Anal. Chem. 2005, 77, 148-154
Array Biosensor for Detection of Ochratoxin A in Cereals and Beverages Miriam M. Ngundi, Lisa C. Shriver-Lake, Martin H. Moore, Michael E. Lassman, Frances S. Ligler, and Chris R. Taitt*
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Center of Bio/Molecular Science & Engineering, Naval Research Laboratory, Washington, D.C. 20375
Contamination of food by mycotoxins occurs in minute quantities, and therefore, there is a need for a highly sensitive and selective device that can detect and quantify these organic toxins. We report the development of a rapid and highly sensitive array biosensor for the detection and quantitation of ochratoxin A (OTA). The array biosensor utilizes a competitive immunoassay format. Immobilized OTA derivatives compete with toxin in solution for binding to fluorescent anti-OTA antibody spiked into the sample. This competition is quantified by measuring the formation of the fluorescent immunocomplex on the waveguide surface. The fluorescent signal is inversely proportional to the concentration of OTA in the sample. Analyses for OTA in buffer and a variety of food and beverage samples were performed. Samples were extracted with methanol, without any sample cleanup or preconcentration step prior to analysis. The limit of detection for OTA in several cereals ranged from 3.8 to 100 ng/g, while in coffee and wine, detection limits were 7 and 38 ng/g, respectively. Mycotoxins are toxic secondary metabolites produced by fungi that often grow in agricultural products prior to harvest or during storage. Mycotoxins of agricultural and health concerns include ochratoxin A (OTA), aflatoxins, trichothecenes (e.g., deoxynivalenol and T-2 toxin), fumonisins, zearalenone, and the ergot alkaloids. OTA is produced by several species of the genera Aspergillus and Penicillium1,2 and occurs in a variety of food commodities of which cereals (e.g., wheat, barley, corn, oats, and rye) are the most vulnerable. OTA is also found in coffee, dried fruits, grape juice, wine, beer, pork kidneys, and blood. Animal studies have shown that OTA is a nephrotoxin, hepatotoxin, carcinogen, and teratogen and is suspected to cause the human disease called “Balkan endemic nephropathy”.2-4 Despite extensive data in the literature concerning the occurrence of OTA, there still exists a need for routine monitoring to acquire enough data for risk assessment and to establish maximum allowable levels. * Corresponding author. Phone: (202) 404-4208. Fax: (202) 767-9594. E-mail:
[email protected]. (1) Weidenborner, M. Encyclopedia of Food Mycotoxins; Springer: Berlin, Germany, 2001. (2) Council for Agricultural Science and Technology. Mycotoxins: Risks in plants, Animals and Human Systems, Report No. 139; Council for Agricultural Science and Technology: Ames, IA., January 2003, (3) Krogh, P. Food. Chem. Toxicol. 1992, 30, 213-224. (4) Stoev, S. D. Vet. Hum. Toxicol. 1998, 40, 352-360.
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Mycotoxin contamination usually occurs in trace amounts ranging from nanograms to micrograms per gram of foodstuff. Detection and identification, therefore, require a highly sensitive technique. Traditional methods for the analysis of OTA are mainly chromatographic techniques including thin-layer chromatography (TLC), gas chromatography, and high-pressure liquid chromatography (HPLC).5-9 HPLC with immunoaffinity columns and fluorescence detection has dominated this class of analytical methods.10 Chromatographic methods generally require multiple steps prior to detection, including extraction, extensive sample cleanup, preconcentration, and sometimes derivatization of the analyte. Such sample treatment not only makes the analysis timeconsuming and costly but also requires trained personnel. To overcome the limitations encountered with the chemical methods of analysis, immunoassay methods have been developed.11-13 The immunological basis of such techniques makes them highly specific and, therefore, less dependent on sample cleanup. So far, enzyme-linked immunosorbent assay (ELISA) methods are the most common immunoassay technique used in OTA analysis due to the capability for parallel analysis of multiple samples. Although conventional ELISA methods take up to several hours to get results, rapid ELISAs are becoming commercially available. One such ELISA test is Veratox (Neogen, Lansing, MI), a competitive ELISA requiring only 20-30 min after sample extraction. New immunosensor devices based on surface plasmon resonance, fiber optics, and microbead-based assays have also been utilized for rapid mycotoxin analysis.14-16 However, many of the reported biosensor procedures for mycotoxin analysis still require some form of sample cleanup to achieve adequate sensitivity and have been applied mainly for fumonisins, aflatoxins, and deoxynivalenol (5) Silvia, C.; Maragos, C. M. J. Agric. Food Chem. 1998, 46, 3162-3165. (6) Soleas, G. J.; Yan, J.; Goldberg, D. M. J. Agric. Food Chem. 2001, 49, 27332740. (7) Santos, E. A.; Vargas, E. A. Food Addit. Contam. 2002, 19, 447-458. (8) Chiavaro, E.; Lepiani, A.; Colla, F.; Bettoni, P.; Pari, E.; Spotti, E. Food Addit. Contam. 2002, 19, 575-581. (9) Shephard, G. S.; Fabiani, A.; Stockenstrom, S.; Mshicileli, N.; Sewram, V. J. Agric. Food Chem. 2003, 51, 1102-1106. (10) Scott, P. M.; Trucksess, M. W. J. AOAC 1997, 80, 941-949. (11) Chu, F. S. In Immunoassays for Residue Analysis: Food Safety; Beier, R. C., Stanker, L. H., Eds.; American Chemical Society: Washington, DC, 1996; pp 294-313. (12) De Saeger, S.; Van Peteghem, C. J. Food Prot. 1999, 62, 65-69. (13) Kwak B. Y.; Shon, D. H. Food Sci. Biotechnol. 2000, 9, 168-173. (14) van der Gaag, B.; Spath, S.; Dietrich, H.; Stigter, E.; Boonzaaijer, G.; van Osenbruggen, T.; Koopal, K. Food Control 2003, 14, 251-254. (15) Maragos, C. M. Adv. Exp. Med. Biol. 2002, 504, 85-93. (16) Maragos, C. M.; Thompson, V. S. Nat. Toxins 1999, 7, 371-376. 10.1021/ac048957y CCC: $30.25
© 2005 American Chemical Society Published on Web 11/30/2004
but not OTA. Moreover, the majority of these devices lack the ability to perform simultaneous analyses of multiple samples. In recent years, array biosensors have been developed and demonstrated for a variety of applications.17-20 The ability of the array biosensors to analyze multiple samples simultaneously for multiple analytes offers significant advantage over other types of biosensors. In particular, the rapid, multianalyte array biosensor developed at the Naval Research Laboratory has demonstrated the potential to be used as a screening and monitoring device for clinical, food, and environmental samples.21-24 The device, which is portable and fully automated,25 can be used with different immunoassay formats.26 In this study, competitive immunoassays were developed and employed for the detection and quantification of OTA in a variety of spiked food and beverage samples. A simple extraction procedure was employed with no need for cleanup or preconcentration of the sample extract. Moreover, methanol was used as the extraction solvent, which eliminates the use of harmful chlorinated organic or acidified solvents employed for extraction in some immunoassay systems and other analytical techniques.27,28 EXPERIMENTAL SECTION Materials. Unless otherwise specified all chemicals were used as received. Borosilicate microscope slides (3 in. × 1 in.) purchased from Daigger (Vernon Hills, IL) were used as waveguides for all assays. Ochratoxin A (from Aspergillus ochraceus), gelatin, bovine serum albumin, sodium hydroxide, sodium bicarbonate, potassium hydroxide, poly(ethylene glycol)ssaverage molecular weight 200 and 3350s(PEG-200 and PEG-3350, respectively), poly(vinylpyrrolidone) (PVP), phosphate-buffered saline (PBS), and PBS containing 0.05% Tween 20 (PBST) were purchased from Sigma (St. Louis, MO). Methanol, anhydrous toluene, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF) were supplied by Aldrich (Milwaukee, WI). (3-Mercaptopropyl)triethoxysilane and N-succinimidyl-4-maleimidobutyrate (GMBS) were obtained from Fluka (St. Louis, MO). NeutrAvidin biotin-binding protein, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and biotin-LC-PEO-amine were obtained from Pierce (Rockford, IL). Rabbit anti-OTA was purchased from (17) Knecht, B. G.; Strasser, A.; Dietrich, R.; Martlbauer, E.; Niessner, R.; Weller, M. G. Anal. Chem. 2004, 76, 646-654. (18) Rowe, C. A.; Tender, L. M.; Feldstein, M. J.; Golden, J. P.; Scruggs, S. B.;MacCraith, B. D.; Cras, J. J.; Ligler, F. S. Anal. Chem. 2000, 71, 38463852. (19) Plowman, T. E.; Durstchi, J. D.; Wang, H. K.; Christensen, D. A.; Herron, J. N.; Reichert, W. M. Anal. Chem. 1999, 71, 4344-4352. (20) Sapsford, K. E.; Shubin, Y. S.; Delehanty, J. B.; Golden, J. P.; Taitt, C. R.; Shriver- Lake, L. C.; Ligler, F. S. J. Appl. Microbiol. 2004, 96, 47-58. (21) Sapsford, K. E.; Rasooly, A.; Taitt, C. R.; Ligler, F. S. Anal. Chem. 2004, 76, 433-440. (22) Taitt, C. R.; Shubin, Y. S.; Angel, R.; Ligler, F. S. Appl. Environ. Microbiol. 2004, 70, 152-158. (23) Shriver-Lake, L. C.; Shubin, Y. S.; Ligler, F. S. J. Food Prot. 2003, 66, 18511856. (24) Ligler, F. S.; Taitt, C. R.; Shriver-Lake, L. C.; Sapsford, K. E.; Shubin, Y. S.; Golden, J. P. Anal. Bioanal. Chem. 2003, 377, 469-477. (25) Golden, J. P.; Taitt, C. R.; Shriver-Lake, L. C.; Shubin, Y. S.; Ligler, F. S. Talanta, in press. (26) Sapsford, K. E.; Charles, P. T.; Petterson, C. H., Jr.; Ligler, F. S. Anal. Chem. 2002, 74, 1061-1068. (27) Scott M. P. In Mycotoxins and Food Safety; DeVries, J. W., Trucksess, M. W., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2002; pp 117-134. (28) Trucksess, M. W.; Pohland, A. E. Mol. Biotechnol. 2002, 22, 287-292.
ImmuneChem Pharmaceuticals Inc. (Burnaby, BC, Canada). Cy5 bisfunctional dye was purchased from Amersham Bioscience Corp. (Arlington Heights, IL). Biotin-SP-conjugated AffiniPure rabbit antichicken IgY and Cy5-conjugated ChromPure chicken IgY used as positive controls were obtained from Jackson ImmunoResearch (West Grove, PA). All foodstuffs were purchased from local grocery stores. A Waring commercial blender used for food preparation was purchased from Fisher Scientific (Pittsburgh, PA). All aqueous solutions were prepared using 18 MΩ Milliporepurified water. Preparation of Biotinylated Ochratoxin A. OTA-biotin conjugate was synthesized based on the procedure reported by Kononenko et al.29 Briefly, 6.3 mg of N-hydroxysuccinimide and 10.3 mg of EDC were added to a test tube containing a small stir bar, which was then capped with a septum. Next, a solution of 10 mg of OTA in 0.4 mL of DMF was added to the test tube, and the resultant mixture was stirred for 1 h. After 1 h, 1 mL of 50 mM biotin-LC-PEO-amine in 0.05 M carbonate/bicarbonate buffer, pH 9.5, was added and then stirred for 24 h. The reaction mixture was transferred into a 500 MWCO dialysis bag (Spectrum Medical Industries, Houston, TX) and dialyzed several times against PBS. The resulting biotin-OTA conjugate was characterized by mass spectrometry, quantified using UV-visible spectroscopy, and stored at 4 °C. Preparation of Fluorescent Antibody. Antibodies used as detection species were labeled with Cy5 bisfunctional dye according to the manufacturer’s instructions. Labeled antibodies were separated from unincorporated dye using size exclusion chromatography using BioGel P10 (Bio-Rad, Hercules, CA). Antibody concentration and protein-to-dye ratio were determined using UV-visible spectroscopy. The labeled antibody reagent was aliquoted and stored at -20 °C. Immobilization of Capture Species. Microscope glass slides (waveguides) were cleaned by immersion for 30 min in 10% KOH (w/v) in methanol followed by copious rinsing with water and drying under nitrogen. The clean slides were then treated under nitrogen with 2% (3-mercaptopropyl)triethoxysilane in anhydrous toluene for 1 h. The slides were washed three times in anhydrous toluene and then dried under a nitrogen stream. The silane-treated slides were incubated for 30 min at room temperature in 1 mM GMBS prepared by first dissolving 12.5 mg GMBS in 250 µL of DMSO and diluting with 43 mL of absolute ethanol. The slides were washed thrice with water and incubated in 30 µg/mL NeutrAvidin in PBS overnight at 4 °C. The NeutrAvidin-functionalized slides were rinsed with PBS and either used immediately or stored in PBS at 4 °C. Patterning of the capture OTA onto the NeutrAvidin-modified waveguide was carried out using poly(dimethylsiloxane) (PDMS) flow channels prepared from liquid silicone elastomers (Nusil Technology, Carpinteria, CA). Each PDMS flow cell was molded to possess 12 channels, each channel of dimensions 21 mm (l) × 1 mm (w) × 2.5 mm (h). The flow cell was first clamped onto the NeutrAvidin-derivatized slide and then biotinylated OTA (2 µg/mL in PBS), biotinylated rabbit anti-chicken IgY (20 µg/mL in PBS, positive controls), and PBS (blank) were introduced into different channels. Typically, at least eight lanes contained (29) Kononenko, G. P.; Burkin, A. A.; Zotova, E. V.; Soboleva, N. A. Appl. Biochem. Microbiol. 2000, 36, 177-180.
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biotinylated OTA. The patterned slides were incubated overnight at 4 °C. The capture species were then aspirated out of each lane, and each channel was rinsed with 1 mL of PBST. After the patterning template was removed, the slides were immersed in a blocking solution of 10 mg/mL gelatin in PBST for 30 min. The slides were rinsed with water, dried under a stream of nitrogen, and assembled for sample analysis or stored at 4 °C. Preparation of Food Samples. Barley, wheat pasta (macaroni), and cornflakes were blended to a fine texture. Several 0.5-g aliquots of ground barley, wheat pasta, cornflakes, cornmeal, and roasted coffee were weighed. OTA was diluted in methanol (0.5 mL) at various concentrations, and the methanolic OTA was spiked into each 0.5-g aliquot of food. The solvent was allowed to evaporate overnight from the open vials in the hood. Each spiked sample aliquot was then extracted with 2 mL of 75% methanol/ water (v/v), first by vortexing for 3 min, followed by shaking on a horizontal shaker for 2 h. The samples were then centrifuged at 3000 rpm at 25 °C for 10 min, and the resulting supernatant (extract) was used for assay against the fluorescent-labeled antibodies. Because of the low pH of the coffee extract, 3 µL of 3 M NaOH was added to 0.6 mL of extract prior to addition of antibodies. An aliquot of wine sample (red wine, 1989, 12% alcohol) was spiked with OTA; then using unspiked wine as diluent, serial dilutions were made to obtain various aliquots containing 0-15.625 µg/mL unlabeled OTA. Three different treatment protocols were employed prior to addition of the detection antibody: (1) pH was adjusted from pH 3.5 to ∼pH 7.5 using NaOH (40 µL of 3 M NaOH for 2 mL of sample); (2) 1:1 dilution with aqueous solution of 5% NaHCO3 containing 1% PEG;30 and (3) treatment of the wine samples with PVP31 followed by pH adjustment as follows. Briefly, 0.4 mL PVP (20 mg/mL in water) was added to each 2-mL wine aliquot. The wine/PVP mix was then shaken for ∼15 min at room temperature, and the pH of each aliquot was then adjusted to pH ∼7.5 by adding 40 µl of 3 M NaOH. Assay Protocol. The supernatants from barley, wheat pasta, cornflakes, cornmeal, and roasted coffee were diluted 3-fold (final concentration of methanol, 25%) with PBST containing detection species, namely, Cy5-anti-OTA and Cy5-conjugated ChromPure chicken IgY (positive control). Detection antibodies were also added to treated wine samples. The final concentrations of Cy5labeled chicken IgY and Cy5-labeled anti-OTA in all samples were 100 ng/mL and 2 µg/mL, respectively. The extract/antibody mix was allowed to sit for 10-20 min at room temperature before analysis. Patterned slides were assembled on assay templates such that the PDMS flow channels (40 mm (l) × 1 mm (w) × 2.5 mm (h)) were oriented orthogonal to the patterned strips of immobilized capture species (i.e., biotin-OTA and biotin-rabbit anti-chicken). The assembled slides were hooked up to a multichannel peristaltic pump (Manostat, Sarah model, Barnant Co., Barrington, IL) by connecting one end (outlet) of each flow channel to the pump tubing via syringe needles. The inlet end of each flow channels was connected to a 1-mL syringe barrel. Each channel was washed with 1 mL of PBST at 0.5 mL/min, which also served as a check for leaks. Next, 0.8 mL of OTA sample containing detector (30) Visconti, A.; Pascale, M.; Centonze, G. J. AOAC Int. 2001, 84, 1818-1827. (31) Ongunjimi, A. A.; Choudary, P. V. FEMS Immununol. Med. Microbiol. 1999, 23, 213-220.
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antibody was passed through the channels at 0.06 mL/min. Each channel was then washed with 1 mL of PBST at 0.5 mL/min. After removing the PDMS, the slides were washed with distilled, deionized water, dried under nitrogen, and imaged using the array biosensor. Fluorescence Imaging, Data Acquisition and Analysis. The array biosensor optical system has been described elsewhere.32,33 Briefly, the biosensor system consists of a 635-nm, 12-mW diode laser excitation source (Lasermax, Rochester, NY), a waveguide support, a GRIN lens array (Nippon Sheetglass, Summerset, NJ), several emission filters (bandwidth 665-730 nm), and a Peltiercooled charge-coupled device (CCD) imaging array (SpectraSource, Teleris, Westlake Village, CA). The excitation beam was launched into the edge of the waveguide at an appropriate angle (∼36°), resulting in the evanescent excitation of the surface-bound fluorescent antibody-antigen complexes. A simple line generator attached at the front of the laser allowed a uniform illumination of the entire waveguide. The GRIN lens array provided a 1:1 imaging of the fluorescent emission onto the CCD. The optical filters mounted between the GRIN lens array and the CCD imager blocked stray excitation light and scattered radiation prior to the CCD imager. Digital images of the waveguide were acquired in the Flexible Image Transport System (FITS) format. Data in the form of fluorescent intensities were extracted from the images using a custom data analysis software.34 The software program generates a mask that consists of squared data spots (corresponding to the area where the capture species were exposed to the detection antibody) and background rectangles on either side of the data spot. The net fluorescent intensity for each data spot is obtained by subtracting the average background value from the data spot value. These values were imported into Microsoft Excel files and analyzed. SAFETY CONSIDERATIONS Cleaning of slides with methanolic KOH was performed in a chemical hood by personnel wearing acid-resistant gloves and appropriate personal protective gear. Silanization procedures were carried out in a nitrogen-filled glovebag placed in a chemical hood. All solutions containing ochratoxin A were handled by personnel wearing appropriate personal protection gear (goggles, lab coat, gloves). All surfaces, glassware, and other containers that were used or came into contact with OTA were treated with 20% bleach before disposal. Food samples spiked with OTA were clearly labeled, kept in a separate fume hood, and placed in biohazard bags for disposal by incineration. RESULTS AND DISCUSSION Detection of OTA in Buffer. Initially, a checkerboard type of assay, whereby different concentrations of immobilized OTA were exposed to various concentrations of Cy5-labeled anti-OTA in PBST, was performed in order to determine the reasonable working concentrations for both capture species and tracer antibody. Competitive assays of unlabeled OTA in buffer were then performed using the optimized concentrations. Figure 1A shows (32) Golden, J. P.; Ligler, F. S. Biosens. Bioelectron. 2002, 17, 719-725. (33) Feldstein, M. J.; Golden, J. P.; Rowe, C. A.; MacCraith, B. D.; Ligler, F. S. J. Biomed. Microdevices 1999, 2, 139-153. (34) Sapsford, K. E.; Liron, Z.; Shubin, Y. S.; Ligler, F. S. Anal. Chem. 2001, 73, 5518-5524.
Figure 1. (A) CCD image of a waveguide assayed with various concentrations of OTA spiked in buffer, each containing 2 µg/mL Cy5labeled anti OTA and 100 ng/mL Cy5-labeled chicken IgY (positive control). (B) Dose-response curve for OTA obtained by plotting the mean percentage inhibition of fluorescent anti-OTA versus the concentration of OTA present in buffer. Error bars are the standard error of the mean with n ) 8.
the CCD image of a typical assay slide showing the presence of positive control squares (anti-chicken IgY) and negative control squares (buffer) in each lane. On each slide was included an additional negative control consisting of Cy5-anti-OTA and Cy5chicken IgY in the sample (no exogenous OTA added, lowest row). The effect of increasing OTA added to the samples is evident from a decrease in fluorescent intensity. These values are plotted as standard dose-response using the percent inhibition of the tracer by unlabeled OTA, as shown in Figure 1B.
% inhibition ) fluorescence of tracer with unlabeled OTA 1× 100 fluorescence of tracer alone (buffer blank)
(
)
The percent inhibition curve for OTA in buffer could be fitted into a similar sigmoidal four-parameter logistic equation described as, Y ) -1.26 + {87.29/[1 + (x/4.96)-0.49]}, with R2 ) 0.996. The limit of detection, calculated as the lowest concentration of OTA
tested at which the mean percentage inhibition was at least 3 standard deviations (n ) 8) above the mean percentage inhibition of buffer blank, was 0.8 ng/mL. The use of aqueous methanol for extraction of OTA from foodstuff has become popular, with various formulations yielding different results.27 To test the effects of methanol on the binding activity of the Cy5-labeled OTA antibodies, competitive assays were performed in PBST as well as PBST containing methanol at concentrations up to 25% (data not shown). The fluorescence intensities for buffer blanks were compared to those measured for buffer/methanol blanks using Student’s t-test and showed no significant difference in the fluorescence intensities between PBST and PBST/methanol blanks for methanol concentrations up to 25% (p > 0.3). This shows that an extract containing up to 25% methanol could safely be assayed without any detriment to the tracer antibody. This is an advantage over most procedures where subsequent steps are employed to remove organic solvents from the extract prior to assay. Such steps include evaporating the organic solvent, then reconstituting the residue with buffer, and solvent-partitioning methods. OTA in Cereals and Cereal Products. As described previously, foods spiked with various concentrations of OTA were extracted using 75% methanol in water (v/v). The extracts were diluted with PBST to yield a final concentration of 25% methanol and were subsequently analyzed using biotinylated OTA-patterned slides. For each experiment using food, two negative controls were included: a buffer blank (PBST, Cy5-anti-OTA, Cy5-anti-chicken), and a matrix blank (matrix, Cy5-anti-OTA, Cy5-chicken, no exogenous OTA). Analysis using Student’s t-test showed that the measured fluorescence of buffer blank was significantly above that of the unspiked food for wheat pasta (p ) 0.0004) and cornmeal (p ) 0.02). However, for cornflakes and barley, the buffer blank and unspiked food samples were not significantly different (p ) 0.06 and 0.9, respectively). The dose-response curves for OTA in four food samplesswheat pasta, cornmeal, cornflakes, and barleysare shown in Figure 2. The curves could be fitted into sigmoidal plots with four parameter logistic equations that relate the percentage inhibition (Y) to OTA concentration (x): Y ) 37.1 + {47.7/[1 + (x/90.0)-0.88]}, R2 ) 0.962 for wheat pasta; Y ) 9.0 + {80.7/[1 + (x/78.6)-0.55]}, R2 ) 0.974 for cornmeal; Y ) 4.0 + {74.4/[1 + (x/186.7)-0.75]}, R2 ) 0.971 for cornflakes; and Y ) 2.8 + {86.9/[1 + (x/594.3)-0.79]}, R2 ) 0.993 for barley. The limits of detection for OTA spiked into the foodstuffs were determined as the concentration of OTA that produced percentage inhibition greater than 3 standard deviations above mean of the unspiked food sample. Limits of detection for OTA in wheat pasta, cornmeal, cornflakes, and barley were 14, 3.8, 25, and 100 ng/g, respectively. All four types of cereals exhibited a wide dynamic range (∼1 ng/g-3.125 µg/g); OTA could be measured as low as 0.1 ng/g in cornmeal, even though this level is below the limit of detection, as currently defined. The detection limits obtained in this study are similar to those reported in the literature for wheat and corn products (Table 1); significant improvements in sensitivity were observed in other systems after a cleanup step, which our system did not use. Although several other systems, some without cleanup procedures, were able to obtain low detection limits in spiked barley samples, we did observe a significant matrix effect. Matrix effects on OTA determination in barley have been Analytical Chemistry, Vol. 77, No. 1, January 1, 2005
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Table 1. Sensitivity of Different Immunoassay Methods Used for OTA in Foods array biosensor (this study)
other immunoassay methods
food
LOD (ng/g)
IC50 (ng)
method
LOD
cleanup
ref
wheat pasta
14
90
ELISA ELISA ELISA membrane immunoassay ELISA affinity column (Ochratest) ELISA (Veratox) membrane immunoassay
30 ng/g 1-2 ng/g 2.5 ng/g 0.2 ng/g 2.5 ng/g 5 ppb 1 ppb nda
none immunoaffinity immunoaffinity none immunoaffinity immunoaffinity none none
11 11 28 12 28 39 neogen.com 12
ELISA ELISA ELISA ELISA (Veratox) ELISA membrane immunoassay
30 ng/g 1-2 ng/g 2.5 ng/g 1 ppb 0.5 ppb nda
none immunoaffinity immunoaffinity none none none
11 11 28 neogen.com 13 12
cornmeal
cornflakes barley
a
3.8
25 100
78
186 594
nd ) not detectable.
Figure 2. Dose-response curves generated by plotting the mean percentage inhibition of Cy5-labeled anti-OTA versus the concentration of OTA spiked in wheat pasta (b), cornmeal (O), cornflakes (1), and barley (3). Various aliquots of each foodstuff were spiked with different concentration of OTA in methanol, extracted with 75% methanol/water (v/v), and subsequently diluted with PBST to a final methanol concentration of 25%.
reported elsewhere; De Saeger and Van Peteghem12 were unable to detect OTA at any of the tested concentrations in barley and maize using a flow-through membrane-based enzyme immunoassay. OTA in Beverages (Coffee and Wine). No change in fluorescent intensities was observed in samples of roasted coffee spiked with OTA unless the pH was adjusted to approximately neutral. Upon adjustment of the pH 5.5 extract to pH 7.5 by addition of NaOH, dose-dependent changes in intensities were observed (Figure 3). The fluorescent intensity of buffer blank was significantly higher than that of unspiked coffee sample (p ) 0.04), demonstrating some inhibition of binding caused by the matrix alone. Due to this inhibition, percentage inhibitions were calcu152 Analytical Chemistry, Vol. 77, No. 1, January 1, 2005
Figure 3. Dose-response curves produced by plotting mean percentage inhibition of fluorescent anti-OTA versus the concentration of OTA in coffee (b) and wine (O). Coffee samples were extracted with 75% methanol/water (v/v) and adjusted to pH ∼7.5, while wine samples were stabilized using PVP followed by pH adjustment using NaOH prior to analysis.
lated using the buffer blank. The dose-response curve could be fitted into a sigmoidal four parameter logistic equation that related percentage inhibition (Y) to spiked OTA (x): Y ) 14.95 + {65.45/[1 + (x/126.7)-0.32]} (R2 ) 0.978). The limit of detection in these assays was determined to be 7 ng/g, similar to that determined determined by Sibanda et al. (4 ng/g)35 using a flowthrough enzyme immunoassay with a cleanup step. Detection limits 10-fold lower than those observed here have been obtained using TLC (0.5 ng/g)7,36 and HPLC (0.1 ng/g)37 methods. However, these analytical methods required use of cleanup or preconcentration steps. (35) Sibanda, L.; De Saeger, S.; Barna-Vetro, I.; Van Peteghem, C. J. Agric. Food Chem. 2002, 50, 6964-6967. (36) Monaci, L.; Palmisano, F. Anal. Bioanal. Chem. 2004, 378, 96-103. (37) Ventura, M.; Vallejos, C.; Anaya, I. A.; Broto-Puig, F.; Agut, M.; Comellas, L. J. Agric. Food Chem. 2003, 51, 7564-7567.
Figure 4. Fluorescent images of waveguides assayed with wine samples after pH adjustment using NaOH (A), treatment with either 5% NaHCO3/1% PEG-200 (B), 5% NaHCO3/1% PEG-3,350 (C), or PVP with pH adjustment (D).
The presence of untreated wine matrix resulted in complete inhibition of fluorescent tracer antibody binding to the immobilized OTA at all concentrations of exogenous OTA tested; controls (wine, no exogenous OTA) were also completely inhibited by the wine (data not shown). Three different methods were therefore investigated for preparation of red wine samples for analysis. Simple adjustment of the pH to neutral, shown previously to be successful with coffee, failed to improve assay results (Figure 4A). To determine whether the amount of alcohol present (12% v/v) inhibited the antibody-antigen binding, control assays were performed using OTA at various concentrations spiked into 12% ethanol/buffer (v/v, no wine matrix); as dose-dependent inhibition was observed in the 12% ethanol, we concluded that the wine effect was not caused by the high ethanol content of the wine (data not shown). A second method tested for preparation of wine samples was a commonly used treatment of wine and beer samples before application to affinity columns.30,35 In this approach, wine samples were treated with aqueous solutions of 5% NaHCO3 containing either 1% PEG-200 or 1% PEG-3350. Although complete inhibition of antibodies was observed in wine samples treated with PEG200 (Figure 4B), those treated with PEG-3350 showed weak
intensities (Figure 4C) that were OTA dose dependent. This shows that although PEG-200 does not enable antibody binding activity, PEG-3350 introduced some stability into the wine samples, which allowed some antibody-antigen binding to be observed. Higher molecular weight PEGs (6000 and 8000 molecular weights) have been employed to facilitate antibody binding activity in wine, beer, fruit juices, and produce extracts.30,31,38 Ogunjimi and Choudary31 have linked inactivation of antibodies in immunoassays to endogenous polyphenols present in fruit juices, wines, and vegetables. The third treatment of wine samples involved the use of PVP, a binding agent commonly used to remove polyphenols from plant extracts, followed by pH neutralization. Addition of PVP to wine samples was effective in stabilizing the wine and therefore facilitated strong antibody binding activity as evidenced in Figure 4D, which allowed a dose-response curve to be determined (Figure 3). As the fluorescent intensity of buffer blank was lower than that of wine samples (p < 0.001), percentage inhibitions were calculated based on unspiked wine. The doseresponse curve for the wine samples (Figure 3) could be fitted into a sigmoidal four-parameter logistic equation, Y ) 4.81 + (38) Visconti, A.; Pascale, M.; Centonze, G. J. Chromatogr., A 1999, 864, 89101.
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{85.98/[1 + (x/58.6)-0.74]} (R2 ) 0.978) with a limit of detection of 38 ng/mL. Although lower detection limits for OTA in wine have been reported,6,38 both of the methods used required significant sample treatment prior to analysis. CONCLUSION We have successfully developed a sensitive and rapid competitive immunoassay-based array biosensor for the detection of OTA in various foodstuffs. Although this and other biosensor systems have achieved low detection limits using competitive immunoassays in buffer and aqueous samples,26,41-44 biosensor analysis of organic extracts from highly complex plant samples has not been demonstrated to date. This is the first demonstration that a rapid biosensor can be used in a competitive assay format to detect a toxin in extracts of relevant foods, with sensitivity similar to data obtained using ELISA and other immunoassay methods. The total time for sample analysis (after extraction) was approximately 30-40 min, including a 10-20-min sample/antibody incubation; this is on the same time scale as other rapid detection systems but without any cleanup procedures or additional manipulation after sample extraction. The ability to perform parallel analyses is an additional advantage; matrix effects can be assessed and quantified. Since our detection limits were determined using a simple onestep extraction method, with methanol extracts simply diluted (39) Pestka, J. J.; Abouzied, M. N. Food Technol. 1995, (February), 120-128. (40) van Bergen, S. K.; Bakaltcheva, I. B.; Lundgren, J. S.; Shriver-Lake, L. C. Environ. Sci. Technol. 2000, 34, 704-708. (41) Shriver-Lake, L. C.; Breslin, K. A.; Charles, P. T.; Conrad, D. W.; Golden, J. P.; Ligler, F. S. Anal. Chem. 1995, 67, 2431-2435. (42) Tschmelak, J.; Kumpf, M.; Proll, G.; Gauglitz, G. Anal. Lett. 2004, 37, 17011718. (43) Tschmelak, J.; Proll, G.; Gauglitz, G. Biosens. Bioelectron. 2004, 20, 743752. (44) Tschmelak, J.; Proll, G.; Riedt, J.; Kaiser, J.; Kraemmer, P.; Barzaga, L.; Wilkinson, J. S.; Hua, P.; Hole, J. P.; Nudd, R.; Jackson, J.; Abuknesha, R.; Barcelo, D.; Rodriguez-Mozaz, S.; Lopez de Alda, M. J.; Sacher, F.; Stien, J.; Slobodnik, J.; Oswald, P.; Hozmenko, H.; Korenkova, E.; Tothova, L.; Krascsenitz, Z.; Gauglitz, G. Biosens. Bioelectron., in press.
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before analysis, these limits could be improved by inclusion of a preconcentration step involving evaporation of the solvent rather than dilution. While such a preconcentration step would significantly increase the sample preparation time, we anticipate that detection limits would be reduced potentially 100-1000-fold if the aqueous methanol solvent used for extraction was evaporated. Limits of detection could be further improved by optimizing the extraction methods for each different sample matrix. One of the benefits of an array-based detection system is the ability to achieve multianalyte detection, with appropriate controls performed in parallel. Although this study did not take advantage of the multiplexing capabilities of the NRL Array Biosensor, we have recently started developing rapid, dual-analyte assays for deoxynivalenol and OTA. Although we have observed some loss in sensitivity in these multiplexed assays, with additional optimization of the concentrations of immobilized antigens and tracer antibodies, we have been able to improve our detection limits to the low-nanogram per milliliter range in buffer. With further optimization, we expect to obtain detection limits similar to those obtained here. The current study is the first significant step in development of rapid, fieldable, multianalyte detection system for mycotoxins that requires no significant sample preparation or preconcentration. ACKNOWLEDGMENT The authors thank Dr. Kim Sapsford for her helpful suggestions. This work was funded by the U.S. Department of Agriculture (USDA) and the Food and Drug Administration (FDA). The views expressed here are those of the authors and do not represent those of the U.S. Navy, the U.S. Department of Defense, or the U.S. Government. Received for review July 16, 2004. Accepted October 14, 2004. AC048957Y
