Attomole Sensitivity of Staphylococcal Enterotoxin B Detection Using

Article pubs.acs.org/ac

Attomole Sensitivity of Staphylococcal Enterotoxin B Detection Using an Aptamer-Modified Surface-Enhanced Raman Scattering Probe Erhan Temur,† Adem Zengin,‡ Iṡ mail Hakkı Boyacı,§ Fahriye Ceyda Dudak,§ Hilal Torul,† and Uğur Tamer*,† †

Department of Analytical Chemistry, Faculty of Pharmacy, and ‡Department of Chemistry, Faculty of Science, Gazi University, 06330 Ankara, Turkey § Department of Food Engineering, Faculty of Engineering, Hacettepe University, Beytepe 06800 Ankara, Turkey S Supporting Information *

ABSTRACT: In this report, we present a new homogeneous detection method for staphylococcal enterotoxin B (SEB) utilizing core−shell-structured iron−gold magnetic nanoparticles and a gold nanorod surface-enhanced Raman scattering (SERS) probe in solution. Peptide ligand (aptamer) functionalized magnetic gold nanorod particles were used as scavengers for target SEB. After the SEB molecules were separated from the matrix, the sandwich assay procedure was tested by gold nanorod particles that act as SERS probes. The binding constant between SEB and peptide−nanoparticle complex was determined as 8.0 × 107 M−1. The correlation between the SEB concentration and SERS signal was found to be linear within the range of 2.5 fM to 3.2 nM. The limit of detection for the homogeneous assay was determined as 224 aM (ca. 2697 SEB molecules/20 μL sample volume). Also, goldcoated surfaces were used as capture substrates and performances of the two methods were compared. Furthermore, the developed method was evaluated for investigating the SEB specificity on bovine serum albumin (BSA) and avidin and detecting SEB in artificially contaminated milk, blood, and urine.

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nanoparticles,16 and quantum dots17 in the immunoassays developed for SEB detection. Immunosensors, based on fiber optics,18,19 quartz crystal microbalance,20 magnetoelastic21 and surface plasmon resonance (SPR),22−27 have been developed as the alternatives of immunological analysis and used for the detection of SEB in a sensitive and rapid manner. All mentioned studies above are related with immunologicalbased techniques, where antibodies were used as recognition agents providing sensitivity and selectivity. However, antibodies also have some limitations as recognition elements in bioassays and biosensors. First, antibody production is quite expensive and time-consuming. Besides, utilizing animals in antibody production causes some ethical problems. Antibodies are functional under specific environmental conditions which can be unfavorable for the analysis of environmental or food samples. These limitations of antibodies led to the development of alternative recognition molecules.28 Peptide aptamers, especially the ones derived from phage displayed peptide libraries, are becoming more popular as novel

he genus Staphylococcus, which is a genus of Grampositive bacteria, can affect human health directly or indirectly by generating toxins known as staphylococcal enterotoxins (SEs).1 SEs are a prominent cause of foodborne diseases, and a very low concentration of SEs can be mortal when they found in food and/or water samples.2 Staphylococcal enterotoxin B (SEB), produced by S. aureus, is one of the exotoxins in the SEs group. SEB is commonly associated with food poisoning and causes nonmenstrual toxic shock syndrome in humans.3 Hence, detection of SEB in food and water samples is a priority process for public health.4 High intoxication, ease of spread, and the high thermal stability of SEB makes it a favorable biological weapon.5 In addition, it is also announced as a restricted agent by the Center for Disease Control and Prevention (CDC).6 Therefore, the reliable, sensitive, and rapid detection of SEB is of great importance. A great number of researchers have reported their works based on different detection methods which are quite sensitive and selective. Among these methods, immunological assays, such as enzyme-linked immunosorbent assay (ELISA)7−10 and the methods based on immunomagnetic separation,11,12 are the most popular ones for the detection of SEB. Higher sensitivity has been obtained by using carbon nanotubes,13−15 gold © XXXX American Chemical Society

Received: July 10, 2012 Accepted: November 9, 2012

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Figure 1. Schematic illustration of SERS-based heterogeneous and homogeneous sandwich immunoassay systems.

recognition agents in bioassay and biosensor applications.29 They have numerous advantages over antibodies, such as high functional stability in extreme environmental conditions and ease of in vitro production. High-affinity peptide ligands can be generated toward various targets, even if they are not immunogenic. In addition, peptides can be easily modified with different functional groups. In spite of all their advantages, the usage of peptides in bioassays is still limited. Although there are a number of studies using whole phages with displayed peptides,30−34 the free peptides have been used as recognition elements only in a few studies.35,36 In this study, we aimed to investigate the performance of peptide ligands selected by phage display in bioassays. For this purpose, 24-mer peptide, which was previously reported by our research group to be a selective ligand for SEB,37 was combined with surface-enhanced Raman scattering (SERS) measurement for the sensitive detection of SEB. SERS, a Raman spectroscopy technique with nanoscale optical phenomena,38 has been used for qualitative and quantitative analyses, providing rapid, reliable, and sensitive measurements. There are many reports

of SERS-based assays, and most of them used antibodies as recognition agents.39−42 To the best of our knowledge, this is the first report of using SERS coupled with peptide aptamers for sensitive and rapid detection of a target molecule. Here, we used the Raman reporter (5,5-dithiobis(2-nitrobenzoic acid))tagged gold nanorods for labeling the target molecule captured on both peptide-immobilized gold-coated glass slides and magnetic gold nanorods (Figure 1). The developed bioassay enables the detection of SEB in attomolar levels. We also demonstrated the ability of the developed system to detect SEB in a complex matrix.

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EXPERIMENTAL SECTION Reagents. Staphylococcal enterotoxin B (SEB), N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxy sulfosuccinimide (NHS), 11-mercaptoundecanoic acid (MUA), 3-mercaptopropionic acid (MPA), 5,5dithiobis(2-nitrobenzoic acid) (DTNB), HEPES, and ethanolamine were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, U.S.A.). SEB binding peptide B

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Figure 2. Transmission electron microscopy (TEM) image of (A) gold nanorod particles and (B) TEM image of the sandwich complex of DTNBlabeled gold nanorod−SEB−peptide−magnetic gold nanorod.

with ethanol, 25 mM MPA in absolute ethanol was used to improve the SAM by covering the blank parts on the surfaces. The surfaces were activated by the solution containing 0.2 M EDC and 0.05 M NHS. Then activated surfaces were incubated with SEB binding peptides (3.6 × 10−6 M) for 1 h, and the surfaces were blocked with 1% (v/v) ethanolamine solution. After washing steps with HEPES buffer (pH 7.4), gold-coated slides and magnetic gold nanoparticles were incubated with different concentrations of SEB in buffer (HEPES, pH 7.4) for 1 h. After capturing, surfaces were washed with buffer to remove unbound or weakly bound molecules. Gold nanorods were used as a Raman label for both magnetic nanorod particles and gold-coated surface studies. The reporter molecule, DTNB (25 mM) in absolute ethanol, was incubated with gold nanorods overnight to compose the SAM. The carboxylate groups of DTNB were activated by using 0.2 M EDC and 0.05 M NHS solution. Activated nanoparticles were incubated with peptide ligands (0.1 mg mL−1) for 1 h, and the surfaces were blocked with 1% (v/v) ethanolamine solution. Peptide-immobilized gold nanorods were incubated with SEBcaptured surfaces and magnetic particles for 1 h, and unbound rods were removed by washing. Surface Plasmon Resonance Measurements. Binding of peptide−nanoparticle complex to SEB was investigated by a Spreeta SPR sensor (Texas Instruments, Dallas, TX, U.S.A.) combined with a three-channel flow cell and 12-bit DSP control box. The flow of the reagents was controlled by syringe pumps (Goldman Pump, Biasis Ltd. Sti., Ankara, Turkey) and four-way switching valves (Upchurch Scientific, Oak Harbor, WA, U.S.A.). Self-assembled monolayer (SAM) was formed on the sensor surfaces by incubating the sensor overnight with 10 mM MUA in absolute ethanol. The SAM on the sensor surfaces was activated by the solution containing 0.2 M EDC and 0.05 M NHS. The activated sensors were coated with SEB by injecting 7.0 μM SEB solution at a flow rate of 25 μL min−1 for 20 min. After the immobilization of SEB, the sensor surfaces were blocked with 1 M ethanolamine. After establishing the baseline with HEPES buffer (pH 7.5), five different concentrations (from 0.5 to 5.0 nM) of nanoparticle−peptide conjugate were injected to the sensor surface at a flow rate of 25 μL min−1. The binding curves were generated by subtracting the control measurement, in which the peptide solutions were injected to

(LLADTTHRPWTLLADTTHRPWT) was synthesized by solid-phase peptide synthesis without any modification at the terminals and purified to 95% purity (GenScript Corp., Piscataway, NJ, U.S.A.). Fabrication of Magnetic Gold Nanorods as Capture Probe. In our previous study, the synthesis of spherical-shaped gold-coated iron oxide nanoparticles was reported.42 Briefly, the seed-mediated synthetic method was performed in a two-step procedure to synthesize magnetic gold nanorod particles. The spherical-shaped gold-coated Fe3O4 nanoparticles were used as seed and different amounts of seed particles were added to 4.75 mL of growth solution containing 0.1 M hexadecyltrimethylammonium bromide (CTAB, capping agent), 0.01 M HAuCl4, 0.01 M silver ions, and 0.1 M ascorbic acid (AA) as a reduction. This final mixture was stirred for a few seconds and allowed to stay 3 h at room temperature for the synthesis of magnetic gold nanorod particles. Fabrication of Gold Nanorods as SERS Tag. Gold nanorods were prepared by using a seed-mediated growth technique. Seed solution was prepared by mixing 7.5 mL of 0.1 M CTAB and 250 μL of 0.01 M HAuCl4 solution. Then, 600 μL of 0.01 M ice-cold NaBH4 was added rapidly to the resulting solution and allowed to stand for 5 min to form seed solution. To prepare rod-shaped gold nanoparticles, 4.75 mL of 0.1 M CTAB, 1 mL of 0.01 M HAuCl4, and 60 μL of 4 × 10−3 M AgNO3 were mixed, and the resulting color was dark orange. When the dark orange color was observed, 250 μL of 0.1 M AA was added dropwise to the resulting solution. The solution turned colorless after adding 250 μL of 0.1 M AA. Then, 5 μL of seed solution was added to the stock solution. The final mixture was stirred for a few seconds and allowed to stand for 3 h at room temperature to form the nanorods. Fabrication of Gold Surfaces. Ti/Au layer (∼200 nm) is sputtered on a silicon wafer by using conventional techniques. Before experiments, the surfaces were cleaned with piranha solution (50% H2SO4 plus 50% H2O2) to remove organic residues. Sample Preparation. Gold-coated slides and magnetic gold nanorod particles were used for capturing target molecule. For this purpose, both slides and magnetic nanoparticles were modified with a self-assembled monolayer (SAM) of 25 mM MUA in absolute ethanol overnight. After washing the surfaces C

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blocked surface, from binding measurement. The curves were fitted to a simple Langmuir binding model to determine the binding constant between SEB and the peptide−nanoparticle complex. Raman Instrumentation. A DeltaNu Examiner Raman microscope (DeltaNu Inc., Laramie, WY) with a 785 nm laser source, a motorized microscope stage sample holder, and a CCD detector was used to detect SEB. During the measurements, a 20× objective was used and the laser spot diameter was 30 μm. Samples were measured with 140 mW laser power, for 50 s acquisition time. Baseline correction was performed for all measurements.

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RESULTS AND DISCUSSION As part of our interest in the area of the construction of SERS probes, we have recently reported magnetic nanoparticles Figure 5. SPR sensorgrams (dashed gray lines) obtained for the binding of nanoparticle−peptide complex in HEPES at pH 7.5 to the SEB-immobilized surface, fit to the 1:1 Langmuir interaction model (solid black line). All runs are overlaid for nanoparticle concentrations at 0.5, 1.0, 2.5, and 5.0 nM.

incorporating specific recognition antibody for E.coli detection in heterogeneous and homogeneous assays.42 The magnetic nanoparticles enable the isolation or extraction of a target molecule or substance by the use of an external magnetic field. The utilization of magnetic nanoparticles not only possesses the advantages of superparamagnetism, but also preconcentrates the analyte in order to amplify the Raman signal efficiently. Figure 1 shows the preparation steps of gold-coated glass slides and magnetic gold nanorod particles for SEB detection. SAMmodified gold-coated glass slides and magnetic gold nanorod particles were covered with peptide ligands, and then, the SEB molecule was captured through the peptides. After washing away the unbound molecules, captured SEB molecules were labeled with DTNB-modified and peptide-immobilized gold nanorods. The DTNB-labeled gold nanorods as the SERS tags were shown to generate a strong Raman scattering.42 The aspect ratio of the gold nanorod particle was about 3 (length, 60 nm; width, 20 nm) as shown in Figure 2A. After the sandwich assay procedure, the magnetic gold nanorod particles

Figure 3. (A) SERS spectra of magnetic gold nanorods, peptideimmobilized magnetic gold nanorods, SEB-captured magnetic gold nanorods, and the sandwich complex of DTNB-labeled gold nanorod−SEB−peptide−magnetic gold nanorod. (B) SERS spectra of magnetic gold nanorods, peptide-immobilized magnetic gold nanorods, and SEB-captured magnetic gold nanorods obtained by adding silver nanoparticles.

Figure 4. (A) SERS spectra of the sandwich complex at different SEB concentrations of 0, 3.5 × 10−10, 3.5 × 10−9, 3.5 × 10−8, 3.5 × 10−7, and 3.5 × 10−6 mol L−1 (from bottom to top) and the plot of the peak height of the DTNB SERS band 1338 cm−1 against the log[SEB] obtained by using gold slides. (B) SERS spectra of the sandwich complex at different SEB concentrations of 0, 2.5 × 10−15, 7.0 × 10−14, 2.8 × 10−12, 1.1 × 10−10, and 3.1 × 10−9 mol L−1 (from bottom to top) and the plot of the peak height of the DTNB SERS band 1338 cm−1 against the log[SEB] obtained by using magnetic gold nanorods. D

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Figure 6. Plot of the peak height of the DTNB SERS band at 1338 cm−1 against log[BSA] and log[avidin] obtained by the developed method using magnetic gold nanorods.

there is a linear relationship between SERS intensity and log[SEB] concentration with correlation coefficients (R2) of 0.990 and 0.988, respectively. The limit of detection (LOD), which corresponds to signal-to-noise ratio of 3, is 2.2 × 10−16 M (ca. 2697 SEB molecule/20 μL) for nanoparticle-based assay. High sensitivity using magnetic gold nanorods can be explained by the superior capturing efficiency of magnetic nanoparticles due to their high surface area.45 Furthermore, nonspherical gold nanorod particles act as electromagnetic hot spots for SERS, and creating hot spots using magnetic nanoparticle−nanorod interactions results in high sensitivity of the developed systems.46 In addition, nanoparticle−peptide ligand complex showed higher affinity than peptide itself. The binding of nanoparticle− peptide complex to SEB was studied by the SPR sensor, immobilizing SEB on the sensor surface at a low surface density and injecting nanoparticle−peptide complex at different concentrations as shown in Figure 5. The binding data were consistent with a simple 1:1 Langmuir interaction model. The adsorption rate and the desorption rate were found to be 4.8 × 105 M−1 s−1 and 6.0 × 10−3 s−1, respectively. The affinity constant of the nanoparticle−peptide complex was calculated as 8.0 × 107 M−1, which is approximately 90-fold higher than the constants obtained for the peptide alone. This enhanced affinity can be the result of multiple binding sites on the surface of the nanoparticle and the multivalent effect of peptide ligands.37 To better assess the performance of the developed system for rapid detection of SEB, we tested the selectivity of the system by using different proteins. For this purpose, the detection procedure was applied for different concentrations of bovine serum albumin (BSA) and avidin and the SERS intensities were measured at 1338 cm−1 as shown in Figure 6. The signals obtained from BSA and avidin indicate very low nonspecific binding of other proteins onto the sensing surface. The SERS intensities of SEB at different concentrations exhibited a noticeable difference from other proteins at 1338 cm−1, which indicates the high selectivity of the developed system. We also investigated the usability of the proposed method for the determination of the toxin in sample matrixes. For this purpose, milk, urine, and blood samples, spiked with different concentrations of SEB, were analyzed by using the nano-

and DTNB-labeled gold nanorods were bound to each other via SEB molecules, forming aggregate structures as shown in Figure 2B. SERS measurements were performed using aggregated nanoparticles. We carried out the control experiments regarding the magnetic gold nanoparticle before and after peptide modification as shown in the Supporting Information, Figure S-1, parts A and B, peptide-modified magnetic gold nanoparticles in the presence of SEB (Supporting Information Figure S-2), and a mixture of capture and SERS probes in the absence of SEB (Supporting Information Figure S-3). As seen in Supporting Information Figure S-3, the nonspecific binding of label to the magnetic particle has not been observed in the absence of SEB. SERS spectra were measured at each step for magnetic gold nanorod particles as indicated in Figure 3. The spectrum of magnetic gold nanorod particles, peptide-immobilized nanorod, and SEB−peptide−nanorod complex without adding silver nanoparticles did not elicit distinctive SERS bands (Figure 3A). SERS spectra of these structures were also collected by adding silver nanoparticles to increase the intensity values as shown in Figure 3B. The spectrum of bare magnetic gold nanorod particles shows a band at 745 cm−1 (Figure 3B). Although a distinct spectral change could not be observed after the immobilization of peptide ligands, the binding of the SEB elicited a different spectrum indicating the formation of the nanorod−peptide−SEB complex. After capturing SEB, some new peaks appeared, especially the ones at 443, 952, and 1295 cm−1. The bands at 668, 1035, and 1174 cm−1 are most likely due to phenylalanine residue,43 amide III C−N stretching, and aliphatic chain C−C stretching,44 respectively. When the complex was labeled with DTNB and peptide-immobilized gold nanorods, a huge peak at 1338 cm−1 and peaks at 1053 and 1553 cm−1 appeared due to the DTNB Raman signal.42 The effect of different concentrations of SEB on the SERS spectra of glass slide and nanoparticle-based complexes was investigated as shown in Figure 4, parts A and B, respectively. The blank signal comes from the nonspecific adsorption, and the SERS intensities of the band at 1338 cm−1 are proportional to the SEB concentrations between 3.5 × 10−10 and 3.5 × 10−6 M for glass slides and 2.5 × 10−15 and 3.2 × 10−9 M for magnetic nanoparticles. As shown in Figure 4, parts A and B, E

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(8) Hahn, I. F.; Pickenhahn, P.; Lenz, W.; Brandis, H. J. Immunol. Methods 1986, 92, 25−29. (9) Khan, A. S.; Cao, C. J.; Thompson, R. G.; Valdes, J. J. Mol. Cell. Probes 2003, 17, 125−126. (10) Wojciechowski, J.; Danley, D.; Cooper, J.; Yazvenko, N.; Taitt, C. R. Sensor 2010, 10, 3351−3362. (11) Yu, H.; Ahmed, H.; Vasta, G. R. Anal. Biochem. 1998, 261, 1−7. (12) Honjoh, K. I.; Kobayashi, H.; Miyamoto, T.; Kamikado, H. J. Food Prot. 2003, 66 (7), 1222−1226. (13) Yang, M. H.; Kostov, Y.; Bruck, H. A.; Rasooly, A. Anal. Chem. 2008, 80, 8532−8537. (14) Yang, M. H.; Sun, S.; Kostov, Y.; Rasooly, A. Lab Chip 2010, 10, 1011−1017. (15) Tang, D. P.; Su, B.; Tang, J.; Chen, G. J. Agric. Food Chem. 2010, 58, 10824−10830. (16) Yang, M.; Kostov, Y.; Bruck, H. A.; Rasooly, A. Int. J. Food Microbiol. 2009, 133, 265−271. (17) 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. (18) Tempelman, L. A.; King, K. D.; Anderson, G. P.; Ligler, F. S. Anal. Biochem. 1996, 233, 50−57. (19) King, K. D.; Anderson, G. P.; Bullock, K. E.; Regina, M. J.; Saaski, E. W.; Ligler, F. S. Biosens. Bioelectron. 1999, 14, 163−170. (20) Lin, H. C.; Tsai, W. C. Biosens. Bioelectron. 2003, 18, 1479− 1483. (21) Ruan, C.; Zeng, K.; Varghese, O. K.; Grimes, C. A. Biosens. Bioelectron. 2004, 20, 585−591. (22) Rasooly, A. J. Food Prot. 2001, 64, 37−43. (23) Naimushin, A. N.; Soelberg, S. D.; Nguyen, D. K.; Dunlap, L.; Bartholomew, D.; Elkind, J.; Melendez, J.; Furlong, C. E. Biosens. Bioelectron. 2002, 17, 573−584. (24) Homola, J.; Dostálek, J.; Chen, S.; Rasooly, A.; Jiang, S.; Yee, S. S. Int. J. Food Microbiol. 2002, 75, 61−69. (25) Slavik, R.; Homola, J.; Brynda, E. Biosens. Bioelectron. 2002, 17, 591−595. (26) Medina, M. B. J. Rapid Methods Autom. Microbiol. 2005, 13, 37− 55. (27) Medina, M. B. J. Rapid Methods Autom. Microbiol. 2003, 11, 225−243. (28) Bamrungsap, S.; Shukoor, M. I.; Chen, T.; Sefah, K.; Tan, W. Anal. Chem. 2011, 83 (20), 7795−7799. (29) Dorst, B. V.; Mehta, J.; Bekaert, K.; Rouah-Martin, E.; De Coen, W.; Dubruel, P.; Blust, R.; Robbens, J. Biosens. Bioelectron. 2010, 26, 1178−1194. (30) Goldman, E. R.; Pazirandeh, M. P.; Charles, P. T.; Balighian, E. D.; Anderson, G. P. Anal. Chim. Acta 2002, 457, 13−19. (31) Goldman, E. R.; Pazirandeh, M. P.; Mauro, J. M.; King, K. D.; Frey, J. C.; Anderson, G. P. J. Mol. Recognit. 2000, 13, 382−387. (32) Olsen, E. V.; Sorokulova, I. B.; Petrenko, V. A.; Chen, I.-H.; Barbaree, J. M.; Vodyanoy, V. J. Biosens. Bioelectron. 2006, 21, 1434− 1442. (33) Huang, S.; Yang, H.; Lakshmanan, R. S.; Johnson, M. L.; Chen, I.-H.; Wikle, H. C., III; Petrenko, V. A.; Barbaree, J. M.; Chin, B. A. Biosens. Bioelectron. 2009, 24, 1730−1736. (34) Nanduri, V.; Balasubramanian, S.; Sista, S.; Vodyanoy, V. J.; Simonian, A. L. Anal. Chim. Acta 2007, 589, 166−172. (35) Cerruti, M.; Jaworski, J.; Raorane, D.; Zueger, C.; Varadarajan, J.; Carraro, C.; Lee, S.-W.; Maboudian, R.; Majumdar, A. Anal. Chem. 2009, 81, 4192−4199. (36) Wu, J.; Cropek, D. M.; West, A. C.; Banta, S. Anal. Chem. 2010, 82, 8235−8243. (37) Dudak, F. C.; Kılıç, N.; Demir, K.; Yasar, F.; Boyacı, I. H. Pept. Sci. 2012, 98, 145−154. (38) Kudelski, A. Talanta 2008, 76, 1−8. (39) Han, X. X.; Zhao, B.; Ozaki, Y. Anal. Bioanal. Chem. 2009, 394, 1719−1727. (40) Tripp, R. A.; Dluhy, R. A.; Zhao, Y. P. Nano Today 2008, 3, 31− 37.

particle-based assay. As shown in Supporting Information Figure S-4, we constructed the calibration curve as SERS intensity versus log[SEB] within the range of 3.6 × 10−15 M to 3.6 × 10−9 M. Selective detection of SEB is enabled by the use of aptamer binding on the capture probe and SERS tag. These standard curves obtained from milk, urine, and serum are quite close to the one obtained from buffer solution, which indicates that the proposed method can be applied to different matrixes with high recovery values.

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CONCLUSION In this study, we have developed a SERS-based assay for fast and sensitive detection of SEB by the use of peptide ligands as recognition agents. Gold-coated glass slides and magnetic gold nanorod particles were both used to capture the target molecule, and the sandwich assay was completed by binding the captured molecules with secondary peptide ligand immobilized and DTNB-labeled gold nanorods. In comparison with the gold slide, the magnetic gold nanorod particle-based assay enables much more sensitive detection of SEB. The proposed method has the ability to detect SEB at attomolar levels, which is better than most of the biosensors and bioassays developed for SEB detection.47,48 In addition, the developed assay can be applied for the quantitative analysis of SEB in complex matrixes, such as milk, urine, and serum. All these results indicate the great potential of peptide ligands in biosensor and bioassay applications. Although a lower LOD value can be achieved by the use of antibodies as recognition elements, peptide ligands provide the development of stable and economical bioassay systems. Furthermore, by using different aptamers, this assay can be used for the detection of a variety of toxins and food contaminants.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +90 312 202 31 05. Fax: +90 312 223 50 18. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial supports provided by The Scientific and Technological Research Council of Turkey, project no. COST-MP091-108T794.

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REFERENCES

(1) Balaban, N.; Rasooly, A. Int. J. Food Microbiol. 2000, 61, 1−10. (2) Yang, M.; Kostov, Y.; Bruck, H. A.; Rasooly, A. Int. J. Food Microbiol. 2009, 133, 265−271. (3) Dinges, M. M.; Orwin, P. M.; Schlievert, P. M. Clin. Microbiol. Rev. 2000, 13, 16−34. (4) Gill, D. M. Microbiol. Rev. 1982, 46, 86−94. (5) Wagner, M. M.; Dato, V.; Dowling, J. N.; Allswede, M. J. Biomed. Inf. 2003, 36, 177−188. (6) Poli, M. A.; Rivera, V. R.; Neal, D. Toxicon 2002, 40, 797−802. (7) Windemann, H.; Luthy, J.; Maurer, M. Int. J. Food Microbiol. 1989, 8, 25−34. F

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(41) Guven, B.; Akgul, N. B.; Temur, E.; Tamer, U.; Boyacı, I. H. Analyst 2011, 136, 740−748. (42) Tamer, U.; Boyacı, I. H.; Temur, E.; Zengin, A.; Dincer, I.; Elerman, Y. J. Nanopart. Res. 2011, 13, 3167−3176. (43) De Gelder, J.; De Gussem, K.; Vandenabeele, P.; Moens, L. J. Raman Spectrosc. 2007, 38, 1133−1147. (44) Nikoobakht, B.; El-Sayed, M. A. J. Phys. Chem. A 2003, 107, 3372−3378. (45) Dudak, F. C.; Bas, D.; Basaran, A. N.; Tamer, U.; Boyacı, I. H. Front. Biosci., Elite Ed. 2011, 3, 1109−1127. (46) Wang, Y.; Lee, K.; Irudayaraj, J. Chem. Commun. 2009, 46, 613− 615. (47) Sapsford, K. E.; Francis, J.; Sun, S.; Kostov, Y.; Rasooly, A. Anal. Bioanal Chem. 2009, 394, 499−505. (48) Quiel, A.; Jürgen, B.; Piechotta, G. Appl. Microbiol. Biotechnol. 2010, 85, 1619−1627.

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