Anal. Chem. 1998, 70, 5047-5053
Immunosensor for 2,4-Dichlorophenoxyacetic Acid in Aqueous/Organic Solvent Soil Extracts Silke Kro 1 ger, Steven J. Setford, and Anthony P. F. Turner*
Cranfield Biotechnology Centre, Cranfield University, Bedfordshire MK43 OAL, U.K.
The development of a simple electrochemical immunoassay procedure for the field-based quantification of the herbicide 2,4-D in methanolic soil extracts is presented. The sensor utilizes a competitive immunoassay format incorporating an immobilized antigen complex at the surface of a disposable screen-printed working electrode element. The extent of glucose oxidase-labeled antibody binding to the antigen-electrode is determined amperometrically and is related to sample analyte concentration. The performance of the sensor is assessed in buffer, 30% methanol, and methanolic soil extracts. The device is capable of quantifying 2,4-D in all three matrixes at the low ppm level with coefficient of variation values of 6.233.6%. The causes of the variation observed in the sensor response in different soil matrixes are examined and improvements proposed. The sensor, tested in parallel with a commercial 2,4-D immunoassay test kit, yields comparable quantitative data and detection limits while exhibiting greater assay simplicity. Conventional herbicide soil analysis procedures are lengthy, requiring organic solvent extraction,1-3 extract purification/ preconcentration,4-6 and chromatographic analysis.5,7,8 Such procedures are also expensive, consume large amounts of solvent, and require trained personnel. A simple in-situ sample prescreening method would reduce the number of samples that have to undergo such analysis and, where appropriate, accelerate the onset of containment or remediation procedures. The combination of simple, portable, lowcost electrochemical measurement systems with the specificity and sensitivity associated with enzyme immunoassay (EIA) * To whom correspondence should be addressed (http://www.cranfield. ac.uk/biotech). (1) U.S. Environmental Protection Agency. Method 3540. Test methods for evaluating solid wastes SW-846. Field Manual Physical/Chemical methods, 3rd ed.; U.S. EPA: Washington, DC, 1986. (2) U.S. Environmental Protection Agency. Method 3550. Test methods for evaluating solid wastes SW-846. Field Manual Physical/Chemical methods, 3rd ed.; U.S. EPA: Washington, DC, 1986. (3) U.S. Environmental Protection Agency. Method 8150. Test methods for evaluating solid wastes SW-846. Field Manual Physical/Chemical methods, 3rd ed.; U.S. EPA: Washington, DC, 1986. (4) Pico´, Y.; Molto´, J. C.; Man ˜es, J.; Font, G. J. Microcolumn Sep. 1994, 6, 331-359. (5) Loconto, P. R. J. Liq. Chromatogr. 1991, 14, 1297-1314. (6) Butz, S.; Stan, H.-J. Anal. Chem. 1995, 67, 620-630. (7) Sa´nchez-Brunete, C.; Garcı´a-Valca´rel, A. J.; Tadeo, A. J. J. Chromatogr. A 1994, 675, 213-218. (8) Kim, I. S.; Sasinos, F. I.; Stephens, R. D.; Wang, J.; Brown, M. A. Anal. Chem. 1991, 63, 819-823. 10.1021/ac9805100 CCC: $15.00 Published on Web 10/24/1998
© 1998 American Chemical Society
procedures represents a promising means to achieve this aim.9-13 This approach is of particular interest given the recent acceptance of immunoassay-based methods for soil analysis by the U.S. EPA.14 Several 2,4-D immunosensors have been reported in the literature.15,16 Kala´b and Skla´dal17 describe the use of screenprinted electrodes in the construction of a disposable amperometric biosensor for detection of 2,4-D in water. A competitive immunoassay format, incorporating direct antigen immobilization to surface-silanized gold working electrodes (WEs) and free horseradish peroxidase-antibody conjugates in solution, was used. A detection limit of ∼0.1 ng mL-1 (0.1 ppb) 2,4-D was achieved with an analysis time of 1 h. Extending the concept, an eight working electrode array, incorporating a 30-µL microwell over each WE, was constructed.18 A competitive immunoassay format was again used, but with free peroxidase-labeled 2,4-D competing with 2,4-D analyte for membrane-immobilized antibody binding sites. A similar detection limit was recorded while allowing several tests to be conducted simultaneously. None of the above approaches considered the analysis of 2,4-D in soil, where analyte solubility necessitates the presence of organic solvent and factors such as soil matrix interferences have to be considered. Field-based assay procedures require simplicity, thus the extraction and analysis procedures should be compatible. (9) Stanley, C. J.; Cox, R. B.; Cardosi, M. F.; Turner, A. P. F. J. Immunol. Methods 1988, 112, 153-161. (10) Aga, D. S.; Thurman, E. M. ACS Symp. Ser. 1997, 657, 1-20. (11) Treloar, P.; Kane, J.; Vadgama, P. In Principles and Practice of Immunoassay, 2nd ed.; Price, C. P., Newman, D. J., Eds.; Macmillan: London, 1997; pp 483-509. (12) Gizeli, E.; Lowe, C. R. Curr. Opin. Biotechnol. 1996, 7, 66-71. (13) Sadik, O. A.; Van Emon, J. M. ACS Symp. Ser. 1996, 646, 127-147. (14) U.S. Environmental Protection Agency. Method 4015. Test methods for evaluating solid wastes SW-846. Field Manual Physical/Chemical methods, 3rd ed.; U.S. EPA: Washington, DC, 1986. (15) Yulaev, M. F.; Sitdykov, R. A.; Dmitrieva, N. M.; Dzantiev, B. B.; Zherdev, A. V.; Askarov, K. A. J. Anal. Chem. 1995, 50, 194-197. (16) Bier, F. F.; Ehrentreich-Fo¨rster, E.; Do¨lling, R.; Eremenko, A. V.; Scheller, F. Anal. Chim. Acta 1997, 344, 119-124. (17) Kala´b, T.; Skla´dal, P. Anal. Chim. Acta 1995, 304, 361-368. (18) Skla´dal, P.; Kala´b, T. Anal. Chim. Acta 1995, 316, 73-78. (19) Dastoli, F. R.; Musto, N. A.; Price, S. Arch. Biochem. Biophys. 1966, 115, 44-47. (20) Hall, G. F.; Best, D. J.; Turner, A. P. F. Enzyme Microb. Technol. 1988, 10, 543-546. (21) Saini, S.; Hall, G. F.; Downs, M. E. A.; Turner, A. P. F. Anal. Chim. Acta 1991, 249, 1-15. (22) Kro ¨ger, S.; Setford, S. J.; Turner, A. P. F. Anal. Chim. Acta 1998, 368, 219-231. (23) Iwuoha, E. I.; Smyth, M. R.; Lyons, M. E. G. Biosens. Bioelectron. 1997, 12, 53-75. (24) Sto ¨cklein, W. F. M.; Scheller, F. Chem.-Ing.-Tech. 1995, 67, 69-77. (25) Mionetto, N.; Marty, J.-L.; Karube, I. Biosens. Bioelectron. 1994, 9, 463470.
Analytical Chemistry, Vol. 70, No. 23, December 1, 1998 5047
Table 1. Soil Characteristics name
clay (%)
silt (%)
sand (%)
dry matter (%)
A. sandy loam soil B. organic-rich topsoil C. high-organic soil D. low-organic soil
15.0 7.7 7.6 5.1
19.0 11.3 11.4 4.5
66 81 81 90
98.0 97.9 99.6
Ideally, the assay procedure should be capable of operating within the sample extract. The fact that enzymes18-25 and antibodies26-28 can retain activities in organic solvent environments indicates thepossibility of using biochemical analysis directly in the presence of soil extracts containing organic solvent. This paper describes the development of a rapid field-based extraction and analysis method for the quantification of 2,4-D in methanol-aqueous buffer mixtures and methanol-buffer soil extracts using screen-printed immunosensor devices. The effect of methanol and different soil matrix components on immunosensor performance is examined. EXPERIMENTAL SECTION Screen-Printed Electrode Fabrication. The screen-printed electrode devices incorporated a rhodinised-carbon WE stabilized in hydroxyethyl cellulose, carbon counter electrode and silver/ silver chloride reference electrode. The electrode design, fabrication and characterization procedure has been described elsewhere.29 Materials were selected to allow device operation in organic solvent environments. General Reagents. Methanol (HiPerSolv) and phosphate buffer salts were purchased from BDH (Atherstone, UK). Polyoxyethylene-sorbitan monolaurate (Tween 20), 2,4-dichlorophenoxyacetic acid (99%), glucose, hydrogen peroxide, and KCl were all purchased from Sigma-Aldrich (Gillingham, UK). Aqueous solutions were prepared with reverse osmosis (RO) water. Biological Reagents. Anti-2,4-D murine monoclonal antibodies were produced by Cell Diagnostica GmbH (Mu¨nster, Germany). Clones tolerant to 30% (v/v) methanol were selected after screening with a resonant mirror biosensor. Glucose oxidase (GOx), specific activity 8350 units mL-1 (340 units mg-1) was purchased from Biozyme Laboratories (Gwent, UK, code GO3S). Antibody-GOx conjugates, also supplied by Cell Diagnostica, were prepared by carbodiimide coupling (∼0.4 mg mL-1). 2,4-D antigen was coupled to bovine serum albumin (BSA) using a carbodiimide/activated ester method at the ICB (Mu¨nster, Germany). The conjugate ratio was assumed to be 11-13 molecules of 2,4-D per molecule BSA, supplied as a 5 mg mL-1 protein solution in phosphate buffer, pH 7.4. Preparation of Antigen-Electrodes. Direct physical adsorption: 4-µL aliquots of 2,4-D-BSA complexes (various dilutions in 0.1 M carbonate buffer, pH 9.6) were dried onto WE pads on an orbital shaker (200 rpm). Control electrodes were prepared (26) Russell, A. J.; Trudel, L. J.; Skipper, P. L.; Groopman, J. D.; Tannenbaum, S. R.; Klibanov, A. M. Biochem. Biophys. Res. Commun. 1989, 158, 80-85. (27) Sto ¨cklein, W. F. M.; Gebbert, A.; Schmidt, R. D. Anal. Lett. 1990, 23, 14651476. (28) Goh, K. S.; Hernandez, J.; Powell, S. J.; Greene, C. D. Bull. Environ. Contam. Toxicol. 1990, 45, 208-214. (29) Kro¨ger, S.; Turner, A. P. F. Anal. Chim. Acta 1997, 347, 9-18.
5048 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
CEC (mequiv/100g)
organic carbon (%)
pH
12
1.0 4.2 11.8 0.7
6.2 4.2 4.1 7.5
33 7
using appropriate dilutions of 5 mg L-1 BSA in buffer. Electrodes were washed to remove loosely bound 2,4-D-BSA. The washing procedure included a drying step (10 min, 60 °C) followed by 20 forward/backward motions in 0.5% Tween/phosphate buffer and rinsing in RO water. Electrodes were redried and stored dry (4 °C over silica). Soils and Soil Spiking. The soil types used in this study are summarized in Table 1. Soil A (type Pt008) was purchased from Levington Agriculture (Ipswich, UK). Other soils were supplied by the Soil Survey and Land Research Centre (Cranfield University, Silsoe, UK). Soil sample histories are not known. Soil B was from the same source as soil C but was dried and sieved to guarantee more homogeneous sample material. Soil samples were weighed into precleaned glass vials with polypropylene screw tops to which were added freshly prepared 2,4-D stock solutions in methanol (10 mL of a 20 ppm 2,4-D solution was used to spike 20 g of soil to the 10 ppm level). The methanol was evaporated at room temperature on an orbital shaker (400 rpm). The dry spiked soil sample was stored at 4 °C for a minimum of 12 h prior to extraction to simulate more realistic field conditions. Soil Extraction. Methanol (30% ((v/v)); 2:1 v/w to soil) in 0.1 M phosphate buffer/0.1 M KCl was added to the samples. The soil slurry was sonicated for 5 min, followed by 5 min of orbital shaking (400 rpm), and this procedure was repeated three times. Samples were left for 1 h to allow soil particles to sediment and then filtered through a 0.2-µm nylon filter. The commercial immunoassay test kit approach employed an Envirogard soil extraction bottle kit containing mixing beads (SDI Europe Ltd., Avon, UK). The sample was shaken in 60% (v/v) methanol in water (2:1 v/w to soil) for 2 min and then incubated for 16 h at 4 °C. Supernatant fractions were filtered through a filter cap and a Millex-HV13 filter unit. Commercial Test Kit Measurements. The immunosensor was tested in parallel with an Envirogard 2,4-D in soil test kit, tube format (SDI Europe Ltd.). The kit uses an indirect competitive assay format with tube-immobilized antibody and complies with U.S. EPA method 4015 (1995) for screening soils and aqueous material for 2,4-D by immunoassay. Filtered samples from the commercial extraction kit were diluted 100-fold in phosphate buffer, and 200-µL volumes were mixed with an equal volume of 2,4-D-enzyme solution in supplied tubes prior to shaking for 10 s and incubation for 20 min. The tubes were washed four times with water, filled with 500 µL of substrate, and incubated for 10 min. The reaction was halted with 500 µL of 1 N HCl and the absorbance measured at 450 nm. Immunosensor Measurements. The selected method was an indirect competitive assay format incorporating preincubation, binding, and washing steps, all performed at room temperature.
Figure 1. 2,4-D extraction and immunosensor analysis procedure.
Ten microliters of appropriately diluted antibody-GOx conjugate was added to 30 µL of sample in an Eppendorf cup, mixed (vortex), and then centrifuged (30-s pulse, 13 000 rpm, bench minicentrifuge) to ensure good contact. After 15 min of preincubation, 10 µL of sample was transferred to the WE. A 15-min binding stage was employed in a sealed box containing wet tissues to ensure a high-humidity environment. The box was orbitally shaken (100 rpm) to promote sample mixing and binding. Electrodes were rinsed in RO water and washed in 0.05% buffered Tween 20 followed by pure buffer (20 forward/backward motions in each). The electrodes were stored in the wet box until tested. Surface-bound GOx activity was measured electrochemically as described earlier.30 The electrodes were equilibrated in unstirred 0.1 M phosphate buffer, pH 7.0, containing 0.5 M glucose and 0.1 M KCl (WE poised at +300 mV vs Ag/AgCl reference). A single-point current measurement was recorded after 2 min using an Autolab electrochemical analyzer with GPES3 software (EcoChemie, Utrecht, The Netherlands).
Figure 2. Binding of antibody-GOx conjugate to catalytic carbon working electrodes modified with either 2,4-D-BSA (specific binding) or BSA (nonspecific binding).
RESULTS AND DISCUSSION Analytical Procedure. The complete analytical procedure for the sample preparation and immunoassay is depicted schematically in Figure 1. The conjugate concentration at which the specific binding of antibody to WE-immobilized antigen was greatest with respect to the nonspecific binding of conjugate was sought. A dilution series of antibody-GOx was prepared in 30% methanol ((v/v) in 0.1 M phosphate buffer) and incubated with 2,4-D-antigen electrodes (4 µL of a 1:100 dilution of 2,4-D-BSA conjugate stock solution; equivalent to 200 ng of material per electrode). Results are depicted in Figure 2. Nonspecific conjugate binding was observed at all conjugate dilutions. The greatest ratio between specific and nonspecific
binding was observed at the 1:40 conjugate dilution. The 1.5-µA response difference was deemed sufficient for immunosensor development. The overall response of 2 µA obtained at this conjugate dilution was within the linear range of the device. Antigen electrodes with varying 2,4-D-BSA quantities were incubated for 15 min in the presence of 10-µL aliquots of 1:40 diluted antibody-GOx conjugate solution. Results are illustrated in Figure 3. The 1:100 2,4-D-BSA dilution (equivalent to 200 ng of protein) gave the highest response and was employed for subsequent experimentation. The response decrease at higher 2,4-D-BSA concentrations was believed to be due to “overloading” of the electrode. Washing of the device after the binding step may result in removal of insufficiently adsorbed 2,4-D-BSA and hence antibody-GOx conjugate. High BSA surface loadings may also result in the creation of a diffusion barrier or insulating layer at the electrode surface.31 BSA, casein, and a commercial blocking
(30) Kro¨ger, S.; Setford, S. J.; Turner, A. P. F. Biotechnol Tech. 1998, 12, 123127.
(31) Blonder, R.; Katz, E.; Cohen, Y.; Itzhak, N.; Riklin, A.; Willner, I. Anal. Chem. 1996, 68, 3151-3157.
Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
5049
Figure 3. Device responses after binding of 1:40 diluted antibodyGOx conjugate to antigen-electrodes containing varying amounts of 2,4-D-BSA. Error bars ) SD, n ) 3.
buffer (SuperBlock, Pierce) were all evaluated as a means of minimizing potential nonspecific binding sites, but with minimal success. Immunosensor Measurements. Immunosensor devices, incorporating 200 ng of physically immobilized 2,4-D-BSA, were incubated in the presence of 10 µL of 1:40 antibody-GOx and 30 µL of sample. The sensor response was tested in three different sample matrixes spiked with 1 ppb-1000 ppm 2,4-D. The matrixes were 0.1 M phosphate buffer, 30% (v/v) methanol in phosphate buffer, and soil type D, extracted with 30% (v/v) methanol by the filtration method and then spiked with 2,4-D. Measurements were performed in triplicate on separate days using fresh 2,4-D preparations. A sigmoidal relationship between the assay response and analyte concentration, characteristic of the competitive immunoassay format, was observed in each matrix (Figure 4). Device reproducibilities are also shown in Figure 4. The data indicate that the immunosensor is capable of distinguishing 2,4-D concentrations in buffer, methanol, and methanolic soil extracts in the low ppm range. While the validity of the immunosensor approach has been proven at this stage, regulatory and commercial requirements would necessitate improvements to device reproducibilities. The logit-log model, the most widely used procedure for immunoassay data evaluation, was employed to further analyze the data.32 The model represents a continuous sigmoidal function with a single inflection point, described by the equation
y ) (a - d)/(1 + (x/c)b) + d where a represents the maximum current at zero analyte (upper asymptote), b the slope of the curve at midpoint (50% signal reduction), c the analyte concentration at midpoint, and d the residual current at infinite dose (lower asymptote; background current + nonspecific binding). These constant values were calculated using the curve fit (math) function of Sigma Plot for Windows and can be seen in Figure 4. Values of c were found to be 3.71, 5.09, and 5.80 ppm in the buffer, 30% (v/v) methanol, and soil D extract matrixes, respectively. Buffer Matrix. The calculated logit-log values for a and d in buffer (1.72 and 0.86 µA, respectively) did not concur with (32) Rodbard, D.; Cooper, J. A. In vitro Procedures with Radioisotopes in Medicine; International Atomic Energy Agency: Vienna, 1970; pp 659-673.
5050 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
Figure 4. Immunosensor response in buffer, 30% (v/v) methanol, and soil D methanolic extracts spiked with varying amounts of 2,4-D.
experimental data (a ) 1.45 µA in unspiked buffer; d ) 0.79 µA using BSA-modified electrodes). This trend was repeatedly observed in subsequent experiments. A possible reason for this phenomenon is proposed, based on the experimental data illustrated in Figure 5. Figure 5 shows that a sigmoidal relationship exists between assay response and free analyte concentration when using electrodes that have been modified with BSA only. Thus, it is possible to discriminate between different 2,4-D concentrations on the basis of nonspecific binding of the analyte to the WE surface. A similar correlation was also observed with caseinmodified electrodes, but not with unmodified electrodes. This phenomenon would account for the discrepancy between the calculated and experimentally determined d values. The exact mechanism by which the concentration of free 2,4-D influences the binding of antibody to the protein immobilized at the electrode surface is not known. Possibly, the adsorptive behavior between the antibody and protein changes upon binding of antigen. Alternatively, there may be a degree of cross-reactivity
Figure 5. Influence of free 2,4-D concentration on the nonspecific binding of antibody-GOx conjugate to BSA-modified electrodes in phosphate buffer, pH 7.
between the antibody and BSA since the antibody was raised to 2,4-D-BSA (given that this phenomenon was observed also in the presence of casein, this cross-reactivity would not be very specific for BSA). The unexpected increase in conjugate binding observed at low concentrations of 2,4-D (d value) can likewise be explained in a number of different ways. It is conceivable that free 2,4-D binds to hydrophobic binding pockets on the BSA molecule, adding further “specific” binding sites. Alternatively, the phenomenon of “cooperative binding”,33,34 whereby the binding of antigen to one binding site on an antibody predisposes the antibody to filling the remaining binding site, may be occurring. At low concentrations of free 2,4-D, a higher proportion of antibodies will have one filled site and, consequently, could bind more favorably to immobilized antigen. Methanol Matrix. Higher current responses were obtained when methanol was present in the incubation step (Figure 4). In quantitative terms, as indicated in Figure 4, a ∼40% increase in device response at zero dose (a) with a minimal change in nonspecific response (d) was recorded. Since previous work has shown that GOx preincubation in methanol does not increase enzyme activity,30 enhanced antibody-immobilized antigen binding in methanol is strongly suggested. For example, changes in the conformation of 2,4-D-BSA conjugate in methanol, allowing more direct interaction of the antibody with immobilized antigen, may be occurring. It is also to be expected that the presence of organic solvent will affect antigen-antibody binding, particularly by altering the electrostatic, hydrophobic, hydrogen-bonding, and van der Waals forces involved in the binding mechanism. The clone used in this study was selected for its antigen binding activity in methanol, and it is possible that the solvent has a positive effect on the affinity interaction. Soil Matrix. The presence of soil extract in the methanol matrix led to a reduction in the current signal when compared with the corresponding signals obtained in 30% (v/v) methanol (Figure 4, zero (constant a) and infinite (constant d) signal responses were reduced by 0.44 and 0.29 µA, respectively, in soil extract). However, the zero response value of 2.21 µA was still (33) Rodbard, D.; Bertino, R. E. Advanced Exper. Med. Biol. 1973, 36, 327341. (34) Carayon, P.; Carella, C. FEBS Lett. 1974, 40, 13-17.
appreciably higher than that recorded in pure buffer (1.72 µA), thus the positive effect of the solvent is still apparent. The 2,4-D concentration at the midpoint of the slope was little affected by the presence of soil extract (5.80 vs 5.09 ppm). The fact that soil extracts can interfere with immunological measurements is well documented35 and is dependent on factors such as organic matter content and the nature of the soil sample. Causes considered for the signal reduction included electrode fouling, inhibition of the enzymatic label by compounds present in the extract, or cross-reactivity of the antibody with extracted soil components such as humic acids. Studies involving the detection of hydrogen peroxide with electrodes preincubated in various soil extracts revealed small changes in the electrode responses, depending on the soil type under investigation. Electrode fouling can thus not be excluded but is unlikely to be the sole cause of the observed decrease in immunosensor response. Experiments undertaken with glucose oxidase adsorbed to the working electrode and measurement of its activity directly in soil extracts (soil B) revealed a 24-27% decrease in response. This decrease was considerably less than that observed when free hydrogen peroxide was injected into this extract (58% decrease) and can, at least in part, be explained by dissolved organic matter or soil particles acting as a catalyst for the degradation of peroxide, resulting in decreased concentration of hydrogen peroxide available for oxidation at the electrode surface. A small degree of enzyme inhibition by soil extracts is also possible. It is concluded that the observed reduction in current signal of the immunosensor when analyzing soil extracts could have been caused by a number of factors and combinations thereof but did not prevent the application of the device in such extracts. To evaluate to which extent different soil extracts influence the sensor response, the immunosensor performance was assessed in methanolic extracts of the four different soil types. Device responses to unspiked extracts and extracts spiked with 10 ppm 2,4-D are shown in Table 2. Similarly spiked and unspiked 30% (v/v) methanol solutions were also tested. Reduction in current signals was variable for the different soil extracts analyzed. A variety of factors were believed responsible, including organic matter content, pH, and soil pretreatment. While such matrix effects are undesirable, they are common to most environmental biosensor systems. The similarity in signal reduction for the 2,4-D spiked extracts indicates the applicability of the device as a field-based screening method. If required, matrix effects could be reduced by complex sample pretreatment, but this would detract from the proposed use of the device as a decentralized testing method. A more attractive alternative would be to use simple field methods to assess soil extract properties to allow more reliable measurements to be made. For example, correlations have been observed between the humic load of an aquatic solution and sample absorptivity at 280 nm.36 This observation suggests that spectroscopic analysis of soil extracts may be useful in estimating the nature and concentration of interferents, thus allowing the generation of more accurate immunoassay data. Standard methods of measuring (35) Lie´geois, E.; Dehon, Y.; De Brabant, B. P. P.; Portetello, D.; Copin, A. Sci. Total Environ. 1992, 123/124, 17-28. (36) Peuravuori, J.; Pihlaja, K. Anal. Chim. Acta 1997, 337, 133-149.
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Table 2. Influence of Soil Type on Immunosensor Response at 0 and 10 ppm 2,4-D 0 ppm
10 ppm
samplea
mean
SD
CV (%)
% reductionb
mean
SD
CV (%)
% reductionb
30% methanol soil D (0.7%) soil A (1.0%) soil B (4.2%) soil C (11.8%)
2.08 1.92 1.69 1.77 2.04
0.18 0.47 0.57 0.51 0.86
8.8 24.2 33.5 29.2 42.0
0.0 7.7 18.8 14.9 1.9
1.38 1.37 1.40 1.04 1.18
0.28 0.37 0.39 0.13 0.10
20.4 26.9 28.1 12.5 8.8
33.7 34.1 32.7 50.0 43.3
a Values in parenthesis refer to organic matter content of soil. b Percent reduction values were based on the current reading in the unspiked 30% (v/v) methanol, taken as 100%.
dissolved organic carbon or total organic carbon are not compatible with methanolic extracts. Electrochemical multianalyte analysis presents another attractive route for quantification of soil extract matrix effects. An array of screen-printed electrodes could be used to quantify the analyte and major interferences by immunoassay (Ulrich et al.37 have described an EIA system for humic acid quantification). Other electrodes could be employed to measure factors such as pH, conductivity, electrode fouling, and electroactive substances. Potentially, this route, combined with sophisticated data analysis techniques, provides a powerful means of minimizing interference problems. Immunosensor versus Envirogard Immunoassay Test Kit. A calibration curve using type D soil filtration extracts, spiked with 2,4-D (1 ppb-1000 ppm) after the extraction step, was obtained with the immunosensor (triplicate measurements). The logit-log values obtained were a, 1.83 µA; b, 0.89 µA/ppm; c, 0.95 ppm; and d, 0.93 µA. The midpoint (c) value was appreciably lower than that previously obtained for the soil extract matrix (5.80 ppm). This discrepancy may be due to soil extract differences, although the changes in the respective currents at zero and infinite dose (a and d values) suggest that interbatch variation in the antigenelectrodes is the most likely cause. The above-described logitlog values were used to determine 2,4-D levels in type D soil filtration extracts prespiked with 2,4-D (0-25 ppm) before extraction. Tests were performed using the same antigen-electrode batch as used to construct the calibration curve in order to maximize assay accuracy. Results are shown in Table 3. Overall, a good correlation was observed between the spiked (column 1) and measured (column 5) 2,4-D levels. The poor correlations (0.01 ppm, 25 ppm 2,4-D) were outside the pseudolinear range of the system, where small variations in current correspond to large differences in concentration readings. The correlation was highest around the midpoint of the assay where, at a nominal concentration of 2.5 ppm, the measured 2,4-D concentration was 74% of this value (1.85 ppm). Interestingly, HPLC analysis of soil samples spiked with 2.5 ppm 2,4-D yielded recovery values of 70-80% after 30% (v/v) methanol extraction.38 These findings support the use of the immunosensor as a screening tool for 2,4-D quantification in soils at the low ppm level. With reevaluation for different soil types, semiquantitative analyses should also be possible. (37) Ulrich, P.; Weller, M. G.; Knopp, D.; Niesser, R. Anal. Sci. 1993, 9, 795797. (38) Electrochemical Biosensors for Monitoring Pesticides in Soil and Water; Final Report to European Commission; MAT1-CT94-0025, April 1997.
5052 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
Table 3. Immunosensor Data for Soil D, Spiked with 2,4-D and then Extracted spiked 2,4-D concn (ppm)a
mean (µA)
SD (µA)
CV (%)
measured 2,4-D concn (ppm)
0 0.01 0.10 0.25 0.50 2.50 25.00
1.98 1.98 1.74 1.60 1.50 1.25 0.96
0.19 0.12 0.05 0.11 0.06 0.11 0.08
9.4 6.1 3.0 6.8 3.7 8.9 8.3
0.00 0.00 0.08 0.29 0.51 1.85 41.80
a The analyte concentration was calculated from the current signal using a calibration curve established by spiking 2,4-D into soil extracts using the same batch of antigen-electrodes.
Figure 6. Envirogard test kit calibration curve for soil D, extracted and then spiked with 2,4-D. Corresponding logit-log values were a, 0.57 OD; b, 0.94 OD/ppm; c, 1.94 ppm; d, 0.07 OD.
Type D soils spiked with 2,4-D (1 ppb-100 ppm) either before or after extraction were extracted using the commercial soil extraction kit and tested using the Envirogard kit. Tests were performed in the presence of 0.3% (v/v) methanol (200-fold dilution of 60% (v/v) methanol). The calibration curve, constructed using postextract spiked soil D data, is shown in Figure 6. Comparison of Figure 6 with Figure 4 shows that both systems operate over a similar analyte concentration range. A comparison of the performances of the immunosensor and commercial test kit for quantification of 2,4-D in the preextract spiked type D soils is given in Figure 7. The commercial kit did not perform as well as expected, underestimating the 2,4-D concentrations of the calibrator and extract solutions. The reason for this was unclear, given that the calibration protocol had performed as expected (Figure 6). It should be noted that the performance of both the assay procedure and the methanol extraction procedure is under test when using preextract spiked
Figure 7. Comparison of immunosensor and Envirogard test kit performance for measurement of extracts from 2,4-D-spiked soils. Response values are recorded as a percentage of the zero dose response. Immunosensor data are recorded as a function of the background-corrected response value.
soil samples. The statistical evaluation of immunoassay and immunosensor data, particularly field-based systems, is the subject of much discussion and controversy.39 Common definitions of immunoassay detection limit are the analyte concentration corresponding to 90% of the zero dose response and the subtraction of three standard deviations from the zero dose response. Using the former definition, the immunosensor had a detection limit of 0.21 ppm 2,4-D, while the latter definition gave a value of 0.67 ppm. The Envirogard test kit had a stated positive response criterion of 0.2 ppm 2,4-D. However, the test kit required a soil extract dilution of 1:200, compared with 3:1 for the immunosensor (the soil extract applied to the working electrode contained 30 µL of sample and 10 µL of conjugate). Thus, further optimization, or alternatively a search for more solvent resistant anti-2,4-D monoclonal antibodies, would allow immunosensor measurements of 2,4-D at the ppb level. Cross-reactivity studies were performed on the antibody by co-workers at the ICB (see Acknowledgments) during the initial clone selection program. Antibody cross-reactivity with structurally similar herbicides was noted. Thus, the device may prove more suitable as a general detector for the phenoxyacetic acid herbicide group and associated degredation products rather than as an exclusive 2,4-D detector.
methanol, and methanolic soil extracts at the ppm level. Enhanced device performance was observed in the presence of 30% (v/v) methanol, believed to be due to enhanced antibody-antigen binding. The presence of soil extract in the methanol matrix yielded a reduction in device performance, although the assay displayed a higher response compared with equivalent tests performed in pure buffer. Device reproducibilities in all of the matrixes tested, expressed as coefficients of variation, were between 6.2 and 33.6%. Device performance varied according to the different soil type tested, due to a range of factors. Proposed routes to circumvent this problem, including simple spectroscopic analysis of the soil extract and the use of multi-analyte arrays coupled to suitable data analysis programs were suggested. The performance of the immunosensor device was tested in parallel with a commercial EIA-based test kit. Both systems were found to operate over a similar analyte range with similar detection limits and assay times (∼30 min). However, the sensor approach utilized a rapid sample extraction procedure (16 h), and did not require excessive sample dilution. Furthermore, the sensor device required only a single-point method, did not require strict timing, and used no harmful stop solution (1 N HCl for the test kit). The electrochemical assay developed, although simple to perform and operate in remote environments, still requires a number of sample-handling steps. A separation-free assay, in which the binding step occurs directly in the measurement solution, would further simplify the process and represents an area of future development. An improved antigen immobilization process, coupled with higher affinity antibodies, would also benefit the assay. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the EC Measurement and Testing Program (MAT1-CT94-0025). We also thank our project partners at the Institute for Chemical and Biochemical Sensor Research and Cell Diagnostica GmbH (both of Mu¨nster, Germany) for the screening and supply of biological reagents and for many helpful discussions.
CONCLUSIONS The screen-printed immunosensor device has proven a valid tool for the quantification of the herbicide 2,4-D in buffer,
Received for review May 8, 1998. Accepted September 11, 1998.
(39) Gerlach, R. W.; Van Emon, J. M. ACS Symp. Ser. 1996, 646, 265-284.
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