Ultrasensitive Magnetic Particle-Based Immunosupported Liquid

Anal. Chem. 2005, 77, 7156-7162

Ultrasensitive Magnetic Particle-Based Immunosupported Liquid Membrane Assay Madalina Tudorache, Michelle Co, Henric Lifgren, and Jenny Emne´us*

Department of Analytical Chemistry, Lund University, P.O. Box 124, 221 00 Lund, Sweden

A magnetic particle-based immuno-supported liquid membrane assay (m-ISLMA) based on chemiluminescence detection of a horseradish peroxidase-labeled hapten tracer that allows sample cleanup, analyte enrichment, and detection in a single analysis unit has been developed. Antibodies were immobilized on magnetic beads, and their position in the acceptor was controlled by two alternating opposing electromagnetic fields generated by a voltage applied to either of two electromagnets placed below and above the acceptor channel of the supported liquid membrane unit. The influence of antibody bead dilution in the acceptor was investigated and found to follow the ISLM theory, that is improved enrichment and sensitivity with increasing antibody concentration. Two different extraction procedures were investigated: procedure 1 (m-ISLMA-P1), which keeps the antibody beads trapped at the bottom of the acceptor during the entire analysis process; and procedure 2 (m-ISLMA-P2), which keeps the antibody beads dispersed and in motion in the acceptor phase during the extraction process. m-ISLMAP2 resulted in 2000 times improved enrichment of simazine and a more than 3 orders of magnitude better limit of detection (LOD10%) (1.29 × 10-5 µg L-1) than for m-ISLMA-P1 (2.00 × 10-2 µg L-1) and corresponding microtiter plate magnetic particle-based ELISA (m-ELISA, LOD10% 1.30 × 10-1 µg L-1). m-ISLMA-P2 and m-ELISA were further applied for the extraction and analysis of simazine-spiked surface water and fruit juice, finding no evidence for matrix influence for the former method; however, indications that trace amounts (nanograms per liter) of simazine or specific cross-reactants were present in both samples. Biorecognition by immunoaffinity binding has in recent years become popular for the development of new, more selective sample pretreatment techniques.1-4 Immunochromatography, immunoaffinity extraction, immunofiltration, and immunoprecipitation are now techniques frequently used in environmental as well * Corresponding author. Phone: +46 +46 222 48 20. Fax: +46 +46 222 45 44. E-mail: [email protected]. (1) Van Emon, J. M.; Gerlach, C. L.; Bowman, K. J. Chromatogr., B 1998, 715, 211-228. (2) Hage, D. S. J. Chromatogr., B 1998, 715, 3-28. (3) Hennion, M.-C.; Pichon, V. J. Chromatogr., A 2003, 1000, 29-52. (4) Delaunay-Bertoncini, N.; Hennion, M.-C. J. Pharm. Biomed. Anal. 2004, 34, 717-736.

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as clinical analysis,1,5 their principle and common feature being simultaneous and selective sample cleanup and analyte enrichment.6 In parallel, superparamagnetic particles (that respond to a magnetic field, but are completely demagnetized when the field is removed)7 are increasingly used in the area of bioscience,8 especially as carriers in separation processes and immunoassay.9-12 Magnetic-particle-based enzyme-linked immunosorbent assays (m-ELISA) are, for example, associated with improved experimental reproducibility and repeatability,13 as compared to classical ELISA14-16 and the uniform dispersion of particles (higher surfacearea-to-volume ratio) throughout the reaction mixture yields faster reaction kinetic.17,18 Recently, we reported the technique immuno-supported liquid membrane (ISLM) extraction,19-21 which essentially is supported liquid membrane (SLM) extraction22-24 using immunoaffinity as the extraction driving force. The technique is based on diffusion of an uncharged analyte from a flowing aqueous sample phase (the donor) into a stagnant antibody-containing aqueous phase (the acceptor) via an organic liquid membrane (the SLM) (5) Hage, D. S. Clin. Chem. 1999, 45, 593-615. (6) Delaunay, N.; Pichon, V.; Hennion, M.-C. J. Chromatogr., B 2000, 745, 1537. (7) Sole´, S.; Merkoc¸ i, A.; Alegret, S. Trends Anal. Chem. 2001, 20, 102-110. (8) Shinkai, M. J. Biosci. Bioeng. 2002, 94, 606-613. (9) Squirrell, D. J.; Price, R. L.; Murphy, M. J. Anal. Chim. Acta 2001, 21675, 1-6. (10) Kassimi, L. B.; Gonzague, M.; Boutrouille, A.; Cruciere, C. J. Virol. Methods 2002, 101, 197-206. (11) Yu, L. S. L.; Reed, S. A.; Golden, M. H. J. Microbiol. Methods 2002, 49, 63-68. (12) Lawruk, T. S.; Gueco, A. M.; Mihaliak, C. A.; Dolder, S. C.; Dial, G. E.; Herzog, D. P.; Rubio, F. M. J. Agric. Food Chem. 1996, 44, 2913-2918. (13) Lawruk, T. S.; Gueco, A. M.; Jourdan, S. W.; Scutellaro, A. M.; Fleeker, J. R.; Herzog, D. P.; Rubio, F. M. J. Agric. Food Chem. 1995, 43, 1413-1419. (14) Lawruk, T. S.; Lachman, C. E.; Jourdan, S. W.; Fleeker, J. R.; Hayes, M. C.; Herzog, D. P.; Rubio, F. M. Environ. Sci. Technol. 1996, 30, 695-700. (15) Itak, J. A.; Selisker, M. Y.; Jourdan, S. W.; Fleeker, J. R.; Herzog, D. P. J. Agric. Food Chem. 1993, 41, 2329-2332. (16) Young, D. L.; Mihaliak, C. A.; West, S. D.; Hanselman, K. A.; Collins, R. A.; Phillips, A. M.; Robb, C. K. J. Agric. Food Chem. 2000, 48, 5146-5153. (17) Hayes, M. A.; Polson, N. A.; Phayre, A. N.; Garcia, A. A. Anal. Chem. 2001, 73, 5896-5902. (18) Luxton, R.; Badesha, J.; Kiely, J.; Hawkins, P. Anal. Chem. 2004, 76, 17151719. (19) Thordarson, E.; Jo ¨nsson, J. Å.; Emne´us, J. Anal. Chem. 2000, 72, 52805284. (20) Tudorache, M.; Rak, M.; Wieczorek, P. P.; Jo¨nsson, J. Å.; Emne´us, J. J. Immunol. Methods 2004, 284, 107-118. (21) Tudorache, M.; Emne´us, J. J. Membr. Sci. 2005, 256, 143-149. (22) Jo ¨nsson, J. Å.; Mathiasson, L. Trends Anal. Chem. 1999, 18, 325-334. (23) Jo ¨nsson, J. Å.; Mathiasson, L. Trends Anal. Chem. 1999, 18, 318-325. (24) Jo ¨nsson, J. Å.; Mathiasson, L. J. Chromatogr., A 2000, 902, 205-225. 10.1021/ac050978k CCC: $30.25

© 2005 American Chemical Society Published on Web 10/14/2005

sandwiched between the donor and acceptor. The presence of antibodies favors the accumulation of immuno complexes in the acceptor, and back-diffusion into the donor is avoided both due to the strength of the complex and because the complex due to size and charge is unable to pass back through the organic membrane.20 In our previous systems, the antibodies were dissolved in the acceptor solution, and the quantification of immuno extracted analyte was performed in a consecutive fluorescence-based flow immunoassay.19-21 In this paper, we report a new magnetic-particle-based immuno-supported liquid membrane assay (m-ISLMA) using chemiluminescence (CL) detection.25-27 The corresponding system allows sample cleanup, analyte enrichment, and detection in a single analysis unit with excellent sensitivity. The novelty is that the antibodies are immobilized on magnetic beads and that their positioning and motion in the acceptor can be controlled by two alternating opposing electromagnetic fields (EMFs) generated by a voltage applied to either of two electromagnets placed below and above the acceptor channel of the SLM unit. The influence of antibody bead “concentration” on the mISLMA sensitivity was investigated, and two different m-ISLM extraction procedures were compared in terms of extraction efficiency. The m-ISLMA was also compared to a corresponding microtiter plate-based m-ELISA with regard to sensitivity and detection of simazine in samples with different matrix complexities (surface water and fruit juice). EXPERIMENTAL SECTION Materials and Solutions. Affinity-purified polyclonal antisimazine immunoglobulin G (IgG) antibody from sheep was kindly provided by Dr. Ram Abuknesha (King’s College, University of London, U.K.).28 The antibody was immobilized on the surface of superparamagnetic particles (3-µm diameter, -COOH active groups on the surface, Micromod, Rostock, Germany) using the following reagents: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), N-hydroxysuccinimide (NHS), and glycine, all from Sigma-Aldrich, Steinheim, Germany. The coupling buffer for activation of -COOH groups on the particle surface was a solution of 0.1 M 2-(4-morpholino)ethanesulfonic acid (MES, Sigma-Aldrich) in purified water (Millipore, Milford, MA) adjusted to pH 6.3 with 0.5 M Na2CO3 (Merck, Darmstadt, Germany). The antibody layer on the magnetic beads was regenerated by washing with 25% methanol (Merck) at pH 11.5 (1:4 dilution of pure MeOH with purified water, following adjustment to pH 11.5 with 1 M NaOH). A 100 mM phosphate buffer saline (PBS) stock solution was prepared from 80 g of NaCl, 2.00 g of KCl, 14.30 g of Na2HPO4‚2H2O, and 3.43 g of KH2PO4 dissolved in 1 L of purified water. A 10 mM PBS was prepared from the stock by dilution in purified water and adjusting the pH to 7.4 with 1 M NaOH solution. All phosphate buffer reagents were purchased from Merck. The horseradish peroxidase (HRP) tracer Ag* (Et/S/(CH2)5CO-HRP) was provided by Prof. Milan Franek (Veterinary (25) Yakovleva, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emne´us, J. Anal. Chem. 2002, 74, 2994-3004. (26) Yakovleva, J.; Davidsson, R.; Bengtsson, M.; Laurell, T.; Emne´us, J. Biosens. Bioelectron. 2003, 19, 21-34. (27) Jain, S. R.; Borowska, E.; Davidsson, R.; Tudorache, M.; Ponte´n, E.; Emne´us, J. Biosens. Bioelectron. 2004, 19, 795-803. (28) Tschmelak, J.; Proll, G.; Gauglitz, G. Anal. Bioanal. Chem. 2004, 379, 10041012.

Research Institute, Brno, Czech Republic).29 Stock solutions of tracer were kept in the freezer, and the working tracer solution was prepared daily by diluting the stock solution with 10 mM PBS at pH 7.4. Bovine serum albumin (0.01%) (BSA, Sigma-Aldrich) was added to the tracer solution for enzyme label conservation and for prevention of unspecific binding of tracer. A 0.8 mg mL-1 simazine (Institute of Industrial Chemistry, Warsaw, Poland) stock solution was prepared in dimethylsulfoxide (DMSO, SigmaAldrich) and diluted with 10 mM PBS to reach concentrations in the range 0-10 mg L-1. The HRP substrate mixture (luminol, p-iodophenol (PIP), and hydrogen peroxide (H2O2), all from Sigma-Aldrich) used for m-ISLMA was prepared by adding 25 µL of luminol (50 mM luminol solution in DMSO), 44 µL of PIP (150 mM PIP solution in DMSO), and 5 µL of H2O2 (30% H2O2) into 50 mL of PBS solution (10 mM, pH 10). Then the mixture was diluted 10 times, and the final substrate used for analysis contained 2.5 µM luminol, 13.2 µM PIP, and 1 mM H2O2. The colorimetric substrate used for m-ELISA contained the following reagents: 1.6 mL of freshly prepared tetramethylbenzidine (TMB, 6 mg mL-1 in DMSO, Merck) and 14.4 µL of hydrogen peroxide (30% H2O2) diluted in 100 mL of citrate buffer pH 5.5 (40 mM trisodium citrate and 40 mM citric acid, Merck). Di-n-hexyl ether (97%, Sigma-Aldrich) was used as the organic solvent for preparation of the SLM. At the end of each day, the donor was cleaned with 0.5 M H2SO4 solution (95-97% H2SO4, Merck) to avoid analyte memory effects. Surface water (Gardstånga, Lund, Sweden) and fruit juice (Kingsway, Dargun, Germany) were filtered (0.45-µm filter, Millipore, Ireland), and the pH was adjusted to 7.4 with NaOH. A centrifugation step prior to filtration of the fruit juice was performed to eliminate the fruit pulp from the juice. Both samples were diluted with 10 mM PBS solution in different proportions and also spiked with simazine at several concentrations (0-10 mg L-1 of simazine). Immobilization of Antibody on Magnetic Beads. Antibodies were immobilized via their amine functional groups onto magnetic beads with carboxylic functionality according to the specific protocol provided by Micromod. In a first step, a 0.5-mL suspension of magnetic beads, placed in an Eppendorf vial, was separated from the solution using a permanent magnet (sintered NdFeB permanent magnet, National Imports LLC, Falls Church, VA). The beads were washed by re-suspension in 1 mL of 0.1 M MES following magnetic separation and removal of the solution. A mixture of EDAC (4 mg) and NHS (8 mg) dissolved in 1 mL of MES buffer (pH 6.3) was added to the beads, vortexed for 1 min, and then incubated for 2 h at room temperature under gentle mixing to activate the -COOH groups on the surfaces of the magnetic beads. The excess of reagents was eliminated, and the magnetic beads were washed by suspending/magnetic separation in/from 1 mL of solution of 0.1 M MES buffer. A 1-mL portion of antibody solution (200 µg mL-1 protein dissolved in 0.1 M PBS) was added to the magnetic beads, and the mixture was continuously mixed for 3 h at room temperature. Finally, to reduce unspecific binding, the antibody-modified beads were resuspended in 1 mL of 25 mM glycine (dissolved in 0.1 M PBS solution) for 30 min, then washed twice with 1 mL of 0.1 M PBS solution and stored in 0.1 M PBS solution at 4 °C. (29) Kolar, V.; Deng, A.; Franek, M. Food Agric. Immunol. 2002, 14, 91-105.

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Figure 1. The m-ISLMA system setup containing (1) the SLM unit together with the electromagnetic system used to change the position of the antibody beads; (2) peristaltic pump; (3) syringe pump; (4) multiposition valve; and (5) PMT detector. Ag*, tracer; S, substrate; and R, regeneration solution.

Instrumentation and Analytical Procedures. The m-ISLMA setup was based on a simple automatic SI (sequential injection) system containing the following components (Figure 1): (1) an SLM extraction unit (donor and acceptor volumes, 10 µL) connected to a homemade electromagnetic device (Chemical Center Workshop, Lund University, Sweden), described in more detail in the next section ; (2) a peristaltic pump (Gilson, Villiersle-Bel, France) to introduce sample through the donor channel; (3) a syringe pump with a 250-µL syringe capacity (P/N 50300, Kloehn, Las Vegas, NV); (4) a multiposition valve (10-position, Valco Instruments Co. Inc., Houston, TX, model CS-1340EMT) used to select and introduce different reagents into the acceptor channel (i.e., antibody beads, tracer (Ag*), and HRP substrate); and (5) a photomultiplier tube (PMT, Hamamatsu Photonics K. K., Japan, model HC135-01, UV to visible). The system was computer-controlled using two different software programs: (i) for controlling the syringe pump and multiposition valve, software was provided by Kloehn (Las Vegas, NV); and (ii) a homemade program for data acquisition based on Microsoft Windows Office ’98. m-ISLM Unit. The m-ISLM unit was composed of two inert blocks of Plexiglas, in which identical 10-µL channels were drilled (78 mm long and 3 mm wide) on one side of each block. On the opposite side of each block, a socket was drilled (6.3 cm long, 0.5 cm wide, and 11 cm deep) for insertion of an electromagnet in each. The unit was assembled by placing an SLM, consisting of a microporous polypropylene membrane support (Celgard 2500 Microporous Membrane, Norderstedt, Germany, 55% porosity, 0.209 × 0.054 µm pore size, 25-µm thickness) impregnated with di-n-hexyl ether (previous immersion of the membrane support in the solvent for 30 min) between the two Plexiglas blocks, which were held together by eight metal screws. When a voltage, controlled with a homemade program through a switching box (Kemo-Electronic, Germany), was applied to any of the two electromagnets, an EMF was generated in the corresponding side of the system (below (EMF1) or above (EMF2) the acceptor channel). Due to development of heat from the electromagnets, an external fan (Kjell & Company, Malmo¨, Sweden) was placed facing the acceptor to keep a constant temperature inside the acceptor. Once the m-ISLM unit was inserted into the SI system, both faces of the SLM were washed with 3 mL of purified water, and the antibody modified magnetic beads could be introduced. m-ISLMA Procedure. The m-ISLMA procedure is described with the following steps: 7158 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

(1) Introduction of antibody beads into the acceptor. A 10-µL suspension of antibody beads (1/10 dilution of antibody bead stock solution, unless otherwise stated) in 10 mM PBS (pH 7.4) was aspirated and dispensed into the acceptor, where the beads were trapped at the bottom of the acceptor by an EMF1. This step was only performed one time per day. (2) Immunoextraction. Analyte (Ag) solution was continuously pumped through the donor channel for 14 min at a flow rate of 100 µL min-1, resulting in the diffusion of the analyte from the donor over the SLM to the acceptor, where it subsequently was captured by antibody beads via two different procedures (see Figure 2, step 1): Procedure 1 (m-ISLMA-P1): the beads were all the time trapped at the bottom of the acceptor by an applied EMF1. Procedure 2 (m-ISLMA-P2): the beads were kept in motion, alternately switching between an applied EMF1 and EMF2, that is, 2 min EMF2; 1 min EMF1; 2 min EMF2; 1 min EMF1; 2 min EMF2; and finally, 6 min EMF1 (total 14 min), the latter to trap the antibody beads at the acceptor bottom, enabling quantification of the amount of immunoextracted analyte through steps 3-5 below. (3) Titration with HRP-tracer (Ag*) (Figure 2, step 2). A 30µL portion of an 80 nM tracer solution was aspirated and then dispensed into the acceptor to saturate the residual free antibody binding sites. (4) Rinsing. A 30-µLportion of 10 mM PBS pH 7.4 was aspirated and then dispensed into the acceptor to remove any excess unbound tracer. (5) Detection of bound Ag* (Figure 2, step 3). A 30-µL portion of HRP substrate (2.5 µM luminol, 13.2 µM PIP, and 1 mM H2O2) was aspirated and then dispensed into the acceptor to react with antibody-bound tracer (Ag-HRP), generating the CL reaction product, which was eluted from the acceptor and detected by the PMT. The obtained CL signal was indirectly proportional to the extracted analyte concentration. (6) Regeneration of the antibody beads. A 30-µL portion of 25% MeOH (pH 11.5), directly followed by 150 µL of 10 mM PBS (pH 7.4), was aspirated and dispensed into the acceptor for antibody regeneration, that is, removing analyte and tracer bound to the antibody beads, thus creating conditions for a new extraction assay cycle to begin at point 2 above. m-ELISA Procedure. The m-ELISA was performed in microtiter plates (Merck Eurolab Ab, Karlskoga, Sweden) similar to the classical ELISA procedure,30,31 but in this case, the antibodies were covalently immobilized on magnetic beads, and a sequential competitive procedure was employed (i.e., the tracer was added after the analyte incubation with the antibody),32-34 to mimic as far as possible the m-ISLMA-P2, as described above. A 10-µL suspension of antibody beads (1/10 dilution from the antibody bead stock solution) in 10 mM PBS (pH 7.4) was added in each (30) Winklmair, M.; Weller, M. G.; Mangler, J.; Schlosshauer, B.; Niessner, R. Fresenius’ J. Anal. Chem. 1997, 358, 614-622. (31) Dzantiev, B. B.; Zherdev, A. V.; Romanenko, O. G.; Sapegova, L. A. Int. J. Environ. Anal. Chem. 1996, 65, 95-111. (32) Lei, C.-X.; Gong, F.-C.; Shen, G.-L.; Yu, R.-Q. Sens. Actuators, B 2003, 96, 582-588. (33) Dı´az-Gonza´lez, M.; Herna´ndez-Santos, D.; Gonza´lez-Garcı´a, M. B.; CostaGarcı´a, A. Talanta 2005, 65, 565-573. (34) Hage, D. S.; Thomas, D. H.; Beck, M. S. Anal. Chem. 1993, 65, 16221630.

Figure 2. Schematic description of two m-ISLMA procedures: m-ISLMA-P1 and m-ISLMA-P2, differing only in the immuno extraction - step 1 ( antibody bead; blue ovals, analyte; squares and solid circles, sample matrix; CL product, chemiluminescent product).

well in which they were trapped at the bottom of the wells by a permanent magnet. The beads were subsequently washed with 50 µL of 10 mM PBS (pH 7.4), and the solution was removed by pipetting. Then 10 µL of analyte standard was added, the magnet was removed, and the antibody beads were resuspended in solution by gently agitating the plate on a shaking board. After a 14-min incubation, the solution was removed, keeping the beads in the well by applying the permanent magnet. Next, 10 µL of 80 nM tracer solution was added to titrate the residual free antibody sites for 1 min with the antibody beads still kept at the bottom of the wells by the magnet. The beads were then rinsed with 150 µL of 10 mM PBS (pH 7.4). Finally, 100 µL of the HRP substrate (TMB and H2O2 in citrate buffer, pH 5.5) was added to the wells, and after a 5-min incubation, the enzymatic reaction was stopped by adding 50 µL of 5% H2SO4 solution. The developed color was detected at 450 nm using a plate reader (EL800, Bio-Tek Instruments, Inc.) with the accompanied software. The analytical signal was inversely proportional to the analyte concentration. RESULTS AND DISCUSSION The use of antibodies immobilized on magnetic beads and two alternating and opposing EMFs surrounding the acceptor in the

presented m-ISLMA systems (Figure 2) have two important advantages, as compared to previously developed ISLM systems: (1) antibodies immobilized on magnetic beads can be reused after regeneration, as opposed to being dissolved directly in the acceptor phase;19-21 (2) the two EMFs make it possible to control the location of the antibody beads in the acceptor, as shown in Figure 2, step 1 (m-ISLMA-P1, keeping the antibody beads trapped at the bottom of the acceptor applying only EMF1; or m-ISLMAP2, keeping the antibody beads in motion by alternating between applying EMF1 and EMF2, forcing the antibody beads to move vertically up and down in the acceptor18). Once the extraction is completed, the voltage is applied so that the antibody beads are trapped at the bottom of the acceptor (EMF1, Figure 2, step 2). At this point, the amount of residual unbound antibodies is titrated by introducing the tracer (Ag-HRP), following the introduction of the HRP substrate mixture (luminol, PIP, and H2O2) and subsequent detection of the produced chemiluminescence (CL) (Figure 2, step 3). All system components and introduction of antibody beads and reagents is computercontrolled in an SI system via a multiposition valve, as depicted in Figure 1. Analytical Chemistry, Vol. 77, No. 22, November 15, 2005

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Figure 3. The influence of the antibody concentration in the acceptor (b, 1/100; 9, 1/50; [, 1/10 dilution of the antibody bead suspension) on the ISLM extraction performance. Conditions: 80 nM tracer concentration, 100 µL min-1 sample flow rate in the donor, and 14-min extraction time; the antibody beads were immobilized at the bottom of the acceptor during the entire extraction assay (m-ISLMA-P1). The error bars represents the RSD (relative standard deviation) calculated for duplicate measurements.

Influence of Antibody Concentration on m-ISLMA. The theory of ISLM extraction can be summarized by the equation

Ee(max) ) 1 + K[Ab] where the maximum enrichment factor (Ee(max)) is linearly dependent on the affinity constant (K) of the antibody for the analyte as well as on the concentration of the antibody ([Ab]) in the acceptor.19,20 In other words, the type and concentration of specific antibodies in the acceptor will from a theoretical viewpoint influence the degree of analyte enrichment in the acceptor and, thus, the overall sensitivity of the assay. The influence of different Abs (affinity constants) was investigated in some of our previous ISLM works.20,21,35 The m-ISLMA using extraction procedure 1 (m-ISLMA-P1) was tested with three different antibody concentrations (1/10, 1/50, and 1/100 dilution of the antibody bead stock suspension). To ensure saturation of the immobilized antibody binding sites at zero analyte dose, the necessary working tracer concentration ([Ag*]w) for the highest antibody concentration (1/10) was established at 80 nM. Figure 3 clearly shows that the limit of detection (LOD10%, calculated as the analyte concentration which produces 10% inhibition of the zero-dose signal) decreases when the antibody concentration in the acceptor increases, that is, the simazine calibration curve is displaced to lower analyte concentrations as the antibody concentration increases, meaning that the analyte is more effectively enriched in the acceptor with increasing antibody concentration. For this reason, an antibody concentration corresponding to 1/10 antibody bead dilution was employed in all further experiments. Comparison of Different Extraction Procedures (m-ISLMA-P1 and -P2) and m-ELISA. The two different immunoextraction procedures (m-ISLMA-P1 and -P2) were investigated to see if and how the location of the antibody beads in the acceptor would influence the extraction efficiency. The apparent enrichment (35) Tudorache, M.; Emne´us, J. Submitted to Biosens. Bioelectron.

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app factor (Eapp e ) and extraction efficiency (E ) for the m-ISLMA-P2, were calculated relative to the m-ISLMA-P120, 21 and are given in Table 1. As previously discussed,20,21 quantitative extraction parameters specific for traditional SLM extractions (Ee and E) can only be characterized with apparent values for ISLM extraction app (Eapp e and E ), since their values are directly influenced by the inherent sensitivity of the immunoassay used for determination of the extracted analyte. The Eapp of 2000 and Eapp of 140% e demonstrate that the m-ISLMA-P2 is significantly more efficient, as compared to the m-ISLMA-P1. The antibody beads are forced by the alternating EMFs to move vertically up and down in the acceptor, that is, alternating between being in close proximity to the SLM surface and far away from the SLM surface, thus nicely accomodating the extraction in m-ISLMA-P2. As visualized in Figure 4, this leads to a displacement of the calibration curve to lower analyte concentrations and results in a LOD10% of 1.29 × 10-5 µg L-1 for simazine, which is more than 3 orders of magnitude lower than for m-ISLMA-P1 (2.00 × 10-2 µg L-1). An m-ELISA12,36 using conditions and procedure as similar as possible to that of the m-ISLMA-P2 was developed for comparative reasons. The same immunoreagents were used, the difference being that the assay was performed in a microtiter plate without any extraction and using colorimetric detection of the HRP-based tracer. As seen in Table 1, the m-ELISA (LOD10% 1.30 × 10-1 µg L-1) was almost 1 order of magnitude less sensitive than the m-ISLMA-P1 and 4 orders less sensitive than m-ISLMA-P2, which demonstrates the excellent performance of the m-ISLMA procedures. Evaluation and Application. Standard immunoassay validation principles require evaluation of the method precision as well as testing of the sample matrix effects on the assay performance.37 The within-assay (intra-assay) precision of the m-ISLMA-P2, calculated on the basis of the use of one SLM, one injected antibody bead portion repeatedly regenerated, and 10 calibration points (0-10 µg L-1 simazine) with two injections for each, was found to be 9%. Similarly, the between assay (interassay) precision of the m-ISLMA-P2, calculated on the basis of three calibration curves performed on three different days, each with a new injected antibody bead portion, a new fresh SLM, and 10 calibration points (0-10 µg L-1 of simazine) with two injections for each, was found to be 16%, results that are well within the recommended precision range (0-20%).37 The sample matrix, constituting everything in the sample other than the analyte of interest, can disturb the immunoassay in several ways: interfering with (i) the antibody-analyte and antibody-tracer interaction, (ii) the separation of bound and free fractions of the tracer, and/or (iii) the detected signal.37 Matrix effects can be evaluated by comparing the analyte standard curve with that of the spiked sample or the diluted sample. A “parallelism” of the calibration curves and a good analyte recovery may demonstrate no influence of the sample matrix on the analysis, whereas different slopes of the calibration curves and low analyte recovery introduce a suspicion of matrix interference.37 Dilution of the sample is one alternative to eliminate the interference, but

(36) Gruessner, B.; Shambaugh, N. C.; Watzin, M. C. Environ. Sci. Technol. 1995, 29, 251-254. (37) Diamandos, E. P.; Chistopoulos, T. K. Immunoassay; Academic Press: San Diego, California, 1996.

Table 1. Comparison of m-ELISA, m-ISLMA Procedure 1, and m-ISLMA Procedure 2

m-ELISA m-ISLMA-P1d m-ISLMA-P2e

LOD10%a (µg L-1)

IC50b (µg L-1)

DRc (µg L-1)

0.130 ( 0.010 0.020 ( 0.001 (1.290 ( 0.06) × 10-5

13.780 ( 1.240 0.250 ( 0.024 (1.070 ( 0.096) × 10-4

0.65-214 0.05-2.44 (2.89-53) × 10-5

a Limit of detection as analyte concentration corresponding to 10% inhibition of the zero-analyte dose signal. b Analyte concentration corresponding to 50% inhibition of the zero analyte dose signal. c Dynamic range between 20 and 80% inhibition of the zero-analyte dose signal. d Procedure 1. e Procedure 2.

Figure 4. Comparison of m-ISLMA-P2 (9) and m-ISLMA-P1 (b), emphasizing the positive role of alternating between EMF 1 and EMF 2, on the extraction efficiency. 1/10 dilution of the antibody bead suspension in the acceptor. Conditions otherwise as in Figure 3.

this simultaneously leads to a reduced analyte concentration in the sample, which might require a more sensitive analysis method.38 The matrix influence of surface water on simazine determination with m-ELISA and m-ISLMA-P2 was investigated by spiking the surface water at different dilutions. Figure 5a shows m-ELISA calibration curves for the standard, spiked undiluted, and 1:2 and 1:4 diluted surface water. The unparallelism of the standard curve and the spiked undiluted and 1:2 diluted sample curves indicates true matrix interference, which, as shown in Figure 5a, is eliminated at a 1:4 dilution of the sample. The same experiment was performed with the m-ISLMA-P2. In this case, the undiluted surface water resulted in such a significant reduction of the zero analyte dose signal that a calibration curve could not be performed. Figure 5b shows m-ISLMA-P2 calibration curves for the standard and for spiked 1:2 and 1:4 dilution of surface water. The curves for the two dilutions coincide almost completely, but are displaced to lower analyte concentrations as compared to the standard curve. The fact that the standard curve and the two spiked sample dilution curves are almost parallel indicates that there is no or very little matrix influence; the surface water seems to contain trace levels of the analyte or some cross-reactant(s) in the lower nanograms-per-liter level (≈0.2 ng L-1). This level is almost 3 orders of magnitude below the LOD10% for the m-ELISA and, thus, undetectable by this method (and in reality, negligible in relation to the acceptable level of 0.1 µg L-1, according to the U.S. EPA39 and the EC).40 In previous work in which ISLM extraction of (38) Nunes, G. S.; Toscano, I. A.; Barce´lo, D. Trends Anal. Chem. 1998, 17, 79-87. (39) http://www.epa.gov/. (40) http://www.eea.eu.int/.

Figure 5. Influence of surface water matrix on (a) m-ELISA and (b) m-ISLMA-P2. Conditions as in Figure 4 (“standard” is simazine in 10 mM PBS at pH 7.4). The error bars represents the RSD (relative standard deviation) calculated for duplicate measurements.

triazines from real samples was described, the extraction was performed directly from undiluted surface water with good analyte recovery.20 The present m-ISLMA is, however, 4 orders of magnitude more sensitive and, as such, likely to be more susceptible to interference.38 On the other hand, simazine and other triazines have recently been found at the nanograms-perliter level in Swedish surface waters (www.naturvardsverket.se/ dokument/mo/modok/export/U933_wfd.pdf and http://www. riksdagen.se/debatt/9798/motioner/jo/Jo14.asp), which increases the probability of their presence in the analyzed surface water. Another sample matrix, fruit juice, was tested in the m-ISLMAP2. Undiluted fruit juice resulted in a complete obliteration of the zero analyte dose signal, again indicating a strong matrix influence or the presence of analyte/cross-reactant in the sample. The experiments were initially performed as above for the surface water; however, for each sample dilution at the same spiking level, the zero analyte dose signal continued to be significantly supAnalytical Chemistry, Vol. 77, No. 22, November 15, 2005

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Figure 6. Testing the capacity of m-ISLMA-P2 to eliminate the matrix effect on the analysis based on successive sample dilution (comparison between standard (b) and successive dilution of juice ()) plot) and the standard addition method (comparison between standard (b) and spiked diluted 1:103 juice (0) plot). Conditions as in Figure 5.

pressed, why a sample dilution experiment was conducted and compared to a standard analyte calibration curve, shown in Figure 6. The almost complete parallelism of the two curves shows that there are no matrix effects, but rather, that some analyte/crossreactant is present in the sample. In the same figure, a spiked 1:103 diluted sample curve completely parallel to the standard curve, but displaced to lower analyte calibration curves, is shown, indicating a simazine or cross-reactant content in the fruit juice at ∼40 ng L-1. CONCLUSIONS A new highly efficient CL-based magnetic particle ISLM extraction assay (m-ISLMA), capable of 2000 times analyte enrichment and detection of picograms-per-liter levels of simazine, which is a substantial improvement compared to previous presented systems,19-21,35 has been presented. This is due to the

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combination of using antibody-coated magnetic beads and their forced motion up and down the acceptor by two alternating but opposing electromagnetic fields, which in this way accommodates the extraction process. Another reason is that detection of extracted analyte was performed using CL and directly on the acceptor surface instead of in a consecutive flow immunoassay, the latter resulting in high dispersion of the sample plug. The problem of high consumption of antibodies was solved, since the same injected portion of antibody beads could be used for at least 30 assay cycles. The assay procedure was, in addition, substantially simplified and automated since a sequential injection configuration with a multiposition valve was used for aspirating and dispensing all necessary reagents, with a total assay cycle of 25 min, the latter of which can be substantially reduced if the extraction time of 14 min is reduced (obviously, with an accompanying loss in sensitivity) and better software for control of the valve and syringe pump is used. Finally, there was no evidence of matrix effects from surface water or fruit juice on the m-ISLMA, indicating that both samples contained trace amounts of simazine or cross-reactant(s) at the nanograms-per-liter level. ACKNOWLEDGMENT The authors kindly acknowledge financial support from the European Commission (ICA2-CT-2000-10033 (BIOFEED) and INTAS contract 99-0995), the Swedish Council for Forestry and Agricultural Research (SJFR), the Swedish Research Council (Vetenskapsrådet), and the Swedish Environmental Protection Agency (NVV-Naturvårdsverket). In addition we espress our thanks for all of the reagents, the antibodies, and analyte hapten derivative from Dr. R. Abuknesha (Kings College, London, U.K.) and Prof. Milan Franek (Veterinary Research Institute, Brno, Czech Republic). Finally, a special thank you to Sven Ha¨gg and Konstantin Droubetski (Department of Analytical Chemistry, Lund University) for their valuable technical input and ideas. Received for review June 3, 2005. Accepted September 2, 2005. AC050978K