Microsoft Word 刘继红-二校

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1 6 12 Vol. 6 No Journal of Food Safety and Quality Dec., 2015 B 1 1, 2, 刘继红 3, Chris Maragos 4 1, 2,, 王敏 3 1, 2,, 尹海燕 3, 张玲 1, 2, 张军锋 3 1, 2,, 洪慧杰 3 1, 2, 3*, 王红旗 (1., ; 2., ; 3., ; 4. (USDA) (NCAUR), 61604) 1,2,3, 摘要 : 目的 B 1 (FB1), 6-(4,6- ) (6-DTAF), 方法, 5-(4,6- ) (5-DTAF), 5-DTAF 结果 5-DTAF FB1 6-DTAF FB1, IC 50 结论 5-DTAF FB1, 6-DTAF FB1,, 关键词 : B 1 ; ; ; Preparation and application of new fluorescein-labeled fumonisins B 1 in fluorescence polarization analysis technique LIU Ji-Hong 1, 2, 3, Chris Maragos 4, WANG Min 1, 2, 3, YIN Hai-Yan 1, 2, 3, ZHANG Ling 1,2,3, ZHANG Jun-Feng 1, 2, 3, HONG Hui-jie 1, 2, 3 1, 2, 3*, WANG Hong-Qi (1. Institute of Quality Standard and Testing Technology for Agro-products, Henan Academy of Agricultural Science, Zhengzhou , China; 2. Henan Key Laboratory of Grain Quality and Safety and Testing, Zhengzhou , China; 3. Laboratory of Quality & Safety Risk Assessment for Agro-Products (Zhengzhou), Ministry of Agriculture, Zhengzhou , China; 4. Bacterial Foodborne Pathogens and Mycology Research Unit, Peoria IL 61604, USA) ABSTRACT: Objective To Prepare a new fluorescent tracer against common mycotoxins such as fumonisins B 1 for replace 6-(4,6-Dichlorotriazinyl) aminofluorescein (6-DTAF) expensive marker, and to develop a technique for quick detection of fumonisins B 1 based on the principle of fluorescence polarization. Methods The one-pot method was used to synthesize 5-(4,6-Dichlorotriazinyl) aminofluorescein (5-DTAF) 基金项目 : (2011-G5) (GJFP ) (2013YQ24) Fund: Supported by the Key Project of the Ministry of Agriculture (2011-G5), Project of Unknown Hazards Factor Identification and the Known Hazard Factor Safety Assessment for Products of Grain and Oil Crops (GJFP ) and Science Technology Foundation for Outstanding Young Scientists of Henan Academy of Agricultural Sciences (2013YQ24) * 通讯作者 :,, huda2000@126.com *Corresponding author: WANG Hong-Qi, Associate Researcher, Institute of Quality Standard and Testing Technology for Agro-products, Henan Academy of Agricultural Science, No. 116, Huayuan Road, Jinshui District, Zhengzhou , China. huda2000@126.com

2 12, : B fluorescent marker, and then fluorescence polarization technique based on 5-DTAF was established to detect fumonisins B 1. Results Fluorescence polarization technology based on 5-DTAF and 6-DTAF fluorescent marker of fumonisins B 1 had almost the same analysis capability and the value of IC 50. Conclusion New fluorescent marker of fumonisins B 1 was successfully synthesized by using 5-DTAF, and fluorescence polarization technology for fumonisins B 1 quick detection was developed. Moreover, the fluorescence polarization technology based on 5-DTAF fluorescent marker had almost the same analysis capability comparing with that of 6-DTAF fluorescent marker, but cut the analysis cost to a half. KEY WORDS: fumonisins B 1 ; aminofluorescein; fluorescent tracer; fluorescence polarization 1 引言 (fumonisin) [1] 15, B 1 (FB1), (IARC) 1993, ; (CAC) JECFA [2], [3] :,, FB1, 60% [4] : 60%,,,,, [5] :,,, [6-11], [12-14], [15], FB1 6-DTAF,, [5], FB1 5-DTAF FB1, 5-DTAF 6-DTAF, IC50, 5-DTAF 6-DTAF,,,, 2 材料与方法 2.1 实验材料 FB1 ( Santa ); FB1 (,, ); 5-DTAF 6-DTAF (, ) ( ) ( 18 MΩ)

3 仪器与设备 (Milli-Q Synthesis, ); (SHZ-22, ); (Comfort, ); (BT6KEL, VIRTIS ); (Hei-VAP Value, Heidolph ) (H1650-W, ); (SENTRY 200, Diachemix ) 2.3 试验方法伏马毒素荧光标记物的制备 6-DTAF : 10 mg FB1, 0.1 ml, 0.1 ml 0.1 mol/l Na 2 CO 3,, 1 mg 6-DTAF( 1), 0.1 ml, 6-DTAF, 8 h 0.3 g 60( µm, ), 0.4 ml 2~3,, 5-DTAF 6-DTAF, : 10 mg FB1, 0.5 ml 0.5 mol/l Na 2 CO 3, 1.0 ml 10 mg/ml 5-DTAF ( 1), g 60( 20~43 µm, ), 0.4 ml 2~3,,, 60( 40~63 µm) 10 g,,, 10 ml,, 50 ml 50 ml ( 30:10:0.5 ), 60 ml ( 30:30:1 ), 40,, FB1 样品的提取 20 g, 100 ml, 1 h, FB1 的荧光偏振检测 900 µl (ph=7.2) 100 µl 100 µl,,,,, 100 µl FB1,,, Fig DTAF 6-DTAF Molecular structural formula of 5-DTAF(A) and 6-DTAF (B) 3 结果与分析 3.1 荧光偏振免疫分析技术原理, 1920 Perrin :,, (P) : P=(I -I )/ (I +I ), I I,

4 12, : B , P,, P ;, P,,,,, 3.2 抗体溶液浓度优化, FB1 FB1 FB2 FB3 100% 38% 50% [16], 0.1% 1:100 1:200 1:300 1:400 1:500 1:1000,,, ( 0 µg/kg µg/kg, mp) 1:100, , 1:1000, 132.9,,, IC 50 1:100, IC µg/kg, 1:1000, IC µg/kg,,,, 1:500, µg/kg 3.3 标准曲线的建立, 1000 mg/kg, 10 µg/kg 50 µg/kg 100 µg/kg 200 µg/kg 500 µg/kg 1000 µg/kg 2000 µg/kg 5000 µg/kg µg /kg 900 µl 100 µl 100 µl,,,,, 100 µl,, 1 min,, (µg/kg) (mp), 2 6-DTAF,,, y =LgstcDoseRsp(a, b, c, d), a= , b= , c= , d= , R DTAF,,, y =LgstcDoseRsp(a, b, c, d), a= , b= , c= , d= , r 表 1 不同浓度抗体溶液荧光偏振强度范围与灵敏度 Table 1 Fluorescence polarization intensity range and sensitivity of different concentration of antibody solution ( mp) IC 50 (µg/kg) 1: : : : : : Fig. 2 Fluorescence polarization analysis standard curve for FB1

5 Fig. 3 Standard curve of FB1 in different matrix 6-DTAF, 5-DTAF, 186.8, IC µg/kg 6-DTAF, 182.3, IC µg/kg, 5-DTAF 6-DTAF 3.4 玉米基质对测定结果的影响,, : 20 g ( <25 µg/kg ), 100 ml, 1 h,,, 3 PBS, 182.3, IC µg/kg, 153.6, IC µg/kg,, 3.5 回收率精密度的考察,,,, mg/kg 1.0 mg/kg 2.5mg/kg,, 97.2% 142.8%, 3.9% 9.8%;, 93.4% 105.1%, 4.4% 9.6%,,,, 1 mg/kg,, 表 2 伏马毒素回收率和精密度 Table 2 Recovery rate and precision of FB1 mg/kg (%) RSD(%) PBS

12, : B 1 4815 4 结论 5-DTAF 6-DTAF, 5-DTAF 5-DTAF FB1,, 2~3 min, 500, 0.4 mg/kg,, 0.5 mg/kg 1.0 mg/kg 2.5 mg/kg, 93.4% 105.1%, 4.4% 9.

[2] Bolger M, Coker RD, DiNovi, M, et al. Fumonisins. WHO/IPCS safety evaluation of certain mycotoxins in food [J]. WHO Food Addit Ser, 2001, 47: 557 680.

Yang LM, Su JM, Lei HY, et al. Progress on fumonisins [J]. Prog Vet Med, 2014, 35:97 100. [5] Chris MM, Michael EJ, Ronald DP, et al.

6 12, : B 结论 5-DTAF 6-DTAF, 5-DTAF 5-DTAF FB1,, 2~3 min, 500, 0.4 mg/kg,, 0.5 mg/kg 1.0 mg/kg 2.5 mg/kg, 93.4% 105.1%, 4.4% 9.6%, 5-DTAF 6-DTAF, 6-DTAF, 5-DTAF 6-DTAF, 参考文献 [1] Guo YC, Liu XM, Liu J. Preparation and identification of anti-fumonisin B1 monoclonal antibody [J]. J Hyg Res, 1999, 28: 297. [2] Bolger M, Coker RD, DiNovi, M, et al. Fumonisins. WHO/IPCS safety evaluation of certain mycotoxins in food [J]. WHO Food Addit Ser, 2001, 47: [3] Marasas WFO, Jackson LS, DeVries JW, et al. Fumonisins: history, worldwide occurrence and impact [M]. In Fumonisins in Food; Plenum Press: New York, 1996; pp [4],,,. [J]., 2014, 35: Yang LM, Su JM, Lei HY, et al. Progress on fumonisins [J]. Prog Vet Med, 2014, 35: [5] Chris MM, Michael EJ, Ronald DP, et al. Fluorescence polarization as a means for determination of fumonisins in maize [J]. J Agric Food Chem, 2001, 49: [6] Shephard GS. Chromatographic determination of the fumonisin mycotoxins [J]. J Chromatogr A, 1998, 815: [7] Plattner RD. Detection of fumonisins produced in fusarium moniliforme cultures by HPLC with electrospray MS and evaporative light scattering detectors [J]. Nat Toxins, 1995, 3: [8] Wilkes JG, Sutherland JB, Churchwell MI, et al. Determination of fumonisins B 1, B 2, B 3, and B 4 by high-performance liquid chromatography with evaporative light-scattering detection [J]. J Chromatogr A, 1995, 695: [9] Plattner RD, Norred WP, Bacon CW, et al. A method of detection of fumonisins in corn samples associated with field cases of equine leukoencephalomalacia [J]. Mycologia, 1990, 82: [10] Musser SM, Jackson LS, DeVries JW, et al. Quantitation and identification of fumonisins by liquid chromatography/mass spectrometry [M]. In Fumonisins in Food; Plenum Press: New York, 1996; pp [11] Maragos CM. Analysis of mycotoxins with capillary electrophoresis [J]. Sem Food Anal, 1998, 3: [12],,, 10 [J]., 2014, 5: Zheng R, Mao D, Wang SM, et al. Determination of 10 mycotoxins in peach kernel by high performance liquid chromatography-tandem mass spectrometry [J]. J Food Saf Qual, 2014, 5: [13],,,. B1 [J]., 2011, 27: Qan Y, Zhan YH, Zhang GH, et al. Chemiluminescent enzyme immunoasssay of fumonisins B 1 in grain [J]. Chin J Public Health, 2011, 27: [14],,. B 1 [J]., 2009, 26: Zhang Y, Zhu L, Chen Y, et al. Ultra-seusitive determination of fumonisin B 1 by time-resolved fluoroimmunoassay [J]. J Environ Health, 2009, 26: [15] Maragos CM. Fluorescence polarization for mycotoxin determination [J]. Mycotoxin Res, 2006, 22: [16] Maragos CM, Bennett, GA, Richard JL. Affinity column cleanup for the analysis of fumonisins and their hydrolysis products in corn [J]. Food Agric Immunol, 1997, 9: 作者简介 ( 责任编辑 : 白洪健 ) 刘继红, 副研究员, 主要研究方向为农产品质量安全检测技术研究及农产品质量安全风险评估 ljha3100@163.com 王红旗, 副研究员, 理学博士, 主要研究方向为农产品质量安全检测技术研究 huda2000@126.com

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.