Highly Sensitive Detection of Zearalenone in Feed Samples Using

Article pubs.acs.org/JAFC

Highly Sensitive Detection of Zearalenone in Feed Samples Using Competitive Surface-Enhanced Raman Scattering Immunoassay Jianzhi Liu, Yongjun Hu,* Guichi Zhu, Xiaoming Zhou, Li Jia, and Tao Zhang Ministry of Education Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, People’s Republic of China ABSTRACT: Accurate and quantitative analysis of mycotoxin (such as zearalenone) is particularly imperative in the field of food safety and animal husbandry. Here, we develop a sensitive and specific method for zearalenone detection using competitive surface-enhanced Raman scattering (SERS) immunoassay. In this assay, a functional gold nanoparticle was labeled with the Raman reporter and the zearalenone antibody, and a modified substrate was assembled with the zearalenone−bovine serum albumin. With the addition of free zearalenone, the competitive immune reaction between free zearalenone and zearalenone− bovine serum albumin was initiated for binding with zearalenone antibody labeled on gold nanoparticle, resulting in the change of SERS signal intensity. The proposed method exhibits high sensitivity with a detection limit of 1 pg/mL and a wide dynamic range from 1 to 1000 pg/mL. Furthermore, this method can be further applied to analyze the multiple natural feed samples contaminated with zearalenone, holding great potential for real sample detection. KEYWORDS: Zearalenone, mycotoxin, surface-enhanced Raman scattering, competitive, immunoassay, quantitative detection

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INTRODUCTION

Surface-enhanced Raman scattering (SERS) is an emerging and powerful optical technology that can provide a nondestructive and ultrasensitive detection even down to a single molecular level.21,22 With several characteristics of tremendous signal enhancement, narrow band widths, and alleviated photobleaching, SERS immunoassay has been widely applied for sensitive detection of nucleic acids, proteins, and microbes.22−27 In SERS immunoassay, a novel labeling technique is employed. Herein, a SERS nanoprobe that relies on gold nanoparticle is labeled by dye molecules (Raman reporter, e.g., 4,4′-dipyridyl) and antibodies, respectively; thus, Raman signal of the reporter molecules can act as the tracer signal.23 However, the conventional SERS immunoassay depends on the formation of a sandwich structure of “antibody−antigen−antibody”, which demands the antigen with multiple recognition sites for binding antibody. For the detection of small molecules with small size and nonmultiple recognition sites, like zearalenone, this sandwich structure is unsuitable. To overcome this defect, the introduction of competitive concept into SERS immunoassay has gained attention.23,28 Dufek et al.29 first applied the competitive SERS immunoassay for the detection of 1,25-dihydroxy metabolite of vitamin D3, but the antibody was chosen to attach to the substrate instead of antigen, which would increase the cost of the assay. Afterward, our group designed a low-cost competitive SERS immunoassay for the detection of adrenergic agonist of clenbuterol in pig urine;30 nevertheless, the analysis of spiked clenbuterol samples could not confirm its presence in real samples.

Contamination of agricultural commodities by mycotoxins is a serious problem worldwide due to their high toxicity and biological persistence. Zearalenone is one of the most important mycotoxins and results from the secondary metabolite of several Fusarium species.1 Because of unusual weather conditions or improper storage, zearalenone contamination is frequent in agricultural commodities and cereal products such as flour, malt, soybeans, and beer.2 Global trade also facilitates the occurrence of zearalenone contamination in food, feed, and feed raw materials.3 Evidence suggests that the consumption of contaminated products might cause various toxic effects on humans and animals, including teratogenesis, carcinogenicity, neurotoxicity, abortion, and estrogenic effect.4−9 Consequently, accurate and quantitative analysis of zearalenone in agricultural commodities is critical for food safety and animal husbandry. Because of the adverse effects and the universal existence of zearalenone, a variety of methods such as chemical, physical, and biological processing approaches have been used to lower its level, and it is necessary to develop effective methods to detect and monitor the trace amount of residual zearalenone.10,11 The traditional analytical methods used for zearalenone detection include high-performance liquid chromatography and ultra high performance liquid chromatography coupled with tandem mass spectrometry, but they usually require expensive instrumentation and complicated sample preparation processes, which might limit their applications in the routine monitoring of zearalenone.12,13 To avoid this limitation, the immunoassay- and aptamer-based methods have been developed with the advantages of simplicity and low cost, but most of them are difficult to reach a satisfactory sensitivity.14−20 Therefore, a simple and sensitive method is highly desirable for zearalenone detection in the real samples. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8325

April 8, 2014 July 21, 2014 July 23, 2014 July 23, 2014 dx.doi.org/10.1021/jf503191e | J. Agric. Food Chem. 2014, 62, 8325−8332

Journal of Agricultural and Food Chemistry

Article

of 30 μL of 0.01 mM 4,4′-dipyridyl was added to 1.0 mL of uncoated gold nanoparticles and the resultant mixture was allowed to react for 10 min. The gold nanoparticles labeled with 4,4′-dipyridyl molecules were then separated from the solution by centrifugation at 12 000 rpm for 5 min and dispersed with 1.0 mL of borate buffer (pH 8.2). Second, zearalenone antibody was immobilized on the 4,4′-dipyridyllabeled gold nanoparticles. Five microliters of 0.5 mg/mL zearalenone antibody was added to 1.0 mL of 4,4′-dipyridyllabeled gold nanoparticles. The mixture was incubated at room temperature for 2 h. Then the SERS nanoprobes were purified by centrifugation for 10 min and resuspension with 1.0 mL of borate buffer (pH 8.2). Finally, 20 μL of 2.5% bovine serum albumin was added into the above SERS nanoprobes to block sites bare of 4,4′-dipyridyl and the mixture was incubated for 1 h at room temperature. After centrifugation and resuspension with 1.0 mL of phosphate-buffered saline (pH 7.4), the SERS nanoprobes were finally obtained and the nanoprobes are usually stable for 2−3 days when stored at 4 °C. Preparation of Capture Substrates. The substrates that we used were concave glass slides and five steps were needed to modify them, which meant that the substrates were modified by piranha solution, 3-aminopropyl trimethoxysilane, glutaraldehyde,35−38 zearalenone−bovine serum albumin, and bovine serum albumin. In the first step, the glass slides were immersed in piranha solution (H2SO4:H2O2, 7:3, v/v) for 30 min to yield hydroxyl on the surface. In the second step, after ultrasonic cleaning, the substrates were dipped in methanol solution containing 5% 3-aminopropyl trimethoxysilane for 12 h at room temperature. In the third step, 2.5% glutaraldehyde was added onto the 3-aminopropyl trimethoxysilane silanized substrates to form an 3-aminopropyl trimethoxysilane− glutaraldehyde surface. The slides were allowed to react for 4 h at room temperature, and the amino groups on the 3aminopropyl trimethoxysilane silanized substrates reacted with the aldehyde groups of glutaraldehyde, thus yielding the 3aminopropyl trimethoxysilane−glutaraldehyde surface. In the fourth step, 100 μL of 50 μg/mL zearalenone−bovine serum albumin were dropped onto the concave surface of the modified substrates. In the last step, the substrates were incubated in 2.5% bovine serum albumin for 1 h to block active sites on the modified substrates. After they were rinsed with ultrapure water and dried under a stream of nitrogen, the capture substrates were eventually obtained. Sample Preparation. Zearalenone-free feed sample was spiked by adding zearalenone standard solution and the mixture was stored overnight at 4 °C. The concentrations of the spiked samples used in the experiment were 0, 1, 10, 100, and 1000 pg/mL for zearalenone. A new extraction method, dispersive liquid−liquid extraction, for SERS immunoassay was investigated. Specifically, 5 g of the spiked ground sample was extracted with 30 mL of methanol and 20 mL of 50 g/L NaCl solution where methanol was used as extractant and NaCl was used to avoid emulsification. The mixture was first shaken for 10 min and then ultrasonically for 30 min. After filtering, the filtrate was placed in the separatory funnel and chloroform was used to extract zearalenone from the mixture. The zearalenone chloroform solution was evaporated to dryness before diluting 30-fold with phosphate-buffered saline (pH 7.4) containing 10% methanol (v/v) and 0.05% Tween 20 (v/v). Three natural feed samples contaminated with zearalenone were also handled according to the procedure above.

Herein, we developed a new method based on a competitive SERS immunoassay, which was carried out between free zearalenone and zearalenone−bovine serum albumin fixed on the substrate, competitively binding with zearalenone antibody conjugated on the gold nanoparticles. In the present report, this method was used to detect the zearalenone in natural feed samples.

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MATERIALS AND METHODS Materials. Zearalenone monoclonal antibody, zearalenone− bovine serum albumin, and zearalenone standard solution were purchased from Beijing Huaan Magnech Bio-Tech Co., Ltd. (Beijing, China). Aflatoxin B1 standard solution and diethylstilbestrol standard solution were from Guangzhou Analysis Center Keli Technology Co., Ltd. (Guangzhou, China). 4,4′Dipyridyl, bovine serum albumin, and trisodium citrate were purchased from Sigma Chemical (St. Louis). 3-Aminopropyl trimethoxysilane and glutaraldehyde were purchased from Aladdin China Ltd. (Shanghai, China). Chloroauric acid (HAuCl4) was received from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The blank feed samples were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China). Sulfuric acid, nitric acid, 30% hydrogen peroxide, sodium chloride (NaCl), boric acid, borax, potassium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride were from Guangzhou Chemical Regent Factory (Guangzhou, China).The following buffer solutions were used: borate buffer (pH 8.2) and phosphate-buffered saline (pH 7.4). The washing buffer was prepared by adding 0.05% Tween-20 to the phosphate-buffered saline. All these reagents were analytical grade or better and all aqueous solutions were prepared with ultrapure water processed by an ultrapure water system (ELGA, London). Equipment. A microscopic Raman spectrometer (Nippon Optical System Co., Tokyo) was used to measure the Raman spectra. The excitation source was a He−Ne laser (Melles Griot, Carlsbad, CA) with a 632.8 nm excitation wavelength and a power of 1 mW was obtained. All spectra were calibrated referring to the 520 cm−1 line of silicon. The typical accumulation time used in this study was 30 s. The Raman spectrum was collected by WinSpec/32 software and could be converted into digital data. Those data were processed with Origin 7.0 software. Thereafter, only a baseline calibration on the spectra was performed to remove the intense fluorescence, which came from the capture substrate. A UV/vis spectrometer (Perkin-Elmer, Waltham, MA) was used to determine the diameter of gold nanoparticles. Preparation of Gold Nanoparticles and SERS Nanoprobes. The gold nanoparticles were prepared according to Frens’ method,31 with slight modification.32 Generally, 100 mL of 1 mM HAuCl4 aqueous solution was heated to boiling, and then 6.0 mL of freshly prepared 38.8 mM trisodium citrate solution was added rapidly. The solution turned deep red quickly and was boiled for another 20 min. Then the solution was allowed to cool down to room temperature with continuous stirring. The resulting red gold nanoparticles were obtained with a diameter of 30 nm and the diameter of the nanoparticles was determined by the UV/vis spectrometer. The SERS nanoprobes were prepared by the procedure reported by Gu and co-workers with slight modification.33,34 It was prepared in a typical two-step process: first, 4,4′-dipyridyl molecules were located on the gold nanoparticles through the spontaneous adsorption on gold nanoparticles surface. A total 8326

dx.doi.org/10.1021/jf503191e | J. Agric. Food Chem. 2014, 62, 8325−8332


Article

Test Procedure. The SERS procedure has been described previously,30 with minor modification, as shown in Figure 1. In

Figure 1. (A) Preparation of SERS nanoprobes. (B) Competitive SERS immunoassay for zearalenone.

the first step, the SERS nanoprobes were prepared as above. In the second step, the competitive SERS immunoassay was carried out. Zearalenone standard solution and SERS nanoprobes in the same volume were simultaneously dropped on the concave surface of the capture substrate, during which zearalenone−bovine serum albumin fixed on the capture substrate and zearalenone antigen suspending in the standard solution competitively conjugated with the zearalenone antibody labeled on the SERS nanoprobes. Finally, the 4,4′dipyridyl Raman signal was obtained from the substrate with a microscopic Raman spectrometer if there are nanoprobes conjugating on the capture substrate. The control experiment was carried out to confirm the antibody and bovine serum albumin were actually labeled on the gold nanoprobes.

Figure 3. (A) SERS signal intensity with different concentrations of zearalenone−bovine serum albumin. (B) SERS signal intensity with different volumes of zearalenone antibody. The Raman intensities are those of the bands at 1612 cm−1 of 4,4′-dipyridyl. Error bars indicate the standard deviations of three independent measurements.

zearalenone antibody or bovine serum albumin. A previous study showed bovine serum albumin could be used to block the surplus active sites of the 4,4′-dipyridyl.33 4,4′-Dipyridyl is able to be perpendicularly adsorbed on the surface of the gold nanoparticles, which makes the amount of the antibodies labeled on gold nanoparticles as large as possible.39,40 Its fastlabeled and nontoxic properties make it an excellent Raman reporter. The characteristic peaks of 4,4′-dipyridyl are at 1612, 1510, 1984, 1229, 1071, 1019, and 772 cm−1, respectively. The characteristic peak at 772 cm−1 is assigned to ring breathing vibrational mode, B1, and the characteristic peak at 1019 cm−1 is related to ring breathing vibrational mode, A1. The characteristic peak at 1071 cm−1 is assigned to ring in-plane deformation vibrational mode and C−H bend vibrational mode, A1. The band at 1229 cm−1 is attributed to C−H inplane bending vibrational mode, A1. The band at 1294 cm−1 is attributed to inter-ring stretching Ω, B1. The band at 1510 cm−1 is assigned to C−H in-plane bending vibrational mode and ring stretching, A1. The band at 1612 cm−1 is assigned to ring stretching vibrational mode, A1.39 From Figure 2, we can see that the number and the locations of the 4,4′-dipyridyl peaks remain invariable, which means that the additions of zearalenone antibody and bovine serum albumin have no effect on 4,4′-dipyridyl adsorption and the 4,4′-dipyridyl is steadily adsorbed on the gold nanoparticles. Because of the long distance between the gold nanoparticles and the zearalenone antibody or bovine serum albumin, no SERS signal of zearalenone antibody or bovine serum albumin was observed. Hence, we chose 4,4′-dipyridyl as the Raman reporter for our experiment. To achieve the optimal Raman spectrum, we carried out a series of experiments to optimize the

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RESULTS AND DISCUSSION Optimization of the SERS Nanoprobes. 4,4′-Dipyridyl is a typical bifunctional Raman reporter, with two nitrogen atoms on both ends of the molecule (Figure 2). One can interact with the gold nanoparticles and the other conjugates with the

Figure 2. (A) SERS spectra of the 4,4′-dipyridyl labeled on gold nanoparticles. (B) SERS nanoprobes labeled with 4,4′-dipyridyl and antibody. (C) SERS nanoprobes labeled with 4,4′-dipyridyl and antibody, and then bovine serum albumin blocking the surplus 4,4′dipyridyl on the gold nanoparticles surface. 8327



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Figure 4. (A) SERS spectra acquired with different zearalenone antigen concentrations. (B) Concentration-dependent SERS intensity of the band at 1612 cm−1 of 4,4′-dipyridyl using the competitive SERS immunoassay. Error bars indicate the standard deviations of three independent measurements. Figure 5. (A) Function of centrifugation on the matrix effect in different dilutions of the feed sample and standard curve of the standard solution. (B) Calibration curve for the competitive SERS immunoassay of zearalenone. B/B0 represents the ratio of SERS intensities of different concentrations versus 0 pg/mL standard solution. Error bars indicate the standard deviations of three independent measurements.

4,4′-dipyridyl amount and the reaction time of 4,4′-dipyridyl and gold nanoparticles. The results indicate that the proper amount is ca. 30 μL of 0.01 mM 4,4′-dipyridyl and the proper incubation time is ca. 10 min, which corresponds with our previous work.30 Optimization of the Concentrations of the Zearalenone−Bovine Serum Albumin and the Zearalenone Antibody. For the sake of the optimal experimental results, determining the proper concentration of zearalenone−bovine serum albumin antigen is very important. As a high-cost reagent, we need to avoid unnecessary waste while making it detectable, so it is important to determine the proper concentration while meeting our experiment demands. To obtain the proper concentration, the same volume of zearalenone−bovine serum albumin antigens with the concentrations of 5, 10, 25, 50, 100, and 250 μg/mL were dropped respectively on the glass slides to incubate for 2 h and the antibody used in this test was sufficient. After 2 h incubation, the SERS calibration curves of different concentrations were obtained as shown in Figure 3A. It is found that the Raman intensity increases with the concentration when the concentrations are lower than 50 μg/mL. At 50 μg/mL, the Raman intensity reaches the maximum and when the concentration is over 50 μg/mL, the Raman intensity is unchanged. This indicates that the glass slide has not been completely occupied by the antigen when the concentrations are lower than 50 μg/ mL, and when the concentration is beyond 50 μg/mL, zearalenone−bovine serum albumin antigens are excessive. Therefore, the optimal concentration of zearalenone−bovine serum albumin antigen used in our experiment is 50 μg/mL.

Table 1. Comparison of Zearalenone Detection by Competitive SERS and HPLC in Feed Samples (n = 3)

competitive SERS

HPLCa a

sample 1 (ng/mL)

sample 2 (ng/mL)

sample 3 (ng/mL)

n=1

36.8

108.4

781.1

n=2 n=3 mean relative standard deviation (%) test

34.1 32.2 34.4 6.7

102.6 124.5 111.8 10.1

1002.4 881.1 888.2 12.5

38.9

133.7

976.1

Data are provided by Beijing Huaan Magnech Bio-Tech Co., Ltd.

The optimal concentration of zearalenone−bovine serum albumin antigen remains at the same level when the sample extract and zearalenone antibodies labeled on the SERS nanoprobes are added together on capture substrates. Apart from zearalenone−bovine serum albumin antigen, zearalenone antibodies labeled on the SERS nanoprobes are vital and the amount of the antibodies also play an important role in this assay, which demands that the amount is neither too much nor too little. To ensure that the competitive 8328



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3 h, which has been confirmed as the optimal incubation time, the Raman spectra of the SERS nanoprobes combining with the zearalenone−bovine serum albumin were obtained. In fact, antibody−antigen reaction is very rapid, and in 30 min, most of the reaction has finished.41,42 We set 3 h as the incubation time to obtain the better sensitivity. Figure 4A indicates that SERS signal intensities change with the concentrations of zearalenone standard solutions. It clearly shows that the higher the concentrations of zearalenone standard solutions, the weaker the SERS signal intensities. This is because when the concentrations of zearalenone standard solutions get higher, the amount of the SERS nanoprobes conjugated on the capture substrate lessens. This implies that the zearalenone−bovine serum albumin fastened on the capture substrate and the zearalenone antigens in the standard solutions have competitively bound with the zearalenone antibodies labeled on the SERS nanoparticles, which corresponds well with our experimental hypothesis about the detection scheme. To investigate the quantitative relation of SERS signal intensities and zearalenone concentrations in the standard solution, we drew a histogram with the band intensity at 1612 cm−1 which is the most intense band. Figure 4B shows that the SERS signal intensity of the control experiment (0 pg/ mL free zearalenone) is the highest, ca. 3595 ± 184 a.u., so we set 3227 a.u. (mean of the control intensity minus 2 times standard deviation) as the detection threshold value.29 Moreover, it is also found that the SERS intensity of 1 pg/ mL can be identified from the threshold value with ease and the intensities of any two adjacent concentrations from 1 to 1000 pg/mL can be easily separated. Therefore, ca.1 pg/mL estimated to be the limit of detection for zearalenone in standard solution is acceptable and reasonable. Furthermore, there is an underlying linear relationship between the SERS intensities and their corresponding zearalenone concentrations, implying that quantitative analysis of zearalenone may be feasible in this experiment. Feed Sample Analysis. Whether this method is practical in the detection of zearalenone in feed sample is a question, so experiments were carried out to verify its feasibility. However, for the sake of fulfilling animal requirement of all kinds of nutrition, feed usually contains various ingredients including numerous additives, thus introducing difficulties in zearalenone detection, due to the matrix effect.43,44 In the present experiment, the matrix is the feed samples diluted with phosphate-buffered saline. Under such circumstances, alleviating the matrix effect is an urgent problem to solve.45 Only in this way can this method realize its practicability in feed detection. First, we directly detected spiked feed samples. Generally, calibration curves of the pure zearalenone standard solution and feed samples diluted with phosphate-buffered saline (with the final dilutions 1:10, 1:20, 1:30, 1:40, and 1:50) were plotted and the zearalenone concentration range was 0.1− 105 pg/mL, during which B/B0 (B/B0 represents the SERS intensity ratios of different concentrations to 0 pg/mL standard solution) versus the zearalenone concentration is the coordinate axis. However, there is a perfect match only when the zearalenone concentration surpassed 10 pg/mL and the diluted multiples is more than 40. When the zearalenone concentration is lower than 10 pg/mL, none of the dilution curves correspond to the standard solution curve, which suggests that the matrix effect is more obvious in the lower concentration range.

Figure 6. Specificity of zearalenone antibody−antigen. (A) The molecular structures of zearalenone, diethylstilbestrol, and aflatoxin B1. (B) Raman spectra of the competitive SERS immunoassay and concentration-dependent SERS intensity of the band at 1612 cm−1 of 4,4′-dipyridyl. Error bars indicate the standard deviations of three independent measurements.

immunoassay is established, the amount of antibodies used in the experiment should be comparable to that of zearalenone− bovine serum albumin. In this experiment, 10, 9, 8, 7, 6, 5, 4, and 3 μL of 0.5 mg/mL zearalenone antibodies were added on the SERS capture substrates with the identical volume 50 μL of 50 μg/mL zearalenone antigens, respectively. About 3 h later, we obtained the calibration curve of SERS spectra with different volumes of antibodies. Figure 3B indicates that the Raman intensity reaches the maximum when the volume is 6 μL. When it is more than 6 μL, the Raman intensities remain unchanged, which means that the antibodies are excessive. When it is lower than 6 μL, the Raman intensities increase sharply with the volumes of antibodies increasing. Thus, we determine 5 μL of 0.5 mg/mL antibodies as the optimal antibodies amount, which indicates that ca. 14 zearalenone antibody molecules are estimated to contact single gold nanoparticle. After we determined the optimal concentrations of the zearalenone− bovine serum albumin antigen and the zearalenone antibody, the free zearalenone concentration was the only variable factor; thus, we could investigate the corresponding relationship between the free zearalenone concentration and the SERS intensity. SERS Detection. In this protocol, a series of zearalenone standard solutions with different concentrations were used to confirm the limit of detection, during which the concentrations of zearalenone standard solutions 1, 10, 100, and 1000 pg/mL, respectively, were used. A control experiment was simultaneously carried out with the solution containing only SERS nanoprobes and phosphate-buffered saline while no zearalenone antigen was added. A blank test was also performed to determine whether nonspecific adsorption existed or not. After SERS nanoprobes and zearalenone standard solutions have been simultaneously incubated on the capture substrate, about 8329



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competitively binding with antizearalenone monoclonal antibody are used for immunorecognition. Aflatoxin B1, which is a commonly occurring mycotoxin, and diethylstilbestrol, which is another kind of estrogen, are selected to investigate the specificity of antibody−antigen−mycotoxin. Specifically, the competitive immunoassays are carried out as follows: the three reagents with the same concentration in the feed sample were dropped on the capture substrate respectively while SERS nanoprobes were simultaneously added on each capture substrate. After 3 h incubation under room temperature with certain humidity, SERS signals were obtained. Figure 6 shows the chemical structures of the three reagents and their SERS signals, and we also drew the histogram with the SERS band intensity at 1612 cm−1 to compare the changes of SERS signal intensity with various concentrations. The SERS signal intensities of aflatoxin B1 and diethylstilbestrol in different concentrations have little changes while that of zearalenone shows a dramatic decline and the histogram shows the changes of the signal intensities along with the concentrations, which indicates that there is a high specificity of zearalenone antibody in recognizing zearalenone antigen. The results of this experiment shown in Figure 6B further establish the viability of our assay concept in natural feed samples detection.

To further alleviate the matrix effect, centrifugal separation has been performed. After the feed sample has been diluted into different concentrations, the solutions were centrifuged for 10 min. Then the supernatant of the feed sample was separated and stored at 4 °C. We set 10 000 rpm as the optimal rotation speed to not only remove the components that are not analyzed but also keep zearalenone undestroyed. Figure 5A shows the function of centrifugation on alleviating the matrix effect and the standard curve of the standard solution which obeys the equation Y = 77.73−13.59 log10 X, R2 = 0.9992. After centrifugation, the calibration curves of the 1:30, 1:40, and 1:50 dilute feed samples were similar to that of zearalenone standard solution with the concentration range from 5 to 5000 pg/mL, which indicates that the matrix effect has been effectively diminished. Hence, all experiments were carried out when the feed samples have been diluted 30 times and centrifuged. Figure 5B shows a linear plot which obeys the equation Y = 67.31−15.44 log10 X, R2 = 0.9969. On the basis of the calibration curve above, recoveries of 99−105.2% (n = 3) and mean relative standard deviation of 8.1% are obtained, which further demonstrates the substantial elimination of the matrix effect compared with the former method. Therefore, this method is appropriate for the quantitative analysis of feed samples. Analysis of Zearalenone in Natural Feed Samples. To validate its availability in natural feed samples, three natural feed samples were analyzed using this method. The feed samples were handled according to the procedure above. However, it was surprising that no SERS signal was obtained. Since matrix effect has been eliminated, the reason may exist in the gold colloid, the buffer pH, or the zearalenone content in the feed sample. The control variable method was used to determine the possible reasons and finally we focused on the zearalenone content, which means the zearalenone content is beyond our detection range. Dilution is a good method to reduce the zearalenone content, so we diluted the samples as 1:30, 1:50, 1:70, and 1:100. When the dilution ratio is 1:50, SERS signal appears, and we calculate the zearalenone content according to the calibration curve. The results are presented in Table 1, with the measured value 111.8 ng/mL (n = 3), and relative standard deviation 10.1%. Another two natural feed samples were also analyzed according to the procedure above. From the data in Table 1, we clearly see that the three feed samples are positive. To further confirm the results above, highperformance liquid chromatography (HPLC) was used and the results of the three feed samples have also been listed in Table 1, respectively. The competitive SERS method has a variation in determining zearalenone, which may be due to the difference of sample preparation, laser light intensity, and the instrument. Although HPLC has an excellent accuracy which is even better than ours,46 the proposed method still has a good enough accuracy with the advantages of high sensitivity, high specificity, and low cost. Moreover, the whole spectrum collection by Raman spectrometry needs only ca. 30 s. It is shorter than that of HPLC, where at least several minutes are needed.46 Therefore, our method has its characteristic advantages and is suitable to detect zearalenone residuue with very low concentration, especially for the trace detection. Investigation of Antibody−Antigen−Mycotoxin Specificity. The experiments described in this section demonstrate the feasibility of combining SERS and competitive method to detect the presence of specific species in aqueous samples. Zearalenone−bovine serum albumin and free zearalenone

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AUTHOR INFORMATION

Corresponding Author

*Telephone: (+86-20) 8521-1436, ext. 8107. Fax: (+86-20) 8521-6052. E-mail: [email protected]. Funding

This research is supported by the NSFC (No. 11079020, 21273083, U1332132) and Guangdong-NSF (No. S2013010016551) grants, the scientific research foundation for the returned overseas Chinese scholars, State Education Ministry, the foundation for introduction of talents by the universities in Guangdong Province, and the project under scientific and technological planning by Guangzhou City. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Beijing Huaan Magnech Bio-Tech Co., Ltd., for providing natural feed samples.

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REFERENCES

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Highly Sensitive Detection of Zearalenone in Feed Samples Using

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