Environ. Sci. Technol. 2008, 42, 1207–1212
Rapid Detection of Picloram in Agricultural Field Samples Using a Disposable Immunomembrane-Based Electrochemical Sensor LIN TANG, GUANG-MING ZENG,* GUO-LI SHEN,† YUAN-PING LI, YI ZHANG, AND DAN-LIAN HUANG College of Environmental Science and Engineering, Hunan University, Changsha 410082, China
Received October 3, 2007. Revised manuscript received November 21, 2007. Accepted December 4, 2007.
Picloram, a widely used chlorinated herbicide, is quite persistent and mobile in soil and water with adverse health and environmental effects. It is essential to establish a rapid and sensitive method for accurate detection of trace picloram in agricultural samples. We employed a disposable, nontoxic, and conductive chitosan/gold nanoparticles composite membrane on electrochemical sensor for the sensitive detection of picloram in several agricultural field samples. A self-synthesized picloram antibody was encapsulated in the immunomembrane to form an immunoconjugate by a competitive immunoreaction in sample solution, followed by the immobilization of horseradish peroxidase (HRP)-labeled secondary antibody. The immunomembrane possessed good reproducibility for fabrication in batch, providing a congenial microenvironment for the immune molecules. The diffused colloidal Au nanoparticles shuttled the electron transfer between the immobilized HRP and the electrode surface. To demonstrate the suitability of the immunosensor for on-site detection, rice, lettuce, and paddy field water were spiked with picloram and assayed without preconcentration. Under optimal conditions, picloram could be detected in the range from 0.005 to 10 µg/mL with the correlation coefficient of 0.9937, and the detection limit is 5 ng/ mL. The proposed immunosensor exhibited good precision, sensitivity, selectivity, and storage stability.
Introduction Chlorinated pesticides are widely distributed anthropogenic environmental pollutants recognized as known or suspected endocrine disruptors because of their persistence and broadspectrum toxicity (1). They are used extensively in agricultural production and rangeland improvements, transferred into soil substrata, surface and ground waters, accumulated in food chains, and ultimately passed on to human beings (2). Although the application of these chemicals has been banned or restricted in many countries, some of them are still frequently used for pest or weed control. Picloram (4-amino3,5,6-trichloro-2-pyridinecarboxilic acid) is a herbicide widely used for the control of broadleaf weeds in rangeland, wheat, barley, and oats, and for woody plant species in other cultures * Corresponding author phone: (86)-731-8822754; fax: (86)-7318823701; e-mail:
[email protected]. † State Key Laboratory for Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China. 10.1021/es7024593 CCC: $40.75
Published on Web 01/18/2008
2008 American Chemical Society
(3). It is a chlorinated pyridine carboxylic acid, mimicking plant auxins or the hormone indoleacetic acid, inhibiting protein synthesis (4). Picloram is quite persistent and mobile in soil and water. As a result of agricultural application and improper waste disposal, it may remain active for more than 300 days depending on the soil and environmental conditions, easily leach into surface and ground waters, move from treated plants to damage nearby nontarget crops, and pose toxic hazard to aquatic species at µg/mL concentrations (5). Human exposure to higher levels of the pesticide can cause damage to the central nervous system, loss of weight, diarrhea, and weakness (6). It is also suspected to be an endocrine disruptor with adverse developmental and reproductive effects. The U.S. Environmental Protection Agency (EPA) has restricted the use of picloram and set the drinking water standard for picloram at 0.5 µg/mL to protect against the risk of these adverse health effects (7). Therefore, a simple and reliable detection method is essential to accurately monitor low concentrations of picloram in agricultural samples. Traditional techniques for identification and quantification of picloram, including gas/liquid chromatography with electron capture detector and capillary electrophoresis/mass spectrometry, are extremely sensitive in pure picloram detection but frequently involve large-scale equipment, large sample volumes, and extensive extraction or derivatization steps (4, 8). Radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) using synthesized picloram antibody were also proved to be sensitive and specific methods for picloram detection (9–11); however, the radiation hazards, large instruments, or complicated washing procedure limit their application in on-site pesticide control. Current concerns about monitoring persistent pollutants have been focused on electrochemical immunosensor techniques that combine the high specificity of immunoreaction with the sensitivity and convenience of electroanalysis and provide fast, on-site, or fully automated detection of environmental samples without pretreatment (12–15). An electrochemical detector can also be arranged as a microcell that allows portability of the immunosensor (16). To date, no immunosensor study has been reported for picloram. In this study, we developed an electrochemical immunosensor based on a disposable chitosan/gold nanoparticles (nano-Au) composite membrane for the detection of low levels of picloram in rice, lettuce, and paddy field water samples. Chitosan is a kind of natural biopolymer with attractive properties of excellent membrane-forming ability, biocompatibility, biodegradability, nontoxicity, and high mechanical strength. The colloidal Au nanoparticles can offer a large specific surface, retain the bioactivity, and promote the electron transfer of immobilized biomolecules (17, 18). They have been employed for the modification of immunosensing electrode surfaces to immobilize biomolecules (19). However, the regeneration times of the electrode for repeated use are quite limited which restrict the mass application in practical samples. Therefore, to improve the convenience and precision of picloram detection, we considered it worth investigating development of a new sensing platform for the construction of immunosensor in this study, establishing a chitosan/nano-Au composite matrix with encapsulation of immunoconjugate as a disposable membrane for the immunosensor assay. The competitive immunoassay and electrochemical measurement were optimized to obtain high sensitivity. The paddy field water and the extracts of rice or lettuce prepared by a simple extraction method were assayed without preconcentration. This composite membrane could VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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also be used efficiently for the entrapment of different biomarkers for the on-site monitoring of trace toxic organic pollutants in the environment.
Experimental Methods Reagents. The analytical standards of picloram, triclopyr, and lontrel were purchased from J&K Chemical Ltd. (Beijing, China). Chitosan with a deacetylation degree of 85%, N-ethylN′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were from SigmaAldrich. Bovine serum albumin (BSA) was from Shanghai Biochemical Reagents (Shanghai, China). Horseradish peroxidase-labeled goat antirabbit immunoglobulin G (HRP-G anti-RIgG, ELISA titer of 1:1000), Freund’s complete adjuvant, and Freund’s incomplete adjuvant were from Dingguo Biotechnology Co., Ltd. (Beijing, China). Colloidal gold (15 nm diameter) was from Sino-America Biotechnology Co. (Beijing, China). Dialysis membrane (15 000 MW cutoff) was from Pierce Biotechnology, Inc. (Rockford, IL). All other chemicals were of analytical grade, and all solutions were prepared in deionized water of 18 MΩ purified from a Milli-Q purification system. Normal saline solution contained (%) 0.9 NaCl, 0.025 CaCl2, and 0.042 KCl. The phosphate buffer solutions (PBS) with 0.07 mol/L (M) KH2PO4 and 0.07 M Na2HPO4 were used in this work. Apparatus. Electrochemical measurements were carried out on CHI660B electrochemistry system (Chenhua Instrument, Shanghai, China). The three-electrode system used in this work consisted of a glassy carbon electrode (GCE, 4 mm diameter) as the working electrode of interest, a saturated calomel electrode (SCE) as the reference electrode, and a Pt foil auxiliary electrode. Scanning electron micrographs (SEM) of the membranes were obtained with a JSM-6700F field emission scanning electron microscope (JEOL Ltd., Japan). A Tecan Sunrise microplate reader (Switzerland) and 96well Costar plates (U.S.) were used to read the absorbance in the ELISA. A 4K15 Sigma centrifuge (Sartorius AG, Germany) and a vacuum freezing dryer were used in the assay. A model CS501-SP thermostat (Huida Instrument, Chongqing, China) was used to control the temperature. All the work was done at room temperature (25 °C) unless otherwise mentioned. Picloram-BSA Conjugate Preparation. Equimolar amounts of picloram (46 mg), NHS (22 mg), and EDC (37 mg) were dissolved in the solution containing 2.5 mL of N,Ndimethylformamide (DMF) and 2.5 mL of 50 mM PBS (pH 5.8) sequentially. The solution was allowed to stand at room temperature for 3 h, and then filtered to remove the precipitate. The filtrate was mixed with a solution of BSA (500 mg) dissolved in 3 mL of 0.1 M borate buffer (pH 9.0), and the mixture was agitated gently for 1 h in the dark. The resulting solution was dialyzed against 3 changes of deionized water over 36 h at 4 °C and lyophilized. Immunization and Antiserum Collection. Conjugated immunogen (8.0 mg) was dissolved in 3 mL of sterile saline solution and emulsified with an equal volume of Freund’s complete adjuvant. New Zealand white rabbits were injected subcutaneously with 2 mL of emulsion each. The injections were repeated 2, 4, and 6 weeks after the initial injection, substituting Freund’s incomplete adjuvant for complete adjuvant. After the second booster injection, blood samples were collected from the rabbit auricular veins for antibody titer determinations by immunodiffusion 1 week after each boost. About 60 mL of rabbit blood was collected from a single bleed through carotid artery after the titer reached 1:32, and allowed to stand at room temperature for 1 h, then centrifugated at 5000 rpm at 4 °C for 10 min to separate out the antiserum, and stored at -20 °C in the presence of 0.1% sodium azide until use. 1208
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To evaluate the reactivity of the antiserum to picloram, each antiserum was titrated against the antigen by immunodiffusion, and indirect ELISA format was carried out with diluted HRP-G anti-RIgG using o-phenylenediamine (OPD) and H2O2 as substrates. See the Supporting Information for details of the immunodiffusion and ELISA procedures. Purification of Antiserum. Ten mL of antiserum was diluted with 10 mL of saline solution, and slowly added with 20 mL of saturated (NH4)2SO4 solution by stirring, and kept at 4 °C for 2 h. The mixture was centrifugated at 9000 rpm at 4 °C for 15 min to obtain a deposit. The deposit was again dissolved in 10 mL of saline solution, and slowly added with saturated 5 mL (NH4)2SO4 solution, and left at 4 °C for 30 min for the next centrifugation. Then the above precipitation treatment was repeated twice. The deposit finally obtained was dissolved in 10 mL of saline solution and dialyzed in 0.1 M PBS (pH 7.4) for 48 h. The concentration of the stock solution of antipicloram IgG was estimated to be 8.72 mg/ mL by measuring the absorbance at 280 nm. The purified immunoglobulin was stored at 4 °C with the addition of sodium azide (0.1% final concentration). Membrane Fabrication. A 2% chitosan solution was prepared by dissolving 0.16 g of chitosan in 8 mL of 2% acetic acid solution (v/v). The solution was maintained in the air and degassed for 30 min before the addition of 1.4 mL of colloidal gold. Then the mixed solution was spread onto a gridded glass plate which was beforehand cleaned in 1:1 (v/ v) HNO3 and water, and dried at 75 °C for 1 h. The chitosan/ nano-Au membrane on plate was washed with 0.1 M NaOH and water, where NaOH was used to neutralize the residual acetic acid. Then it was cut into circular segments with the diameter of 6 mm. Each membrane was soaked in 100 µL of 10 mg/mL picloram-BSA conjugate solution at 4 °C for 24 h, then treated with 100 µL of 0.25% BSA at 37 °C for 1 h to prevent nonspecific adsorption, followed by being rinsed with PBS (pH 7.38) and stored at 4 °C. Immunoreaction on Membrane and Electrochemical Measurement. Scheme 1 is a schematic diagram of the immunosensor based on competitive immunoreaction and electrochemical detection. The immunomembrane was soaked in 100 µL of PBS containing different concentrations of picloram antibody and target picloram at 37 °C for a certain time. The immobilized picloram-BSA and target picloram in PBS would compete to react with picloram antibody. Then the membrane was immersed into 100 µL of HRP-G antiRIgG diluted with PBS (pH 6.98) at 37 °C for a certain time, followed by being rinsed with water. The GCE was polished thoroughly with 0.5 mm alumina paste, sonicated in 1:1 (v/v) HNO3, acetone, and water successively, and rinsed with water before use. The membrane containing the immobilized antigen/antibody/HRP-G anti-RIgG was laid on the GCE surface, covered by a dialysis membrane and secured by means of a rubber O-ring. The electrolyte was PBS containing certain concentrations of hydroquinone and H2O2. The cyclic voltammetry was performed between -0.4 and +0.8 V vs SCE at 100 mV/s, and the chronoamperometry was carried out to determine the catalytic activity of the HRP with the reduction current recorded as Ix. When no picloram existed in the competitive immunoreaction, the reduction current was recorded as I0. The decreased percentage (DP %) of current is given by DP% )
I0 - Ix × 100 I0
(1)
After each immunoassay and detection, the immunomembrane was dismantled and another new one was attached on the GCE surface for the next detection. Extraction of Picloram from Samples. The pesticidefree samples of rice, lettuce, and paddy field water were taken
SCHEME 1. Schematic Diagram of the Immunosensor Based on Competitive Immunoreaction and Electrochemical Detection
from a field in a suburb of Changsha (Hunan Province, China). Two g of lettuce leaves or rice which had been chopped or ground in fine pieces, respectively, was spiked with different concentrations of picloram (0.5 and 8 µg/g), and then left for 24 h. Each sample was incubated in 10 mL of MeOH for 10 min, shaking at 120 r/min on a mechanical vibrator, and filtered. The container and the residue were rinsed with 10 mL of MeOH and filtered, and the filtrate was combined with the previous filtrate. MeOH was evaporated under reduced pressure, and the residue was extracted with 20 mL of a 3% MeOH-PBS (pH 6.98). The extract was diluted and analyzed by the supposed immunosensor. Paddy field water samples were spiked with 0.05 and 0.8 µg/mL picloram, filtered through 0.22 µm filter membrane, and directly submitted to analysis. Blank samples were prepared as described above but not added with picloram. Safety Considerations. Picloram is highly toxic if inhaled and can cause severe eye damage under dangerous exposure. Product labels and the material safety data sheet (MSDS) should be reviewed prior to use. Proper ventilation and protective equipment including a laboratory coat, a face mask, gloves, and shoes plus socks are required when handling this chemical.
Results and Discussion Characterization of Antiserum. The titer of the antiserum increased with the immunization schedule and reached 1:32 in the immunodiffusion test after 7 weeks from the first injection (see Figure S1, Supporting Information). The ELISA results indicated that the antiserum displayed a high level of affinity for picloram with the favorable titer of 1:100 000. Morphologies of the Immunomembrane. Figure 1 shows the morphologies of different membranes characterized respectively by SEM. The micrograph of chitosan membrane shows a relatively clean homogeneous structure (Figure 1A). The entrapment of colloidal gold nanoparticles in the chitosan membrane led to the narrow particle size distribu-
FIGURE 1. SEM images of chitosan membrane (A), chitosan/ nano-Au membrane (B), and picloram-BSA doped chitosan/ nano-Au membrane (C). tion (Figure 1B). These densely distributed gold nanoparticles significantly increased the effective adsorbility for antigen. When the picloram-BSA conjugate was encapsulated in the chitosan/nano-Au membrane, the SEM image showed a network-like structure with highly cross-linking larger-sized aggregates of trapped biomolecules (Figure 1C). Because of the excellent properties of the colloidal Au doped chitosan membrane, it retained the bioactivity of picloram-BSA conjugate to produce good analytical performance of the immunosensors. Electrochemical Behaviors of the Immunosensor for Enzymatic Reaction. After the immunomembrane was successively incubated in 28 µg/mL picloram antibody solution and 1:100 diluted HRP-G anti-RIgG solution each for 1 h at 37 °C, it was laid on the GCE surface for cyclic voltammetry study shown in Figure 2. The immunosensor displayed a low background current without observable electrochemical response in blank PBS (pH 6.81). Upon the addition of hydroquinone, a pair of stable and well-defined redox peaks was observed for the redox of hydroquinone on the electrode. After H2O2 was added into the solution VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Cyclic voltammograms of HRP-G anti-RIgG/antibody/ picloram-BSA/nano-Au/chitosan membrane in blank PBS (pH 6.81) (a), with addition of 1 mM hydroquinone (b), and in the presence of 0.5 mM H2O2 and 1 mM hydroquinone (c) at 50 mV/s. containing hydroquinone, the reduction peak current significantly increased, and the peak potential shifted slightly in the negative direction, which was attributed to the enzymatic catalysis of the immobilized HRP to the oxidation reaction of hydroquinone by H2O2. The mechanism of enzymatic electrocatalysis was reported by us previously (20). From the reduction peak potential of the enzymatic system, an applied potential of -0.08 V was chosen for amperometric detection of the immunosensor for picloram. Optimization of Detection System. The pH value and the substrate concentrations of the detection solution are important influence factors of the immunosensor performance. Most enzymes display good activity only in a limited range of pH. The maximum current response of the immunosensor occurred at pH 6.98 (see Figure S2A, Supporting Information), which was chosen for the detection, indicating that the chitosan/nano-Au matrix did not alter the native optimum pH value for redox behavior of immobilized enzyme. In the presence of 0.5 mM H2O2, the current response increased with an increasing hydroquinone concentration and reached a maximum at 1 mM, indicating the saturated reaction concentration (see Figure S2B, Supporting Information). In the presence of 1 mM hydroquinone, the change in current with the addition of H2O2 measured by chronoamperometry reached a steady state in less than 1 min at low H2O2 concentrations due to the catalytic reaction, and increased linearly with the H2O2 concentration in the range of 0 to 1 mM (see Figure S2C, Supporting Information). A midpoint concentration of 0.5 mM in the linear range was chosen for the detection precision and stability. Optimization of Immunoassay Procedure. The factors that influenced the immunoreaction for the immunoassay included pH value of the incubation solution, incubation time, picloram antibody concentration, and dilution ratio and volume of HRP-G anti-RIgG. During incubation, the maximum current response of the immunosensor occurred at pH 6.98 (see Figure S3A, Supporting Information), which was just the optimal pH value for living organisms. The response current increased with the increasing incubation time, and reached a platform at 40 and 60 min for antibody incubation and HRP-G anti-RIgG incubation, respectively (see Figure S3B, Supporting Information). Seven µg/mL antibody was observed as the optimal picloram antibody concentration, indicating the saturated formation of immunocomplex in the competitive reaction (see Figure S3C, Supporting Information). In addition, the optimum dilution rate and volume of HRP-G anti-RIgG were found to be 1:50 and 75 µL, respectively, in the immunoassay under the optimal detection conditions. Response of the Immunosensor to Picloram Concentration. Under the optimal immunoreaction and detection 1210
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FIGURE 3. Calibration curve of immunosensor for picloram determination under the optimal experimental conditions. Inset: linear regression of decrease percentage vs the natural logarithm of picloram concentration. The vertical bars designate the standard deviations for the mean of three replicative tests. conditions, the target picloram molecules in sample solution would compete with the immumomembrane to react with the limited molecules of dissociative picloram antibody to form immunocomplex. The decrease percentage of amperometric current response attributed to the competitive immunoreaction increased with an increasing picloram concentration (Figure 3). The average decrease percentage DP (%) is linearly related to the natural logarithm of picloram concentration C (µg/mL) in the range from 0.005 to 10 µg/ mL with the following regression equation DP% ) (5.130 ( 0.205) × lnC + (32.460 ( 0.551) (2) The correlation coefficient is 0.9937. Each of the calibrations was done three times, and the relative standard deviations (RSDs) were 4.18 and 2.10% for five replicates at the picloram concentrations of 0.05 and 1 µg/mL, respectively, which guaranteed the precision of the immunosensor. The detection limit of picloram in the incubation solution is 5 ng/mL. It can be inferred that the immunosensor held superior sensitivity compared with the reported detection methods for picloram (6, 8). Detection of Picloram in Samples. Picloram is reported to be quite mobile in soil, and may easily leach into waters or move from treated plants through the roots to nearby nontarget crops (5). Rice, lettuce, and paddy field water were spiked with picloram analyzed by the immunosensor. Immunoassay usually requires no sample cleanup in the case of water samples, but it usually requires some sort of sample pretreatment for agricultural products to circumvent the matrix effect (21). The procedure for rice or lettuce sample preparation adopted in this study was extraction of spiked sample with MeOH, followed by evaporation of the solvent, extraction of the residue in 3% MeOH-PBS, and dilution by PBS (pH 6.98). Evaporation of MeOH can reduce the matrix effect significantly without influencing the sample volume. Dilution of the sample is also an effective means to circumvent matrix effect, while it requires sufficiently high sensitivity of the assay (22). Without dilution by PBS (pH 6.98), unspiked extracts of rice and lettuce samples which were not originally contaminated by pesticides were also applied in the immunoreaction with certain amount of antibody, while the amperometric response current of the immunomembrane decreased obviously due to the matrix effect. This resulted in the overestimation of picloram concentrations in the spiked samples without dilution. Therefore, the extracts of rice and lettuce samples were diluted 10-fold by PBS (pH 6.98) before detection. The recoveries of picloram from the samples were generally satisfactory, presented in Table 1. There were little or no matrix effects observed in paddy field water, rice, and lettuce
TABLE 1. Recovery of Picloram from Paddy Field Water, Rice Extract, and Lettuce Extract Samples Using the Proposed Immunosensor
sample paddy field watera rice lettuce
immunoassay procedures, and the molecular structures of interferents. This information is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited
concentration added (µg/g)
concentration found (µg/g)
0.05
0.053 ( 0.004
106.6
8.0
0.8 0.5 8 0.5 8
0.763 ( 0.083 0.56 ( 0.07 7.70 ( 0.68 0.54 ( 0.05 8.29 ( 0.97
95.3 111.9 96.3 108.7 103.6
10.9 12.9 8.8 9.9 11.7
recovery (%) CV (%)
a Picloram concentration (added and determined) unit for paddy field water samples is µg/mL.
TABLE 2. Comparison of the Immunosensor Response to Picloram and Other Structurally Related Compounds under the Same Conditions interferent
concentration (µg/mL)
decrease percentage of current (%)
picloram pyridine lontrel triclopyr
0.5 0.5 0.5 0.5
26.96 1.96 4.70 1.43
samples, which indicated the suitability of the immunosensor as a simple, fast, and reliable determination method of picloram in the environmental samples. Specificity and Stability. One of the concerns about immunosensor analysis of contaminated environmental samples is the resistance to interference from irrelevant cocontaminants. The structurally similar pesticides, such as lontrel and triclopyr, as well as pyridine (see Figure S4, Supporting Information), were tested under the same optimal conditions as picloram (see Table 2). It can be inferred that the interference of these chemicals to the immunosensor is negligible although they all contain the same pyridyl structure as that of picloram. The performance stability of the immunosensor was examined by storing the chitosan/nano-Au/antigen/BSA membrane in PBS (pH 6.98) at 4 °C when not in use. The amperometric response to 7 µg/mL picloram antibody retained 91% of its initial response after a 15-day storage. In conclusion, a disposable immunomembrane-based sensor has been developed for the detection of low levels of picloram in practical environmental samples. The immunomembrane had good reproducibility for fabrication in batch and exhibited satisfactory results for picloram detection in real agricultural field samples. All of these observations clearly illustrate that with the entrapment of different biomarkers in the composite membrane, this immunosensor could be readily extended toward the on-site monitoring for other trace toxic organic pollutants in environmental matrices in further studies.
Acknowledgments This study was financially supported by the National Natural Science Foundation of China (50608029), the Chinese National Basic Research Program (973 Program; 2005CB724203), the National 863 High Technology Research Program of China (2004AA649370), and the Program for Changjiang Scholars and Innovative Research Team in University.
Supporting Information Available More details of the immunodiffusion and ELISA procedures, optimization results of the influence factors in detection and
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