Anal. Chem. 1996, 68, 3364-3369
Microformat Imaging ELISA for Pesticide Determination Anatoli Dzgoev,* Michael Mecklenburg, Per-Olof Larsson, and Bengt Danielsson*
Department of Pure and Applied Biochemistry, The Chemical Center, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
A flat-well microformat competitive enzyme-linked immunosorbent chemiluminescent assay for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film technology was used to pattern a hydrophobic layer 100 µm thick onto glass microscope slides to form an array of 2 × 2 mm2 squares. These flat wells were able to hold 2 µL of reagents, corresponding to a height of ∼500 µm, with minimal contamination risk. The hydrophobic ink used to pattern the surfaces allowed significantly larger volumes of samples to be applied when compared with surfaces patterned with nonhydrophobic inks. This reduced evaporation effects and permitted greater pipetting accuracy, thereby improving assay reproducibility. A competitive immunoassay was developed based on the ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal antibodies to 2,4-D-bovine serum albumin conjugate adsorbed onto the glass support. The support was subsequently incubated with alkaline phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate bound was determined by quantitating the chemiluminescent emission produced from the enzymatic breakdown of CSPD substrate by AP using a cooled CCD camera. The detection limit of the singlesample microformat assay was 2.7 × 10-11 M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10-8-4.5 × 10-11 and 4.5 × 10-7-1.66 × 10-10 M, respectively. In comparison, the detection limits of a tube-based chemiluminescent assay using standard luminometer and of a colorimetric ELISA were 45 × 10-11 and 9.9 × 10-8 M, respectively. The ability to scale the thick-film-based microformat assay makes it an ideal candidate for the development of affinity arrays and high-throughput assay formats. Prospects for further improvements of this imaging ELISA strategy will be discussed. There is an ever-increasing demand for high-throughput (HTP) assay technology for large-scale assaying of natural products (biodiversity), chemical and biological combinatorial libraries, genetic screening, and genome mapping, as well as for specific analyte detection.1 In addition, the proliferation of new diseases such as HIV and bovine spongiform encephalitis (BSE) increasingly requires rapid and flexible screening technology. The development of HTP assays based on current assay formats would * Address correspondence to these authors. E-mail: Anatoli.Dzgoev@ tbiokem.lth.se and
[email protected]. (1) Brown, R. K. Automated and accelerated high-throughput screening; International Business Communications, Inc.: Southborough, MA, 1996.
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require vast increases in laboratory space, labor, and reagents, which would make them economically and environmentally unfeasible. Thus, assay miniaturization is of central importance to all real-world HTP strategies. The microtiter footprint is the most widely used HTP assay format. Advances in injection molding technology have allowed the development of high-quality plastic products for a wide range of applications. The HTP 386 and 720 well formats, using the microtiter footprint, are examples of the more recent advancements in this area. Indeed, injection molding has numerous advantages and will continue to be the method of choice for many applications. However, this strategy will clearly become limiting as miniaturization continues. Thick- and thin-film patterning technology provides a more scalable strategy, which can be rapidly adapted to incorporate new materials and/or assay needs. A variety of techniques have been used to pattern surfaces, including thick-film, inkjet, and elastomer printing, with a variety of “inks”.2,3 Thick-film patterning is the most widely used technique for this purpose.4 The characteristics of the film can be modified to provide a wide range of surface qualities. A prime example is the development of hydrophobic surface coatings, which can be patterned onto hydrophilic surfaces. The sharp contrast in hydrophilicity results in the strong adhesion of aqueous solutions, which provides accurate positioning and minimizes crosscontamination. This allows aqueous samples to extend several times higher than the physical height of the“flat well”, which greatly increases the sample size, thereby reducing evaporation effects and increasing pipetting reproducibility. The planar nature of the format lends itself to automated deposition as well as surface-based detection techniques. Furthermore, the format provides a flexible manufacturing strategy, which allows rapid development/adaptation to new assay requirements, which can be applied to a wide range of materials. Chemiluminescent assays have several advantages, including excellent sensitivity, speed, absence of hazardous reagents, and compatibility with current assay formats. In addition, substrates are available for most of the enzymes employed in ELISAs, such as horse radish peroxidase (HRP), alkaline phosphatase (AP), and β-galactosidase. The use of 1,2-dioxetane phenyl phosphate substrates for the chemiluminescent detection of AP and AP conjugates is rapidly increasing.5 A detection limit of 0.001 amol (2) Hart, A. L.; Turner, A. P. F.; Hopcraft, D. Biosens. Bioelectron. 1996, 11, 263. (3) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87. (4) Swallen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Israelashvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (5) Voyta, J. C. Lumin. Top. 1994, 3, 1. S0003-2700(96)00129-1 CCC: $12.00
© 1996 American Chemical Society
of enzyme has been reported.6 Currently, the most sensitive enzymatic chemiluminescent assay is AP and the recently developed 1,2-dioxetane substrate, CSPD.7-10 This extreme sensitivity is largely due to improved substrates and advancements in photon sensing technology such as photomultiplyer tubes (PMTs) and in charge-coupled device (CCD) cameras.11-13 New cooled CCD cameras have very low background current, which allows the signal to be integrated over longer periods of time, thereby increasing sensitivity. Moreover, the array-based construction of CCD cameras is well suited for the simultaneous determination of multiple samples. In addition, the imaging area can easily be adjusted from millimeters to micrometers by simply exchanging the optics. Surface imaging provides a powerful strategy for the development of multiarray microanalytical devices. The pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) was chosen as a model compound for the development of a chemiluminescent assay using the flat-well microformat assay. The pesticide 2,4-D is a suspected cancer-causing agent.14 A maximum allowable concentration of 0.1 ng/mL 2,4-D is recommended.15 This compound is most commonly determined by gas chromatography (GC) after derivatization to an ester or by gas chromatography coupled with mass spectrometry (GC/MS).16 Although these methods have high sensitivity, they are complex and time consuming and employ toxic chemicals. As a result, numerous immunoassays for detecting 2,4-D and other pesticides have been described.17-22 For general screening purposes, these assays have adequate sensitivities. However, in some instances, such as studying the spread of pesticides deposited in soil and groundwaters as well as for monitoring their breakdown, the development of rapid, sensitive, HTP assays are still needed. The purpose of this study was to determine if significant increases in sensitivity could be obtained by combining thick-film technology with newly developed chemiluminescent substrates and rapid imaging-based CCD camera detection. Here, we report the development of a highly sensitive microformat immunoassay for 2,4-D based on this strategy. (6) Schaap, A. P.; Akhavan, H.; Romano, L. J. Clin. Chem. 1989, 35 (9), 18631864. (7) Bronstein, I.; Edwards, B.; Voyta, J. C. J. Biolumin. Chemilum. 1989, 4, 99-111. (8) Gillespie, P. G.; Hudspeth, A. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2563-2567. (9) O’Connor, K. L.; Culp, L. A. Biotechniques 1994, 17, 502-509. (10) Bronstein, I.; Fortin, J. J.; Voyta, J. C.; Juo, R. R.; Edwards, B.; Olesen, C. E. M.; Lijam, N.; Kricka, L. J. Biotechniques 1994, 17, 172-178 (11) Bunce, R. A.; Carter, T. J. N.; Kricka, L. J.; Whitehead, T. P.; Kennedy, J. H. Brit. Pat. 2025609A, 1980. (12) Jansen, E. H. J. M.; Buskens, C. A. F.; Van den Berg, R. H. J. Biolumin. Chemilumin. 1989, 3, 53-57. (13) Kricka, L. J.; Ji, X.; Nozaki, O.; Wilding, P. J. Biolumin. Chemilum. 1994, 9, 135-138. (14) Hoar, S. K.; Blair, A.; Holmes, F. F.; Boysen, C. D.; Robel, R. J.; Hoover, R.; Frasumeni, J. F. J. Am. Med. Assoc. 1986, 256, 1141-1147. (15) Wittmann, C.; Hock, B. J. Agric. Food Chem. 1991, 39, 1194-1200. (16) Sirons, G. J.; Chau, A. S.; Smith, A. E. Analysis of Pesticides in Water, Vol. II, CRC Press, Inc.: Boca Raton, FL, 1982; pp 155-227. (17) Van Emon, J.; Lopez-Avila, V. Anal. Chem. 1992, 64, 79-88. (18) Hall, J. C.; Deschamps, R. J. A.; Krieg, K. K. J. Agric. Food Chem. 1989, 37, 981-984. (19) Minnuni, M.; Mascini, M. Anal. Lett. 1993, 26, 1441-1460. (20) Khomutov, S. M.; Zherdev, A. V.; Dzantiev, B. B.; Reshetilov, A. N. Anal. Lett. 1994, 27, 2983-2995. (21) Lukin, Yu. V.; Dokuchaev, I. M.; Polyak, I. M.; Eremin, S. A. Anal. Lett. 1994, 27, 2973-2982. (22) Millipore Laboratory Catalogue; Millipore Corp.: Bedford, MA, 1995; 198.
EXPERIMENTAL SECTION Materials. Chemicals, Immunoreagents and Standards. The 2,4-dichlorophenoxyacetic acid (2,4-D), N-hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), Tween 20, and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). The diethanolamine was obtained from Merck-Schuchardt (Hannover, Germany). The 1,4-dioxan was bought from Riedel-De Haen AG (Hannover, Germany). The disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.13,7]decan]-4-yl)phenyl phosphate (CSPD), 11.6 mg/mL stock solution, and Emerald II enhancer, 10 mg/mL stock solution, were purchased from Tropix Inc. (Bedford, MA). Calf intestinal alkaline phosphatase (EIA grade) and 4-nitrophenyl phosphate (4-NPP) were obtained from Boehringer Mannheim (Mannheim, Germany). The anti-2,4-D monoclonal antibodies (clone 1/F6/C10) were raised23 at the Veterinary Research Institute (Brno, Czech Republic) and kindly provided by Dr. Sergei Eremin, Moscow State University, Russia. Rabbit anti-mouse IgG-AP was purchased from DAKO (Copenhagen, Denmark). Buffers and standards were prepared using distilled and deionized water. Phosphate-buffered saline (PBS), pH 7.4, contained 0.13 M NaCl, 2.6 mM KCl, 4.0 mM Na2HPO4‚7H2O, and 1.0 mM KH2PO4. Washing buffer solution (PBST) contained PBS with 0.1% Tween-20. Blocking solution contained PBST with 0.2% BSA. Sodium carbonate buffer was used for coating and contained 13 mM Na2CO3 and 85 mM NaHCO3, pH 9.6. A stock solution of 2,4-D (10.0 mg/mL) was prepared in methanol. For calibration, a serial dilution of the stock solution with PBS was prepared from 0.001 to 1000 ng/mL. The CCD camera employed in this study was a Photometrix 200 (Photometrix, Tuscon, AZ). The camera was thermoelectrically cooled to -45 °C, equipped with a Thompson TH 7895 chip which has 512 × 512 pixels, dark current 0.3 electrons s-1 pixel-1, a well capacity of 366 000 electrons, a 14 bit AD converter, and a quantum efficiency at 542 nm of 0.36. Microscope slides patterned with a proprietary hydrophobic layer using thick-film technology were purchased from Cel-Line (Newfield, NJ). The rectangular pattern consisted of 4 × 20, 2 mm2 squares (hydrophilic), separated by 1 mm lines (hydrophobic). The film is ∼100 µm thick. Methods. Synthesis of 2,4-D-BSA Conjugate. The synthesis was carried out as previously described.24 Briefly, a solution of 0.19 mM DCC in 0.5 mL of dioxane was added to a solution of 0.19 mM 2,4-D and 0.19 mM NHS in 2 mL of dioxane. The solution was left to react overnight at room temperature and then filtered to remove the precipitated dicyclohexylurea. The solvent was removed under reduced pressure at 35 °C. To the residue was added a solution of 500 mg of BSA in 3 mL of 0.15 M sodium borate buffer, pH 9.0. After 45 min at room temperature, the reaction mixture was dialyzed against PBS and then freeze-dried. Assay and Imaging Procedure for the CCD Camera-Based Chemiluminescence System. Each microwell was incubated with 2 µL of the 2,4-D-BSA conjugate (1 µg/mL in 0.1 M sodium carbonate buffer solution, pH 9.6) for 1 h at room temperature. The plate was washed for 4 min with a slow stream of the PBSTween solution. A solution containing 40 µL of the 2,4-D standard and 20 µL of the diluted antibody (1:1000 in PBST) was preincu(23) Franek, M.; Kolar, V.; Granatova, M.; Nevorankova, Z. J. Agric. Food Chem. 1994, 42, 1369-1374. (24) Fleeker, J. J. Assoc. Off. Anal. Chem. 1987, 70, 874-878.
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Figure 1. Schematic overview of the immunoassay and detection procedure. (a) Antibody is preincubated with antigen. (b) The mixture is added to flat-well support coated with 2,4-D-BSA conjugate. (c) Anti-mouse-AP is added. (d) The substrate, CSPD, is added. (e) The surface is imaged using CCD camera. (f) The image is analyzed and intensities quantitated using image analysis software (actual image).
bated. Aliquots of the preincubated mixture were transferred to the microwells (2 µL/well) and incubated for 1 h at room temperature. The slide was washed, and 2 µL of diluted rabbit anti-mouse IgG-AP conjugate (1:1000 in PBS) was added. The slide was washed again, and 2 µL of the substrate solution was added. The substrate solution contained 10 µL of enhancer (Emerald II), 1 µL of CSPD stock solution, and 90 µL of 0.1 M diethanolamine buffer, pH 10.0. The plate was imaged with the Photometrix 200 CCD camera, fitted with a 50 mm AF NIKKOR (1:1.8) objective (Nikon, Japan). The readout noise was 16.19 e-, and the system integrating gain was 23.61 e-/ADU (analog to digital units). To combine the signal from adjacent pixels into superpixels, we used the binning factor 2 × 2, i.e., the final image contained 256 × 256 superpixels. The exposure time was 90 s throughout these studies. Samples were imaged individually, unless stated otherwise. Imaging and quantification of the signal intensities were performed using the PMIS Photometrix software (Photometrics Ltd., Tuscon, AZ). A region of interest (ROI) was defined, and the corresponding pixel intensities were combined. A value for the background was obtained by imaging an equally sized region outside the image area and subtracted from each measurement, typically 239 ADU. These ADU values were then plotted as a function of concentration to give the standard curve. Luminometer-Based Chemiluminescent ELISA. One milliliter of coating antigen, 20 mg/mL in 0.1 M sodium carbonate buffer solution, pH 9.6, was added to each glass tube and incubated overnight. The tubes were washed, and 1 mL of a preincubated mixture containing 800 µL of 2,4-D standard and 200 µL of diluted antibody (1:150 in PBST) was added to the hapten-coated tubes. After 2 h, the tubes were washed, and rabbit anti-mouse IgG-AP conjugate (1:500) was added. After an additional 30 min, the tubes were washed, and 1 mL of substrate solution (170 µL of Emerald II amplifier, 150 µL of CSPD stock solution, and 15 mL of 0.1 M diethanolamine buffer, pH 10) was added. The signal was measured with an LKB 1250 luminometer (Bromma, Sweden). Colorimetric ELISA. Microtiter dishes were coated with the 2,4-D-BSA conjugate, 10 mg/mL in 0.1 M sodium carbonate buffer, pH 9.6, for 1 h at 37 °C. After washing, the plate was 3366
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blocked with 0.2% solution of BSA in PBST for 1 h. After a second washing, 100 µL of a preincubated mixture containing 800 µL of 2,4-D standard and 200 µL of diluted antibody (1:150 in PBST) was added. After 1 h, the plate was washed, and rabbit anti-mouse IgG-AP conjugate (1:500 in PBS) was added. The plate was incubated for 30 min and washed, and substrate solution (75 mg of 4-NPP in 20 mL of 1.3 M diethanolamine buffer solution, pH 9.8) was added. After an additional 30 min, the absorbance of each well was measured at 405 nm with plate reader Multiskan MCC/340 (Helsinki, Finland). RESULTS AND DISCUSSION An overview of the assay procedure is shown in Figure 1. Briefly, the standard or unknown sample is incubated with a moderate excess of specific antibodies (empirically determined) in the solution phase. The mixture is transferred to a 2,4-DBSA conjugate-coated well, where unreacted antibodies bind. After washing, the AP-labeled rabbit anti-mouse IgG is added. The bound AP is detected enzymatically using the chemiluminescent substrate, CSDP. The light emitted from the reaction is detected by imaging the sample with a CCD camera and quantitated using computer software. Microformat Chemiluminescent ELISA. The assay conditions were optimized with a mock assay using unconjugated AP. In these studies, 1 µL of the diluted enzyme solution was added to 4 µL of the substrate solution. A 2 µL aliquot of the mixture was transferred to the glass support and imaged. A number of parameters were optimized, including mixing conditions, substrate and enhancer concentrations, buffer composition, assay timing, and CCD camera positioning and exposure settings. For reasons of economy, a lower than optimal CSPD concentration was employed. These studies also showed that addition of enhancer in appropriate concentrations significantly increased the sensitivity of the assay by raising the efficiency of the chemiluninescent process and by shifting the wavelength into a range in which the quantum efficiency of the CCD camera is higher. The manual addition of multiple samples to the microformat support reduced assay reproduciblity, thereby complicating assay optimization.
Figure 2. Standard curve showing the sensitivity of the CSPD/AP assay. A background value of 49 units was obtained by imaging an appropriately diluted substrate solution. This has been subtracted and the curve plotted.
Figure 3. Plot of light intensity development as a function of time at the following concentrations: 8 × 10-8 (4), 8 × 10-9 (2), 8 × 10-10 (0), 8 × 10-11 (9), 8 × 10-12 (]), 8 × 10-13 ([), and 8 × 10-14 M (x).
Thus, in order to reduce these effects, each sample was imaged individually (see Materials and Methods sections for experimental details). The absolute sensitivity of the CCD camera for the detection of AP using the chemiluminescent substrate CSPD was determined by analyzing a serial dilution of AP ranging from 0.4 × 10-9 to 0.4 × 10-15 M. A graph showing the relation between AP concentration and light intensity is given in Figure 2. A linear range between 2 × 10-9 and 2 × 10-12 M and a detection limit of 8 × 10-13 or 1.6 × 10-19 mol of AP was obtained. The kinetics and stability of the chemiluminescent emission from the AP/CSPD reaction were analyzed by taking multiple images and plotting this as a function of time over a range of enzyme concentrations (Figure 3). As can be seen, the chemiluminescent signal from the CSPD/AP reaction rises rapidly and remains stable over the 20 min time course. The substrate becomes limiting only at the very highest AP concentrations, which are far in excess of those used in the 2,4-D immunoassay studies. For practical purposes, a relatively short exposure time of 90 s was employed throughout these studies. However, the long-term stability of the signal indicates that it should be possible to increase the sensitivity by a factor of 10 or more by merely extending the exposure times. Indeed, initial experiments bear this out (data not shown).
Figure 4. Standard calibration curve for 2,4-D determination using the competitive assay format. The curve has been corrected using a background value (see Experimental Section) of 34 relative units.
This long-term stability is somewhat surprising if one takes into account the considerable evaporation that occurs over the 20 min time period. In this respect, it is important to note that the concentrations of buffer, enhancer, and substrate have been optimized for the microformat and differ significantly from those used in the tube-based chemiluminescent assay (see Experimental Section for details). In any case, the stability and reproducibility of our data show that, by judiciously choosing assay conditions, evaporation related effects can be minimized. These factors will become increasingly important as immunoassay miniaturization proceeds. This CCD-based imaging protocol for the chemiluminescent CSPD/AP reaction was combined with a competitive immunoassay employing mAb against the pesticide 2,4-D (see Experimental Section for details). A typical standard curve using the mean values (n ) 4) is shown in Figure 4. The calibration curve was linear from 10 to 0.01 ng/mL. The precision within this range varied between 3 and 14% (Figure 4, inset). That curve was created by performing all measurements four times and expressing the data in terms of coefficient of variation (CV, %). The detection limit of the assay was 6 pg/mL, or 2.7 × 10-11 M, which corresponds to 5.4 × 10-17 mol of 2,4-D. The detection limit was defined as 3 times the standard deviation of the blank value. The CV for this concentration was 10.5%. The accuracy was studied by recovery experiments in which increasing amounts of 2,4-D (0.05-50 ng/mL) were added to samples having pesticide levels from 0.5 to 100 ng/mL. The samples were repeatedly analyzed (n ) 10) using the microassay format. The results showed an 84-112% recovery over the assay range. The precision of the assay was determined by measuring of 2,4-D by replicate analysis of water samples. Within-assay precision was assessed from replicate measurements in one assay, and day-to-day assay precision was assessed from repeated analysis in the following assays. The results are presented in Table 2. Both the within-assay and, especially, the day-to-day assay CVs of the results were >10% for the samples containing more than 1 ng/mL of 2,4-D, while the CVs for the samples containing
