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Development of an immunochromatographic assay for the rapid detection of bromoxynil in water
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Environmental Pollution 156 (2008) 136e142 www.elsevier.com/locate/envpol
Development of an immunochromatographic assay for the rapid detection of bromoxynil in water Jiang Zhu a, Wenchao Chen b, Yitong Lu a,*, Guohua Cheng a a
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China b University of Nottingham, School of Biosciences, Leicestershire LE12 5RD, United Kingdom
Received 19 July 2007; received in revised form 13 December 2007; accepted 15 December 2007
One-step strip test is a rapid method for qualitative detection of bromoxynil residues in water. Abstract A rapid immunochromatographic one-step strip test was developed to specifically determine bromoxynil in surface and drinking water by competitive inhibition with the nano colloidal gold-conjugated monoclonal antibody (mAb). Bromoxynil standard samples of 0.01e10 mg L1 in water were tested by this method and the visual limit was 0.06 mg L1. The assay only required 5 min and one-step by dispensing a drop of sample solution onto a strip. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA. Therefore, the one-step strip test is very useful as a screening method for qualitative detection of bromoxynil in water. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Bromoxynil; Gold immunochromatographic assay (GICA); Colloidal gold; One-step strip test; Pesticide residue
1. Introduction Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) is a nitrile herbicide to inhibit the photosynthesis of weeds. It has some trade names such as Brominal, Bromotril, Bronate, Buctril, Certrol B, Litarol, M & B 10064 (Kidd and James, 1991; Breton et al., 2007). Bromoxynil is vital for cropland (corn, sorghum, onions, flax, mints, turfs, etc.) and non-cropland. However, it is one of Restricted Use Pesticides (RUPs) that may be purchased and used only by certified applicators (Edwards et al., 1991; Jan et al., 2005). It is categorized as toxicity class II e moderately toxic. Anomalies found in rat fetuses exposed to bromoxynil suggest it a teratogen. Workers chronically exposed to bromoxynil over a year long span showed symptoms of weight loss, fever, vomiting, headache and urinary problems (Cao et al., 2005). As a result of widespread and improper use of bromoxynil, serious problems have turned up in China. Bromoxynil could * Corresponding author. Tel./fax: þ86 21 3420 6143. E-mail address: [email protected] (Y. Lu). 0269-7491/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2007.12.020
be present in air, food, and soil as well as in surface water and contaminate groundwater by runoff and leaching. Bromoxynil in water is likely to pose health hazards to non-target aquatic organisms including wildlife and human. Acute pesticide poisoning exposed to bromoxynil was reported (Wang, 2005; Liang et al., 2006). Surface water in Yangtze River has seen bromoxynil (Li et al., 2005). Moreover, bromoxynil has been found widespread to contaminate soil (Sheng et al., 2005). Currently, the major analysis method for bromoxynil is multiresidue method by high performance liquid chromatography (HPLC), chemiluminescent determination, SPME and GCtandem mass spectrometry (Dijkman et al., 2001; Hogendoorn et al., 2001; Jan et al., 2005; Pawlicova et al., 2005; Shah et al., 2005; Jaber et al., 2007; Scheyer et al., 2007). However, chromatographic methods are expensive and time-consuming due to the complicated sample preparation and sophisticated equipments. On the other hand, enzyme-linked immunosorbent assays (ELISA) based on antigeneantibody reaction have been increasingly reported for residual pesticide analysis recent years. Generally, immunochemical methods are suitable
J. Zhu et al. / Environmental Pollution 156 (2008) 136e142
for high sample throughput and on-site screening analysis and have been proved simple, cost effective, and rapid (Harris et al., 1995; Ellis, 1996). ELISA with polyclonal antibody have been used successfully for the quantitative analysis of bromoxynil in water since it is both specific and sensitive to bromoxynil (Cao et al., 2005). High-affinity monoclonal antibody (mAb) against bromoxynil has enhanced the development of immunoassay technique dramatically in that single and homogeneous mAb can be unlimitedly prepared (Zhu et al., 2006). Colloidal gold has been introduced into immunochemistry and the nano colloidal gold particles could replace the enzyme to be labeled to antibody (Hayat, 1989; Dykman and Bogatyrev, 1997; Nagatani et al., 2006). Immunoglobulin with colloidal gold particles responds to the corresponding antigen and leads to a visible color reaction (Horton et al., 1991; Dequaire et al., 2000; Zhang et al., 2002). Therefore, a rapid test method, simply by one-step strip, has been developed and applied increasingly to various research fields. It was first introduced to target human chorionic gonadotropin (hCG) for the detection of pregnancy (Nagainis et al., 1986). This method has been mainly applied to human diagnostics so far (Garrote et al., 1999; Soroka et al., 2003; Hasegawa et al., 2004; Lee et al., 2006; Sithigorngul et al., 2007). It can also be applied to low molecular mass analyses, e.g., detection of drugs abuse (Pattarawarapan et al., 2007; Silva-Colombeli and Falkenberg, 2007), prediction of steroid-based ovulation, and determination of progesterone, antibiotic in milk and pesticide residue according to competitive immunoassay protocols (Verheijen et al., 1998; Weller, 2000; Oku et al., 2001; Zhou et al., 2004). In this study, based on the highly specific immunochemical reactions between antigens (bromoxynil) and antibodies, we developed a one-step assay for the detection of bromoxynil by colloidal gold immunochromatography, which takes just 5 min to detect bromoxynil in various water samples. Such test is suitable for qualitative detection of bromoxynil. 2. Materials and methods 2.1. Reagents and equipments All reagents used in the present study were of analytical grade. Bromoxynil standard (99.9%) was obtained from the Institute of Environmental Protection, Ministry of Agriculture (Tianjin, China). Anti-bromoxynil mAb was produced in our laboratory according to previous methods (Cao et al., 2005; Zhu et al., 2006). The mAb was purified with protein A affinity column (Pharmacia, Uppsala, Sweden). Bovine serum albumin (BSA), ovalbumin (OVA), polyethylene glycol (PEG MW: 20 000) and gold chloride were from Sigma (Shanghai, China). Goat anti-mouse IgG was from Sino-American Biotechnology Co., Shanghai Branch (Shanghai, China). NC membranes (pore size 5 mm) and glass fiber membranes were provided by Schleicher & Schuell Co. (Dassel, Germany). The BioDot system consisted of two BioJets Quanti 3000 and one Airjet Quanti 3000 attached to BioDot XYZ-3000 (Irvine, CA, USA) dispensing platform. The guillotine cutter (Model: CM 4000) was supplied by BioDot (Irvine, CA, USA). The centrifuge (Heraeus multifuge 3 S-R) was purchased from Kendro Laboratory Products GmbH (Hamburg, Germany). The UV-2401 (pc)s was from SHIMADZU corporation (Tokyo, Japan). Transmission electron microscope (JEM-100CX __) was from JEOL (Tokyo, Japan).
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2.2. Preparation of colloidal gold Nanometer colloidal gold with an average particle diameter of 40 nm was prepared according to the procedures described by Frens (1973). The size of nanometer colloidal gold depended on the amount of trisodium citrate as reducing agent. The colloidal gold suspension was prepared as follows: about 100 mL of 0.01% (m/v) AuCl3$HCl$4H2O was heated to boiling point. One percentage of trisodium citrate with various volumes of 0.3, 0.45, 0.7, 1, 1.5 and 2 mL was added rapidly by constant stirring. After the color of solution changed (in 2 min), the mixture was boiled for another 15 min. After cooling, deionized water was added till the initial volume. The average particle diameter was checked with UV and a transmission electron microscopy. The colloidal gold suspensions supplemented with 0.05% (m/v) of sodium azide was stored at 4 C for several months.
2.3. Conjugation of anti-bromoxynil mAb and colloidal gold The antibody to stabilize the colloidal gold particles was prepared according to the methods of Roth (1982a,b). Fresh 0.2 M K2CO3 solution was added to colloidal gold and pH was adjusted to 8.2. Purified anti-bromoxynil mAb (0.1 mg mL1) of 6 mL was added to 94 mL of dispersed gold solution to give a final concentration of 6 mg L1 by fast stirring and then incubated for 15 min at room temperature. It was followed by PEG (20 000, 1% as the final concentration) added by another 15-min stirring. The mixture was centrifuged for 30 min at 10 000 rpm and the supernatant was removed. In the end, the precipitate of the gold-labeled antibodies was washed with PBS (pH 8.2), centrifuged for twice, resuspended in PBS (pH 8.2) and then stored at 4 C.
2.4. Immobilization of reagents Biojet (XYZ3000) (Irvine, CA, USA) was used to dispense two lines on the NC membrane strips (25 300 mm). OVA-bromoxynil (0.5 mg mL1) was dispensed around the bottom as the test line (1 mL per 1 mm line) while 1 mg mL1 goat anti-mouse IgG was dispensed at the upper position as the control line (1 mL per 1 mm line). The NC membrane was dried for 2 h at 37 C, blocked with 1% BSA for 30 min, washed three times with 0.01 M PBS (pH 7.4) and then dried again at 37 C for 3 h. Finally, the strips were stored under dry conditions at room temperature until required. Gold-labeled anti-bromoxynil mAb (detective reagent) was dispensed onto a glass fiber filter with Airjet (XYZ3000) (Irvine, CA, USA) and then dried at 37 C for 3 h. The optimal concentration of the dispensed mixture was selected with OD value of 10 at 520 nm. Its dispensed volume could also be calculated as 1 mL for 1 mm line.
2.5. Development of the one-step strip The one-step strip was composed of three pads (sample, conjugate and absorbent pads) and an NC membrane. They were pasted onto an adhesive plastic backing. In particular, the NC membrane containing the OVA-bromoxynil and goat anti-mouse IgG was pasted at the center of the backing plate. The conjugate pad containing the gold-labeled mAb was pasted by over-crossing 2 mm with NC membrane. The sample pad (glass fiber) was also pasted by over-crossing 4 mm with the conjugated pad. The absorbent pad was pasted on the other side of the plate. The whole assembled plate was cut lengthways and divided into strips with a guillotine cutter (5 60 mm).
2.6. Analysis of water samples Water samples containing Bromoxynil (0.01e10 mg L1) were prepared from double distilled water, tap water, a commercial mineral water, and surface water of Huangpu River (Shanghai, China) with a known amount of bromoxynil derived from 100 mg L1 stock solution in 0.01 M PBS (pH 7.4). Particularly, the river water was collected in 1-L bottles and stored at 4 C until required. Tap water and river water samples were filtered through a 0.45 mm nylon filter. Each 100 mL sample supernatant was applied to
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J. Zhu et al. / Environmental Pollution 156 (2008) 136e142
one-step strip. Negative controls were water samples without bromoxynil. After 5 min, the test results were determined as positive or negative visually (Fig. 2). The control region (c) and test region (t) on the strip were made from the goat anti-mouse IgG and the OVA-bromoxynil individually. The mAb labeled with colloidal gold particles tended to react with bromoxynil by competition inhibition. When applied to a negative sample, the test and control lines turned red separately while a positive sample resulted in a red control line only. The test was invalid if no line was shown in the control region.
3. Results and discussion 3.1. Size selecting of colloidal gold particle Nano colloidal gold particles have a promising application such as immuno-analysis, biosensor and gene therapy (Deng et al., 2000; Nagatani et al., 2006). Since immunoglobulins could be labeled with nano colloidal gold particles instead of enzymes, the substrate is not needed in the reaction system. The strength of color reaction is closely related to the size and quality of the colloidal gold particles. The size of colloidal gold particles directly depends on the amount of trisodium citrate in its preparation process. Trisodium citrate is the key factor that influences the formation of particular cores. Changing the amount of trisodium citrate could result in different colloidal gold particles with different colors. In our study, six different types of colloidal gold particles were made. When more than 1.6 mL trisodium citrate (1%) was added in a 100 mL (0.01%) of gold chloride solution, the colloidal gold particles obtained were reddish and small with a diameter of less than 25 nm. They were found too small to indicate a clear and bright color. When less than 0.8 mL trisodium citrate was added, the color was between purple and blue with the diameter more than 55 nm. However, those large size colloidal gold particles were found not stable. Only when 1 mL trisodium citrate was added did the colloidal gold particles show a red color with a diameter of 40 nm. They were found stable when combined with immunoglobulins. The indicating color was obvious and easy to tell. The immunoreaction compound moved fast on the NC membrane. The reaction could be completed and remained stable. Therefore, the 40 nm colloidal gold particles were selected in this test (Fig. 1). 3.2. Analytical parameters of the optimized GICA To determine the appropriate concentration of OVA-bromoxynil coated on the membrane, the OVA-bromoxynil was prepared in 0.01 M PBS (pH 7.4) by serial dilutions ranging from 0.05 mg mL1 to 1.0 mg mL1. We found that the higher the OVA-bromoxynil concentration was, the more bromoxynil was needed in the sample to compete with OVA-bromoxynil. But below 0.5 mg mL1, the signal was too faint to be seen. As a result, 0.5 mg mL1 of OVA-bromoxynil was used to dispense on the NC membrane as the test line. The different buffers including 0.01 M PBS (pH 7.4), 0.1 M PBS (pH 7.4), carbonate buffer (pH 9.6) and borate buffer (pH 9.0) used for gold-labeled antibody were compared as to their
Fig. 1. The size of valid colloidal gold particles observed by transmission electron microscopy (120 000). The diameter of those particles was 40 nm.
effects on this assay. Generally, 0.01 M PBS (pH 7.4) was used in various immunoassays as a working solution (Table 1). A 0.01 M PBS (pH 7.4) and deionized water resulted in a lower detection limit in the one-step strip test. We found that the pH has significant effects on the assay and 0.01 M PBS (pH 7.4) was the best for the sensitivity.
3.3. Sensitivity, specificity and reliability of a one-step strip test The detection limit was defined as the amount of bromoxynil in the sample solution that just causes a complete invisibility of the test line. A series of dilutions of standard bromoxynil (0.01e10 mg L1) were assayed by strip tests (Table 2 and Fig. 2). The results were recorded 5 min after the reactions. Two lines detected on the NC membrane presented that the bromoxynil concentration was below the detection limit while only one line detected around the control zone showed that the bromoxynil concentration was above the detection limit. To ensure the detection, the lower detection limit of the optimized one-step strip was at 0.06 mg L1 since the concentration of 0.03 mg L1 of bromoxynil was found to cause a considerable decrease in the assay signal. The result of one-step strip test was very similar to that of ELISA developed by Yongsong Cao (Cao et al., 2005). However, one-step strip test usually offered lower sensitivity than ELISA. It was possible to improve the sensitivity by adjusting relative factors, which affect the assay system (Shyu et al., 2002). In particular, it was feasible to significantly improve the sensitivity of our method by silver staining. It was a key that samples were preconcentrated before the analysis by dichloromethane extraction. Another important way was to rectify the quantity of OVA-bromoxynil or mAb, which were dispensed on the strip by adjusting their concentration. Therefore, this assay has a great application potential. The specificity of this assay was evaluated in comparison to other analog compounds. The strips were dipped into water
J. Zhu et al. / Environmental Pollution 156 (2008) 136e142
139
Table 1 Influence of deionized water, 0.01 M PBS (pH 7.4) and different organic solvents as working solutions on the GICA of bromoxynil Bromoxynil standard concentration (mg L1)
Working solution
0.01
0.03
0.06
0.12
0.25
0.5
1
2
5
10
0.01 M PBS(pH 7.4)
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
0.1 M PBS (pH 7.4)
Test line Control line
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
Carbonate buffer(pH 9.6)
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
Borate buffer (pH 9.0)
Test line Control line
þ þ
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
Deionized water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
20% Methanol
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
20% Acetone
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
20% Acetonitrile
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
þ: Red line appeared. : Red line appeared but very weak. : Red line did not appear.
samples containing different compounds for 10 s, taken out and laid on a flat table and the results were judged visually after 5 min. As shown in Table 3 and Fig. 2, the qualitative working range of the strip test was from 0.06 mg L1 to 5 mg L1 with a LOD of 0.06 mg L1 and the probability of occurring of cross-reaction was lower than 0.06 mg L1. This indicates that this assay had a specific reaction with bromoxynil and had not cross-reactivity with other related pesticides and herbicides in the qualitative working range. This method was not only rapid and simple, but also did not require intensive labor and expensive equipment for the sample analysis, especially for fast on-site detection. Also, it provided only a preliminary, semi-quantifying result simply with
positive or negative results. The results could help determine whether the pesticide concentrations in the samples were higher than the detection limit or not. In addition, a conventional and standard analysis was necessary for precise determination of the concentration of the pesticide. The reliability of the assay was determined by the tests with the standard water samples containing bromoxynil. Among those 110 samples, 55 samples were at 0.01 mg L1 and the other 55 samples were at 0.06 mg L1. The concentrations of bromoxynil in the treated samples were detected with GICA method and the ELISA method at the same time. The results obtained by the two methods were compared (Table 4). The overall agreement was 98%.
Table 2 Results of water samples by GICA Spiked bromoxynil concentration (mg L1)
Sample source
0.01
0.03
0.06
0.12
0.25
0.5
1
2
5
10
Double distilled water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Tap water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
A commercial bottled mineral water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Surface water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ: Red line appeared. : Red line appeared but very weak. : Red line did not appear.
J. Zhu et al. / Environmental Pollution 156 (2008) 136e142
140
Fig. 2. Immunochromatographic detection of bromoxynil. A series of dilutions (0.01e10 mg L1) of bromoxynil were prepared. Details of the preparation of the test device and assay procedures are described in the text. Bromoxynil (0.03 mg L1) led to a fainter test line. However, pure water and other analog compounds except 2,6-dibromophenol, chloroxynil and ioxynil presented an obvious test line even though analog compounds reached at a high level of 100 mg L1.
3.4. Sample analysis We analyzed prepared water samples by the one-step strip test. Fifty-five water samples were prepared as
mentioned above. One-step strip test showed two red lines for samples at the concentration of 0.01 mg L1. However, those samples of 0.06 mg L1 presented only one line (Table 4).
Table 3 Cross-reactivity (CR) with bromoxynil and related compounds by the one-step strip test Each compound concentration (mg L1)
Compounds
0.01
0.03
0.06
0.12
0.25
0.5
1
2
5
10
Bromoxynil
Test Control
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Chloroxynil
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
Ioxynil
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
2,6-dibromophenol
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
þ
p-hydroxybenzonitrile
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
2,4-D
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
Dichlorprop
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
2,4-DB
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
MCPA
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
Mecoprop
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
MCPB
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ: Red line appeared. : Red line appeared but very weak. : Red line did not appear.
J. Zhu et al. / Environmental Pollution 156 (2008) 136e142 Table 4 Reliability of the test strip compared to ELISA GICA Positive
Negative
ELISA Positive Negative
Total
53 0
2 55
55 55
Total
53
57
110
Comparison of the results between gold immunochromatagraph assay (GICA) and ELISA. Fifty-five samples for each concentration, a total of 110 samples were tested. The overall agreement is 98%.
4. Conclusions We presented the development and evaluation of a GICA for the qualitative detection of bromoxynil residue in the water. Bromoxynil standard sample of 0.01e10 mg L1 in water were determined by the one-step strip test. The limit was determined visually as 0.06 mg L1 and the probability of cross-reactions was lower than 0.06 mg L1. The results showed this assay led to a special reaction with bromoxynil without cross-reactions to other related pesticides and herbicides in the qualitative working range. Several factors affecting the immunochemical specificity have been demonstrated. What is more, pH was shown to have significant influence on the system and 0.01 M PBS (pH 7.4) was the best for the sensitivity. OVA-bromoxynil (0.5 mg mL1) was used to dispense on the NC membrane as the test line. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA and the overall agreement was 98%. The one-step strip test was rapid and simple. Also, it did not require intensive labor and expensive equipments for the sample analysis. The proposed GICA turned out to be a powerful tool for monitoring residual bromoxynil in water. In particular, the one-step strip test was suitable for on-site detection of water samples, e.g., for a water inspection center and a beverage company. In addition, the assay can be applied for the rapid detection of bromoxynil in agricultural and environmental samples. Acknowledgements This work was supported by National Natural Science Foundation of International Cooperation (NSFC-RFBR) (No. 20611120017), National Key Basic Research Program of China (No. 2004CB18503), National Natural Science Foundation of China (No. 20337010) and Key Program of Basic Research of Shanghai City (No. 04JC14051). We express our sincere gratitude to Dr. Yongsong Cao for supplying the hapten used for the production of anti-bromoxynil mAb. References Breton, F., Rouillon, R., Piletska, E.V., Karim, K., Guerreiro, A., Chianella, I., Piletsky, S.A., 2007. Virtual imprinting as a tool to design efficient MIPs
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Liang, H., Liu, X., Lu, Z.Q., Qiu, Q.M., Hu, G.X., 2006. Experimental study on toxicity and changes of biochemical indicator in acute bromoxynil poisoning. Chinese Journal of Industrial Hygiene and Occupational Diseases 24, 494e495 (in Chinese). Nagainis, P.A., Nakagawa, C.H., Baron, S.L., Fuller, S.A., Chandler, H.M., Hurrell, J.G.R., 1986. A rapid quantitative capillary tube enzyme immunoassay for human chorionic gonadotropin in urine. Clinica Chimica Acta 160, 273e279. Nagatani, N., Tanaka, R., Yuhi, T., Endo, T., Kerman, K., Takamura, Y., Tamiya, E., 2006. Gold nanoparticle-based novel enhancement method for the development of highly sensitive immunochromatographic strip tests. Science and Technology of Advanced Materials 7, 270e275. Oku, Y., Kamiya, K., Kamiya, H., Shibahara, Y., Ii, T., Uesaka, Y., 2001. Development of oligonucleotide lateral-flow immunoassay for multi-parameter detection. Journal of Immunological Methods 258, 73e84. Pattarawarapan, M., Nangola, S., Cressey, T.R., Tayapiwatana, C., 2007. Development of a one-step immunochromatographic strip test for the rapid detection of nevirapine (NVP), a commonly used antiretroviral drug for the treatment of HIV/AIDS. Talanta 71, 462e470. Pawlicova, Z., Albert-Garcia, J.R., Sahuquillo, I., Garcia Mateo, J.V., Catala Icardo, M., Martinez Calatayud, J., 2005. Chemiluminescent determination of the pesticide bromoxynil by on-line photodegradation in a flowinjection system. Analytical Sciences 22, 29e34. Roth, J., 1982a. The protein A-gold (pAG) techniqueda qualitative and quantitative approach for antigen localization on thin sections. In: Bullock, G.R., Petrusz, P. (Eds.), Techniques in Immunocytochemistry, vol. 1. Academic Press, London, pp. 107e133. Roth, J., 1982b. The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labeling multiple antigens on ultrathin sections. Histochemical Journal 14, 791e801. Scheyer, A., Briand, O., Morville, S., Mirabel, P., Millet, M., 2007. Analysis of trace levels of pesticides in rainwater by SPME and GC-tandem mass spectrometry after derivatisation with PFFBr. Analytical and Bioanalytical Chemistry 387, 359e368. Shah, J., Jan, M.R., Bashir, N., 2005. Determination of starane (fluroxypyr) herbicide using flow injection spectrophotometry. Analytical Sciences 22, 145e146.
Sheng, G.Y., Yang, Y.N., Huang, M.S., Yang, K., 2005. Influence of pH on pesticide sorption by soil containing wheat residue-derived char. Environmental Pollution 134, 457e463 (in Chinese). Shyu, R.H., Shyu, H.F., Liu, H.W., Tang, S.S., 2002. Colloidal gold-based immunochromatographic assay for detection of ricin. Toxicon 40, 255e258. Silva-Colombeli, A.S., Falkenberg, M., 2007. Analytical interferences of drugs in the chemical examination of urinary protein. Clinical Biochemistry 40, 1074e1076. Sithigorngul, W., Rukpratanporn, S., Sittidilokratna, N., Pecharaburanin, N., Longyant, S., Chaivisuthangkura, P., Sithigorngul, P., 2007. A convenient immunochromatographic strip test for rapid diagnosis of yellow head virus infection in shrimp. Journal of Virological Methods 140, 193e199. Soroka, S.D., Granade, T.C., Phillips, S., Parekh, B., 2003. The use of simple, rapid tests to detect antibodies to human immunodeficiency virus types 1 and 2 in pooled serum specimens. Journal of Clinical Virology 27, 90e96. Verheijen, R., Stouten, P., Cazemier, G., Haasnoot, W., 1998. Development of a one step strip test for the detection of sulfadimidine residues. Analyst 123, 2437e2441. Wang, C.W., 2005. Investigation on an accident of death induced by bromoxynil poisoning. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 23, 376e377 (in Chinese). Weller, M.G., 2000. Immunochromatographic techniques e a critical review. Fresenius’ Journal of Analytical Chemistry 366, 635e645. Zhang, C., Zhang, Z., Yu, B., Shi, J., Zhang, X., 2002. Application of the biological conjugate between antibody and colloid Au nanoparticles as analyte to inductively coupled plasma mass spectrometry. Analytical Chemistry 74, 96e99. Zhou, P., Lu, Y.T., Zhu, J., Hong, J.B., Li, B., Zhou, J., Gong, D., Montoya, A., 2004. Nanocolloidal gold-based immunoassay for the detection of the N-methylcarbamate pesticide carbofuran. Journal of Agricultural and Food Chemistry 52, 4355e4359. Zhu, J., Lu, Y.T., Cheng, G.H., 2006. Production, purification and identification of bromoxynil monoclonal antibody. Journal of Agro-Environment Science 25, 527e530 (in Chinese).
Environmental Pollution 156 (2008) 136e142 www.elsevier.com/locate/envpol
Development of an immunochromatographic assay for the rapid detection of bromoxynil in water Jiang Zhu a, Wenchao Chen b, Yitong Lu a,*, Guohua Cheng a a
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China b University of Nottingham, School of Biosciences, Leicestershire LE12 5RD, United Kingdom
Received 19 July 2007; received in revised form 13 December 2007; accepted 15 December 2007
One-step strip test is a rapid method for qualitative detection of bromoxynil residues in water. Abstract A rapid immunochromatographic one-step strip test was developed to specifically determine bromoxynil in surface and drinking water by competitive inhibition with the nano colloidal gold-conjugated monoclonal antibody (mAb). Bromoxynil standard samples of 0.01e10 mg L1 in water were tested by this method and the visual limit was 0.06 mg L1. The assay only required 5 min and one-step by dispensing a drop of sample solution onto a strip. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA. Therefore, the one-step strip test is very useful as a screening method for qualitative detection of bromoxynil in water. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Bromoxynil; Gold immunochromatographic assay (GICA); Colloidal gold; One-step strip test; Pesticide residue
1. Introduction Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) is a nitrile herbicide to inhibit the photosynthesis of weeds. It has some trade names such as Brominal, Bromotril, Bronate, Buctril, Certrol B, Litarol, M & B 10064 (Kidd and James, 1991; Breton et al., 2007). Bromoxynil is vital for cropland (corn, sorghum, onions, flax, mints, turfs, etc.) and non-cropland. However, it is one of Restricted Use Pesticides (RUPs) that may be purchased and used only by certified applicators (Edwards et al., 1991; Jan et al., 2005). It is categorized as toxicity class II e moderately toxic. Anomalies found in rat fetuses exposed to bromoxynil suggest it a teratogen. Workers chronically exposed to bromoxynil over a year long span showed symptoms of weight loss, fever, vomiting, headache and urinary problems (Cao et al., 2005). As a result of widespread and improper use of bromoxynil, serious problems have turned up in China. Bromoxynil could * Corresponding author. Tel./fax: þ86 21 3420 6143. E-mail address: [email protected] (Y. Lu). 0269-7491/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2007.12.020
be present in air, food, and soil as well as in surface water and contaminate groundwater by runoff and leaching. Bromoxynil in water is likely to pose health hazards to non-target aquatic organisms including wildlife and human. Acute pesticide poisoning exposed to bromoxynil was reported (Wang, 2005; Liang et al., 2006). Surface water in Yangtze River has seen bromoxynil (Li et al., 2005). Moreover, bromoxynil has been found widespread to contaminate soil (Sheng et al., 2005). Currently, the major analysis method for bromoxynil is multiresidue method by high performance liquid chromatography (HPLC), chemiluminescent determination, SPME and GCtandem mass spectrometry (Dijkman et al., 2001; Hogendoorn et al., 2001; Jan et al., 2005; Pawlicova et al., 2005; Shah et al., 2005; Jaber et al., 2007; Scheyer et al., 2007). However, chromatographic methods are expensive and time-consuming due to the complicated sample preparation and sophisticated equipments. On the other hand, enzyme-linked immunosorbent assays (ELISA) based on antigeneantibody reaction have been increasingly reported for residual pesticide analysis recent years. Generally, immunochemical methods are suitable
J. Zhu et al. / Environmental Pollution 156 (2008) 136e142
for high sample throughput and on-site screening analysis and have been proved simple, cost effective, and rapid (Harris et al., 1995; Ellis, 1996). ELISA with polyclonal antibody have been used successfully for the quantitative analysis of bromoxynil in water since it is both specific and sensitive to bromoxynil (Cao et al., 2005). High-affinity monoclonal antibody (mAb) against bromoxynil has enhanced the development of immunoassay technique dramatically in that single and homogeneous mAb can be unlimitedly prepared (Zhu et al., 2006). Colloidal gold has been introduced into immunochemistry and the nano colloidal gold particles could replace the enzyme to be labeled to antibody (Hayat, 1989; Dykman and Bogatyrev, 1997; Nagatani et al., 2006). Immunoglobulin with colloidal gold particles responds to the corresponding antigen and leads to a visible color reaction (Horton et al., 1991; Dequaire et al., 2000; Zhang et al., 2002). Therefore, a rapid test method, simply by one-step strip, has been developed and applied increasingly to various research fields. It was first introduced to target human chorionic gonadotropin (hCG) for the detection of pregnancy (Nagainis et al., 1986). This method has been mainly applied to human diagnostics so far (Garrote et al., 1999; Soroka et al., 2003; Hasegawa et al., 2004; Lee et al., 2006; Sithigorngul et al., 2007). It can also be applied to low molecular mass analyses, e.g., detection of drugs abuse (Pattarawarapan et al., 2007; Silva-Colombeli and Falkenberg, 2007), prediction of steroid-based ovulation, and determination of progesterone, antibiotic in milk and pesticide residue according to competitive immunoassay protocols (Verheijen et al., 1998; Weller, 2000; Oku et al., 2001; Zhou et al., 2004). In this study, based on the highly specific immunochemical reactions between antigens (bromoxynil) and antibodies, we developed a one-step assay for the detection of bromoxynil by colloidal gold immunochromatography, which takes just 5 min to detect bromoxynil in various water samples. Such test is suitable for qualitative detection of bromoxynil. 2. Materials and methods 2.1. Reagents and equipments All reagents used in the present study were of analytical grade. Bromoxynil standard (99.9%) was obtained from the Institute of Environmental Protection, Ministry of Agriculture (Tianjin, China). Anti-bromoxynil mAb was produced in our laboratory according to previous methods (Cao et al., 2005; Zhu et al., 2006). The mAb was purified with protein A affinity column (Pharmacia, Uppsala, Sweden). Bovine serum albumin (BSA), ovalbumin (OVA), polyethylene glycol (PEG MW: 20 000) and gold chloride were from Sigma (Shanghai, China). Goat anti-mouse IgG was from Sino-American Biotechnology Co., Shanghai Branch (Shanghai, China). NC membranes (pore size 5 mm) and glass fiber membranes were provided by Schleicher & Schuell Co. (Dassel, Germany). The BioDot system consisted of two BioJets Quanti 3000 and one Airjet Quanti 3000 attached to BioDot XYZ-3000 (Irvine, CA, USA) dispensing platform. The guillotine cutter (Model: CM 4000) was supplied by BioDot (Irvine, CA, USA). The centrifuge (Heraeus multifuge 3 S-R) was purchased from Kendro Laboratory Products GmbH (Hamburg, Germany). The UV-2401 (pc)s was from SHIMADZU corporation (Tokyo, Japan). Transmission electron microscope (JEM-100CX __) was from JEOL (Tokyo, Japan).
137
2.2. Preparation of colloidal gold Nanometer colloidal gold with an average particle diameter of 40 nm was prepared according to the procedures described by Frens (1973). The size of nanometer colloidal gold depended on the amount of trisodium citrate as reducing agent. The colloidal gold suspension was prepared as follows: about 100 mL of 0.01% (m/v) AuCl3$HCl$4H2O was heated to boiling point. One percentage of trisodium citrate with various volumes of 0.3, 0.45, 0.7, 1, 1.5 and 2 mL was added rapidly by constant stirring. After the color of solution changed (in 2 min), the mixture was boiled for another 15 min. After cooling, deionized water was added till the initial volume. The average particle diameter was checked with UV and a transmission electron microscopy. The colloidal gold suspensions supplemented with 0.05% (m/v) of sodium azide was stored at 4 C for several months.
2.3. Conjugation of anti-bromoxynil mAb and colloidal gold The antibody to stabilize the colloidal gold particles was prepared according to the methods of Roth (1982a,b). Fresh 0.2 M K2CO3 solution was added to colloidal gold and pH was adjusted to 8.2. Purified anti-bromoxynil mAb (0.1 mg mL1) of 6 mL was added to 94 mL of dispersed gold solution to give a final concentration of 6 mg L1 by fast stirring and then incubated for 15 min at room temperature. It was followed by PEG (20 000, 1% as the final concentration) added by another 15-min stirring. The mixture was centrifuged for 30 min at 10 000 rpm and the supernatant was removed. In the end, the precipitate of the gold-labeled antibodies was washed with PBS (pH 8.2), centrifuged for twice, resuspended in PBS (pH 8.2) and then stored at 4 C.
2.4. Immobilization of reagents Biojet (XYZ3000) (Irvine, CA, USA) was used to dispense two lines on the NC membrane strips (25 300 mm). OVA-bromoxynil (0.5 mg mL1) was dispensed around the bottom as the test line (1 mL per 1 mm line) while 1 mg mL1 goat anti-mouse IgG was dispensed at the upper position as the control line (1 mL per 1 mm line). The NC membrane was dried for 2 h at 37 C, blocked with 1% BSA for 30 min, washed three times with 0.01 M PBS (pH 7.4) and then dried again at 37 C for 3 h. Finally, the strips were stored under dry conditions at room temperature until required. Gold-labeled anti-bromoxynil mAb (detective reagent) was dispensed onto a glass fiber filter with Airjet (XYZ3000) (Irvine, CA, USA) and then dried at 37 C for 3 h. The optimal concentration of the dispensed mixture was selected with OD value of 10 at 520 nm. Its dispensed volume could also be calculated as 1 mL for 1 mm line.
2.5. Development of the one-step strip The one-step strip was composed of three pads (sample, conjugate and absorbent pads) and an NC membrane. They were pasted onto an adhesive plastic backing. In particular, the NC membrane containing the OVA-bromoxynil and goat anti-mouse IgG was pasted at the center of the backing plate. The conjugate pad containing the gold-labeled mAb was pasted by over-crossing 2 mm with NC membrane. The sample pad (glass fiber) was also pasted by over-crossing 4 mm with the conjugated pad. The absorbent pad was pasted on the other side of the plate. The whole assembled plate was cut lengthways and divided into strips with a guillotine cutter (5 60 mm).
2.6. Analysis of water samples Water samples containing Bromoxynil (0.01e10 mg L1) were prepared from double distilled water, tap water, a commercial mineral water, and surface water of Huangpu River (Shanghai, China) with a known amount of bromoxynil derived from 100 mg L1 stock solution in 0.01 M PBS (pH 7.4). Particularly, the river water was collected in 1-L bottles and stored at 4 C until required. Tap water and river water samples were filtered through a 0.45 mm nylon filter. Each 100 mL sample supernatant was applied to
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one-step strip. Negative controls were water samples without bromoxynil. After 5 min, the test results were determined as positive or negative visually (Fig. 2). The control region (c) and test region (t) on the strip were made from the goat anti-mouse IgG and the OVA-bromoxynil individually. The mAb labeled with colloidal gold particles tended to react with bromoxynil by competition inhibition. When applied to a negative sample, the test and control lines turned red separately while a positive sample resulted in a red control line only. The test was invalid if no line was shown in the control region.
3. Results and discussion 3.1. Size selecting of colloidal gold particle Nano colloidal gold particles have a promising application such as immuno-analysis, biosensor and gene therapy (Deng et al., 2000; Nagatani et al., 2006). Since immunoglobulins could be labeled with nano colloidal gold particles instead of enzymes, the substrate is not needed in the reaction system. The strength of color reaction is closely related to the size and quality of the colloidal gold particles. The size of colloidal gold particles directly depends on the amount of trisodium citrate in its preparation process. Trisodium citrate is the key factor that influences the formation of particular cores. Changing the amount of trisodium citrate could result in different colloidal gold particles with different colors. In our study, six different types of colloidal gold particles were made. When more than 1.6 mL trisodium citrate (1%) was added in a 100 mL (0.01%) of gold chloride solution, the colloidal gold particles obtained were reddish and small with a diameter of less than 25 nm. They were found too small to indicate a clear and bright color. When less than 0.8 mL trisodium citrate was added, the color was between purple and blue with the diameter more than 55 nm. However, those large size colloidal gold particles were found not stable. Only when 1 mL trisodium citrate was added did the colloidal gold particles show a red color with a diameter of 40 nm. They were found stable when combined with immunoglobulins. The indicating color was obvious and easy to tell. The immunoreaction compound moved fast on the NC membrane. The reaction could be completed and remained stable. Therefore, the 40 nm colloidal gold particles were selected in this test (Fig. 1). 3.2. Analytical parameters of the optimized GICA To determine the appropriate concentration of OVA-bromoxynil coated on the membrane, the OVA-bromoxynil was prepared in 0.01 M PBS (pH 7.4) by serial dilutions ranging from 0.05 mg mL1 to 1.0 mg mL1. We found that the higher the OVA-bromoxynil concentration was, the more bromoxynil was needed in the sample to compete with OVA-bromoxynil. But below 0.5 mg mL1, the signal was too faint to be seen. As a result, 0.5 mg mL1 of OVA-bromoxynil was used to dispense on the NC membrane as the test line. The different buffers including 0.01 M PBS (pH 7.4), 0.1 M PBS (pH 7.4), carbonate buffer (pH 9.6) and borate buffer (pH 9.0) used for gold-labeled antibody were compared as to their
Fig. 1. The size of valid colloidal gold particles observed by transmission electron microscopy (120 000). The diameter of those particles was 40 nm.
effects on this assay. Generally, 0.01 M PBS (pH 7.4) was used in various immunoassays as a working solution (Table 1). A 0.01 M PBS (pH 7.4) and deionized water resulted in a lower detection limit in the one-step strip test. We found that the pH has significant effects on the assay and 0.01 M PBS (pH 7.4) was the best for the sensitivity.
3.3. Sensitivity, specificity and reliability of a one-step strip test The detection limit was defined as the amount of bromoxynil in the sample solution that just causes a complete invisibility of the test line. A series of dilutions of standard bromoxynil (0.01e10 mg L1) were assayed by strip tests (Table 2 and Fig. 2). The results were recorded 5 min after the reactions. Two lines detected on the NC membrane presented that the bromoxynil concentration was below the detection limit while only one line detected around the control zone showed that the bromoxynil concentration was above the detection limit. To ensure the detection, the lower detection limit of the optimized one-step strip was at 0.06 mg L1 since the concentration of 0.03 mg L1 of bromoxynil was found to cause a considerable decrease in the assay signal. The result of one-step strip test was very similar to that of ELISA developed by Yongsong Cao (Cao et al., 2005). However, one-step strip test usually offered lower sensitivity than ELISA. It was possible to improve the sensitivity by adjusting relative factors, which affect the assay system (Shyu et al., 2002). In particular, it was feasible to significantly improve the sensitivity of our method by silver staining. It was a key that samples were preconcentrated before the analysis by dichloromethane extraction. Another important way was to rectify the quantity of OVA-bromoxynil or mAb, which were dispensed on the strip by adjusting their concentration. Therefore, this assay has a great application potential. The specificity of this assay was evaluated in comparison to other analog compounds. The strips were dipped into water
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139
Table 1 Influence of deionized water, 0.01 M PBS (pH 7.4) and different organic solvents as working solutions on the GICA of bromoxynil Bromoxynil standard concentration (mg L1)
Working solution
0.01
0.03
0.06
0.12
0.25
0.5
1
2
5
10
0.01 M PBS(pH 7.4)
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
0.1 M PBS (pH 7.4)
Test line Control line
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
Carbonate buffer(pH 9.6)
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
Borate buffer (pH 9.0)
Test line Control line
þ þ
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
Deionized water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
20% Methanol
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
20% Acetone
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
20% Acetonitrile
Test line Control line
þ þ
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
þ: Red line appeared. : Red line appeared but very weak. : Red line did not appear.
samples containing different compounds for 10 s, taken out and laid on a flat table and the results were judged visually after 5 min. As shown in Table 3 and Fig. 2, the qualitative working range of the strip test was from 0.06 mg L1 to 5 mg L1 with a LOD of 0.06 mg L1 and the probability of occurring of cross-reaction was lower than 0.06 mg L1. This indicates that this assay had a specific reaction with bromoxynil and had not cross-reactivity with other related pesticides and herbicides in the qualitative working range. This method was not only rapid and simple, but also did not require intensive labor and expensive equipment for the sample analysis, especially for fast on-site detection. Also, it provided only a preliminary, semi-quantifying result simply with
positive or negative results. The results could help determine whether the pesticide concentrations in the samples were higher than the detection limit or not. In addition, a conventional and standard analysis was necessary for precise determination of the concentration of the pesticide. The reliability of the assay was determined by the tests with the standard water samples containing bromoxynil. Among those 110 samples, 55 samples were at 0.01 mg L1 and the other 55 samples were at 0.06 mg L1. The concentrations of bromoxynil in the treated samples were detected with GICA method and the ELISA method at the same time. The results obtained by the two methods were compared (Table 4). The overall agreement was 98%.
Table 2 Results of water samples by GICA Spiked bromoxynil concentration (mg L1)
Sample source
0.01
0.03
0.06
0.12
0.25
0.5
1
2
5
10
Double distilled water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Tap water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
A commercial bottled mineral water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Surface water
Test line Control line
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ: Red line appeared. : Red line appeared but very weak. : Red line did not appear.
J. Zhu et al. / Environmental Pollution 156 (2008) 136e142
140
Fig. 2. Immunochromatographic detection of bromoxynil. A series of dilutions (0.01e10 mg L1) of bromoxynil were prepared. Details of the preparation of the test device and assay procedures are described in the text. Bromoxynil (0.03 mg L1) led to a fainter test line. However, pure water and other analog compounds except 2,6-dibromophenol, chloroxynil and ioxynil presented an obvious test line even though analog compounds reached at a high level of 100 mg L1.
3.4. Sample analysis We analyzed prepared water samples by the one-step strip test. Fifty-five water samples were prepared as
mentioned above. One-step strip test showed two red lines for samples at the concentration of 0.01 mg L1. However, those samples of 0.06 mg L1 presented only one line (Table 4).
Table 3 Cross-reactivity (CR) with bromoxynil and related compounds by the one-step strip test Each compound concentration (mg L1)
Compounds
0.01
0.03
0.06
0.12
0.25
0.5
1
2
5
10
Bromoxynil
Test Control
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
Chloroxynil
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
Ioxynil
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
2,6-dibromophenol
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
þ
p-hydroxybenzonitrile
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
2,4-D
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
Dichlorprop
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
2,4-DB
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
MCPA
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
Mecoprop
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
MCPB
Test Control
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ: Red line appeared. : Red line appeared but very weak. : Red line did not appear.
J. Zhu et al. / Environmental Pollution 156 (2008) 136e142 Table 4 Reliability of the test strip compared to ELISA GICA Positive
Negative
ELISA Positive Negative
Total
53 0
2 55
55 55
Total
53
57
110
Comparison of the results between gold immunochromatagraph assay (GICA) and ELISA. Fifty-five samples for each concentration, a total of 110 samples were tested. The overall agreement is 98%.
4. Conclusions We presented the development and evaluation of a GICA for the qualitative detection of bromoxynil residue in the water. Bromoxynil standard sample of 0.01e10 mg L1 in water were determined by the one-step strip test. The limit was determined visually as 0.06 mg L1 and the probability of cross-reactions was lower than 0.06 mg L1. The results showed this assay led to a special reaction with bromoxynil without cross-reactions to other related pesticides and herbicides in the qualitative working range. Several factors affecting the immunochemical specificity have been demonstrated. What is more, pH was shown to have significant influence on the system and 0.01 M PBS (pH 7.4) was the best for the sensitivity. OVA-bromoxynil (0.5 mg mL1) was used to dispense on the NC membrane as the test line. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA and the overall agreement was 98%. The one-step strip test was rapid and simple. Also, it did not require intensive labor and expensive equipments for the sample analysis. The proposed GICA turned out to be a powerful tool for monitoring residual bromoxynil in water. In particular, the one-step strip test was suitable for on-site detection of water samples, e.g., for a water inspection center and a beverage company. In addition, the assay can be applied for the rapid detection of bromoxynil in agricultural and environmental samples. Acknowledgements This work was supported by National Natural Science Foundation of International Cooperation (NSFC-RFBR) (No. 20611120017), National Key Basic Research Program of China (No. 2004CB18503), National Natural Science Foundation of China (No. 20337010) and Key Program of Basic Research of Shanghai City (No. 04JC14051). We express our sincere gratitude to Dr. Yongsong Cao for supplying the hapten used for the production of anti-bromoxynil mAb. References Breton, F., Rouillon, R., Piletska, E.V., Karim, K., Guerreiro, A., Chianella, I., Piletsky, S.A., 2007. Virtual imprinting as a tool to design efficient MIPs
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