Multiplexed Microimmunoassays on a Digital Versatile Disk

Anal. Chem. 2009, 81, 5646–5654

Multiplexed Microimmunoassays on a Digital Versatile Disk Sergi Morais, Luis A. Tortajada-Genaro, Tania Arnandis-Chover, Rosa Puchades, and Angel Maquieira* Instituto de Quı´mica Molecular Aplicada, Departamento de Quı´mica, Universidad Politećnica de Valencia, Camino de Vera s/n, 46071 Valencia, Spain Multiplexed microimmunoassays for five critical compounds were developed using a digital versatile disk (DVD) as an analytical support and detecting technology. To this end, coating conjugates were adsorbed on the polycarbonate face of the disk; a pool of specific antibodies, gold labeled secondary antibodies, and silver amplification were addressed for developing the assays. The detection principle is based on the capture of attenuated analog signals with the disk drive that were proportional to optical density of the immunoreaction product. The multiplexed assay achieved detection limits (IC10) of 0.06, 0.25, 0.37, 0.16, and 0.10 µg/L, sensitivities of (IC50) 0.54, 1.54, 2.62, 2.02, and 5.9 µg/L, and dynamic ranges of 2 orders of magnitude for atrazine, chlorpyrifos, metolachlor, sulfathiazole, and tetracycline, respectively. The features of the methodology were verified by analyzing natural waters and compared with reference chromatographic methods, showing its potential for high-throughput multiplexed screening applications. Analytes of different chemical nature (pesticides and antibiotics) were directly quantified without sample treatment or preconcentration in a total time of 30 min with similar sensitivity and selectivity to the ELISA plate format using the same immunoreagents. The multianalyte capabilities of immunoassaying methods developed with digital disk and drive demonstrated the competitiveness to quantify targets that require different sample treatment and instrumentation by chromatographic methods. Given the growing demand for effective multianalysis, operationally easy protocols, good sensitivity assays, and faster readouts, the development of methods comprising these key requirements is of great analytical interest. Classic multiple analytical determinations have usually been made by dividing a sample in aliquots and each one is analyzed for a single analyte. The standard highsensitivity multianalyte methods are mainly based on chromatographic techniques. Both types of methodologies have limitations, for example, in determining compounds of different chemical nature at one time. Indeed, specific and laborious sample pretreatment, enrichment, or even derivatization steps for each * To whom correspondence should be addressed. E-mail: amaquieira@ qim.upv.es. Phone: +34-963877342. Fax: +34-963879349.

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chemical family are needed; these methods being time-consuming and frequently relegated to confirmatory purposes. Immunochemical methods were historically developed for single-analyte determination. Several approaches have attempted to overcome this limitation performing selective assays in different wells of a single microtiter plate.1,2 Other more sophisticated approaches have been proposed to perform simultaneous multianalyte immunoassays,3-5 including label and label-free methods.6 Labeled probe methods, such as multilabel and spatially resolved assays, can provide amplified signals.7 At present, together with suspension based assays,8 planar microarrays are the major representative of multiplexed analysis techniques.9-11 With this method, each analyte is identified by its spatial location, without use of recognition codes. Although a variety of assay configurations and reagents have been tested,12-16 the widespread use of these methods in routine analysis is still impaired by the lack of suitable measurement platforms for fast and accurate determination in laboratory or in situ.17 An innovative and promising approach is to use a compact disk based microarray system. Compact disk technology as an analytical tool has experienced a considerable advance since Kido and (1) Strahan, J. In Environmental Immunochemical Methods; Van Emon, J. M., Gerlach, C. L., Johnson, J. C., Eds.; American Chemical Society: Washington, DC, 1996; pp 65-73. (2) Adrian, J.; Pinacho, D. G.; Granier, B.; Diserens, J.-M.; Sańchez-Baeza, F.; Marco, M.-P. Anal. Bioanal. Chem. 2008, 391, 1703–1712. (3) Ekins, R.; Chu, F. W. Clin. Chem. 1991, 37, 1955–1967. (4) Kricka, L. J. Clin. Chem. 1992, 38, 327–328. (5) Wilson, R.; Cossins, A. R.; Spiller, D. G. Angew. Chem., Int. Ed. 2006, 45, 6104–6117. (6) Hoheisel, J. D. Nat. Rev. Genet. 2006, 7, 200–210. (7) Wu, J.; Yan, F.; Tang, J.; Zhai, C.; Ju, H. Clin. Chem. 2007, 53, 1495–1502. (8) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R. Clin. Chem. 1997, 43, 1749–1756. (9) Moreno-Bondi, M. C.; Taitt, C. R.; Shriver-Lake, L. C.; Ligler, F. S. Biosens. Bioelectron. 2006, 21, 1880–1886. (10) Moser, C.; Mayr, T.; Klimnat, I. Anal. Chim. Acta 2006, 558, 102–109. (11) Dupuy, A. M.; Lehmann, S.; Cristol, J. P. Clin. Chem. Lab. Med. 2005, 43, 1291–1302. (12) Rowe, C. A.; Scruggs, S. B.; Feldstein, M. J.; Golden, J. P.; Ligler, F. S. Anal. Chem. 1999, 71, 433–439. (13) Sapsford, K. E.; Charles, P. T.; Patterson, C. H. J.; Ligler, F. S. Anal. Chem. 2002, 74, 1061–1068. (14) Knecht, B. G.; Strasser, A.; Dietrich, R.; Martlbauer, E.; Niessner, R.; Weller, M. G. Anal. Chem. 2004, 76, 646–654. (15) Rucker, V. C.; Havenstrite, K. L.; Herr, A. E. Anal. Biochem. 2005, 339, 262–270. (16) Goldman, E. R.; Clapp, A. R.; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684–688. (17) Nichkova, M.; Dosev, D.; Gee, S. J.; Hammock, B. D.; Kennedy, I. M. Anal. Biochem. 2007, 369, 34–40. 10.1021/ac900359d CCC: $40.75  2009 American Chemical Society Published on Web 06/12/2009

coauthors18 used a standard compact disk as a molecular screening platform. After that, different approaches have been reported for analytical applications up to finally exploiting the advantages of integrating an audio-video digital disk as a support with a disk drive as the chemical detector.19,20 In these approaches, the polycarbonate surface of the compact disks is used to attach probes by adsorption. Compact disks (CDs) and digital versatile disks (DVDs) are the most frequently mass-produced and cost-effective optical platforms available in the market for high-capacity data storage. Despite the essential physical differences between CDs and DVDs, the base substrate of both is made of polycarbonate, an alternative material to glass to design microanalytical devices.21,22 In regard to optical disk technology, DVD drives achieve a sharp focus using a 650 nm wavelength laser, whereas CD players use a nearinfrared (780 nm) laser. Besides, because objects of 1/4 λ size are needed to induce significant disruption of the laser reflection of a standard optical drive, sensitivity is higher in DVD drives. Further, DVD readers employ a focusing lens with a higher numerical aperture than the CD players, improving optical resolution and signal-to-noise ratios.20 The advantage of the compact disk platform for immunoanalysis is three-fold. First, the surface of a single standard disk (94 cm2) can hold thousands of spots, so thousands of replicates can be made simultaneously in a sample or a huge number of analytes can be quantified in the same sample, including the calibration standards. Second, commercial disks have demonstrated good properties for passive adsorption and covalent immobilization of coating probes (proteins and nucleic acids) in high-density scales to develop bioanalytical assays.23,25 Third, microarrays developed on optical disks are attractive primarily because of their cost efficiency, minimal reagent consumption, and the ability both to conduct assays and to record and/or read data from the same disk using standard drives. The interest in using standard drives as detectors arises from their low cost, ubiquity, portability, reliability, and easy utilization.24 Given that the compact disk based applications dealt with quantitative measurements based on individual determinations,25-31 the development of quantitative assays on raw compact disks for (18) Kido, H.; Maquieira, A.; Hammock, B. D. Anal. Chim. Acta 2000, 411, 1–11. (19) Morais, S.; Carrascosa, J.; Mira, D.; Puchades, R.; Maquieira, A. Anal. Chem. 2007, 79, 7628–7635. (20) Morais, S.; Tamarit-Lo´pez, J.; Puchades, R.; Maquieira, A. Anal. Bioanal. Chem. 2008, 391, 2837–2844. (21) McCarley, R. L.; Vaidya, B.; Wei, S.; Smith, A. F.; Patel, A. B.; Feng, J.; Murphy, M. C.; Soper, S. A. J. Am. Chem. Soc. 2005, 127, 842–843. (22) Morais, S.; Marco-Mole´s, R.; Puchades, R.; Maquieira, A. Chem. Commun. 2006, 2368–2370. (23) Ban ˜uls, M. J.; Garcıá-Pin õn, F.; Puchades, R.; Maquieira, A. Bioconjugate Chem. 2008, 19, 665–672. (24) Ligler, F. S. Anal. Chem. 2009, 81, 519–526. (25) Alexandre, I.; Houbion, Y.; Collet, J.; Demarteau, J.; Gala, J. L.; Remacle, J. Biotechniques 2002, 33, 435–439. (26) La Clair, J. J.; Burkart, M. D. Org. Biomol. Chem. 2003, 1, 3244–3249. (27) Lange, S. A.; Roth, G.; Wittermann, S.; Lacoste, T.; Vetter, A.; Grassle, J.; Kopta, S.; Kolleck, M.; Breitinger, B.; Wick, M.; Horber, J. K. H.; Dubel, S.; Bernard, A. Angew. Chem., Int. Ed. 2006, 45, 270–273. (28) Potyrailo, R. A.; Morris, W. G.; Leach, A. M.; Sivavec, T. M.; Wisnudel, M. B.; Boyette, S. Anal. Chem. 2006, 78, 5893–5899. (29) Li, Y.; Wang, Z.; Ou, L. M. L.; Yu, H.-Z. Anal. Chem. 2007, 79, 426–433. (30) Li, Y.; Ou, L. M. L.; Yu, H.-Z. Anal. Chem. 2008, 80, 8216–8223. (31) Tamarit-Lo´pez, J.; Morais, S.; Puchades, R.; Maquieira, A. Anal. Chim. Acta 2008, 609, 120–130.

simultaneous multianalyte measurements deserves further investigation. The aim of this research is to set up a compact disk based methodology for the simultaneous and high-sensitivity multianalysis of several compounds from different chemical families. For detection, two disk reading strategies were compared. One aims to acquire attenuated analog signals and the other is based on the analysis of reading errors. Also, the calibration is studied in a practical and simple mode to achieve a powerful multianalyte capabilities. As proof of concept, very different analytes (pesticides, atrazine, chlorpyrifos, and metolachlor, and antibiotics, sulfathiazole and tetracycline) were chosen,32-36 the selection criteria being two-fold. First, there is an increasing need for highly sensitive, effective, and simple screening techniques to determine chemical residues in different scenarios. Second, the current standard protocol for their quantification is off-site laboratory analysis by SPE-LC tandem MS37-40 and SPE-GC/MS41-43 for antibiotics and pesticides, respectively. MATERIALS AND METHODS Chemicals. Printing buffer, 0.1 M sodium carbonate buffer, pH 9.6 containing 10% glycerol (v/v), total ionic strength adjustment buffer (TSAB), 200 mM sodium phosphate buffer, 1.5 M NaCl, 0.5% Tween 20, pH 7.0 containing 10 mg/L biotin and washing solutions were filtered through a 0.22 µm pore size disk before use. For multianalyte determination of atrazine, chlorpyrifos, metolachlor, sulfathiazole, and tetracycline, previously characterized polyclonal antibodies R12, C2-II, R48, S3-I, and KOTC3-III as well as coating conjugates OVA-2d, OVA-C5, OVAmetolachlor, OVA-S2, and OVA-TC1, respectively, were used as immunoreagents. Rabbit polyclonal antibiotin antibody (pBT) was from Abcam (Cambridge, U.K.). Standard atrazine, chlorpyrifos, and metolachlor were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Standard sulfathiazole, tetracycline, biotin, biotin N-succinimidyl ester (used to prepare OVA-biotin conjugate; OVA-BT), bovine serum albumin (BSA), ovalbumin (OVA), gold labeled goat antirabbit immunoglobulins (GAR-Au), silver enhancer solutions (A and B), dichloromethane, ethyl acetate, methanol, acetonitrile, and phenyldodecane were from SigmaAldrich (Madrid, Spain). Keyhole limpet hemocyanin (KLH) was from Pierce (Rockford, IL). Note: Pesticides, antibiotics, silver enhancer solutions, and solvents should be handled following their material safety data sheets. (32) Gascoń, J.; Oubin ã, A.; Ballesteros, B.; Barcelo´, D.; Camps, F.; Marco, M. P.; Gonza´lez-Martıńez, M. A.; Morais, S.; Puchades, R.; Maquieira, A. Anal. Chim. Acta 1997, 347, 149–162. (33) Brun, E. M.; Garce´s-Garcıá, M.; Puchades, R.; Maquieira, A. J. Agric. Food Chem. 2005, 53, 9352–9360. (34) Casino, P.; Morais, S.; Puchades, R.; Maquieira, A. Environ. Sci. Technol. 2001, 35, 4111–4119. (35) Pastor-Navarro, N.; Garcıá-Bover, C.; Maquieira, A.; Puchades, R. Anal. Bioanal. Chem. 2004, 379, 1088–1099. (36) Pastor-Navarro, N.; Morais, S.; Maquieira, A.; Puchades, R. Anal. Chim. Acta 2007, 594, 211–218. (37) Kaufmann, A.; Roth, S.; Ryser, B.; Widmer, M. J. AOAC Int. 2001, 85, 853–860. (38) Carlson, K.; Cha, J.; Yang, S. J. Chromatogr., A 2005, 1097, 40–53. (39) Dıáz- Cruz, M. S.; Garcıá-Galań, M. J.; Barcelo´, D. J. Chromatogr., A 2008, 1193, 50–59. (40) Davis, J. G.; Truman, C. C.; Kim, S. C.; Ascough, J. C.; Carlson, K. J. Environ. Qual. 2006, 35, 2250–2260. (41) Sabik, H.; Jeannot, R.; Rondeau, B. J. Chromatogr., A 2000, 885, 217–236. (42) Quintana, J.; Martı´, I.; Ventura, F. J. Chromatogr., A 2001, 938, 3–13. (43) Fillion, J.; Sauve´, F.; Selwyn, J. J. AOAC Int. 2000, 83, 698–713.


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Figure 1. DVD layout scheme. The disk holds 8 arrays of 10 columns of 10 spots each. From left to right, the columns correspond to (1) BSA, (2) OVA, (3) OVA-atrazine, (4) OVA-biotin, (5) OVA-chlorpyrifos, (6) OVA-metolachlor, (7) KLH, (8) OVA-sulfathiazole, (9) OVA-tetracycline, and (10) rabbit serum.

Microarraying. Bulk DVD-R disks were purchased from MPO Ibe´rica (Madrid, Spain). The disks were first conditioned by gentle ethanol washing, water rinsing, and dried by centrifugation. The coating conjugates, diluted in printing buffer, were dispensed in a 384-well plate (20 µL/well) and transferred to the disk (50 nL) with a noncontact printing device (AD 1500 BioDot, Inc., Irvine, CA) in microarray format. The array layout consists in 10 columns of 10 dots each; 5 of the 10 columns correspond to single-target systems; 3 are positive controls; 1 is the negative control, and the final column is the internal standard. A total of eight arrays (800 spots) were printed on the polycarbonate surface of the DVD-R separated from each other by 45° (Figure 1). In this configuration, spots are 500 µm in diameter with a track pitch (center to center distance) of 1.5 mm, achieving an array density of 1.0 spot/mm2. The reproducibility of the delivered volume was ensured by the steady-state pressure inside the dispensing channel. Also, the working temperature and relative humidity were controlled (25 °C and 90%, respectively) because these parameters have dramatic effects on the printed microarray quality. Microimmunoassay Protocol. The microimmunoassays on DVDs were based on an indirect competitive format. First, coating conjugate solutions (OVA-2d, OVA-C5, and OVA-metolachlor at 0.5 mg/L, OVA-S2 at 1.0 mg/L, and OVA-TC1 at 10.0 mg/L) were arrayed in columns on the polycarbonate surface of a DVD-R disk. As an internal standard, OVA-BT at 0.2 mg/L was used whereas BSA (5 mg/L), KLH (1 mg/L), and a 1/4000 dilution of nonimmunized rabbit sera (RIgG) were arrayed as positive controls for the first and second immunoreaction steps, respectively. Ovalbumin solution (5 mg/L) was included as a negative control. After 16 h at 4 °C, the disk was thoroughly washed with PBS-T, rinsed with deionized water, and dried by centrifugation at 800 rpm. For single-target assay optimization, 1.0 mL of polyclonal antibody solution (1/1000 dilution for R12, C2-II, R48, pBT, and 5648


KOTC3-III and 1/2000 for S3-I) in 2× PBS-T, with or without analyte, was dispensed onto the disk and covered with a 12 cm diameter 0.6 mm thick dummy plastic surface (DPS). After 5 min incubation at room temperature, the cover was removed, and the disk was washed with PBS-T buffer and then rinsed with deionized water. Next, 1.0 mL of gold-labeled secondary antibody solution (1/50 in PBS-T) was dispensed onto the disk, and it was evenly distributed using a DPS. After 8 min at room temperature, the cover surface was removed, and the disk washed and dried as before. The immunoreaction was developed by homogenously dispensing 1.0 mL of silver enhancer solution onto the disk, and the reaction was stopped by washing the disk with water after 8 min. For multiplexed assays, 0.9 mL of deionized water, with and without analyte, conditioned with 0.1 mL of TSAB was used. Then, 7.5 µL of the mixed antibody solution (cocktail) were added and systematically mixed for 2 min. Then, the solution was dispensed onto the disk. Next, the immunoassay was performed as described above. As is shown in Table 1 the total assay takes 30 min. Compact Disk Scanning and Data Acquisition. The DVD drive used in this study was from LG Electronics Inc. (Englewood Cliffs, NJ), which was controlled by custom software (Diskpick), running on a personal computer and connected to it through a USB2.0 universal serial bus interface as described.20 Briefly, during the DVD reading, the laser strikes the immunoreaction product which modifies the reflection properties of the DVD surface, attenuating the laser beam intensity that reaches the photodiode of the pickup. The analog signals are directly acquired from the photodiode of the CD/DVD drive, being related to optical density of the reaction product which is inversely proportional to analyte concentration. This reading strategy was named AAS. To scan the surface of the DVD completely (5 min at 16× speed), the software simulates the writing process of a 3.8 GB

Table 1. Assay Steps and Time Taken for a Competitive Multiplexed Microimmunoassay on Precoated DVD assay steps

time (min)

standard or sample-antibody mixing analyte-antibody reaction on disk washing/rinsing secondary antibody application and reaction on disk washing/rinsing signal amplification (reagents application and development) washing/rinsing disk reading total assay time

2 5 0.5 8 0.5 8 0.5 5 29.5

size file. During the disk scanning, only signals coming from selected areas are processed for digitization, stored in the computer (5 MB size file) and deconvoluted into an image. Diskpick software was written in Visual C++ to control the optical disk drive, the data acquisition board (sampling rate, detector gain, spatial resolution, and scanning speed) and identifies spots with SNR g2 to then calculate the mean signal intensity by averaging data points from a circular area of 150 µm in diameter. Moreover, this software allows for exporting the results. For sample analysis, the measured signal intensity was related to that of the internal standard response. Inhibition curves were mathematically analyzed by fitting experimental results to a sigmoidal four-parameter logistic equation. To compare the results obtained by external and internal standards, a representative water sample, containing the analytes and biotin, was diluted at different levels. Each aliquot represents a theoretical lost of analytes and internal standard. The samples were analyzed several times in different days by external standard and internal standard calibration modes and compared. In relation with the disk reading mode, a second approach was studied. For that, disks were scanned with the DVD drive using a diagnostic analysis program (Nero DiscSpeed V.4.10.3.0, Nero Inc., Glendale, CA) that generates an error distribution plot as function of playtime following the named error reading detection (ERD) strategy. Analysis of Water Samples. A total of 4 river water samples from the Valencia region and 12 Spanish spring waters were analyzed with the developed method. Samples were filtered through a 0.22 µm pore size disk and stored at 4 °C prior to use. The samples were then spiked with a mixture of targeted compounds at different levels, covering the analytical working ranges, and determined directly without previous extraction. Each sample was analyzed in three replicates on separate disks (240 replicate spots measured per sample) as described above. Finally, the disk was read by the DVD drive and the results quantified. For comparison purposes, simultaneous determination of sulfathiazole and tetracycline residues in water samples also was carried out by high-performance liquid chromatography-tandem mass spectrometry (LC-MS2),37 whereas simultaneous determination of atrazine, chlorpyrifos, and metolachlor residues in water samples was carried out by solid-phase extraction-gas chromatography/mass spectrometry (SPE-GC/MS).41 The analytical procedure is given in detail in the Supporting Information.

RESULTS AND DISCUSSION Assay Setup. In order to integrate the larger number of systems and demonstrate the multianalyte capabilities of the methodology, previously in-house-developed immunoreagents32-36 were used to set up competitive microimmunoassays on an indirect format. For that, a set of protein-hapten conjugates to pesticides and antibiotics were microarrayed on DVDs and tested against a pool of rabbit polyclonal antibodies raised to 2,4,5-TP, acetochlor, alachlor, atrazine, chlorpyrifos, diazinon, fention, malathion, metolachlor, sulfathiazole, and tetracycline. Coating conjugate concentrations and antibody dilutions were selected on the basis of signal intensity, signal-to-noise ratio (SNR), and specificity by check-board titration in a competitive format. For these experiments, antibody concentrations ranged from 1/500 to 1/16 000, whereas coating conjugate concentrations varied from 0.01 to 10 mg/L. Of all the possible 121 coating/antibody pair combinations, only those giving an acceptable SNR g 30 for the blanks, negligible cross-reactivity, and highest sensitivity for 10 µg/L were screened. Of the results obtained, 5 out of 11 initial systems were selected. The pool of antibodies of a particular antiserum recognizes a specific moiety of the chemical structure of the analyte and a fraction of the chemical structure of both the carrier protein and arm spacer. The spacer was common for diazinon, fenthion, and malathion haptens. Thus, coating conjugates prepared for these analytes share the thiophosphate group and the aliphatic chain through the carrier protein is attached, showing false positive results when testing in homologous and heterologous formats. These similarities in hapten structure might be responsible for these findings. The most unexpected false positive result was that obtained using atrazine antisera tested against malathion and 2,4,5TP coating conjugate. In this case, the similarity in hapten structure comes exclusively from the aliphatic chain of the spacer arm. Besides, some of these systems showed low sensitivity in comparison to those obtained using the same immunoreagents in ELISA format, as was the case for fenthion and diazinon, achieving values in the hundreds of part per billion. For all these reasons, the integration of diazinon, fenthion, malathion, and 2,4,5TP with the other analytes was unfeasible. In the case of analytes belonging to the chloroacetanilide group (acetochlor, alachlor, and metolachlor), the best results in terms of sensitivity and specificity were obtained using the pair coating conjugate/antisera for the metolachlor assay. It is worth mentioning that both antisera for alachlor and acetochlor did recognize the OVA-metolachlor conjugate, which only permitted the integration of one chloracetanilide compound in the final disk array layout. The coating conjugate-antibody pairs OVA-2d-R12 (0.5 mg/ L-1/1000), OVA-C5-C2-II (0.5 mg/L-1/1000), OVA-metolachlor-R48 (0.5 mg/L-1/1000), OVA-S2-S3-I (1.0 mg/L-1/ 2000), and OVA-TC1-KOTC3-III (10 mg/L-1/1000) were selected for atrazine, chlorpyrifos, metolachlor, sulfathiazole, and tetracycline assays, respectively. The chemical structures of selected analytes, coating, and immunization conjugates are shown in Figure 2. In regard to the selectivity, all the chosen single-target systems were specific, since each antiserum only recognized its particular Analytical Chemistry, Vol. 81, No. 14, July 15, 2009

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Figure 2. Chemical structures of the selected analytes, coating, and immunization conjugates. ATZ, atrazine; CLP, chlorpyrifos; MTL, metolachlor; STZ, sulfathiazole; TC, tetracycline. *The letters a, b, and c correspond to the substituent for analyte, coating, and immunization conjugates, respectively. The carrier protein used to prepare the conjugates is shortened as follows: Bovine serum albumin, BSA; ovalbumin, OVA; and keyhole limpet hemocyanin, KLH.

coating conjugate, as shown in Table 2. Thus, SNR values ranged from 31.3 to 76.1 for tetracycline and atrazine assays, respectively. In contrast, nonspecific recognition was negligible showing SNR values below the limit of quantification (SNR ranged from 2 to 5). These results are at the same sensitivity level as those reported by the respective ELISA plate methods in terms of specificity. In a second set of experiments, cross-reactivity studies were performed by measuring signal intensity variation to the presence of mixed analytes. In these experiments, single-target assays were carried out and cross-reactivity calculated as percentage of inhibition. In all cases, signal variation was below 0.01%, indicating high selectivity to the mixed analytes. This fact is essential when considering single assay candidates to be applied in multiplexed analysis. In summary, several points should be considered before using a single-target in multiplexed assays. First, to have a broad collection of immunoreagents, second the antisera specificity and sensitivity, and third, the carrier protein used in the coating conjugates is another variable to be marked. In our case, since immunogens used to raise the antibodies were prepared with KLH or BSA, a different carrier protein was employed for coating the conjugate preparation, avoiding nonspecific interactions and false positives. This criterion was matched by preparing ovalbuminbased coating conjugates. Multiplexed Competitive Microimmunoassays. Simultaneous determination of the five analytes was performed by an all5650


in-one reaction assay (see Figure 1). For these experiments, the above-mentioned specific single assays were integrated into one by using a mixed-antibody solution. For cocktail preparation, the concentration of each antibody was kept the same as that used in single assays. In this way, each particular antibody would recognize its own coating conjugate without affecting other immunoreactions. As shown in Table 2 (last row), SNRs were very similar to those for the corresponding single-target assays. In particular, SNR variation ranged from 1.4% to 5.0%, indicating an acceptable difference between single and multiplexed assays. Also, interdisk relative standard deviation (RSD) ranged from 7.8% (atrazine) to 17% (tetracycline). Similar precision was achieved in single assays using the same reagents and concentrations. In another set of experiments, different competition times (1, 5, 10, 15, 30 min) were tested to study the effect on signal intensity and sensitivity. It was observed that long competition times reduced assay sensitivity (higher IC50 values), while optical density at 650 nm was similar for all incubation tested times. The best results in terms of signal intensity and sensitivity were achieved after a 5 min competitive incubation time. The standard calibration curves are shown in Figure 3, displaying the mean of 8 curves performed on different days; in total, 640 spots per concentration of analyte were averaged. The intradisk RSD for all the analyte concentrations ranged from 7.5% to 9.1%, while the mean interdisk RSD varied from 7.9% to 22.0%. As analyte concentration increased, the signal-acquired intensity varied from the highest (absence of analyte) to the background signal. The resulting silver deposit from the highest analyte concentration was minimal (SNR

Multiplexed Microimmunoassays on a Digital Versatile Disk

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