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1 5 12 Vol. 5 No Journal of Food Safety and Quality Dec., 2014 SPR B 12 1, 孙明君 2*, 陈雍硕 3, 郑小龙 1, 陈启 (1., ; 2., ; 3. GE, ; 4., ) 4 摘要 : 目的 (surface plasmon resonance, SPR), B 12 方法 CM5, B 12, B 12,, 10 结果, 50 (relative standard deviation, RSD) 10% 0.54%, μg/100 g, 92.1%~104.1% 10 B 12 结论 6 h, 关键词 : ; ; B 12 ; Method establishment of testing vitamin B 12 in milk powder based on surface plasmon resonance SUN Ming-Jun 1, 2*, CHEN Yong-Shuo 3, ZHENG Xiao-Long 1, CHEN Qi 4 (1. Food and Agricultural Products Testing Agency, Technical center of Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao , China; 2. Ocean University of China College of Food Science and Engineering, Qingdao , China; 3. GE Healthcare, Shanghai , China; 4. Physicochemical Analysis Center of Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou , China) ABSTRACT: Objective To establish a method of fast-testing vitamin B 12 in milk by using the technique of surface plasmon resonance (SPR). Methods Vitamin B 12 was covalently coupled to laboratory prepared CM5 chip to obtain vitamin chip, the optimal antibody working concentration and chip regeneration conditions were investigated. Meanwhile, the stability of vitamin chip was measured. A series of different concentrations of vitamin B 12 was added to blank milk samples to construct a standard curve for vitamin B 12 quantification according to the principle of competitive immune inhibition assay. Ten commercial milk samples were determined by the developed method. Results Vitamin chip displayed a good stability, and the relative standard deviation (RSD) for 50 cycles was less than 10%. The limit of detection (LOD) of vitamin B 12 was μg/100 g, and recoveries were 92.1%~104.1%. Vitamin B 12 content below the required threshold level was not observed in 10 milk samples. Conclusion Less than 6 h was consumed for the determination of vitamin B 12 by the developed method, this method could offer a simple and efficient approach for vitamin B 12 quantification. KEY WORDS: surface plasmon resonance; biochip; vitamin B 12 ; microbiological method * 通讯作者 :,, sun_mingjun@hotmail.com *Corresponding author: SUN Ming-Jun, Assistant Engineer, Technical Center of Shandong Entry-Exit Inspection and Quarantine Bureau, No.70, Qutangxia Road, Shinan District, Qingdao , China. sun_mingjun@hotmail.com

2 引言 B 12 B,,, B 12 B 12 [1], B 12 B 12, Vitamin B 12 -AOAC Determination of Vitamin B 12 in Infant Formula and Adult Nutritions by HPLC; GB B 12 B 12, GB GB B 12, B 12, HPLC [2],, (surface plasmon resonance, SPR) ( ) [3] Kretschmann [4], (surface plasma wave, SPW), SPR Caelen [5] Biacore Q SPR B 2 ( ), SPR 70 ng/ml Haughey [6] Biacore QSPR B 5 ( ) B 5 (,,,, ), 4.4 ng/ml Indyk [7] SPR B 8 ( ) B 9 ( ) Biacore Q SPR 2 ng/ml SPR, SPR [8-9] [10-11] [12-13] Biacore SPR,, SPR [14],,,,, HPLC-MS HPLC GC GC-MS, SPR, SPR,, [15], 2 材料与方法 2.1 材料试剂 ( ) B 12 (GE : CM5) B 12 (GE : ) ( ) Pall 0.22 μm 1 ml ( ml) 10.0 ml 96 ( ) Na 2 HPO 4 (, )(, ) BSA(AMResco, ) 0.2% NaCN(, ) 0.5% SDS(, ) 0.2M NaOH(, ) () (1) mol/l : 35.6 g Na 2 HPO 4, 1000 ml 0.1 mol/l : 21.0g, 1000 ml - : 450 ml 0.2 mol/l 550 ml 0.1 mol/l, ph 4.5 (2) calibrant buffer 0.5 gbsa, 50 ml min, 0.22 μm

3 12, : SPR B (3) 1 0.2% NaCN 40 - ( 0.005%) (, NaCN ) (4) 2 ml0.5% SDS, 1.5 ml 0.2 mol/l NaOH, 0.5 ml, (5) IM I(2.4 μg/ml):100 μl VB 12, 10 ml, 10 ml IM II(24 ng/ml):100 μl(im I) 10 ml, 10 ml 2.2 仪器 (, BT 224S)(eppendorf, 5810R) Biacore 3000 (General Electric, GE) (, MLS-3750) ph (, SG2) 2.3 样品的前处理 ( 避光 ) 0.2 g 15 ml, 9 ml, min( ),, 10 ml, 2 ml 2 ml, r/min 10 min, 1 ml 1 ml ( ) 0.22 μm, 200 μl 1.5 ml 2.4 标准曲线的构建 B 12 (0.24 mg/ml) ng/ml, 2.5 优化芯片 Sensor Chip Vitamin B 12, (0.01 mol/l HEPES 0.15 mol/l NaCl 3 mmol/l EDTA 0.005% Surfactant p20) Buffer, Prime(Buffer ),, 20 μl/min, 200 μl 1.5 ml,, 回收率试验 10 3, 2.3, II0.8 ng/ml, I: IM II 2 ml+8 ml, (4.8 ng/ml) II: I 6 ml+30 ml, (0.8 ng/ml) 2.7 样品检测 File New Application Wizard, Use Template, VB 12 assay draft.blw(vb 12 ) Edit, Detection1, Cycle Settings Detection (Fc1 Fc2 Fc3 Fc4), Next ( Sample1-20 ),, ( Sample1-1,Sample1-2) Rack Position, 96 EP (,, ) EP, VB 12 (VBP), 2070 μl 230 μl VBP 2.7.7,, 2.7.8, 96, EP, 96 () 96 ( ) 2.7.9,, Next Save as, Start,

4 结果与分析 3.1 再生条件的优化与芯片稳定性, (ph 1.5, ph 2.0, ph 2.5 ph 3.0) ( mmol/l) (1 2 mol/l), ( μl/min) ( s) 75 mmol/l NaOH, 0.25%SDS, 20 μl/min, 50 s 480 s, 20 μl/min 75 mmol/l NaOH, 0.25%SDS, 20 μl/min, 50 s, 50, 1 2, 1216 RU, 50 (RSD) 2.2%,, 1 Fig. 1 Stability test of chip ( Y :, Y : ) 2 Fig. 2 Chip test reaction process A: ; B: ; C:

5 12, : SPR B 标准曲线的建立, 4, 3 logistic, (Y=Rhi-(Rhi-Rlo)/(1+(Conc./A 1 )^A 2 )), R 2 = , 3.3 不同方法的样品测定结果的比较, GB B 12 [1] B 12, () 10 B 12, 1, RSD 10%, 10%, B 12, B 12 ;, B 12, GB GB [1] B 12,, ;, 3 B 12 Fig. 3 Calibration curve or vitamin B 12 Table 1 表 1 两种方法检测维生素 B 12 的数据结果 (n=3) Comparison of the data of vitamin B 12 assay in 2 different methods (n=3) Biacore (μg/100 g) (μg/100 g) (AVG) (RSD) (AVG) (RSD) (μg/100 g) % % % % % % % % % % % % % % % % % % % % 2.0

6 回收率 10 3, 3 6 8, 0.8 ng/ml,, % % %,, 3 Table 2 (μg/100 g) 表 2 日内精密度数据 (n=7) Data of the intra-day precision (n=7) (SD) (RSD) % 表 3 样品回收率数据 (n=6) Table 3 Data of sample recovery rate(n=6) (%) (SD) (RSD) % 5.69% 6.19% % 7.75% 8.04% % 3.01% 3.52% 3.5 检测限 3, 3, 0.06 ng/ml : 10%, RSD 0.54%, % 104.1%, 0.08~2.4 ng/ml 18 min, 6 h 20 HPLC GC,,, 4 结论 Biacore 3000,, B 12 B 12 (Vitamin B 12 binding protein, VBBP), B 12 B 12 VBBP, VBBP B 12, B 12, VBBP,, B 12, B 12 (LOD)=0.06 ng/ml, (LOQ)=0.2 ng/ml,,,, 10,, 10%, RSD 0.54%, %~104.1%, B ~2.4 ng/ml 18 min, 6 h 20 HPLC GC,,, B 12,,, 参考文献 [1] GB B 12 [S]. GB Determination of vitaminb 12 in foods for infants and young children,milk and milk products [S]. [2],,,. B 12 [J]., 2013, 23(11): Liu H, Zhang Y, Xue ZM, et al. Research progress of the determination methods of vitamin B 12 in the food [J]. Chin J Health Lab Technol, 2013, 23(11): [3] Homola J, Yee SS, Gauglitz G. Surface plasmon sensors:review [J]. Sensors and Actuators B, 1999, 54: [4] Kretschmann E, Raether H. Radiative decay of non-radiative surface plasmons excited by light [J]. Z Naturforsh, 1968, 23A: [5] Caelen I, Kalman A, Wahlstrom L. DNA aptamer-based surface plasmon resonance biosensor for the detection of retinol binding protein 4 for the early diagnosis of type 2 diabetes [J]. Anal Chem, 2004, 76: 137. [6] Haughey SA, O Kane AA, Baxter GA, et al. Determination of pantothenic acid in foods by optical biosensor immunoassay [J].

7 12, : SPR B J AOAC Int, 2005, 88: [7] Indyk HE, Evans EA, Bostrom Caselunghe MC, et al. Determination of biotion and folate in infant formula and milk by optical biosensor-based immunoassay [J]. J AOAC Int, 2000, 83(5): [8] Thillaivnayagalingam P, Newcombe AR, Donovank O, et al. Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance [J]. Biotechnol Appl Biochem, 2007, 48(2): [9] Li J, Wu H, Hong J, et al. Odorranalectin is a small peptide lection with potential for drug delivery and targeting [J]. PLoS One, 2008, 3(6): [10] Rebe RS, Bremer MG, Giesbers M, et al. Development of a biosensor microarray towards food screening using imaging surface plasmon resonance [J]. Biosens Bioelectron, 2008, 24(4): [11] Labib M, Hedstrom M, Amin M, et al. A capacitive immunosensor for detection of cholera toxin [J]. Anal Chim Acta, 2009, 634(2): [12] Zhang WW, Chen YC, Luo ZF, et al. Analysis of 17β-estadiol from sewage in coastal marine environment by surface plasmon resonance technique [J]. Chem Res Chin Univ, 2007, 23(4): [13] Khalavka Y, Becker J, Sonnichsen C. Synthesis of rod-shaped gold nanorattles with improved plasmon sensitivity and catalytic activity [J]. J American Chem Soc, 2009, 131(5): [14],. [J]., 2010, 31(6): Zhou HM, Ou HC. Using surface Plasmon resonance technology for rapid detection of sulfa methoxazole in milk [J]. Food Sci, 2010, 31(6): [15]. [J]., 2004, 38(11): Guo WL. Veterinary Inspection of animal tissue surface plasmon resonance biosensor residues utilization [J]. China J Vet Med, 2004, 38(11): 作者简介 ( 责任编辑 : 白洪健 ) 孙明君, 助理工程师, 主要研究方向为进出口食品农产品质量与安全检验 sun_mingjun@hotmail.com

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较