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Simultaneous Determination of Pesticides Using a Four-Band Disposable Optical Capillary Immunosensor Christos Mastichiadis,† Sotirios E. Kakabakos,*,† Ion Christofidis,† Michael A. Koupparis,‡ Caroline Willetts,§ and Konstantinos Misiakos|
Institute of Radioisotopes & Radiodiagnostic Products, Immunoassay Laboratory and Institute of Microelectronics, NCSR “Demokritos”, 15310 Athens, Greece, Department of Chemistry, Laboratory of Analytical Chemistry, University of Athens, Panepistimiopolis, 15771 Athens, Greece, and Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire, RG426EY, United Kingdom
The development of a four-band capillary optical immunosensor for the simultaneous determination of mesotrione, hexaconazole, paraquat, and diquat is described. Four distinct bands (each corresponding to a different analyte) are created in the internal walls of a plastic capillary by immobilizing protein conjugates of the analytes. To perform the assay, the capillary is filled with a mixture of antianalyte-specific antibodies together with a standard or sample containing the analyte(s). After a short incubation, a mixture of the appropriate second antibodies labeled with fluorescein is introduced into the capillary. To measure the fluorescence intensity bound onto each band, the capillary was scanned, perpendicularly to its axis, by a laser light beam. Part of the emitted photons were trapped into the capillary walls and waveguided to a photomultiplier placed at the one end of the capillary. The analytical characteristics of the assays of mesotrione, paraquat, diquat, and hexaconazole were as follows: detection limits of 0.04, 0.06, 0.09, and 0.10 ng/mL, respectively; dynamic ranges up to 9, 6, 12, and 15 ng/ mL, respectively; intra- and interassay CVs less than 10%. The analytical characteristics of the assays were comparable with those of the corresponding single-analyte fluoroimmunoassays performed in microtitration wells, proving the ability of the proposed immunosensor for reliable multianalyte determinations. Moreover, the combination of low-cost disposable plastic capillary tubes with the low consumption of reagents, the short assay time, and the multianalyte feature of the proposed immunosensor indicates its potential for environmental analysis. Analytical techniques based on antibody-antigen interaction are well established in biomedical analysis.1 During past years, * Corresponding author. E-mail:
[email protected]. Fax: ++30106515573. † Institute of Radioisotopes & Radiodiagnostic Products, NCSR “Demokritos”. ‡ University of Athens. § Syngenta. | Institute of Microelectronics, NCSR “Demokritos”. (1) Christopoulos, T. K.; Diamandis, E. P. Past, Present, and Future of Immunoassays. In Immunoassay; Diamandis, E. P., Christopoulos, T. K., Eds.; Academic Press: San Diego, CA, 1996; pp 1-3.
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these techniques were successfully implemented for the screening of pesticides in food, agricultural, and environmental samples.2,3 Immunoassay methods are characterized by several advantages4 such as the following: specificity, sensitivity, ease of use, reduced assay time, cost-effectiveness, wide applicability, and reduced sample size requirements. Immunosensors represent an improvement in immunochemical techniques in the sense that they combine the advantages of immunoassays with a greater potential for automation, miniaturization, and multianalyte determination. This last characteristic of the immunosensors is expected to lead to a further reduction of both the time and cost of analysis. So far, two different approaches have been adopted in order to realize the goal of multianalyte determination. A multilabel approach was the first successful strategy reported in the literature.5,6 However, the restrictions in number of labels and, therefore, of analytes that could be determined simultaneously, have limited its application.7 The second strategy, proposed by Ekins and his colleagues, is based on the spatial resolution of different bioactive species on the same substrate that creates an array,8 permitting the detection of all the analytes through the same universal tracer. Due to the simplicity it offers, this approach seems to be well suited for the development of both multianalyte immunoassays9-11 and immunosensors.12-19 (2) Schobel, U.; Barzen, C.; Gauglitz, G. Fresenius J. Anal. Chem. 2000, 366, 646-658. (3) Ahmed, F. E. Trends Anal. Chem. 2001, 20, 649-661. (4) Sherry, J. Chemosphere 1997, 34, 1011-1025. (5) Gutcho, S.; Mansbach, L. Clin. Chem. 1977, 23, 1609-1615. (6) Xu, Y. Y.; Pettersson, K.; Blomberg, K.; Hemmila¨, I.; Mikola, H.; Lo ¨vgren, T. Clin. Chem. 1992, 38, 2038-2043. (7) Kricka, L. J. Clin. Chem. 1992, 38, 327-328. (8) Ekins, R. P.; Chu, F. W.; Biggart, E. M. J. Clin. Immunoassay 1990, 13, 169-181. (9) Kakabakos, S. E.; Christopoulos, T. K.; Diamandis, E. P. Clin. Chem. 1992, 38 (3), 338-342. (10) Parsons, R. G.; Kowal, R.; LeBlond, D.; Yue, V. T.; Neargarder, L.; Bond, L.; Garcia, D.; Slater, D.; Rogers, P. Clin. Chem. 1993, 39, 1899-1903. (11) Silzel, J. W.; Cerek, B.; Dodson, C.; Tsay, T.; Obremski, R. J. Clin. Chem. 1998, 44, 2036-2043. (12) Narang, U.; Gauger, P. R.; Kusterbeck, A. W.; Ligler, F. S. Anal. Biochem. 1998, 255, 13-19. (13) Klotz, A.; Brecht, A.; Barzen, C.; Gauglitz, G.; Harris, R. D.; Quingley, G. R.; Wilkinson, J. S.; Abuknesha, R. A. Sens. Actuators, B 1998, 51, 181187. 10.1021/ac020330x CCC: $22.00
© 2002 American Chemical Society Published on Web 10/31/2002
Glass slides,14-16 optical fibers,17 and capillaries12,18,19 have been used as solid substrates for the fabrication of multianalyte immunosensors. Among them, capillaries offer several advantages, including improved assay kinetics due to the higher surface-tovolume ratio and restricted diffusion of reacting compounds.20 In addition, consumption of the immunoreagents is minimized, due to the small dimensions of capillaries. Most of the capillary immunosensors reported so far are made of glass due to the satisfactory optical properties of this material. However, chemical modification of the glass surface is required for the proper immobilization of the sensing probes. On the contrary, although biomolecules can be readily immobilized onto plastic supports through physical adsorption, the development of optical immunosensors based on such materials is fairly limited. 19, 21 In a previous report,21 we demonstrated, in principle, the use of polystyrene capillaries for the development of an optical immunosensor based on the formation of distinct analyte bands on the internal surface of the capillaries. The different bands of the biotinylated reagents were detected after reaction with Eulabeled streptavidin by scanning the capillary with a light beam. Part of the photons emitted by the bound fluorescent molecules were trapped into the capillary walls and waveguided to a photon detector placed at the one of its ends. The use of Eu-labeled streptavidin permitted measurement of the fluorescence intensity in time-resolved mode that excluded the background short-lived fluorescence originated, mainly, in the plastic capillary itself. More recently, we presented an optical immunosensor for the simultaneous determination of three peptide hormones in serum samples19 based on the aforementioned detection principle.21 The three hormones were determined through noncompetitive sandwich immunoassay configuration using streptavidin labeled with Rphycoerythrin, as universal tracer. Single-analyte and multianalyte capillary immunosensors based on fluorescent label detection have been described in the past by other research groups, too.12,22,23 However, in all cases, fully coated fused-silica capillaries were employed, whereas the assays were performed under flow conditions. In a recent report, a singleanalyte capillary-based integrating waveguide biosensor23 was presented. Although the detection principle of this sensor is similar to the one proposed by the authors in ref 21, the excitation of the fluorescent molecules was achieved through illumination of most of the length of the capillary and not through stepwise scanning with a light beam. The claimed advantage of full capillary (14) Row: C. A.; Scruggs, S. B.; Feldstein, M. J.; Golden, J. P.; Ligler, F. S. Anal. Chem. 1999, 71, 433-439. (15) Plowman, T. E.; Durstchi, J. D.; Wang, H. K.; Christensen, D. A.; Herron, J. N.; Reichert, W. M. Anal. Chem. 1999, 71, 4344-4352. (16) Weller, M. G.; Schuetz, A. J.; Winklmair, M.; Niessner, R. Anal. Chim. Acta 1999, 393, 29-41. (17) Anderson, G. P.; King, K. D.; Gaffney, K. L.; Johnson, L. H. Biosens. Bioelectron. 2000, 14, 771-777. (18) Koch, S.; Wolf, H.; Danapel, C.; Feller, K. A. Biosens. Bioelectron. 2000, 14, 779-784. (19) Petrou, P. S.; Kakabakos, S. E.; Christofidis, I.; Argitis, P.; Misiakos, K. Biosens. Bioelectron. 2002, 17, 261-268. (20) Cousino, M.; Jarbawi, T. B.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1997, 69, 544A-549A (21) Misiakos, K.; Kakabakos, S. E. Biosens. Bioelectron. 1998, 13, 825830. (22) Narang, U.; Gauger, P. R.; Ligler, F. S. Anal. Chem. 1997, 69, 27792785. (23) Ligler, F. S.; Breimer, M.; Golden, J. P.; Nivens, D. A.; Dodson, J. P.; Green, T. M.; Haders, D. P.; Sadik, O. A. Anal. Chem. 2002, 74, 713-719.
illumination, compared to the scanning approach, relies on the increased fluorescence signal collected from a considerably increased surface area, whereas the electronic noise is not integrated, resulting in an improved signal-to-noise ratio. However, when the background signal originating in the material used for the fabrication of the capillary is not negligible, it will be also integrated, canceling, therefore, the possible signal-to-noise ratio improvement. This waveguide biosensor was used for the determination of mouse IgG and staphylococcal enterotoxin B (SEB) adopting the noncompetitive flow immunoassay configuration. In a different report, a single-analyte displacement flow immunosensor22 was used for the determination of 2,4,6 trinitrotoluene (TNT). The displaced labeled antigen was detected downstream using a fluorometer. In an ensuing report, the use of this displacement immunosensor in single- and dual-analyte format (employing two capillaries in parallel) for the determination of TNT and hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) analytes was described.12 The aim of the present work was to exploit the previously described sensor design21 for the development of a disposable multiband optical plastic capillary fluoroimmunosensor, based on the competitive immunoassay configuration and using detection antibodies labeled with fluorescein, for the simultaneous determination of four different pesticides in the same sample. Pesticides are low molecular mass molecules lacking multiple epitopes. Their determination is, therefore, based on the competitive immunoassay format that, in contrast to the noncompetitive sandwich immunoassay, demands limited concentrations of antibody and analyte conjugate in order to achieve high analytical sensitivity of the determination. These demands, however, may negatively affect the analytical signal of the assay. Fluorescein, the most common fluorescent compound, was selected as label in this work in order to expand the applications of the proposed sensor. This is expected since a great variety of low-cost fluoresceinated biomolecules and fluorescein derivatization reagents are available on the market. On the other hand, compared with other fluorescent compounds that are excited at longer wavelengths or present a larger Stokes’ shift,24,25 fluorescein with excitation maximum at ∼494 nm, relative broad emission spectrum, and small Stokes’ shift of 25-30 nm, presents higher background fluorescence signals, mainly due to scattering.26 The case is even worse when plastics are used as solid supports since, besides scattering, fluorescence originating in the material itself is added to the background fluorescence signal and affects the measurement.27 This is the main reason that the use of fluorescein in solid-phase fluoroimmunoassays is very limited.27 Thus, both the competitive immunoassay format and the use of fluorescein as label combined with the plastic substrate raise extra analytical challenges, not encountered when the proposed immunosensor operates in noncompetitive immunoassay configuration using other labels or solid substrates that do not fluoresce (e.g., glass). The ability of the proposed immunosensor to meet the objectives of this work, permitting accurate, highly sensitive, and simultaneous determination of pesticides is demonstrated through a panel of analytes (24) Soini, E.; Hemmila¨, I. Clin. Chem. 1979, 25, 353-361. (25) Patonay, G.; Antoine, M. D. Anal. Chem. 1991, 63, 321A-327A. (26) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes, Inc.: Eugene, OR, 1996; Chapter 1. (27) Hemmila¨, I. Clin. Chem. 1985, 31, 359-370.
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Figure 1. Schematic representation of the multianalyte capillary immunosensor optoelectronic setup. The excitation source was a pulsed nitrogen dye laser. An interference filter at 480 nm for the excitation and an emission filter at 540 nm were used. The detector was a photomultiplier, the photocurrent of which was fed to a lownoise transimpedance amplifier. The output voltage of the amplifier was digitized by a signal oscilloscope, which also performed digital signal processing. A linear stage, controlled by a PC, moved the capillary detection system so that the laser beam could scan the capillary.
comprising mesotrione (a triketone herbicide), hexaconazole (a triazole fungicide), paraquat, and diquat (bipyridyl herbicides). EXPERIMENTAL SECTION Materials. Capillary tubes (6 cm long, 1 mm internal diameter) made of polystyrene (PS), poly(methylpentene) (PMP), and poly(methyl methacrylate) (PMMA) were kindly donated by Dr. S. E. Rasmussen and Dr. M. Strange (Nunc A/S, Denmark). White opaque polystyrene microtitration wells were obtained from the same company. Bovine serum albumin (BSA), bovine γ-globulins (BgG), and casein were from Sigma (St Louis, MO). Diquat (DQ), paraquat (PQ), mesotrione (MES), and hexaconazole (HEX) analytical standards, rabbit anti-DQ, anti-HEX, and anti-MES antisera, mouse monoclonal anti-PQ antibody, and MES-, HEX-, DQ-BgG, and PQ-BSA conjugates were from Syngenta (Bracknell, U.K.). Fluorescein-labeled anti-rabbit IgG and anti-mouse IgG (FITC conjugates) were from Chemicon. All the other chemicals and reagents were obtained from Merck (Darmstadt, Germany). All reagents were used as received, without further purification. Instrumentation. A simplified schematic representation of the optoelectronic setup is depicted in Figure 1. A more detailed description of the setup has been presented previously.19 Fluorescence intensity measurements in microtitration plates were carried out using the Ascent Fluoroscan plate reader (Labsystems OY). Preparation of the Patterned Capillaries. An ordered array of four distinct analyte bands, 4 mm long each, was created by sequentially introducing with microsyringes 3.2 µL of PQ-BSA (1 µg/mL), DQ-BgG (5 µg/mL), MES-BgG (2 µg/mL), and HEX-BgG (20 µg/mL) solutions in 0.05 M sodium carbonate buffer, pH 9.2 (coating buffer), at distinct parts of the capillary lumen. Prior to the application of the coating solution, one of the ends of the capillaries was marked. The different analyte conjugates were positioned in a standard order, with respect to the marked end of the capillary. The capillaries were incubated 6066
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overnight at room temperature (RT) and then washed with 3 mL of 0.15 M phosphate-buffered saline, pH 7.0 (PBS), containing 1% (w/v) casein (blocking solution), filled with this solution, and incubated for 1 h at room temperature (washing and filling of the capillaries were carried out by syringes). Afterward, the capillaries were washed twice with 2 mL of 0.01 M Tris-HCl buffer, pH 8.25, containing 0.05% (v/v) Tween 20 (washing solution) and dried with a stream of nitrogen, prior to use. Fully coated capillaries were prepared similarly, but the entire capillary was filled with the respective analyte-protein conjugate coating solution. Standard and Sample Preparation. Standard stock solutions (1.00 mg/mL) for each individual analyte were prepared in either doubly distilled water (PQ and DQ), ethyl acetate (MES), or methanol (HEX). A mixture standard stock solution was prepared by mixing appropriate volumes of the individual stock solutions. Multianalyte standards were obtained by serial dilution of the mixture stock solution with doubly distilled water. Single-analyte standards were used in microtitration wells as well as in fully coated and single-band capillary based assays. In the multiband capillary immunosensor-based experiments, multianalyte standards were employed. Samples were prepared by spiking various volumes of the individual analyte stock solutions in deionized water. Assay Protocol for the Capillary Immunosensor. Two volumes of multianalyte standards or samples were mixed with one volume of mixed antibody solution containing monoclonal antiPQ antibody (600 ng/mL), rabbit anti-DQ (dilution 1:500), antiMES (dilution 1:8000), and anti-HEX (dilution 1:500) antisera in PBS containing 0.05% (v/v) Tween 20 (PBS/T). The capillaries were, then immediately filled with this mixture (∼50 µL) and incubated for 5 min at room temperature. After the capillaries were washed four times with ∼50 µL of washing solution, the remaining droplets were removed by blowing with nitrogen. Then, the capillaries were filled with a solution containing a mixture of antirabbit IgG-FITC (5.0 µg/mL) and anti-mouse IgG-FITC (10.0 µg/ mL) conjugates in 0.15 M Tris-HCl buffer, pH 8.25, with 0.1% (w/ v) BSA and 0.05% (w/v) BgG (second antibody dilution buffer) and incubated for 30 min at room temperature. Afterward, the capillaries were washed as above and filled with washing solution, prior to the measurement of the fluorescence intensity bound to the solid by the described optical setup. Single-band or fully coated single-analyte capillaries were assayed similarly using only the corresponding analyte standard solution, analytespecific antibody, and corresponding labeled antispecies-specific antibody. Data Processing. Detailed information on data processing in single band and multiband capillary assays and on the construction of the calibration curves has been provided previously.19 In the case of fully coated capillaries, the blank measurement was obtained by performing the assay procedure using an uncoated, blocked capillary. The net signal was calculated by subtracting the blank value from the total analytical signal, which was the average of all 40 signal values, obtained by scanning the coated capillary. Assay Protocol for the Microtitration Wells. White opaque polystyrene microtitration wells were coated with 100 µL of either PQ-BSA, DQ-BgG, or MES-BgG or HEX-BgG conjugate solutions at concentrations of 0.5, 1, 1 and 5 µg/mL, respectively, in
coating buffer, overnight at room temperature. Then, the wells were washed twice with 0.3 mL of washing solution per well, incubated with blocking solution (0.3 mL/well) for 1 h at room temperature, and washed three times with washing solution prior to use. To perform the assay, 0.1 mL of single-analyte standard solution or sample and 0.05 mL of the respective antibody solution, at concentrations or dilutions used for the capillaries, were added in each well and incubated under shaking for 30 min at room temperature. The wells were washed four times with washing solution, and 0.1 mL of a 10.0 µg/mL anti-mouse IgG-FITC solution (PQ assay) or a 5.0 µg/mL anti-rabbit IgG-FITC solution (DQ, HEX, MES assays) in the second antibody dilution buffer was added per well. The wells were incubated under shaking, for 2 h at room temperature and then washed five times with 0.3 mL of washing solution. The fluorescence intensity bound on the bottom of the wells was measured at 538 nm after excitation at 485 nm, using the Ascent Fluoroscan plate reader. RESULTS AND DISCUSSION In this work, we present the development of an optical plastic capillary fluoroimmunosensor capable of the simultaneous determination of different low molecular mass analytes, such as pesticides, in the same sample using fluorescein as label. The capillary serves as solid support for immobilization of recognition elements and as waveguide that carries part of the photons emitted by the fluorescent compounds to the detector. Besides, adoption of plastic material for the fabrication of the sensor offers lowcost disposable capillaries that permit direct immobilization of immunoreagents through physisorption. Selection of the Appropriate Plastic Material. Since the material employed for the fabrication of the sensor has direct impact on its analytical performance, several issues concerning the selection of the appropriate plastic support should be addressed. The appropriate plastic material should be characterized by the following: (a) optical clarity, since it is destined to serve as waveguide, (b) high efficiency to bind immunoreagents through physisorption, (c) low background fluorescence when illuminated, and (d) appropriate mechanical rigidity, in order to be easily handled during the immunoassay steps and the measurement. Certain optically clear and mechanically rigid plastic capillaries made of PS, which is a long-established material for the fabrication of immunoassay solid supports, PMP, or PMMA were evaluated in terms of protein binding capacity and background fluorescence. The protein binding capacity of the different types of capillaries was evaluated by determining their surface saturation pattern through a rabbit γ-globulin/anti-rabbit IgG-FITC conjugate model assay. As is shown in Figure 2, PMMA capillaries yielded poor fluorescence signals for the whole range of the rabbit γ-globulin concentrations used. On the other hand, PS and PMP capillaries provided similar fluorescence values using coating concentrations up to 5 µg/mL. At higher coating concentrations, where maximum plateau signals for both materials were observed, PS capillaries yielded 10-15% higher fluorescence values. Background fluorescence of the plastic materials can be attributed to the existence of fluorescent groups in the polymer molecule itself, to the initiator molecules added during the monomer polymerization process, or even to the additives used for the molding/extrusion process. It is well known that PS
Figure 2. Comparison of net fluorescence signals obtained using PS (0), PMP (O), and PMMA (4) capillaries coated with different concentrations of rabbit γ-globulins for 24 h, blocked with 1% BSA solution, and then assayed with a 5 µg/mL anti-rabbit IgG-FITC conjugate solution for 30 min. Fluorescence intensity measurements were carried out with the described setup. Each point corresponds to the mean value of three different capillaries; the error bars represent (SD.
molecule contains aromatic groups that fluoresce. On the other hand, the rest of the plastic materials tested lack such groups in their molecule and, therefore, were expected to yield low background fluorescence values. The background fluorescence of the different capillaries was measured by scanning plain capillaries using a set of filters appropriate for either fluorescein (excitation 480 nm, emission 540 nm) or R-phycoerythrin (excitation 480 nm, emission 585 nm). The R-phycoerythrin filters were tested since, as reported earlier,19 they provided 2 times lower background signal compared with fluorescein filters when polystyrene capillaries were used. It was found that, using fluorescein filters, PMP capillaries presented approximately 2.5 and 3 times lower background fluorescence values compared with PS and PMMA capillaries, respectively. More interestingly, the PMP capillaries employed provided similar background fluorescence values using either the fluorescein or R-phycoerythrin set of filters. The net specific-to-background signal ratio obtained using either FITC or R-phycoerythrin as labels was evaluated through the rabbit γ-globulin/anti-rabbit IgG model assay. It was found that this ratio was 9.5 for the FITC-labeled antibody, whereas the corresponding value for R-phycoerythrinantibody was 6.7. This finding was, to some extent, unexpected considering the fluorescent molecule per protein molecule ratio (4.5 and 1 for FITC- and R-phycoerythrin-labeled antibodies, respectively), the extremely high extinction coefficient of Rphycoerythrin compared to FITC, and the quantum yield of each fluorescent molecule.28 However, saturation experiments indicated that the R-phycoerythrin-labeled antibody was considerably defective as compared with the FITC-labeled one (data not shown). Based on these results, PMP capillaries in combination with FITClabeled antibody were selected as the most appropriate solid support and fluorescent label for our application. (28) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes, Inc.: Eugene, OR, 1996; Chapter 6.
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Capillary Patterning. Multianalyte immunosensors based on the immobilization of different recognition molecules on the same support offer several advantages over those relying on the use of independent biologically activated substrates. These advantages mainly include the following: (a) incorporation of controls in order to correct for substrate or detection variations, (b) lower sample and immunoreagent volume consumption, and (c) easier automation due to less complicated microfluidics required. In this work, we adopted the immobilization of different recognition molecules on the same substrate. Patterning of biomolecules on the internal capillary surface was accomplished through successive applications of the coating solutions, containing different analyte conjugates, at predefined positions using microsyringes. The band length and the betweenband distance were investigated and optimized, using the setup available, to achieve maximum signal and avoid cross-talk effects. It should be noted that although the capillary length was 6 cm, its utilizable part was limited to 4.2 cm, since the capillary holder occupied 0.9 cm from each end. The optimization was performed through a rabbit γ-globulin/anti-rabbit IgG-FITC conjugate model assay. To determine the optimum band size, which can be defined as the minimum length of a band that provides signal equal with that obtained using fully coated capillaries run under the same conditions, several capillaries were coated with single bands of varying length (2-10 mm). The coated capillaries were scanned using the 2-mm optical aperture, and the scanning step was 1 mm. It was found that 3-10-mm bands yielded signals equal to those provided by fully coated capillaries. To determine the least between-band distance that provides good band isolation, several capillaries were coated with two bands each and scanned. The between-band space tested differed from 2 to 10 mm. The least distance between bands that excluded cross-talk effects was found to be 3 mm. Since, in our application, four immunoreactive bands were required, we adopted 4-mm-long bands separated by 5-mm between-band distance that ensured maximum signal from each band and efficient baseline discrimination. Additional analyte bands could be introduced and measured, however, by using 2-mm-long bands, 3-4-mm between-band distances, a 1-mm optical aperture, and a scanning step of 0.5 mm. As shown in Figure 3, using this arrangement, seven bands of rabbit γ-globulins could be determined without cross-talk problems. Concerning the patterning of biomolecules onto solid supports, several methodologies have been proposed in the literature during the last years. However, only a few of them such as caged biotinBSA photopatterning,29 photoactivated site-specific immobilization using either photobiotin,30 or photoactivatable linkers,31 as well as techniques based on biocompatible and plastic-compatible lithographic materials32 and processes,33 seem to be suitable for both capillary geometry and plastic materials. However, these methods require special apparatus and photoreagents that, in most cases, are not commercially available and accomplish patterning (29) Blawas, A. S.; Oliver, T. F., Pirrung, M. C.; Reichert, W. M. Langmuir 1998, 14, 4243-4250. (30) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chim. Acta 1995, 310, 251-256. (31) Nivens, D. A.; Conrad, D. W. Langmuir 2002, 18, 499-504. (32) Douvas, A.; Argitis, P.; Diakoumakos, C. D.; Misiakos, K.; Dimotikali, D.; Kakabakos, S. E. J. Vac. Sci. Technol. B 2001, 19, 2820-2824. (33) Douvas, A.; Argitis, P.; Misiakos, K.; Dimotikali, D.; Petrou, P. S.; Kakabakos, S. E. Biosens. Bioelectron. 2002, 17, 269-278.
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Figure 3. Fluorescence intensity obtained by scanning a capillary coated with seven rabbit γ-globulins bands and assayed with antirabbit IgG-FITC conjugate. The length of each band and the between-band distances were approximately 2 and 3-4 mm, respectively. The apparent broadening of the bands observed at the baseline in this scanning (approximately 3-3.5 mm instead of 2 mm) is attributed to the use of a 1-mm optical aperture, whereas the scanning step was 0.5 mm.
through successive coating steps that extend the duration of the surface activation procedure. These drawbacks restrict the wide application of the above-mentioned methodologies. Here, we propose the use of microsyringes for the patterning of the internal surface of the plastic capillary tubes, which is a fast and straightforward procedure that takes advantage of the binding properties of the plastic surface. Liquid-dispensing techniques are powerful tools for the fabrication of protein arrays on planar surfaces.34 Two advantages can be named in favor of capillary geometry as compared to planar regarding patterning through liquid-dispensing techniques: (a) one-dimensional and linear geometry inherently facilitates the definition of the activated sites; (b) its small internal diameter retains the liquid drop at the exact point of its application and, moreover, prevents the evaporation of the liquid (a considerable drawback of planar surfaces patterning). Given the recent advances in microdispensing techniques, there is no doubt that development of automated instrumentation for patterning of capillaries is within near-future perspectives. Capillary Format and Reaction Rates. To compare the immunoreaction rate in band-coated capillaries with that in fully coated capillaries and microtitration wells, a set of time study experiments was carried out. These experiments included immunoassays for each one of the panel analytes, performed in a) singleband capillaries, (b) fully coated capillaries, (c) microtitration wells under shaking, and (d) microtitration wells without shaking. Since all the analytes studied behaved in a similar way in these experiments, only the results for mesotrione are presented in Figure 4. As is shown, maximum plateau signal values were obtained after 60 min of immunoreaction using fully coated capillaries, whereas about 6-7 h of incubation was required when the immunoassay was performed in microtitration wells without (34) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763.
Table 1. Molecular Structures of All the Panel Analytes
Figure 4. Time evolution of the fluorescence intensity for MES zero standard, obtained by the optical capillary immunosensor using singleband coated capillaries (9) or fully coated capillaries (0) and by microtitration wells under shaking (b) or without shaking (O) during immunoreaction.
shaking. In both cases, mass transfer was limited to diffusion of the reactive species; therefore, results were expected to reflect the differences in the surface-to-volume ratio between capillaries and microtitration wells. In fact, in our application, the surfaceto-volume ratio for fully coated capillaries was 42 cm-1, whereas for microtitration wells, the corresponding value was 6 cm-1 (0.9 cm2 corresponding to the surface of microtitration well covered by 100 µL of coating solution/150 µL of immunoreaction volume). Therefore, in fully coated capillaries, surface-to-volume ratio was 7 times higher than in microtitration wells, and thus, our experimental data were in good agreement with the aforementioned theoretical calculations. As was expected, shaking of the microtitration wells increased the immunoreaction rate. Under these conditions, equilibrium was reached in 2-3 h, which is about 2.5-3 times earlier than static wells. However, compared with fully coated capillaries, the time required for the wells under shaking to reach equilibrium was doubled. The signal yielded by the single-band capillary was approximately equal to the signal obtained by the fully coated capillary, until equilibrium of the latter was reached (60 min). Beyond this point, the immunoreaction in single-band capillaries progressed slowly, requiring several hours to reach equilibrium (data not shown). This was due to the ability of the band to attract immunoreagents not only within the band but also residing in its close vicinity and diffusing toward the band. Our results indicated that, for reaction times less than 60 min, single-band capillaries behaved like fully coated ones in terms of reaction rate and therefore, practically, only the species lying above or quite close to each analyte band have the chance to react with it. Thus, for short incubation periods, the active immunoreaction volume could be considered equal to that surrounded by the analyte band. Cross-Reactivity Studies. Cross-reactivity is a crucial analytical parameter regarding specificity and, thus, reliability of immunoassays. The typical way to assess cross-reactivity in competitive immunoassays is to determine the percent inhibition
of binding of each analyte-specific antibody to its corresponding analyte caused by the presence of the compound in question. In multianalyte configuration, however, one more parameter, which is the binding of one analyte-specific antibody to the protein conjugates of the co-determined analytes, should be also evaluated. Cross-reacting molecules usually present structural homology with the analyte of interest. Thus, similarities in molecular structures provide a clue for potential cross-reacting action of a chemical substance. As demonstrated in Table 1, the molecular structures of PQ and DQ present an increased homology. Therefore, at least a minimum cross-reactivity level was expected. The rest of the co-determined pesticides differ significantly in terms of chemical structure, and thus, insignificant cross-reactivity effects should be expected. In the context of this work, several antibodies were tested, using single-analyte fluoroimmunoassays performed in microtitration wells. The selected antibodies provided adequate analytical signal and assay sensitivity and presented minimal crossreactivity values. Once the appropriate antibodies were selected, both aforementioned types of cross-reactivity were investigated by employing the capillary fluoroimmunosensor. As shown in Table 2, no cross-reactivities were observed in the HEX and MES assays when tested with all the other analytes. Additionally, HEX and MES did not cross-react in both PQ and DQ assays. Concerning the PQ and DQ assays, it was determined that PQ cross-reactivity in the DQ assay was 0.04%, whereas, the respective DQ cross-reactivity in the PQ assay was 0.006%. According to these values, to detect an apparent concentration of 0.1 ng/mL DQ, 250 ng/mL PQ concentration was required, whereas, 1670 ng/mL DQ was required to provide an apparent concentration of 0.1 ng/mL PQ. However, since these analytes are determined simultaneously with the proposed immunosensor, it is possible to correct the concentrations found, based on the cross-reactivity data in case one of the analytes is in great excess. The binding of each analyte-specific antibody to the conjugates of the co-determined analytes, immobilized at distinct bands in Analytical Chemistry, Vol. 74, No. 23, December 1, 2002
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Table 2. Cross-Reactivitya Values (%) of the Panel Analytes
Table 3. I50 Values (ng/mL) Obtained for HEX, MES, PQ, and DQ Assays by Varying the Immunoreaction Time
assay
immunoreaction time
analyte
MES
HEX
PQ
DQ
analyte
2 min
5 min
10 min
15 min
30 min
MES HEX PQ DQ
100 ndb nd nd
nd 100 nd nd
nd nd 100 0.006
nd nd 0.04 100
HEX MES PQ DQ
1.7 0.8 0.9 1.1
1.6 0.8 0.9 1.3
1.9 1.1 1.1 1.8
2.2 1.5 1.1 2.1
3.1 1.9 1.2 2.9
a Cross-reactivity was determined using single-band capillaries, single-analyte standards, and single interfering substance solutions. b nd, not detectable inhibition was observed for cross-reactant concentrations up to 10 000 ng/mL.
the internal surface of the capillary, was also determined. Different four-band capillaries were filled with solutions consisting of a mixture of zero standard (doubly distilled water) with a different analyte-specific antibody. In all cases, only one peak at the corresponding analyte band was obtained, whereas no detectable antibody binding to the bands corresponding to the rest of the analytes was observed. Optimization of the Multianalyte Assay Conditions. The optimum assay conditions are described in detail in the Experimental Section. Certain parameters in multianalyte assays such as buffer composition and immunoreaction time were optimized on the understanding that they ought to be the same for all assays performed simultaneously in the same capillary tube. Therefore, optimization of all assays was based on the achievement of the highest possible sensitivity for all analytes with minimal compromises on the analytical signal levels acquired. A critical parameter affecting both the analytical signal and sensitivity of an assay is the pH. Therefore, to select the optimum pH for all four assays, several different assay buffers covering a pH range of 6.5-8.25 were investigated. The assay buffers examined were as follows: 150 mM PBS, pH 6.5, pH 7.0, and pH 7.4; 50 mM Tris-HCl, pH 7.8 and pH 8.25. All the buffers tested were used with or without 0.05% Tween 20 and 0.1% BSA. In any case, the 150 mM PBS containing 0.05% Tween 20, pH 7.0, provided 14-40% higher signal than the rest of the buffers tested, whereas sensitivities obtained by this buffer were superior or comparable with those attained by all the other buffers. Consequently, this buffer was chosen as the most appropriate common assay buffer. Immunoreaction time is another parameter that should be carefully optimized. Concerning the competitive immunoreaction format, when an antigen-antibody pair is given enough time to react, it may yield sufficient analytical signal but sensitivity may be negatively influenced. To address this problem, the sensitivities of all four assays were assessed for immunoreaction times of 2, 5, 10, 15, and 30 min. In all cases, the signal was adequate to perform the immunoassay. In Table 3, the concentrations of analytes causing 50% inhibition of the signal compared to zero standard (I50) are presented. According to these results, similar I50 values were observed for 2 and 5 min of immunoreaction; however, 5-min reaction provided higher analytical signals. On the other hand, increase in immunoreaction time from 5 to 30 min resulted to an increase of the I50 values observed for all four assays, indicating a deterioration of the assay sensitivity. This effect was more obvious for MES, HEX, and DQ assays. The PQ 6070 Analytical Chemistry, Vol. 74, No. 23, December 1, 2002
assay was less affected by increased immunoreaction times, possibly due to the use of monoclonal antibody instead of polyclonal antisera. Similar trend of the I50 values was observed for fluoroimmunoassays in microtitration wells; in that case, however, deterioration of the assay sensitivity was observed when the immunoreaction time exceeded 30 min. To spot the specific antibodies bound on the respective bands, a solution containing a mixture of FITC-labeled anti-mouse IgG and anti-rabbit IgG antibodies at excessive concentrations was employed. As was found, 30 min of incubation with this solution yielded maximum signals, whereas at least 120 min was required for the assays performed in microtitration wells. Consequently, the total incubation time for the single-analyte assays performed by the immunosensor was 35 min, whereas the corresponding time for each assay in microtitration wells was at least 150 min. Thus, concerning single-analyte assays, the immunosensor provided an approximately 75% reduction in assay time, compared to microtitration wells. However, taking into account that using the capillary immunosensor, four analytes were determined simultaneously, the reduction in assay time achieved approximated 95%. Analytical Characteristics. Typical calibration curves of the four different assays, performed by the multianalyte immunosensor and by the single-analyte fluoroimmunoassays in microtitration wells, are illustrated in Figure 5. The detection limits (calculated as the concentrations of analytes corresponding to the mean value of 20 replicates of zero calibrator minus three times the standard deviation) for MES, PQ, DQ, and HEX assays using the optical immunosensor were 0.04, 0.06, 0.09 and 0.10 ng/mL, respectively. In addition, the linear region of standard curves, defining the working range of each assay, was extended up to 9, 6, 12, and 15 ng/mL for MES, PQ, DQ, and HEX assay, respectively. Although similar results in terms of dynamic range were determined for the assays in microtitration wells, immunosensor assays seem to be more sensitive since the respective dose-response curves are steeper. However, the detection limits calculated in both cases were similar, due to the lower CVs observed for the assays performed in microtitration wells. Both the intra- and interassay CVs of the multianalyte immunosensor assays were estimated based on the consecutive determination of three samples covering the whole range of the assays. The within-run precision (intra-assay CV), estimated by eight replicate determinations of each sample, in a single run, was 2.5-7%, whereas, the between-run precision (interassay CV), estimated by the triplicate determinations of the same samples in 10 different days was between 4.5 and 10%. The corresponding values for the assays in microtitration wells were 0.5-4 (intraassay CV) and 1.5-5% (interassay CV). The increased CVs
Figure 5. Typical dose-response curves for MES, HEX, PQ, and DQ obtained using either the four-band capillary immunosensor (b) or single-analyte fluoroimmunoassays performed in microtitration wells (0). Each point corresponds to the mean value of three different capillaries; the error bars represent (SD.
observed for the assays in the capillary immunosensor can be ascribed to the fact that, in contrast to PS microtitration wells, which is a standard solid support for immunoassays, PMP capillaries are a novel solid support for this kind of applications. Therefore, improvements in fabrication methodology of the capillaries are expected to minimize imperfections in their optical qualities, which in turn, would increase the assays sensitivity. The accuracy of the assays performed in the capillary immunosensor was evaluated by carrying out recovery and dilution tests. The recovery test was performed in water samples prepared with deionized water and containing the analytes at concentrations lying within the linear ranges of the corresponding assays. Increasing amounts of pesticides were added to these samples, and their concentration was determined before and after the addition. Recovery values were expressed as the percent ratio of the concentration of analyte observed in the spiked sample minus the initial analyte concentration to the analyte concentration added. The recoveries for MES, PQ, DQ, and HEX assays ranged between 97 and 128, 85 and 122, 83 and 126, and 79 and 117%, respectively. Dilution test was performed by successive dilutions of three different samples containing all analytes with concentrations lying at the higher level of each assay dynamic range. The results demonstrated in Table 4 indicated that all four assays presented good linearity (r > 0.988). Other capillary sensors have been employed in the past for the determination of various analytes.12,22,23 The integrating wave-
Table 4. Resultsa of Dilution Linearity Test of Samples. Linear Regression Correlation between Expected and Determined Values (n ) 12) analyte
slope
intercept
r
p
HEX MES PQ DQ
1.11 1.04 0.952 1.05
+0.0447 -0.0338 +0.0970 +0.0373
0.993 0.995 0.988 0.991
