon (GC) electrodes has been developed for the detection of antibiotin IgG.27 It is demonstrated that SWNTs are a good alternative to MWs. First, carbon nanotubes are small (as small as 1 nm in diameter), rigid, and simple to produce in large quantities. Their small size and conductivity means that they can be regarded as the smallest possible electrodes, with diameters of less than 1 nm.28 Second, SWNTs are known to have a number of carboxylic acid groups at each end of the tubes, which are important for further fabrication, after being cut in a 3:1 v/v mixture of concentrated H2SO4/HNO3.29 Third, the assembly of SWNTs increases the electronic communication between the electrode and the environmental solutions.26 However, to the best of our knowledge, no report of using SWNTs as the MW on a sensing interface for the detection of small molecules by a displacement assay has been published to date. In this work, we aim to develop a label-free immunosensor based on an SWNT−aryldiazonium salt-modified sensing interface for the detection of endosulfan (Scheme 1). In this design, four key components are introduced to the sensing interface: (a) mixed layers of aryldiazonium salts, (b) SWNTs, (c) PEG molecules, and (d) endosulfan monoclonal antibodies. Due to the aryldiazonium salt chemistry, the SWNT assemblies are expected to have stability advantages over those based on SAMs of alkanethiols on gold and those where the anchoring layer is simply chemisorbed to the surface.30 In addition, the mixed layers of aryldiazonium salts aid the assembly of SWNTs on the surface.26 Binding of the antibody to the surface-bound epitope immerses the redox probe ferrocene in a protein medium. A consequence of this change in the environment is the attenuation of electron transfer to the ferrocene due to the inaccessibility of a counterion. The PEG antifouling layer

Scheme 1. SWNT-Modified Sensing Interface for the Detection of Endosulfana

a

The structures for one isomer of endosulfan and its hapten are shown.

ensured that the antibody only interacted with the interface when a specific biorecognition event occurred. Furthermore, the rigidity of SWNTs was essential to allow access of the antibody to the surface epitope without hindrance from the surface.

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EXPERIMENTAL SECTION Materials and Reagents. Sodium nitrite, potassium ferricyanide, hydrochloric acid, 4-phenylenediamine, aniline, N,N′-dicyclohexylcarbodiimide (DCC), horseradish peroxidase (HRP), keyhole limpet hemocyanin (KLH), Freund’s adjuvants, bovine serum albumin (fraction V, BSA), and myoglobin were purchased from Sigma-Aldrich. Endosulfan and other endosulfan derivatives were purchased from Fluka. Hybridoma Fusion and Cloning Supplement (HFCS) was from Roche Applied Science (Indianapolis, IN). Peroxidase-labeled rabbit antimouse immunoglobulins and goat antimouse immunoglobulins were obtained from eBioscience (San Diego, CA). Culture media (high-glucose Dulbecco’s modified Eagle’s medium with Glutamax I and sodium pyruvate), fetal calf serum (Myoclone Super Plus), and supplements were from Gibco (HaoranBio, Shanghai). Culture plastic ware was from Bibby Sterilin Ltd. (Stone, UK). Flat-bottom polystyrene

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clones were expanded and cryopreserved in liquid nitrogen. The achieved antibodies were classified as antiendosulfan IgG which were purified on a small scale directly from late stationary phase culture supernatants by saline precipitation with saturated ammonium sulfate followed by affinity chromatography.36 Preparation of the Immunosensor Interface for Detection of Endosulfan. Prior to modification, GC electrodes were polished successively with 1.0, 0.3, and 0.05 μm alumina slurries (alumina: Buehler, Lake Bluff, IL) on microcloth pads (Buehler). The electrodes were thoroughly rinsed and sonicated in Milli-Q water for 1 min between polishing steps. Before modification, the electrodes were dried with a stream of argon. The derivatization of the clean GC electrode with mixed layers of 4-aminophenyl and phenyl was conducted with in situ-generated aryldiazonium cations that involved the electrochemical reduction of the corresponding anilines in acidic media to achieve 4-aminophenyl/phenylmodified surfaces (GC-Ph-NH2).37,38 Specifically, 0.5 mM pphenylenediamine and 0.5 mM aniline were solubilized in 0.5 M aqueous HCl, and 1 mM sodium nitrite was added to generate the aryldiazonium salts in the electrochemical cell (in situ). The mixture was degassed and left to react for about 10 min at 0 °C. The electrochemical reductive modification of the GC surface with an in situ-generated mixture of 4-aminophenyl- and phenyldiazonium salts was carried out by applying a potential to the electrode between 1.0 V and −1.0 V for two cycles at a scan rate of 100 mV s−1. After surface derivatization, the electrodes were rinsed with copious amounts of Milli-Q water and dried under a stream of argon prior to the next step. Modification of SWNTs with carboxylic acid-terminated groups was carried out by immersing GC-Ph-NH2-modified GC electrodes in the ethanol solution of cut SWNTs (0.2 mg mL−1) in the presence of 0.5 mg mL−1 DCC. For resisting the nonspecific protein adsorption, the SWNT-modified GC surfaces (GC-Ph-NH2/SWNT) were modified with PEG molecules through aryldiazonium treatment of SWNTs reported by Tour et al.32 For coupling FDMA to the open ends of the SWNTs assembled on GC substrates, SWNT- and PEG-modified GC surfaces (GC-Ph-NH2/SWNT/PEG) were incubated in an absolute ethanol solution containing 40 mM DCC and 5 mM FDMA for 6 h at room temperature to achieve the surface of GC-Ph-NH2/SWNT/PEG/FDMA. Endosulfan hapten was coupled to the ferrocene by an amide coupling reaction with 1 mg mL−1 hapten in 0.1 M PBS for 2 h at 4 °C. The endosulfan hapten-modified electrodes were rinsed with copious amounts of water and PBS before immersion into a PBS solution of endosulfan monoclonal IgG for 30 min at 4 °C. The electrodes were transferred to phosphate buffer solution for measurement using cyclic voltammetry (CV) and square wave voltammetry (SWV).

ELISA high binding plates were from Costar (Cambridge, MA). SWNTs prepared by the HiPco process were purchased from Carbon Nanotechnologies Incorporated. Cut SWNTs were prepared as reported previously.31 The aryldiazonium cations for 2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethanol (PEG) was custom synthesized by following the modified procedures of Bahr et al.17,32 Ferrocenedimethylamine (FDMA) was synthesized by the literature method.33 The synthesis of endosulfan haptens was accomplished by following the procedures of Lee et al.10 N-Hydroxysuccinimide activated esters of haptens were conjugated to HRP and KLH using the method described earlier.34 All other reagents were used as received. Aqueous solutions were prepared using Mill-Q water (>18 MΩ cm). Phosphate-buffered saline (PBS) solutions were 0.15 M NaCl and 0.1 M phosphate buffer, pH 7.3. Phosphate buffer solution for electrochemistry was prepared using 0.1 M buffer with added 0.05 M KCl (pH 7.0). Apparatus. All electrochemical experiments were conducted using a GaossUnion EC510 potentiostat (GaossUnion, China). GC electrodes were 3 mm disks embedded in epoxy resin (GaossUnion). All experiments utilized a Pt secondary electrode and a Ag/AgCl (3.0 M NaCl) reference electrode. All voltammetric measurements were obtained with a scan rate of 100 mV s−1. X-ray photoelectron spectra (XPS) were collected from GC plates on a VG multilab 2000 spectrometer with a monochromated Al Kα source (1486.6 eV), hemispherical analyzer, and multichannel detector. The spectra were calibrated on the C1s peak (285.0 eV). Spectra were analyzed using XPSPEAK41 software. The quoted percentage coverage for the different elements and subgroups were estimated from the corresponding fitted areas and associated sensitivity factors. Atomic force microscopy (AFM) images were taken on GC plates using a Digital Instruments Dimension 3100 scanning probe microscope. All images were acquired in tapping mode using commercial Si cantilevers/tips (Olympus) at their fundamental resonance frequencies, which typically varied between 275 and 320 kHz. Preparation of Endosulfan Monoclonal Antibodies IgG. BALB/c female mice were used for the production of endosulfan monoclonal antibodies according to the method described by Manclus et al.15 Antibodies were raised by intraperitoneal injections of endosulfan hapten conjugated to KLH. The immunogens (100 μg of conjugate) were diluted in PBS and emulsified in Freund’s complete (first immunization) or incomplete adjuvant (subsequent immunizations). After three initial fortnightly intervals, booster injections were given monthly. Blood was collected from mouse tail one week after each monthly booster injection, and the antiserum was tested for antihapten antibody titer by indirect ELISA and for analyte recognition properties by competitive indirect ELISA. After 4 weeks from the last injection in adjuvant, mice selected to be spleen donors for hybridoma production received a final soluble intraperitoneal injection of 100 μg of conjugate in PBS four days prior to cell fusion. HB-10744 SH-3 marine myeloma cells (ATCC, Xiangf bio, Shanghai) were cultured in high-glucose culture media supplemented with 2 mM L-glutamine, 1 mM nonessential amino acids, 25 μg mL−1 gentamicin, and 15% fetal bovine serum. Cell fusion procedures were carried out as described by Nowinski et al.35 Ten days after cell fusion, culture supernatants were screened for the presence of antibodies that recognized the analyte. Selected hybridomas were cloned by limiting dilution using HT (hypoxanthine−thymidine) medium supplemented with 2% HFCS (v/v). Stable antibody-producing

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RESULTS AND DISCUSSION Characterization of the SWNT-Modified GC Surfaces by AFM. SWNTs were assembled on the mixed layers of 4aminophenyl/phenyl-modified GC surface by reacting the substrate with SWNTs for 24 h in the ethanol solution of cut SWNTs (0.2 mg mL−1) in the presence of 0.5 mg mL−1 DCC. Bond formation involving the multiple carboxylic acid groups at the end of each SWNT is expected to result in vertical alignment of SWNTs, as demonstrated in studies using other amine-functionalized substrates.29,39,40 The so-fabricated surfaces are referred to as GC-Ph-NH2/SWNTs and were imaged by 3923

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Figure 1. AFM images of (a) the GC-Ph-NH2-modified GC plate and (b) self-assembled SWNTs on the GC-Ph-NH2-modified GC plates after 24 h incubation in SWNT solution.

researchers.25,40,41,44,45 The authors concluded that the SWNTs act as Coulombic islands (electron transfer stations), ‘tunneling’ electrons between the electrode surface and redox species in solution. The present results provide another example of the accelerating effect of SWNTs assembled on tethered layers. It is well-known that aryldiazonium salt layers on GC electrodes are very stable toward sonication in water.30 After sonication of the GC-Ph-NH2/SWNT surface in water for 10 min and scanning in 1 mM Fe(CN)63−/4− solution for 50 cycles, the electrochemistry of GC-Ph-NH2/SWNT surfaces in Fe(CN)63−/4− solution did not show significant change. This observation indicates that the GC-Ph-NH2/SWNT surface is very stable, which is essential for the sensor construction in the following studies. XPS Characterization of Stepwise-Modified GC Surfaces. As shown in Scheme 1, the fabrication of the sensing interface of the electrochemical immunosensor involves multisteps. First, XPS was used for characterization of the GC-Ph-NH2 surface. The N1s spectrum of the bare GC surface does not show any obvious peaks (Figure 3 a), confirming that the substrate does not contain detectable nitrogen at the surface. For the GC-Ph-NH2-modified surface (mixed layers of 4-aminophenyl/phenyl), a strong N1s peak at 400.2 eV was observed (Figure 3 b), which is attributed to the amino groups (NH2).38 However the presence of azo groups (NN) is also possible.46,47 To clarify the possibility of introducing azo groups, N1s spectra of the phenyl-modified GC (GC-Ph) was investigated. No significant N1s signal was detected on the GCPh surface (Figure 3 b), indicating that no significant nitrogen species such as azo groups were introduced during the modification step. In addition, incorporation of phenyl groups to form mixed layers of GC-Ph-NH2 is helpful to prevent the diazo coupling.26 The appearance of a significantly stronger N1s peak for the GC-Ph-NH2 surface further confirms that the NH2 groups are predominant on GC-Ph-NH2 surfaces. The C1s scan of GC-Ph-NH2/SWNT/PEG (Figure 3 d) is dominated by the graphitic carbon peak at 284.4 eV together with a peak at the high binding energy 285.2 eV due to the presence of low levels of oxidized carbon species on the electrode surface. Both the ether carbon species at 286.3 eV and peptide carbon species at 287.3 eV are observed, indicating the successful attachment of PEG molecules. A peak at 288.8 eV, which is typical of the carbon of the carboxylic acid group, in the C1s spectra is observed as well, indicating the successful immobilization of SWNTs on the GC surfaces. After the further

AFM as shown in Figure 1. Compared with the AFM image of the GC-Ph-NH2-modified GC substrates, the surface of the GC substrates after modification with SWNTs was much rougher as illustrated, showing the shortened SWNTs aligned normal to the electrode surface. AFM images clearly show that SWNTs are perpendicularly oriented with only one end anchored on the GC surface. The observed lengths of the SWNTs from the AFM image are about 100−150 nm with a diameter of 4−10 nm. The observed lengths of the SWNTs from the AFM image are similar to those observed using TEM analysis.26 Electrochemistry of the GC-Ph-NH2/SWNT Surface in Redox Probe Solutions. The electrochemistry of the GC electrodes from step-by-step modifications was studied in solutions containing the redox species Fe(CN)63−/4−. As shown in Figure 2, GC-Ph-NH2 surfaces prevent access of ferricyanide

Figure 2. Cyclic voltammograms of bare GC electrode (blue), GC-PhNH2 (black), GC-Ph-NH2/SWNT (red) in Fe(CN)63−/4− (1 mM; 0.05 M KCl; 0.05 M phosphate buffer; pH 7.0) at a scan rate of 100 mV s−1.

to the electrode. As frequently reported for organic films grafted by aryldiazonium cation grafting, the film acts as an insulating barrier, slowing the rate of electron transfer between the underlying electrode and solution species.37,41−43 After the assembly of aligned SWNTs to GC-Ph-NH2 surfaces, welldefined redox peaks from Fe(CN)63−/4− similar to that from bare GC electrodes were observed (Figure 2), indicating that the SWNT assembly can restore the electron transfer rate to that observed prior to film grafting. An increase in the rate of electron transfer to solution redox probes after assembly of SWNTs on insulating tether layers has been reported by many 3924

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Figure 3. N1s core level spectra for (a) bare GC, (b) GC-Ph-NH2, and (c) GC-Ph surfaces; C1s core level spectra for the (d) GC-Ph-NH2/SWNT/ PEG surface and (e) the GC-Ph-NH2/SWNT/PEG/FDMA/endosulfan hapten surface; (f) XP survey spectra for stepwise modification of the sensing interface.

attachment of FDMA, the peptide peak at 287.3 eV increased, but the peak at 288.8 eV shifted to 288.5 eV and decreased significantly in magnitude, suggesting that not all carboxylic acid groups had been converted to amide bonds after the attachment of FDMA (Figure 3 e). The atomic percentage of C1s at 288.8 eV and at 288.5 eV is 2.4% and 1.3%, respectively. Thus, conversion efficiency can be calculated to be about 46% ((C288.8% − C288.5%)/C288.8%). The appearance of Fe2p species with an atomic percentage of 0.99% was observed in the XP survey spectra of the GC-Ph-NH2/SWNT/PEG/FDMA surface (Figure 3 f), further suggesting the successful attachment of FDMA. Except for the difference in magnitude, XP survey spectra of bare GC, GC-Ph-NH2, and GC-Ph-NH2/SWNT surfaces are similar to that of the GC-Ph-NH2/SWNT/PEG surface (Figure 3 f). After incubation of the GC-Ph-NH2/ SWNT/PEG/FDMA surface in endosulfan hapten, a new S2p peak was observed in the XP survey spectra for the GC-PhNH2/SWNT/PEG/FDMA/endosulfan hapten surface, indicating the attachment of endosulfan hapten. Antifouling Properties of the Sensing Interface. The efficacy of the PEG molecules to suppress nonspecific adsorption of proteins was studied by measuring the electrochemistry of the electrodes incubated in solutions of myoglobin. Figure 4 shows the cyclic voltammetries of GC-

Figure 4. CVs of the GC-Ph-NH2/SWNT surface before (long dashed line) and after (dashed line) incubation in myoglobin−PBS solution for 1 h at a scan rate of 100 mV s−1, and CVs of the GC-Ph-NH2/ SWNT/PEG surface before (dotted line) and after (full line) incubation in myoglobin−PBS solution for 1 h at a scan rate of 100 mV s−1.

Ph-NH2/SWNT- and GC-Ph-NH2/SWNT/PEG-modified surfaces before and after incubation in myoglobin (1 mg mL−1 in PBS) for 1 h. Before incubation of GC-Ph-NH2/SWNT in myoglobin−PBS solution, no electrochemistry was observed in the phosphate buffer solution. After incubation in myoglobin− 3925

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PBS solution, a pair of obvious redox peaks at −0.15 V appeared, which is the typical formal potential of myoglobin.48 The appearance of redox peaks indicates that a considerable amount of myoglobin had adsorbed onto the GC-Ph-NH2/ SWNT surfaces. Importantly, after incubation of GC-Ph-NH2/ SWNT/PEG surfaces in myoglobin solution under the same conditions, the CV in Figure 4 does not show any electrochemistry of myoglobin. Controls with GC-Ph-NH2/ SWNT and GC-Ph-NH2/SWNT/PEG in background solvent PBS for 1 h showed no redox peaks. Therefore, in conclusion, GC-Ph-NH2/SWNT/PEG surfaces provide sufficient resistance to nonspecific adsorption of protein, and the observed electrochemistry should be assigned to protein attached to the end of the SWNTs as depicted in Scheme 1. Characterization of SWNT-Modified Immunosensors for the Detection of Endosulfan. The construction of the label-free immunosensor is shown in Scheme 1. The antiendosulfan IgG-modified GC electrodes were used as the sensing interface for the detection of endosulfan. The electrochemical response of the attached ferrocene group is modulated by the binding event between antiendosulfan IgG and endosulfan to reach the detection purpose. Figure 5 shows the electrochemistry of GC-Ph-NH2/ SWNT/PEG-modified GC surfaces after the stepwise binding of FDMA, epitope (endosulfan hapten), antiendosulfan IgG, and endosulfan. There is no significant change in the ferrocene peak current after attachment of epitope; however, there is a pronounced decrease in peak current after incubation of GC-

Ph-NH2/SWNT/PEG/FDMA/endosulfan hapten-modified surface with 0.5 μg mL−1 antiendosulfan IgG. The FDMA peak current before and after incubation of 0.5 μg mL−1 antiendosulfan IgG is 0.29 ± 0.05 μA (95% confidence, n = 5) and 0.11 ± 0.02 μA (95% confidence, n = 5), respectively. Control experiments showed no significant change in peak current when the GC-Ph-NH2/SWNT/PEG/FDMA-modified GC electrodes were incubated in antiendosulfan IgG solution without prior attachment of endosulfan hapten. In addition, the OSWV peak currents were almost the same before and after treatment of the GC-Ph-NH2/SWNT/PEG/FDMA/endosulfan hapten-modified surfaces with other proteins which are not specific to endosulfan. Before incubation with 1 μg mL−1 BSA, the FDMA current (Ibefore) was 0.22 ± 0.03 μA (95% confidence, n = 3), and after incubation, the FDMA current (Iafter) was 0.20 ± 0.04 μA (95% confidence, n = 3). For 1 μg mL−1 antipig IgG, the corresponding values were Ibefore= 0.26 ± 0.04 μA (95% confidence, n = 3) and Iafter = 0.29 ± 0.02 μA (95% confidence, n = 3). These results indicate that the antiendosulfan IgG specifically associates with the endosulfan hapten and only a biospecific interaction leads to a change of electrochemistry. The FDMA redox peaks increased after incubation of GCPh-NH2/SWNT/PEG/FDMA/endosulfan hapten/antiendosulfan IgG-modified GC surfaces in endosulfan solution for 10 min (Figure 5 a). In addition, the higher the concentration of endosulfan, the higher the current increases (Figure 5 b). These results are consistent with those reported in our previous study.16 Decreased current density upon antiendosulfan IgG binding reflects changes in the interfacial microenvironment which arises from formation of an immunocomplex on the electrode surface. Formation of the complex blocks counterions from accessing the ferrocene probe with a corresponding decrease in current. Because of the affinity between the antiendosulfan IgG and endosulfan, the antiendosulfan IgG can dissociate from endosulfan hapten on the electrode surface and then bind with the endosulfan in the solution, resulting in the increase of FDMA current. However, the current (Iendosulfan) after incubation of the GC-Ph-NH2/SWNT/PEG/FDMA/ endosulfan hapten/antiendosulfan IgG surface with endosulfan solution for 10 min is lower than the current (Iendosulfan hapten) before incubation of the GC-Ph-NH2/SWNT/PEG/FDMA/ endosulfan hapten surface with antiendosulfan IgG solution, which indicates that not all antiendosulfan IgG can be dissociated from the sensing interface. Thus, the relative current (Iendosulfan/Iendosulfan hapten) is lower than 1. The specificity of antiendosulfan IgG against other chlorinated cyclodiene pesticides was also investigated by incubation of the antiendosulfan IgG-modified sensing interface with a solution of different endosulfan derivatives for 10 min, respectively. Five popular endosulfan derivatives such as heptachlor, endrin, dieldrin, chlordane, and aldrin were tested, and the current changes of FDMA before and after incubation of these possible interfering compounds were monitored as shown in Table 1. The current increased dramatically after incubation of the antiendosulfan IgG-modified sensing interface with 20 ppb endosulfan. However, there was no obvious current change after incubation with the other chlorinated cyclodiene pesticides. This further indicates that the antiendosulfan IgG shows the highest affinity for endosulfan and almost no affinity for the other cyclodiene insecticides in the concentration range of interest.

Figure 5. (a) CVs of GC-Ph-NH2/SWNT/PEG-modified GC surfaces after the stepwise binding of FDMA, epitope (endosulfan hapten), antiendosulfan IgG, and endosulfan in 0.05 M phosphate buffer (0.05 M KCl, pH 7.0) at a scan rate of 100 mV s−1. (b) SWV curves for GCPh-NH2/SWNT/PEG/FDMA/endosulfan hapten/antiendosulfan IgG-modified GC surfaces after incubation in endosulfan solutions with concentrations of 0, 0.02, 0.05, 0.1, 0.2, 0.4, 0.8, 1, 2, and 4 ppb, respectively. 3926

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immunosensor showed good reproducibility, with a relative standard deviation of 6.8% for the response of five independently prepared immunosensors to 1 ppb endosulfan. As determined from the midpoint of the calibration curve, the affinity constant between endosulfan and antiendosulfan IgG is calculated to be ca. 109 M−1, which is within the range of typical affinity constants for antigen−antibody reactions (108 to 1012 M−1). The application of negative voltage can hasten the dissociation of reversible antibody−antigen binding.49,50 To investigate the repeatability of the fabricated immunobiosensor, the biorecognition interface was regenerated by applying a potential of −0.9 V for 5 min to remove as much surfacenondissociated antibody as possible. The displacement assay can provide a similar electrochemical signal after six repeated measurements with a relative standard deviation of 7.2%. An obvious advantage of the displacement assay is that it requires no washing or rinsing steps or addition of reagents to perform; hence, it is user intervention-free, requiring the end user to simply put the analytical device in a sample solution and get the reading. The fabricated immunosensor was used for the detection of endosulfan (10 ppb) spiked in different environmental waters by a displacement assay (Figure 6 b). Three types of natural waters were used. Field water was collected from a farm growing many kinds of crops, the East lake water was collected from the East lake located at Wuhan city, and tap water is the drinking water. Both the field water and East lake water contain suspended particles, microorganisms, leaf debris, algae, and contaminants from wildlife resulting in humic substances. The recoveries obtained for endosulfan detection with three endosulfan-spiked environmental water samples were 79%, 85%, and 93%, respectively. The highest recovery was obtained for the tap water. The lower recovery for other two samples might be due to the matrix sample effect as observed previously.50

Table 1. Specificity of the Antiendosulfan IgG-Modified Sensing Interface for Chlorinated Cyclodiene Pesticides currenta (μA) pesticides

Ibefore

Iafter

endosulfan heprachlor chlordane aldrin endrin dieldrin

0.02 0.02 0.02 0.03 0.02 0.03

0.18 0.02 0.02 0.02 0.04 0.04

a

Values correspond to the average of three separate measurements. Ibefore and Iafter mean the FDMA current before and after incubation of GC-Ph-NH2/SWNT/PEG/FDMA/endosulfan hapten/antiendosulfan IgG surfaces with the corresponding pesticide at a concentration of 20 ppb.

Using the displacement assay, GC-Ph-NH2/SWNT/PEG/ FDMA/endosulfan hapten/antiendosulfan IgG-modified GC surfaces can be used as the sensing interface for the specific detection of endosulfan. The calibration curve (Figure 6 a) for

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CONCLUSIONS Vertically aligned SWNTs can be assembled on amine-tethered mixed layers that are covalently attached to GC substrates by aryldiazonium salt chemistry. The formed SWNT-modified GC electrodes have desirable properties for biosensor construction, as evidenced by the preparation of an immunosensor based on the modulation of voltammetric signals of surface-bound redox species FDMA. The GC-Ph-NH2/SWNT/PEG/FDMA/endosulfan hapten/antiendosulfan IgG-modified sensing interface shows good antifouling properties and high specificity to endosulfan and is functional for the detection of endosulfan by a displacement assay. It was demonstrated that there is a linear relationship between the FDMA current and the concentration of endosulfan over the range of 0.01−20 ppb with a lowest detected limit of 0.01 ppb. There is potential to use a similar sensing interface for the detection of a spectrum of pesticides. The concept of using SWNTs as rigid conduits of electron transfer can be readily extended to the construction of other novel immunosensor systems. Furthermore, in the present work, the aryldiazonium salt layers were electrografted on carbon substrates only; actually, grafting of aryldiazonium salts is applicable to a wide range of other substrates. Grafting can be achieved on the substrate electrochemically or spontaneously at room temperature by use of chemical reducing agents. Hence, the approach we established here is a research prototype that is both versatile and attractive for practical applications in sensing and bioelectronics, especially for the on-site detection of small molecules in environmental monitoring.

Figure 6. (a) A calibration curve showing the variation in relative current (I endosulfan/Iendosulfan hapten ) with the logarithm of the concentration of endosulfan in 0.05 M phosphate buffer (0.05 M KCl, pH 7.0). (b) Recovery results for the detection of endosulfan (10 ppb) spiked in different water samples using the SWNT-fabricated immunobiosensor. The results are an average of three separate experiments.

the detection of endosulfan demonstrates a classical sigmoidal shape with relative current versus the logarithm of the concentration of endosulfan in 0.05 M phosphate buffer (0.05 M KCl, pH 7.0). For the displacement assay, the linear concentration range was from 0.01 to 20 ppb. The lowest detected concentration of endosulfan was 0.01 ppb, which is lower than that of an immunoassay (0.1 ppb).10 This 3927

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Analytical Chemistry

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Article

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-27-6786 7535. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (grant 209237057). REFERENCES

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