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Rapid detection method for aflatoxin B1 in soybean sauce based on fluorescent microspheres probe
Food Control 50 (2015) 659e662
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Rapid detection method for aflatoxin B1 in soybean sauce based on fluorescent microspheres probe Daofeng Liu 1, Yanmei Huang 1, Minghui Chen, Shuying Wang, Kun Liu, Weihua Lai* State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 24 August 2014 Accepted 8 October 2014 Available online 16 October 2014
Soybean sauce, a Chinese traditional and daily condiment, is often contaminated by aflatoxin B1. An extract-free immunochromatographic assay was proposed based on fluorescent microspheres probe for the' detection of aflatoxin B1 in soybean sauce. The probe was prepared by coupling fluorescent microspheres with anti-aflatoxin B1 antibody by the 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride-mediated method. The background from the soybean sauce sample on strip was eliminated because of the optical property of the probe. The sample without extracting procedure was directly detected by diluting with 10% methanol solution. The visible detection limit for the qualitative analysis of aflatoxin B1 in the proposed method was 2.5 mg/L, which was lower than the maximum level of 5 mg/L set by the Chinese government. The results were well agreed with those obtained by liquid chromatographymass spectrometry (LC-MS). The method showed satisfactory characteristics, such as rapid detection, easy operation, and high sensitivity, and can thus be applied for the large-scale and on-site screening of soybean sauce contaminated with aflatoxin B1. To our knowledge, this report is the first one on the qualitative detection of aflatoxin B1 in dark colored food samples directly by fluorescent microspheres probe-based immunochromatography. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Fluorescent microspheres probe Aflatoxin B1 Immunochromatography Soybean sauce Detection
1. Introduction Soybean sauce is generally produced from soybean by fermentation and is one of the China's traditional and daily condiment. The annual production of soybean sauce in China is approximately six million tons, which accounts for more than 65% of the total world production (Zhao, Wang, Zou, & Zhao, 2013). Aflatoxin B1 (AFB1) is a secondary metabolite produced by various genera of fungi (Pleadin et al., 2014). AFB1 is classified as a Group I carcinogen by World Health Organization (WHO) because of its highly hepatotoxic, teratogenic, mutagenic, and carcinogenic effects on humans and animals (Matumba et al., 2014; Mehrzad, Devriendt, Baert, & Cox, 2014; Zegura, Straser, & Filipic, 2011). AFB1 can be found in soybean sauce when the raw material used for processing is contaminated by some genera of fungi. A study analyzing the AFB1 content in 203 soybean sauce samples from five provinces in China showed that AFB1 contamination was widespread in soybean sauce and the average contamination rate was 95.06%, as detected by ELISA (Sun, Yan, Xu, Sun, & Zhu, 2010).
* Corresponding author. Tel.: þ86 791 83969526; fax: þ86 791 88157619. E-mail address: [email protected] (W. Lai). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.foodcont.2014.10.011 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
China's Ministry of Health has set 5 mg/L as the maximum permitted level of AFB1 in soybean sauce (Chinese National Standard, 2011) because of consumer safety concerns. Therefore, a rapid detection method for large-scale and on-site screening of soybean sauce contaminated with AFB1 should be developed. Several analytical methods, such as HPLC (Ding, Li, Bai, & Zhou, 2012), LCeMS/MS (Soleimany, Jinap, & Abas, 2012) and ELISA (Yu, Gribas, Vdovenko, & Sakharov, 2013), are currently available for AFB1 determination. However, these methods involve tedious pretreatment, which is not suitable for non-laboratory applications. Thus, an analytical system involving simple pretreatment procedures should be developed to decrease the time for on-site analysis and screening of soybean sauce. Immunochromatographic assay (ICA) has always been used for the detection of liquid samples. In ICA, nitrocellulose membrane is used as a carrier, whereas probe-labeled antigen or antibody is used as a tracer, and the result is indicated by the test (T) line. The assay has several advantages, such as simplicity, speediness, low cost, and easy operation. Previous studies have reported the use of colloidal gold (CG) ICAs for the qualitative detection of AFB1 in food (Liu, Hsu, Lu, & Yu, 2013; Sun et al., 2006). In the two previous studies, CG was used as biomarker for different samples, but this biomarker was unsuitable
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for soybean sauce. The strip is covered with black background because of the color of the soybean sauce, and the CG is not easily observed by naked eye. Fortunately, luminescent nanoparticles such as fluorescent microspheres (FM) can solve the problem of dark background on the strip. The visible fluorescence emitted by FM can be observed using a portable instrument. With the optical property of FM, the interference of black background from soybean sauce is eliminated. The schematic of FM-based ICA (FM-ICA) is shown in Fig. 1. In present study, an extract-free FMeICA for detecting AFB1 in soybean sauce was developed. Sensitivity was defined by the visible detection limit (VDL), which was lower than the maximum permitted level of AFB1 in China. By this proposed assay, soybean sauce containing AFB1 was effectively screened on a large scale. 2. Materials and methods 2.1. Reagent and instruments FM was purchased from Merck company (Darmstadt, Germany), nitrocellulose membrane, absorbent pad, sample pad, and conjugate pad were obtained from Millipore (Bendford, USA), anti-AFB1 monoclonal antibody was prepared by our laboratory (State Key Laboratory of Food Science and Technology, Nanchang, China). AFB1 standard sample was purchased from SigmaeAldrich Co. LLC (St. Louis, USA), soybean sauce was purchased from a local grocery store in Nanchang, China. The digital scanner was from Shanghai Huguo Science Instrument Co., Ltd (Shanghai, China), and the portable reader was from Jiangxi Zodolabs Biotech Co., Ltd (Nanchang, China). 2.2. FM probe preparation The probe was obtained by 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC)-mediated method using the
procedures referred in earlier study (Xie, Lai, et al., 2014, Xie, Wu, et al., 2014). Anti-AFB1 antibody was added to a 2.6 mL PB solution containing 0.15 mg of FM and 2.7 mL of EDC (5 mg/mL). The solution was agitated for 2 h. Subsequently, 300 mL of bovine serum albumin (BSA; 10%, w/v) was added as a blocking buffer, and the solution was agitated for 30 min. After centrifugation (14,000 r/min at 4 C for 15 min), the supernatant solution was discarded, and the precipitate was resuspended. Moreover, a digital scanner was used to collect signals of T to optimize numerically coupling conditions of probe. 2.2.1. Optimizing pH for coupling The pH-adjusted PB solutions were used for coupling to optimize the labeling pH (pH 4.0, 5.0, 6.0, 7.0, and 8.0). Prepared probes were dissolved in phosphate buffered saline (PBS) and added to strips to collect the T line signal by a digital scanner. One strip caused the strongest fluorescence intensity in the T line and was used for subsequent optimization. 2.2.2. Optimizing mass ratio between anti-AFB1 antibody and FM Probes were prepared with different mass ratios between the antibody and the FM (0, 10, 20, 30, 40, 50, and 60 mg/mg) in PB solution (pH 7.0). Prepared probes were dissolved in PBS and drop on the strip. To study the effect of mass ratio on FM-ICA, T line signals were read. 2.3. Assembling strip for FM-ICA AFB1eBSA (0.5 mg/mL) and goat anti-mouse IgG antibody (0.5 mg/mL) were sprayed onto the nitrocellulose membrane to form the T and C lines, respectively. The prepared nitrocellulose membrane and the absorption, conjugate, and sample pads were assembled to create the strip.
Fig. 1. The schematic of FM-ICA. Test (T) lines and control (C) lines or only C line were produced by negative sample (A) or positive sample (B), respectively. Signals could be observed under a portable instrument with a light source and a filter.
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2.4. FM-ICA procedure
3.2. Effect of mass ratio between anti-AFB1 antibody and FM
FM probes were pre-mixed with 100 mL of PBS or sample at ELISA wells for 10 min. The mixture was transferred on the strip. Result could not be read until clear bands appeared on the reaction regions. No T line can be observed because the antibody on FM combined with AFB1 in sample instead of the AFB1-BSA immobilized on the T line (Fig 1(A)). However, the T line was observed because of FM aggregation (Fig 1(B)). In addition, C line invariably appeared because either combined or uncombined FMs reacted with the goat anti-mouse IgG antibody.
The mass ratio between antibody and FM was also a key factor for coupling. As shown in Fig. S2, no signal was observed at 0 mg/mg mass ratio, which indicated that uncombined FM did not react with the AFB1-BSA that was immobilized on the T line. A fall in signal from 50 mg/mg to 60 mg/mg followed the sharp signal increase from 0 mg/mg to 40 mg/mg. A peak in signal was observed at 40 mg/mg, which means that minimum amount of probe was used for one strip. The antibody numbers on FM showed an increasing trend, according to ELISA (data not shown), but signals were not expected to show an increasing trend because of steric hindrance. The steric hindrance was caused by the overload of antibody on the surface of FM, which led to a reduction in the combination of antibody for AFB1-BSA. Thus, based on these optimal results, FM probe (labeling pH 7.0 and mass ratio of 40 mg/mg) was used to characterize the strip for detecting AFB1 in soybean sauce.
2.5. Sample pretreatment for FM-ICA Soybean sauce sample was diluted fivefold with methanol solution, and 100 mL of the solution was dropped onto the strip. To study the effect of methanol solution concentration on the strip, the T line signals produced by 10%, 20%, 30%, and 40% methanol in PBS solution (v/v) were analyzed, respectively. After comparing the different signals, the related concentration of methanol solution with the strongest signal was used as the optimal diluent. 2.6. FM-ICA evaluation 2.6.1. Sensitivity of FM-ICA Soybean sauce spiked with AFB1 at different concentrations (0, 0.5, 1.0, 2.5, and 5.0 mg/mL) was diluted with methanol solution and then analyzed by FM-ICA to study the sensitivity of FM-ICA. For onsite detection application, the results were judged by naked eye. Sensitivity was defined as the VDL. The lowest concentration of AFB1 causing observable absence of the T line was recorded as the VDL of the method. 2.6.2. Validation of the optimized FM-ICA by LC-MS To evaluate the practicability of proposed method for detecting AFB1 in different soybean sauce samples, the strips were used to analyze 36 samples gathered from grocery stores, and the results were validated by LC-MS method referred in earlier study (Xie, Lai, et al., 2014, Xie, Wu, et al., 2014). A procedure of extraction followed as Chinese National Standard (2004) was carried out prior to LC-MS testing.
3.3. Effect of methyl alcohol solution on strip Even if soybean sauce, being fluid, is appropriate for the test, its high viscosity adversely affects the ability to flow on the strip. Preliminary experiments confirmed that the sample did not reach the absorbent pad unless diluted fivefold (data not shown). Reports by Dzantiev (Dzantiev, Byzova, Urusov, & Zherdev, 2013) and Beloglazova (Beloglazova et al., 2012) showed that using organic solvents as extracting or diluting buffer had adverse effects on immunochromatography. Interestingly, in our study, methyl alcohol solution at a certain concentration promoted signal intensity of the T line. As shown in Fig. S3, the strongest signal was obtained at 10% methanol, and this effect was possibly due to the alcohol solubility of AFB1. Alcohol-soluble AFB1 maintained the native conformation required to react with the antibody at 10% methanol. Nevertheless, 40% methyl alcohol had a negative effect on the strip. The denaturation of the antibody may have induced the loss of bioactivity at a high methyl alcohol concentration. 3.4. The visible detection limit
3.1. Effect of labeling pH
In Fig. 2, a “cut off” of the T line was revealed when the sample, diluted fivefold and originally containing 2.5 mg/L AFB1, was tested. AFB1 (0.5 mg/L) can be detected by the strip. The ideal VDL of 2.5 mg/ L can be observed by naked eye (in a portable reader), and this value was lower than the maximum permitted level of AFB1 in soybean sauce by Chinese standards. The FM probe-based assay was suitable for on-site screening of soybean sauce contaminated
Optimizing the labeling pH to develop an ICA was necessary because the labeling pH can affect both antibody activity and coupling efficiency (Chen et al., 2013). According to a report by Kang (Kang, Kan, Yeung, & Liu, 2006), COOH on the particle surface was neutralized gradually to COOewith the increase of labeling pH. The hydrophilicity of polymer chains with COOe was higher than the hydrophilicity of those with eCOOH. Increasing affinity of particles to target resulted in the increase of coupling efficiency. However, extreme pH had adverse effect on antibody activity. T line signals were significantly weaker at pH 4.0 and 8.0 than at pH 5.0, 6.0, and 7.0. In addition, a peak was present at labeling pH 7.0 (Fig. S1), and in this labeling pH, the coupling efficiency and antibody activity reached a satisfactory combination because of the generation of the strongest signal on the T line. Therefore, labeling pH 7.0 was regarded as the optimal pH for coupling.
Fig. 2. The visible detection limit of FM-ICA for detection of AFB1 in soybean sauce. The strips were used to detect diluted spiked soybean sauce (originally contained AFB1 standards 0.5, 1.0, 2.5, 5.0 mg/L, respectively). NF and NG represent result of negative sample by FM-ICA and CG-ICA, respectively.
3. Results and discussion
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Table 1 Comparison between FM-ICA and LC-MS for detecting AFB1 in soybean sauce. “＋,” positive; “－,” negative; “ND,” not detected. Sample
FM-ICA
LC-MS (mg/L)
Sample
FM-ICA
LC-MS (mg/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
e e e e e e e e e ＋ e e e e e e e e
0.7028 0.0843 0.1387 ND 0.0741 0.4013 0.8725 0.0619 0.6628 2.9679 0.3610 0.0041 0.0192 ND 0.2033 0.0391 0.4130 ND
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
e e e e e e e e e e e e e e e e e e
0.3560 0.8141 ND ND 0.5103 0.0521 ND 0.2619 1.0339 0.2840 0.7319 0.0912 0.1783 0.5273 ND 0.2810 0.4921 0.0098
rapid, sensitive, simple, convenient, and on-site detection of contaminated AFB1 soybean sauce on a large scale. In case of positive samples observed by FM-ICA which is a screen method, confirmatory methods have to be implemented to confirm the result. Furthermore, FM-ICA can also be used for the detection of other substances with background interference. Acknowledgments This work was supported by Jiangxi province main science and technology leader project (20113BCB22007), and the Jiangxi education bureau technology put into use the project (KJLD13009), and earmarked fund for Jiangxi Agriculture Research System (JXARS-03). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodcont.2014.10.011. References
with AFB1. The method has certain advantages, as follows: extractfree, sensitive, convenient equipment, and user-friendly operation. In addition, compared with CGeICA, FMeICA is more beneficial for the detection of AFB1 in soybean sauce (as shown in Fig. 2). FMeICA provided lower background interference. FMs can emit visible fluorescence (520 nm) using a portable instrument, which has a 470 nm blue light that can excite FMs and a filter that can eliminate the light below 500 nm, whereas no fluorescence was generated by the soybean sauce sample. CGeICA showed inevitable interference by the dark color of the soybean sauce, which may decreased sensitivity. Thus, the FM probe is much more suitable than CG for detecting analytes by immunochromatography in darkcolored samples. However, FM has a larger diameter than CG. When used as a label, the aggregation of a larger-sized probe on both T and C lines can be easily observed, which indicates higher signal intensity. Moreover, researchers (Goryacheva, Lenain, & De Saeger, 2013; Wang et al., 2009) found that the size of the probe is correlated with the flow rate and affects the reaction time in ICA. Large probe size is associated with a long detection time for sufficient reaction of antigen and antibody. Thus, compared with CG probe, the larger-sized FM probe can be designed for a higher sensitivity. 3.5. Validation by LC-MS As shown in Table 1, 36 soybean sauce samples were analyzed by LC-MS, and 29 of these samples were contaminated with AFB1. Among the 29 contaminated samples, only one sample contained AFB1 at a concentration that exceeded 2.5 mg/L (the VDL of FM-ICA). Similar to results of FM-ICA, one sample revealed a positive result. Therefore, the results measured by the VDL of FM-ICA were in good agreement with those measured by LC-MS. FM-ICA can increase work efficiency for large-scale, on-site soybean sauce evaluation and can be used for rapid screening of samples containing high concentrations of AFB1. 4. Conclusions A sensitive, extract-free, FM probe-based ICA method for AFB1 determination in soybean sauce was proposed. Compared with CG, FM showed greater advantage as a biomarker for immunochromatography in soybean sauce. In this study, samples can be directly detected. As expressed by the VDL, an ideal sensitivity of 2.5 mg/L for detecting AFB1 was achieved. Results of FM-ICA and LC-MS were in good agreement. Thus, FM-ICA is an alternative analytical tool for
, S., & Beloglazova, N. V., Speranskaya, E. S., De Saeger, S., Hens, Z., Abe Goryacheva, I. Y. (2012). Quantum dot based rapid tests for zearalenone detection. Analytical and Bioanalytical Chemistry, 403, 3013e3024. Chen, Y., Xin, Y., Yang, H. L., Zhang, L., Zhang, Y. R., Xia, X. L., et al. (2013). Immobilization and stabilization of cholesterol oxidase on modified sepharose particles. International Journal of Biological Macromolecules, 56, 6e13. Chinese National Standard. (2004). GB/T 5009.22-2003 determination of aflatoxin B1 in foods. Chinese National Standard. (2011). GB 2761-2011 maximum levels of mycotoxins in foods. Ding, X. X., Li, P. W., Bai, Y. Z., & Zhou, H. Y. (2012). Aflatoxin B1 in post-harvest peanuts and dietary risk in China. Food Control, 23, 143e148. Dzantiev, B. B., Byzova, N. A., Urusov, A. E., & Zherdev, A. V. (2013). Immunochromatographic methods in food analysis. TrAC Trends in Analytical Chemistry, 55, 81e93. Goryacheva, I. Y., Lenain, P., & De Saeger, S. (2013). Nanosized labels for rapid immunotests. TrAC Trends in Analytical Chemistry, 46, 30e43. Kang, K., Kan, C., Yeung, A., & Liu, D. (2006). The immobilization of trypsin on soap-free P (MMA-EA-AA) latex particles. Materials Science and Engineering C, 26, 664e669. Liu, B. H., Hsu, Y. T., Lu, C. C., & Yu, F. Y. (2013). Detecting aflatoxin B1 in foods and feeds by using sensitive rapid enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip. Food Control, 30, 184e189. Matumba, L., Monjerezi, M., Biswick, T., Mwatseteza, J., Makumba, W., Kamangira, D., et al. (2014). A survey of the incidence and level of aflatoxin contamination in a range of locally and imported processed foods on Malawian retail market. Food Control, 39, 87e91. Mehrzad, J., Devriendt, B., Baert, K., & Cox, E. (2014). Aflatoxin B1 interferes with the antigen-presenting capacity of porcine dendritic cells. Toxicology in Vitro, 28, 531e537. (2014). Aflatoxin Pleadin, J., Vuli c, A., Persi, N., Skrivanko, M., Capek, B., & Cvetni c, Z. B1 occurrence in maize sampled from Croatian farms and feed factories during 2013. Food Control, 40, 286e291. Soleimany, F., Jinap, S., & Abas, F. (2012). Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry. Food Chemistry, 130, 1055e1060. Sun, X. L., Yan, L., Xu, D., Sun, T. T., & Zhu, Y. (2010). Risk assessment of AFB1 in soy sauce. Chinese Journal of Microecology, 22, 748e753. Sun, X. L., Zhao, X. L., Tang, J., Gu, X. H., Zhou, J., & Chu, F. S. (2006). Development of an immunochrom atographic assay for detection of aflatoxin B1 in foods. Food Control, 17, 256e262. Wang, Y., Xu, H., Wei, M., Gu, H., Xu, Q., & Zhu, W. (2009). Study of superparamagnetic nanoparticles as labels in the quantitative lateral flow immunoassay. Materials Science and Engineering: C, 29, 714e718. Xie, F., Lai, W. H., Saini, J., Shan, S., Cui, X., & Liu, D. F. (2014). Rapid pretreatment and detection of trace aflatoxin B1 in traditional soybean sauce. Food Chemistry, 150, 99e105. Xie, Q. Y., Wu, Y. H., Xiong, Q. R., Xu, H. Y., Xiong, Y. H., Liu, K., et al. (2014). Advantages of fluorescent microspheres compared with colloidal gold as a label in immunochromatographic lateral flow assays. Biosensors and Bioelectronics, 54, 262e265. Yu, F. Y., Gribas, A. V., Vdovenko, M. M., & Sakharov, I. Y. (2013). Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin B1 in food products. Talanta, 107, 25e29. Zegura, B., Straser, A., & Filipi c, M. (2011). Genotoxicity and potential carcinogenicity of cyanobacterial toxinsea review. Mutation Research/Reviews in Mutation Research, 727, 16e41. Zhao, H. F., Wang, Y. Q., Zou, Y., & Zhao, M. (2013). Natural occurrence of deoxynivalenol in soy sauces consumed in China. Food Control, 29, 71e75.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Rapid detection method for aflatoxin B1 in soybean sauce based on fluorescent microspheres probe Daofeng Liu 1, Yanmei Huang 1, Minghui Chen, Shuying Wang, Kun Liu, Weihua Lai* State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 June 2014 Received in revised form 24 August 2014 Accepted 8 October 2014 Available online 16 October 2014
Soybean sauce, a Chinese traditional and daily condiment, is often contaminated by aflatoxin B1. An extract-free immunochromatographic assay was proposed based on fluorescent microspheres probe for the' detection of aflatoxin B1 in soybean sauce. The probe was prepared by coupling fluorescent microspheres with anti-aflatoxin B1 antibody by the 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride-mediated method. The background from the soybean sauce sample on strip was eliminated because of the optical property of the probe. The sample without extracting procedure was directly detected by diluting with 10% methanol solution. The visible detection limit for the qualitative analysis of aflatoxin B1 in the proposed method was 2.5 mg/L, which was lower than the maximum level of 5 mg/L set by the Chinese government. The results were well agreed with those obtained by liquid chromatographymass spectrometry (LC-MS). The method showed satisfactory characteristics, such as rapid detection, easy operation, and high sensitivity, and can thus be applied for the large-scale and on-site screening of soybean sauce contaminated with aflatoxin B1. To our knowledge, this report is the first one on the qualitative detection of aflatoxin B1 in dark colored food samples directly by fluorescent microspheres probe-based immunochromatography. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Fluorescent microspheres probe Aflatoxin B1 Immunochromatography Soybean sauce Detection
1. Introduction Soybean sauce is generally produced from soybean by fermentation and is one of the China's traditional and daily condiment. The annual production of soybean sauce in China is approximately six million tons, which accounts for more than 65% of the total world production (Zhao, Wang, Zou, & Zhao, 2013). Aflatoxin B1 (AFB1) is a secondary metabolite produced by various genera of fungi (Pleadin et al., 2014). AFB1 is classified as a Group I carcinogen by World Health Organization (WHO) because of its highly hepatotoxic, teratogenic, mutagenic, and carcinogenic effects on humans and animals (Matumba et al., 2014; Mehrzad, Devriendt, Baert, & Cox, 2014; Zegura, Straser, & Filipic, 2011). AFB1 can be found in soybean sauce when the raw material used for processing is contaminated by some genera of fungi. A study analyzing the AFB1 content in 203 soybean sauce samples from five provinces in China showed that AFB1 contamination was widespread in soybean sauce and the average contamination rate was 95.06%, as detected by ELISA (Sun, Yan, Xu, Sun, & Zhu, 2010).
* Corresponding author. Tel.: þ86 791 83969526; fax: þ86 791 88157619. E-mail address: [email protected] (W. Lai). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.foodcont.2014.10.011 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
China's Ministry of Health has set 5 mg/L as the maximum permitted level of AFB1 in soybean sauce (Chinese National Standard, 2011) because of consumer safety concerns. Therefore, a rapid detection method for large-scale and on-site screening of soybean sauce contaminated with AFB1 should be developed. Several analytical methods, such as HPLC (Ding, Li, Bai, & Zhou, 2012), LCeMS/MS (Soleimany, Jinap, & Abas, 2012) and ELISA (Yu, Gribas, Vdovenko, & Sakharov, 2013), are currently available for AFB1 determination. However, these methods involve tedious pretreatment, which is not suitable for non-laboratory applications. Thus, an analytical system involving simple pretreatment procedures should be developed to decrease the time for on-site analysis and screening of soybean sauce. Immunochromatographic assay (ICA) has always been used for the detection of liquid samples. In ICA, nitrocellulose membrane is used as a carrier, whereas probe-labeled antigen or antibody is used as a tracer, and the result is indicated by the test (T) line. The assay has several advantages, such as simplicity, speediness, low cost, and easy operation. Previous studies have reported the use of colloidal gold (CG) ICAs for the qualitative detection of AFB1 in food (Liu, Hsu, Lu, & Yu, 2013; Sun et al., 2006). In the two previous studies, CG was used as biomarker for different samples, but this biomarker was unsuitable
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D. Liu et al. / Food Control 50 (2015) 659e662
for soybean sauce. The strip is covered with black background because of the color of the soybean sauce, and the CG is not easily observed by naked eye. Fortunately, luminescent nanoparticles such as fluorescent microspheres (FM) can solve the problem of dark background on the strip. The visible fluorescence emitted by FM can be observed using a portable instrument. With the optical property of FM, the interference of black background from soybean sauce is eliminated. The schematic of FM-based ICA (FM-ICA) is shown in Fig. 1. In present study, an extract-free FMeICA for detecting AFB1 in soybean sauce was developed. Sensitivity was defined by the visible detection limit (VDL), which was lower than the maximum permitted level of AFB1 in China. By this proposed assay, soybean sauce containing AFB1 was effectively screened on a large scale. 2. Materials and methods 2.1. Reagent and instruments FM was purchased from Merck company (Darmstadt, Germany), nitrocellulose membrane, absorbent pad, sample pad, and conjugate pad were obtained from Millipore (Bendford, USA), anti-AFB1 monoclonal antibody was prepared by our laboratory (State Key Laboratory of Food Science and Technology, Nanchang, China). AFB1 standard sample was purchased from SigmaeAldrich Co. LLC (St. Louis, USA), soybean sauce was purchased from a local grocery store in Nanchang, China. The digital scanner was from Shanghai Huguo Science Instrument Co., Ltd (Shanghai, China), and the portable reader was from Jiangxi Zodolabs Biotech Co., Ltd (Nanchang, China). 2.2. FM probe preparation The probe was obtained by 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC)-mediated method using the
procedures referred in earlier study (Xie, Lai, et al., 2014, Xie, Wu, et al., 2014). Anti-AFB1 antibody was added to a 2.6 mL PB solution containing 0.15 mg of FM and 2.7 mL of EDC (5 mg/mL). The solution was agitated for 2 h. Subsequently, 300 mL of bovine serum albumin (BSA; 10%, w/v) was added as a blocking buffer, and the solution was agitated for 30 min. After centrifugation (14,000 r/min at 4 C for 15 min), the supernatant solution was discarded, and the precipitate was resuspended. Moreover, a digital scanner was used to collect signals of T to optimize numerically coupling conditions of probe. 2.2.1. Optimizing pH for coupling The pH-adjusted PB solutions were used for coupling to optimize the labeling pH (pH 4.0, 5.0, 6.0, 7.0, and 8.0). Prepared probes were dissolved in phosphate buffered saline (PBS) and added to strips to collect the T line signal by a digital scanner. One strip caused the strongest fluorescence intensity in the T line and was used for subsequent optimization. 2.2.2. Optimizing mass ratio between anti-AFB1 antibody and FM Probes were prepared with different mass ratios between the antibody and the FM (0, 10, 20, 30, 40, 50, and 60 mg/mg) in PB solution (pH 7.0). Prepared probes were dissolved in PBS and drop on the strip. To study the effect of mass ratio on FM-ICA, T line signals were read. 2.3. Assembling strip for FM-ICA AFB1eBSA (0.5 mg/mL) and goat anti-mouse IgG antibody (0.5 mg/mL) were sprayed onto the nitrocellulose membrane to form the T and C lines, respectively. The prepared nitrocellulose membrane and the absorption, conjugate, and sample pads were assembled to create the strip.
Fig. 1. The schematic of FM-ICA. Test (T) lines and control (C) lines or only C line were produced by negative sample (A) or positive sample (B), respectively. Signals could be observed under a portable instrument with a light source and a filter.
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2.4. FM-ICA procedure
3.2. Effect of mass ratio between anti-AFB1 antibody and FM
FM probes were pre-mixed with 100 mL of PBS or sample at ELISA wells for 10 min. The mixture was transferred on the strip. Result could not be read until clear bands appeared on the reaction regions. No T line can be observed because the antibody on FM combined with AFB1 in sample instead of the AFB1-BSA immobilized on the T line (Fig 1(A)). However, the T line was observed because of FM aggregation (Fig 1(B)). In addition, C line invariably appeared because either combined or uncombined FMs reacted with the goat anti-mouse IgG antibody.
The mass ratio between antibody and FM was also a key factor for coupling. As shown in Fig. S2, no signal was observed at 0 mg/mg mass ratio, which indicated that uncombined FM did not react with the AFB1-BSA that was immobilized on the T line. A fall in signal from 50 mg/mg to 60 mg/mg followed the sharp signal increase from 0 mg/mg to 40 mg/mg. A peak in signal was observed at 40 mg/mg, which means that minimum amount of probe was used for one strip. The antibody numbers on FM showed an increasing trend, according to ELISA (data not shown), but signals were not expected to show an increasing trend because of steric hindrance. The steric hindrance was caused by the overload of antibody on the surface of FM, which led to a reduction in the combination of antibody for AFB1-BSA. Thus, based on these optimal results, FM probe (labeling pH 7.0 and mass ratio of 40 mg/mg) was used to characterize the strip for detecting AFB1 in soybean sauce.
2.5. Sample pretreatment for FM-ICA Soybean sauce sample was diluted fivefold with methanol solution, and 100 mL of the solution was dropped onto the strip. To study the effect of methanol solution concentration on the strip, the T line signals produced by 10%, 20%, 30%, and 40% methanol in PBS solution (v/v) were analyzed, respectively. After comparing the different signals, the related concentration of methanol solution with the strongest signal was used as the optimal diluent. 2.6. FM-ICA evaluation 2.6.1. Sensitivity of FM-ICA Soybean sauce spiked with AFB1 at different concentrations (0, 0.5, 1.0, 2.5, and 5.0 mg/mL) was diluted with methanol solution and then analyzed by FM-ICA to study the sensitivity of FM-ICA. For onsite detection application, the results were judged by naked eye. Sensitivity was defined as the VDL. The lowest concentration of AFB1 causing observable absence of the T line was recorded as the VDL of the method. 2.6.2. Validation of the optimized FM-ICA by LC-MS To evaluate the practicability of proposed method for detecting AFB1 in different soybean sauce samples, the strips were used to analyze 36 samples gathered from grocery stores, and the results were validated by LC-MS method referred in earlier study (Xie, Lai, et al., 2014, Xie, Wu, et al., 2014). A procedure of extraction followed as Chinese National Standard (2004) was carried out prior to LC-MS testing.
3.3. Effect of methyl alcohol solution on strip Even if soybean sauce, being fluid, is appropriate for the test, its high viscosity adversely affects the ability to flow on the strip. Preliminary experiments confirmed that the sample did not reach the absorbent pad unless diluted fivefold (data not shown). Reports by Dzantiev (Dzantiev, Byzova, Urusov, & Zherdev, 2013) and Beloglazova (Beloglazova et al., 2012) showed that using organic solvents as extracting or diluting buffer had adverse effects on immunochromatography. Interestingly, in our study, methyl alcohol solution at a certain concentration promoted signal intensity of the T line. As shown in Fig. S3, the strongest signal was obtained at 10% methanol, and this effect was possibly due to the alcohol solubility of AFB1. Alcohol-soluble AFB1 maintained the native conformation required to react with the antibody at 10% methanol. Nevertheless, 40% methyl alcohol had a negative effect on the strip. The denaturation of the antibody may have induced the loss of bioactivity at a high methyl alcohol concentration. 3.4. The visible detection limit
3.1. Effect of labeling pH
In Fig. 2, a “cut off” of the T line was revealed when the sample, diluted fivefold and originally containing 2.5 mg/L AFB1, was tested. AFB1 (0.5 mg/L) can be detected by the strip. The ideal VDL of 2.5 mg/ L can be observed by naked eye (in a portable reader), and this value was lower than the maximum permitted level of AFB1 in soybean sauce by Chinese standards. The FM probe-based assay was suitable for on-site screening of soybean sauce contaminated
Optimizing the labeling pH to develop an ICA was necessary because the labeling pH can affect both antibody activity and coupling efficiency (Chen et al., 2013). According to a report by Kang (Kang, Kan, Yeung, & Liu, 2006), COOH on the particle surface was neutralized gradually to COOewith the increase of labeling pH. The hydrophilicity of polymer chains with COOe was higher than the hydrophilicity of those with eCOOH. Increasing affinity of particles to target resulted in the increase of coupling efficiency. However, extreme pH had adverse effect on antibody activity. T line signals were significantly weaker at pH 4.0 and 8.0 than at pH 5.0, 6.0, and 7.0. In addition, a peak was present at labeling pH 7.0 (Fig. S1), and in this labeling pH, the coupling efficiency and antibody activity reached a satisfactory combination because of the generation of the strongest signal on the T line. Therefore, labeling pH 7.0 was regarded as the optimal pH for coupling.
Fig. 2. The visible detection limit of FM-ICA for detection of AFB1 in soybean sauce. The strips were used to detect diluted spiked soybean sauce (originally contained AFB1 standards 0.5, 1.0, 2.5, 5.0 mg/L, respectively). NF and NG represent result of negative sample by FM-ICA and CG-ICA, respectively.
3. Results and discussion
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Table 1 Comparison between FM-ICA and LC-MS for detecting AFB1 in soybean sauce. “＋,” positive; “－,” negative; “ND,” not detected. Sample
FM-ICA
LC-MS (mg/L)
Sample
FM-ICA
LC-MS (mg/L)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
e e e e e e e e e ＋ e e e e e e e e
0.7028 0.0843 0.1387 ND 0.0741 0.4013 0.8725 0.0619 0.6628 2.9679 0.3610 0.0041 0.0192 ND 0.2033 0.0391 0.4130 ND
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
e e e e e e e e e e e e e e e e e e
0.3560 0.8141 ND ND 0.5103 0.0521 ND 0.2619 1.0339 0.2840 0.7319 0.0912 0.1783 0.5273 ND 0.2810 0.4921 0.0098
rapid, sensitive, simple, convenient, and on-site detection of contaminated AFB1 soybean sauce on a large scale. In case of positive samples observed by FM-ICA which is a screen method, confirmatory methods have to be implemented to confirm the result. Furthermore, FM-ICA can also be used for the detection of other substances with background interference. Acknowledgments This work was supported by Jiangxi province main science and technology leader project (20113BCB22007), and the Jiangxi education bureau technology put into use the project (KJLD13009), and earmarked fund for Jiangxi Agriculture Research System (JXARS-03). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodcont.2014.10.011. References
with AFB1. The method has certain advantages, as follows: extractfree, sensitive, convenient equipment, and user-friendly operation. In addition, compared with CGeICA, FMeICA is more beneficial for the detection of AFB1 in soybean sauce (as shown in Fig. 2). FMeICA provided lower background interference. FMs can emit visible fluorescence (520 nm) using a portable instrument, which has a 470 nm blue light that can excite FMs and a filter that can eliminate the light below 500 nm, whereas no fluorescence was generated by the soybean sauce sample. CGeICA showed inevitable interference by the dark color of the soybean sauce, which may decreased sensitivity. Thus, the FM probe is much more suitable than CG for detecting analytes by immunochromatography in darkcolored samples. However, FM has a larger diameter than CG. When used as a label, the aggregation of a larger-sized probe on both T and C lines can be easily observed, which indicates higher signal intensity. Moreover, researchers (Goryacheva, Lenain, & De Saeger, 2013; Wang et al., 2009) found that the size of the probe is correlated with the flow rate and affects the reaction time in ICA. Large probe size is associated with a long detection time for sufficient reaction of antigen and antibody. Thus, compared with CG probe, the larger-sized FM probe can be designed for a higher sensitivity. 3.5. Validation by LC-MS As shown in Table 1, 36 soybean sauce samples were analyzed by LC-MS, and 29 of these samples were contaminated with AFB1. Among the 29 contaminated samples, only one sample contained AFB1 at a concentration that exceeded 2.5 mg/L (the VDL of FM-ICA). Similar to results of FM-ICA, one sample revealed a positive result. Therefore, the results measured by the VDL of FM-ICA were in good agreement with those measured by LC-MS. FM-ICA can increase work efficiency for large-scale, on-site soybean sauce evaluation and can be used for rapid screening of samples containing high concentrations of AFB1. 4. Conclusions A sensitive, extract-free, FM probe-based ICA method for AFB1 determination in soybean sauce was proposed. Compared with CG, FM showed greater advantage as a biomarker for immunochromatography in soybean sauce. In this study, samples can be directly detected. As expressed by the VDL, an ideal sensitivity of 2.5 mg/L for detecting AFB1 was achieved. Results of FM-ICA and LC-MS were in good agreement. Thus, FM-ICA is an alternative analytical tool for
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