Advanced Homogeneous−Heterogeneous Immunosensing Format

Nov 17, 2007 - A rapid immunosensing methodology that employs the so-called homogeneous−heterogeneous assay mode is presented. The immunosensor ...
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Anal. Chem. 2007, 79, 9331-9339

Advanced Homogeneous-Heterogeneous Immunosensing Format Employing Restricted Access Supports Dolors Jornet, Miguel A Ä ngel Gonza´lez-Martı´nez, A Ä ngel Maquieira,* and Rosa Puchades*

Departamento de Quı´mica, Instituto de Quı´mica Molecular Aplicada, Universidad Polite´ cnica de Valencia, Camino de Vera s/n, 46071 Valencia, Spain

A rapid immunosensing methodology that employs the socalled homogeneous-heterogeneous assay mode is presented. The immunosensor is based on the homogeneous competition among the analyte, a fluorescent tracer, and the antibody, followed by separation of free and bound species by means of a restricted access alkyl-diol silica C18 reversed-phase chromatographic support. In order to develop a general labeling methodology, fluorescent tracers are synthesized from oligonucleotides covalently bound to the hapten in 3′ position and the marker in 5′. The immunosensor principle is demonstrated by determining atrazine in a completely automated manner at 2 min/ sample without regeneration of the support and a limit of detection of 1.0 µg/L with the optimized system. Preliminary assays employing multilabeled tracers indicate that sensitivity can be improved. Organic solvents 2-propanol and acetonitrile up to 15% (v/v) are well tolerated, while methanol can be added to 50%. The sensor capabilities are demonstrated through the analysis of natural waters. Within the spectrum of immunochemical techniques, flow immunosensors are sensitive, cost-effective, and robust. Immunosensing systems have now developed to an advanced stage,1 and notable achievements have been accomplished in their properties, especially sensitivity, portability, and multianalyte capacity.2-6 However, response time and reusability are still parameters to be drastically improved, since immunosensing devices find special application in continuous monitoring, so simplicity and rapidity can be of highest importance. * To whom correspondence should be addressed. E-mail: amaquieira@ qim.upv.es; [email protected]. (1) Gonza´lez-Martı´nez, M. A.; Puchades, R.; Maquieira, A. Anal. Bioanal. Chem. 2007, 387, 205-218. (2) Tschmelak, J.; Proll, G.; Gauglitz, G. Talanta 2005, 65, 313-323. (3) Tschmelak, J.; Kumpf, M.; Ka¨ppel, N.; Proll, G.; Gauglitz, G. Talanta 2006, 69, 343-350. (4) Ciumasu, I. M.; Kra¨mer, P. M.; Weber, C. M.; Kolb, G.; Tiemann, D.; Windisch, S.; Frese, I.; Kettrup, A. A. Biosens. Bioelectron. 2005, 21, 354364. (5) Gonza´lez-Martı´nez, M. A.; Puchades, R.; Maquieira, A. Anal. Chem. 2001, 73, 4326-4332. (6) Tschmelak, J.; Proy., G.; Riedt, J.; Kaiser, J.; Kraemmer, P.; Ba´rzaga, L.; Wilkinson, J. S.; Hua, P.; Hole, J. P.; Nudd, R.; Jackson, M.; Abuknesha, R.; Barcelo´, D.; Rodrı´guez-Mozaz, S.; Lo´pez, de Alda, M. J.; Sacher, F.; Stien, J.; Slobodnı´k, J.; Oswald, P.; Kozmenko, H.; Korenkova´, E.; To´thova´, L.; Krascsenits, Z.; Gauglitz, G. Biosens. Bioelectron. 2005, 20, 1499-1508. 10.1021/ac071427s CCC: $37.00 Published on Web 11/17/2007

© 2007 American Chemical Society

Many developed immunosensors, mainly those using immobilized antigen formats, show a total assay time between 15 and 20 min,7 although much more rapid systems are also found in the literature. For instance, an immunosensor based on antibodies entrapped in a sol-gel matrix8 needs only 5 min, including enzymatic signal display and sensor regeneration. The most rapid flow immunosensors are those based on the displacement assay format;1 analysis times as short as 2 min have been reported in an immunosensor for explosives.9 Nevertheless, displacement-based immunosystems are difficult to set up because the displacement is managed by association and dissociation kinetics, and sensitivity reached is in general lower than in competition-based immunosensing, so this format is hardly used. More recently, the development of microanalytical devices for biosensing has resulted in very high analyte detectability,10 although assay times have not been improved. A case in point is the microchip displacement immunoassay for staphylococcal enterotoxin B,11 with a limit of detection of 1 fM in a 20-min assay, with additional 30-min sample preparation. In general, flow immunosensors are based on heterogeneous competition formats performed in several steps (competition, tracing reaction, regeneration, etc.).7 Simplifying the assays to maintain high sensitivity is a challenging task because it is necessary to work each step of the immunoassay process. A good approach may involve carrying out the reaction in solution, which takes place almost instantaneously.7 Homogeneous phase-based immunoassays have been studied and developed, on the basis on a competition mode.12,13 However, they lack sensitivity if compared to the competitive heterogeneous format. The ideal approach would therefore combine the speed and simplicity of homogeneous assays and the sensitivity inherent to heterogeneous formats. On the other hand, one of the keys for achieving rapid and well-performing immunosensing analyses is to avoid immu(7) Gonza´lez-Martı´nez, M. A.; Puchades, R.; Maquieira, A. Trends Anal. Chem. 1999, 18, 204-218. (8) Pulido-Tofin ˜o, P.; Barrero-Moreno, J. M.; Pe´rez-Conde, M. C. Anal. Chim. Acta 2006, 562, 122-127. (9) Holt, D. B.; Gauger, P. R.; Kusterbeck, A. W.; Ligler, F. S. Biosens. Bioelectron. 2002, 17, 95-103. (10) Sheehan, P. E.; Whitman, L. J. Nano Lett. 2005, 5, 803-807. (11) Haes, A. J.; Terray, A.; Collins, G. E. Anal. Chem. 2006, 78, 8412-8420. (12) Goldman, E. R.; Cohill, T. J.; Patterson, C. H., Jr.; Anderson, G. P.; Kusterbeck, A. W.; Mauro, J. M. Environ. Sci. Technol. 2003, 37, 47334736. (13) Yang, X.; Janatova, J.; Andrade, J. D. Anal. Biochem. 2005, 336, 102-107.

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nosorbent regeneration, because this process is time-consuming and implies exposure of the immunocomplex to conditions of protein denaturation, with the resulting loss of activity of the immobilized reagent.14 All these considerations lead us to propose a new flow immunosensing format, which employs competition in homogeneous phase, heterogeneous separation of free and bound products, and readily detectable fluorescent labels, avoiding the regeneration step but maintaining reproducibility and the concept of long-use surface. In the basic studies of this type of sensor, three aspects should be considered: the separation of the reaction products, the label to be used, and finally the detection, which must be compatible with the separation and tracing strategies. For separation purposes, an issue to be addressed is the employment of restricted access material (RAM) supports.15 These sorbents separate the analytes by combining size exclusion and conventional reversed-phase partition or ion-exchange mechanisms, allowing the interaction with small molecules but restricting the access of macromolecules. The pore size of these materials is typically ∼10 nm, with a cutoff of 10 000-20 000 Da.16 Thus, large molecules such as proteins are excluded in the void volume while the small and more hydrophobic substances are retained. The most common application of RAM is in cleanup precolumns for HPLC analysis of biological fluid samples.17 In another application of RAM columns, described in a classic work by Oosterkamp et al.,18 the analyte eluting from liquid chromatography is titrated with an excess of a macromolecular ligand, and the free binding sites are further saturated with analytefluorescein conjugate so that the RAM support separates free and bound fluorophore. A more recent work from the above-mentioned research group describes the use of two serial RAM columns for drug purification. This application involves associating-dissociating the analyte to a specific antibody and RAM separation of free and bound species, prior to MS analysis.19 In the herein proposed homogeneous-heterogeneous (HH) immunoassay format, the immunoreaction takes place in solution, and the RAM support captures the free analyte and tracer in its hydrophobic cavity but excludes the large-sized immunocomplex (bound analyte and tracer), which directly reaches the detector, the generated signal being related to the analyte concentration. The proposed format should eliminate the regeneration step after each immunoreaction. This means avoiding chromatographic conditions but retaining the free tracer. Therefore, support capacity must be high in order to carry out many analyses prior to saturation. Alkyl-diol-silica (ADS) is one of the most common RAM supports available with different reversed (C4, C8 and C18) phases.20 Indeed, a physical barrier (pore size 6 nm) excludes macromol(14) Gonza´lez-Martı´nez, M. A.; Puchades, R.; Maquieira, A. Food Technol. Biotechnol. 1997, 35, 193-204. (15) Boos, K. S.; Grimm, C. H. Trends Anal. Chem. 1999, 18, 175-180. (16) Souverain, S.; Rudaz, S.; Veuthey, J. L. J. Chromatogr., B 2004, 801, 141156. (17) Cassiano, N. M.; Lima, V. V.; Oliveira, R. V.; de Pietro, A. C.; Cass, Q. B. Anal. Bioanal. Chem. 2006, 384, 1462-1469. (18) Oosterkamp, A. J.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1994, 66, 4295-4301. (19) van Elswijk, D. A.; Tjaden, U. R.; van der Greef, J.; Irth, H. Int. J. Mass Spectrom. 2001, 210/211, 625-636. (20) Boos, K. S.; Rudolphi, A. LC-GC 1997, 15, 84-95.

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ecules (Mr > 15 kDa), which are not adsorbed onto the support due to the hydrophilic groups (glycerylpropyl, i.e., diol moieties) bound at the outer surface of particles. Smaller molecules can interact with the inner bead surface and are partitioned by a conventional reversed-phase mechanism. The development of the HH sensor introduces an important issue associated with the chemical nature and molecular size of tracers to be used: molecular mass of competition tracers is limited by the support cutoff, so hapten must be covalently bound to a small label, such as a fluorescent dye. Coupling the hapten to the marker is a synthesis task, with the troubleshooting associated with this kind of operation: reaction conditions, product purification, yields, etc. The process could be unsuccessful, as reported in the past,21 or phenomena such as quenching and resonance energy transfer may substantially modify the fluorescence performances of the label.22 On the other hand, the preparation of a tracer with a different hapten or label implies developing a new, yet untested and laborious synthesis and purification process. In this sense, it is of considerable interest to use a general methodology to attach haptens to fluorescent dyes. Oligonuclotides are promising bifunctional molecules for conjugation purposes. An oligonucleotide is a single DNA chain with a variable number of nucleosides. It is easy to prepare, quite versatile, and has molecular mass under 15 kDa. The chemistry of oligonucleotides has been widely studied, so there are numerous possibilities for coupling other molecules (haptens and markers) at any position.23 Currently, oligonucleotides derivatized with functional groups or attached to different labels are commercially available, relatively inexpensive, and can be ordered a` la carte for the specific marker and base sequence. In this sense, once a method for preparing a hapten-label conjugate by coupling the hapten to a labeled oligonucleotide is developed, changing the label only requires purchasing and using the same oligonucleotide derivatized with the new label. The research presents the development of a fluorescent HH format flow immunosensor to target small-molecule residues. Atrazine is chosen as model analyte because there are many data of all types of immunoanalysis for comparative purposes, so immunoreagents for this herbicide are readily available. Further, atrazine is of great interest for screening studies given its widespread use, alone or with other herbicides in formulations, even though environmental control restricts the use and quantities of herbicides.24 Determination of atrazine by means of chromatographic methods continues to be of interest; examples described in the recent literature include those employing GC/MS,25 HPLC,26,27 HPLC-MS,28 and capillary electrophoresis.29 In all (21) Gonza´lez-Martı´nez, M. A.; Penalva, J.; Rodrı´guez-Urbis, J. C.; Brunet, E.; Maquieira, A.; Puchades, R. Anal. Bioanal. Chem. 2006, 384, 1540-1547. (22) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (23) Beaucage,S. L.; Radhakrishnan, P. I. Tetrahedron 1993, 49, 1925-1963. (24) Graymore, M.; Stagnitti, F.; Allinson, G. Environ. Int. 2001, 26, 483-495. (25) Perreau, F.; Einhorn, J. Anal. Bioanal. Chem. 2006, 386, 1449-1456. (26) Katsumata, H.; Kaneco, S.; Suzuki, T.; Ohta, K. Anal. Chim. Acta 2006, 577, 214-219. (27) Cha´fer-Perica´s, C.; Herra´ez-Herna´ndez, R.; Campı´ns-Falco´, P. J. Chromatogr., A 2006, 1125, 159-171. (28) Garcı´a-Reyes, J. F.; Ferrer, C.; Thurman, E. M.; Ferna´ndez-Alba, A. R.; Ferrer, I. J. Agric. Food Chem. 2006, 54, 6493-6500. (29) Carabias-Martı´nez, R.; Rodrı´guez-Gonzalo, E.; Miranda-Cruz, E.; Domı´nguezAÄ lvarez, J.; Herna´ndez-Me´ndez, J. J. Chromatogr., A 2006, 1122, 194-201.

examples, extraction, mainly SPE, and preconcentration steps are carried out prior to the analysis. MATERIALS AND METHODS Chemicals and Biochemicals. Analytical standards of atrazine and s-triazine derivatives were purchased from Dr. Ehrenstorfer (Augsburg, Germany) and Riedel de Ha¨en (SeelzeHannover, Germany). HPLC-grade methanol and acetonitrile were obtained from Sharlab (Barcelona, Spain). Deionized water was generated by means of a Millipore Milli-Q system (Bedford, MA). All other common reagents were analytical grade. The buffers employed were PBS (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl and HCl until pH 7.0) and PBS with 0.8 M NaCl added, also called “phase A” and “phase B”, respectively, in the chromatographic separation of tracers. Twofold concentrated PBS was used as carrier solution in the immunosensor assay. Oligonucleotides with sequences 5′ FL-TGGTGGATCG-C7-NH2 3′, 5′ FL-TAG-C7-NH2 3′, and 5′ FL-TAGTA-C7-NH2 3′, named SYM16, SYM18, and SYM20, respectively, were purchased from Molbiol (Berlin, Germany). Immunoreagents for atrazine were obtained as reported in a previous work.30 First, hapten 2d (N-(4-chloro-6-isopropylamino[1,3,5]triazine-2-yl)aminohexanoic acid) was synthesized and coupled to keyhole limpet hemocyanin by means of the active ester method.31 The conjugate was employed for immunizing four rabbits according to the described immunization protocol,30 and after the usual titration controls, four high-affinity anti-atrazine antisera were obtained. Instrumentation. All analyses were performed using a 1100 Series HPLC from Agilent Technologies (Santa Clara, CA), equipped with a fluorescence detector (excitation and emission wavelengths set at 495 and 519 nm, respectively). The column employed for tracer purification was a HP Zorbax Oligo analytical column 6.2 × 80 mm, 5-µm particle size, from Hewlett-Packard (larger size columns for higher preparative scale are available). The RAM columns consisted of RP 18 and RP 4 LiChrospher ADS materials, 25 × 4 mm i.d. (Merck, Darmstadt, Germany). Tracer Preparation. Hapten 2d and amino acids (Gly, Asp, Trp, His, and Val; Sigma, St. Louis, MO) were covalently linked to the oligonucleotides via their amino derivatization, according to a modification of the active ester method. Briefly, 20 µmol of hapten was incubated for 4 h with 20 µmol of N-hydroxysuccinimide (NHS) and 20 nmol of dicyclohexylcarbodiimide (DCC), all in 80 µL of anhydrous DMF. The final mixture was centrifuged at 11000g for 5 min, and 1 µL of the supernatant was added to 25 nmol of the oligonucleotide dissolved in 100 µL of 0.05 M sodium carbonate buffer, pH 9.6. The mixture was stirred for 2 h at room temperature and then overnight at 4 °C. Finally, the conjugate was purified by preparative HPLC employing the HP Zorbax Oligo column, in five injections of 20 µL each. The flow rate was 1 mL/ min, and the mobile phase was in gradient, from 100% phase A to 100% phase B in 60 min. The purified product was collected in six to eight 1-mL fractions. Homogeneous-Heterogeneous Immunoassay. To carry out the analyses, 70 µL of analyte standard or sample in PBS was (30) Gasco´n, J.; Oubin ˜a, A.; Ballesteros, B.; Barcelo´, D.; Camps, F.; Marco, M. P.; Gonza´lez-Martı´nez, M. A.; Morais, S.; Puchades, R.; Maquieira, A. Anal. Chim. Acta 1997, 347, 149-162. (31) Bauminger, S.; Wilcheck, M. Methods Enzymol. 1980, 70, 151-159.

mixed with 5 µL of the antibody solution (1:5 v/v dilution in water) and 25 µL of the tracer solution. When working with organic mixtures, the analyte solution was 50:50 v/v methanol-PBS, resulting in a methanol concentration of 35% v/v. Then, 20 µL of the mixture was injected through the RAM column, using 2-fold concentrated PBS as carrier at 0.5 mL/min, and the fluorescence peak was registered. Each measurement was carried out in triplicate. At the end of a working day, the column was washed with 20 mL of methanol, thus eluting all the retained material, and kept in this solvent when not in use. Water Sample Treatment. Ten natural waters were sampled in different fountains and wells from Comunidad Valenciana (Spain). Samples were analyzed as native and spiked with atrazine at 0.5 µg/L, after solid-phase extraction in 47-mm-diameter, 0.05mm-thick Empore C18 disks (Varian, Harbor City, CA) employing vacuum filtration. The disks were first conditioned with 10 mL of methanol followed by 5 mL of water. Samples (until 1500 mL) were percolated through the disk at 50 mL/min flow rate, and analyte was further eluted with 4 mL of methanol using fractions of 1 mL. The extract was then diluted with 4 mL of PBS, and 70 µL was mixed with antibody and tracer as described in the previous section and analyzed. RESULTS AND DISCUSSION Synthesis and Purification of Tracers. To develop a general method for the fluorescent labeling of haptens, commercially available bifunctionalized oligonucleotides were employed. The oligonucleotide can be used as derivatized in 5′ and 3′ depending on the desired marker and the functional group of the hapten to be labeled. The 5′ end was bound to fluorescein as marker, and the 3′ end was derivatized with an amino group to attach to the hapten. Since in this case the hapten for atrazine (2d) is carboxylic acid-functionalized, the coupling reaction was exactly the same as that employed for conjugating carboxylic acid haptens to carrier proteins or enzyme labels. When carrying out the procedure, two problems arose: the coupling reaction and the purification of the final product. The general method is based on the activation of the hapten with NHS and DCC, and this reaction takes place in DMF. As some organic solvents are harmful for oligonucleotides, it was necessary to modify the procedure minimizing the amount of DMF. The final procedure used the solvent at 1% v/v, maintaining an initial haptenoligonucleotide molar ratio of 10, lower than that usually employed to attach haptens to proteins, but high enough to couple the hapten to the oligonucleotide. Separating the conjugate hapten-oligonucleotide from the free hapten and oligonucleotide was also troublesome. Conventional preparative gel permeation chromatography (Sephadex G-25 or analogue supports) is not useful for effective conjugate purification. Thus, the separation was accomplished by HPLC, employing a mixed-mode ion-exchange and reversed-phase bonded phase, developed to isolate highly pure oligonucleotides.32 For the purification, isocratic and gradient elution were assayed, employing PBS at different concentrations as mobile phase, with and without organic modifiers (methanol and acetonitrile). No peak separation was observed when applying isocratic (32) Agilent ZORBAX Bio Series Oligo Column datasheet. Hewlett-Packard Company, Publication (23) 5695-7512E, 1999.

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Figure 1. HPLC purification of the 2d-oligonucleotide tracer. (a) Chromatogram obtained with the native SYM16 oligonucleotide in solution. (b) Chromatogram corresponding to the 2d-SYM16 reaction product. See text for details.

elution, and the presence of organic solvents in the mobile phase damaged the tracer. The final separation, with suitable peak resolution, was done with a gradient from 100% phase A (PBS) to 100% phase B (PBS with 0.8 M NaCl added), in 60 min at 1 mL/ min. In order to identify the products of the conjugation reaction and estimate the yield, the reaction mixture was diluted 1/500 v/v with phase A, and 20 µL was injected through the column. Figure 1a shows a chromatogram obtained with a solution containing SYM16 oligonucleotide alone, being eluted at tr 34.4 min. After the conjugation, the chromatogram (Figure 1b) shows two fluorescence peaks, one is the 2d-SYM16 conjugate (tr 46.4 min) and the other is the unconjugate oligonucleotide (tr 34.4 min). Moreover the total area of the two peaks from chromatogram b is approximately the same as that of the peak from chromatogram a, and the area of the conjugate peak is ∼90% of the total area, which indicates that the coupling yield is 90%. After identifiying the peaks, the original concentrated reaction mixture was chro9334

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matographied and 1-mL fractions containing the separated conjugate were collected (6-8 fractions). The product was diluted during chromatography, because the entire product was obtained in a total volume of 6-8 mL. However, the final concentration was high enough to be employed in the immunosensor assay, because tracers are used at very low concentrations. Similar results concerning product separation and coupling yields were obtained for the conjugation of 2d hapten to oligonucleotides SYM18 and SYM20. Preliminary Assays. In the HH immunoassay format, first analyte, antibody, and tracer are mixed for competition, giving rise to several resulting species: free analyte, free tracer, free antibody, analyte-antibody complex, and tracer-antibody complex. The competition is nearly instantaneous, so the reaction times30 s approximatelysis the time required for mixing the solutions. Immunocomplexes are further separated from the small species in the RAM column (Figure 2). The basic binding and separation properties of the RAM support were studied prior to

Figure 2. Scheme of the RAM-based separation used in the homogeneous-heterogeneous immunoassay format. When the reaction mixture is injected through the RAM support, the uncomplexed species are retained in the hydrophobic stationary phase, while the free antibody and the immunocomplexes pass through and reach the detector. Model based on an image from the LiChrospher ADS manual (Merck; http://es.vwr.com/ app/Header?tmpl)/cromatografia/prep_muestra.htm).

assaying the final immunoreagents (tracers and antibodies) to be used in the sensor. Lichorspher RP18 ADS material was employed in the preliminary assays, since both fluorescein and atrazine are hydrophobic (Koc higher than 600 at pH between 3 and 9, for the two compounds), so a C18 surface is expected to retain them properly. First, exclusion features of the RAM C18 column were assayed using fluorescein solutions and a nonspecific immunoglobulin covalently bound to this molecule. The IgG-fluorescein conjugate was seen to elute completely from the column with the void volume, while the fluorescein remained inside the column. A different experiment was carried out to verify the retention or elution of other small molecules with different hydrophobicity and charge status, as well as to examine the behavior of the oligonucleotide conjugates in the support. Free amino acids Gly, Asp, Trp, His, and Val (with isolectric point of 6.06, 2.98, 5.88, 7.60, and 6.01, respectively), at 100 mg/L concentration, were injected through the C18 column (20 µL). All of them were completely retained, regardless of their size, charge, or hydrophobicity. When using SYM16 conjugates, they were also retained in the column, but not 100%, and a small peak was registered, which in some cases reached 30% of the total tracer signal. This behavior of SYM16 was repeated when using it bound to the hapten. Even the unconjugated SYM16 oligonucleotide (10-mer) was not completely retained in the column. Due to the results obtained with SYM16, the experiment was repeated with the same amino acids coupled to SYM18 oligonucleotide (3-mer). In this case, the conjugates were retained completely inside the column, with no differences between the amino acids. Consequently, in terms of RAM separation using this support, for small molecules the exclusion mechanism of the support prevails over the reverse phase one and all of them are strongly bound to the support, while for oligonucleotide-hapten conjugates, the retention-elution profile depends on oligonucleotide moiety, which dominates the hapten one.

In another preliminary experiment, the capacity of the C18 column to retain small molecules was studied by injecting fluorescein at 1 mg/L repeatedly through the column until detecting saturation. Fluorescein leaching was not observed at all, even after 170 sequential injections of 500 µL each (total 85 µg, 0.256 µmol). This is equivalent to more than 4000 20-µL injections of 1 mg/L fluorescein. This result indicated that the column is able to retain large amounts of small molecules, considering that concentrations employed in immunoanalysis are usually very low, in the microgram per liter magnitude order. Finally, the retention and elution of the tracer and the tracerantibody immunocomplex was studied, employing the antibodyatrazine antiserum, 2d-SYM16 conjugate as tracer and the C18 RAM column. Experiments of injecting the tracer alone and a mixture of tracer-antibody, with and without column, were carried out. It was noticed that the peak area corresponding to the immunocomplex was the same with or without column, which indicates that all the immunocomplex passes unretained through the column and the separation is effective. The tracer alone was retained in the column but not at 100%, as it has been described above. As the fraction of tracer not retained in the column represents a background signal, the first optimization was devoted to minimize it, always keeping the complete elution of the immunocomplexes. Immunosensor Setup. (1) Carrier Influence. Flow rate affects not only the total assay time but also the working life of the column. The 0.5 mL/min was found to be a suitable compromise to avoid overpressure in the system and minimize assay time, set at 2 min as routine. The optimization of the carrier solution was also performed in terms of maximum adsorption of the tracer and no retention of the immunocomplex on the RAM support. Different concentrations of PBS were assayed, and the best results were obtained for the carrier containing a total salt concentration higher than that of PBS, but no significant differences were found among 2-, Analytical Chemistry, Vol. 79, No. 24, December 15, 2007

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3-, and 4-fold concentrated PBS. Twofold concentrated PBS was selected due to the lower salt concentration. The pH of the buffer was also examined. Values lower than 6.0 were discarded due to a decrease in the fluorescence intensity of the label, while pH higher than 7.5 is detrimental for the RAM support. Within this interval, pH 7.0 was selected as optimal because the fraction of unretained tracer was the lowest. (2) Tracer Study. Tracer properties determine the final immunosensor performances. In the HH immunosensor format, the labeled hapten should be hydrophobic and small enough to be completely retained on the inner surface of the RAM support. In this sense, the oligonucleotide linker should not be very large. Using a single-label 10-mer, Mr ≈ 3000 Da oligonucleotide (SYM16) conjugated to 2d, the tracer alone was not captured 100% into the RAM columnssee previous itemswhile the immunocomplex was eluted completely with the void volume, the same peak area at the same time with and without RAM column, as expected. Atrazine-tracer-antibody competition was observed, but there was always a notable background signal due to the unretained tracer. As results with SYM16-based tracer were not acceptable, lower size tracers were assayed. 2d hapten was conjugated to 3-mer Mr ≈ 1700 Da, the smallest available, and 5-mer Mr ≈ 2300 Da oligonucleotides (SYM18 and SYM20, respectively). The smallest conjugate was completely adsorbed on the column, and no background signal at all was displayed in the chromatograms, while the SYM20-based conjugate produced a low background signal, less than 5% of the total, which allowed for the practical use of this oligonucleotide. The concentrations of tracer and antibody employed were adjusted in order to obtain the best sensitivityslowest I50 values but maintaining tall and well-shaped peaks, using as a starting point a sample volume of 20 µL in the C18 RAM column. High fluorometric signals were obtained when the purified tracers were diluted 1/25 (v/v) or in a lower factor, so the concentration interval for this reagent always ranged between 1/25 and 1/5. For antibody, concentrationssexpressed as serum dilutionsthat produced acceptable fluorescence, peaks were in the range from 1/1000 to 1/50 (v/v). The first assay carried out with SYM16-based tracer led to an optimal combination of tracer dilution 1/10 and antibody dilution 1/500, which produced competition curves with I50 around 100 µg/L. When assaying SYM18- and SYM20-based tracers, the best results were obtained, in both cases, for antibody dilution 1/200 and tracer dilution 1/10. However, the I50 value for the SYM20 tracer (5-mer oligonucleotide) was always higher than 50 µg/L, while competition with SYM18 tracer (3-mer) resulted in I50 values between 20 and 30 µg/L. Hence, SYM18 oligonucleotide was selected for hapten labeling (1:1 marker ratio) in all subsequent experiments, since the competition was the most sensitive. (3) Supports. In the HH immunoassay format, the RAM support is essential. Lichrospher ADS was chosen first because this is a very common commercial support described in the literature,16 mainly applied for biological fluid cleanup. Initially, a study into the influence of the support on assay performance was carried out with two kinds of Lichrospher ADS: C4 and C18 inner reversed phase. Using a SYM18-based tracer, complete calibrations were run with each support under the optimized conditions. 9336 Analytical Chemistry, Vol. 79, No. 24, December 15, 2007

Table 1. Homogeneous-Heterogeneous Immunosensor for Atrazine

support C18 C4

(a) Sensitivity Features in C18 and C4 Supports blank signal I50 LOD DR (AU)a (µg/L)c (µg/L)d (µg/L)b 139 127

29.3 ( 3.6 32.7 ( 4.7

1.5 2.2

4.1-181.0 6.4-151.3

(b) Influence of Sample Volume (Using C18 Support) blank volume signal I50 LOD DR (µL) (A.U.) (µg/L) (µg/L) (µg/L) 20 50 200

139 286 1530

29.3 ( 3.6 29.3 ( 5.8 27.3 ( 8.3

1.5 0.80 2.40

4.1-181.0 3.6-153.9 6.0-115.4

RSD (%) 0.8 1.9

RSD (%) 0.8 2.8 4.3

a AU, arbitrary units. b I 50 analyte concentration that inhibits the binding of the tracer to the antibody by 50%. c LOD, limit of detection, analyte concentration that inhibits the tracer binding by 10%. d DR, dynamic range, analyte concentration range for which tracer binding is inhibited between 20 and 80%.

Data from Table 1a show the results obtained with the two supports. Additionally, I50, LOD, and dynamic range values are comparable; the repeatability of measurements is higher for the C18 support, so this column was chosen. The two types of RP supports work properly because the small molecules (analyte, tracers) are not extremely hydrophobic or hydrophilic, so they are retained by both C4 and C18 inner surfaces. Employing a C18 support, different sample volumes (20, 50, and 200 µL) were assayed in order to optimize the competition process. As expected, fluorescence peak areas are higher for larger sample volumes (Table 1b). However, if comparing competition curves using normalized signals, the obtained plots are similar as are the sensitivity parameters. The 20-µL sample loop was therefore selected on the basis on lower assay time and reagent consumption. Additionally, this experiment indicates that the support capacity was very high, confirming the previously stated in the preliminary experiments. This property allowed us to work for a complete day (more than 200 assays of 20-µL injection each) without a cleaning/ regeneration step, since no column saturation was observed after more than 20 200-µL injections. In any case, when the system was left on standby, the complete regeneration of the support was carried out by washing with methanol. Figure 3 shows the register displayed for a complete calibration of the HH immunosensor for atrazine, employing the optimized conditions for the SYM18-based tracer and the C18 RAM support. Analytical peaks are tall and narrow, also showing good repeatability in the replicates. The sensitivity parameters obtained in different competition curves are as follows: an I50 value ranging from 28 to 35; LOD from 1.0 to 2.0; and the dynamic range (DR) varies from a minimum of 3.0-6.0 to a maximum of 150-180, all data in micrograms per liter. These data indicate that sensitivity reached with the HH immunosensor is worse than that achieved by a previously developed capture immunosensor,33 which produced an I50 value of 0.42 and a LOD of 0.014 µg/L employing (33) Gonza´lez-Martı´nez, M. A.; Morais, S.; Puchades, R.; Maquieira, A.; Marco, M. P.; Barcelo´, D. Fresenius’J. Anal. Chem. 1998, 361, 179-184.

Figure 3. Fluorometer display of the calibration data for atrazine. Numbers over peaks are analyte concentration (in µg/L).

the same immunoreagents. Further, the HH immunosensor was less sensitive than batch ELISA using the same antibody and hapten, with sensitivity parameters of 0.50 and 0.030 µg/L for I50 and LOD, respectively (unpublished data). It is clear that the application of the HH immunosensor to highly diluted samples requires a preconcentration treatment, although the direct determination of atrazine in the 10 µg/L range and lower is possible. However, sensitivity should be improved and new research is in progress with this purpose. Finally, it should be noted that the entire calibration is carried out in less than 40 min. This is less than the time needed to assay a standard in triplicate in other immunosensors using enzyme labeling with optical (20 min/ assay)7 or electrochemical (>30 min/assay),34 as well as than other sensors employing label-free SPR (24 min/assay)35 and piezoelectric (15-min assay)36 detection. The selectivity of the antiserum and the HH immunoassay for atrazine was examined by measuring the cross-reactivity of a panel of s-triazine derivatives. Data are shown in Table 2. It is noteworthy that most compounds are not recognized at all by the antibody, and only propazine and terbuthylazine are able to bind the antibody due to the structural similarity of these two s-triazines with the immunizing hapten. This result is similar to that obtained for atrazine in ELISA30 and competitive immunosensing,33 and it is indicative that the selectivity depends mainly on the immunoreagents employed, rather than the assay format. Influence of Organic Solvents. As SPE (see below Application to Water Samples section) is frequently required to analyze real samples, it is of interest to study the effect of organic solvents in the sample on assay performance, in order to employ the extracts directly eluted from SPE supports. The practical advantage is that solvents would not need to be removed completely before analysis, thus facilitating automated on-line sample processing. The entire study was carried out with the most frequently (34) Zacco, E.; Pividori, M. I.; Alegret, S.; Galve, R.; Marco, M. P. Anal. Chem. 2006, 78, 1780-1788. (35) Mauriz, E.; Calle, A.; Montoya, A.; Lechuga, L. M. Talanta 2006, 69, 359364. (36) Hala´mek, J.; Makower, A.; Kno ¨sche, K.; Skla´dal, P.; Se´ller, F. W. Talanta 2005, 65, 337-342.

Table 2. Cross-Reactivity (CR) of Different Atrazine-Related Compoundsa

a

compound

I50 (µg/L)

CR (%)

atrazine irgarol 1051 atrazine mercapturic acid deethylatrazine ametrin prometrin deisopropilatrazine terbutylazine terbutrin simazine terbumeton propazine

28.8 >1000 >1000 >1000 >1000 >1000 >1000 61.7 >1000 >1000 >1000 41.7

100