A new strategy for the analysis of tetracycline residues in foodstuffs by a surface plasmon resonance biosensor

A new strategy was developed for the establishment of an indirect tetracycline assay using surface plasmon resonance (SPR). The principle for the assay is based on the most important resistance mechanism against tetracycline in gram-negative bacteria. Tetracyclines release the 46.6 kDa Tet repressor protein (TetR) from the tet operator (tetO), a biotinylated short double strand DNA sequence bound to a streptavidin biosensor chip. Tetracyclines present in a sample solution bind to the repressor protein, inducing a conformational change accompanied with a reduction of the affinity constant of TetR to tetO. We were able to quantify tetracycline residues in spiked raw milk samples in concentrations corresponding to the maximum residue limit (MRL) set by the European Union. Honey samples could also be analysed but with lower sensitivity. The assay detects seven of the most commonly used tetracyclines in veterinary medicine. Nine antibiotics of five other antibiotic classes were tested and no interference with the SPR assay was found. Thus, the described principle forms the basis for screening assays to routinely test for tetracycline residues in foodstuffs.     

Comparison of a fluoroquinolone surface plasmon resonance biosensor screening assay with established methods

The performance of a previously developed immunochemical biosensor screening method for fluoroquinolone (FQ) antibiotics in poultry muscle, fish and egg was compared with established methods. Blank sample material of the target matrices was individually spiked with the FQs at half maximum residue levels. Homogeneity of the test materials was confirmed by liquid chromatography–mass spectrometry (LC–MS/MS). Identical sets of spiked samples as well as incurred samples from a previous feeding experiment were sent to three independent laboratories and analysed by LC–MS/MS, a microbiological assay and the new biosensor assay. The new method correctly identified all contaminated samples and demonstrated advantages in sensitivity and analysis time compared to the microbiological screening assay.     

表面等离子共振法传感器快速检测农药残留的研究进展

为确保农产品质量安全, 发展高灵敏检测技术对农药残留进行检测尤为重要。本文简要介绍了表面等离子共振(surface plasmon resonance, SPR)传感器的原理和增强SPR检测灵敏度的方法, 重点综述了国内外应用SPR传感器检测农药残留的研究现状, 分析了SPR检测农药残留的优势和发展趋势。     

莱克多巴胺ELISA检测假阳性原因分析

目的对酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测莱克多巴胺假阳性结果产生的原因、影响因素和质量控制方法进行分析研究。方法应用经验证可靠的ELISA方法对进口猪肉产品进行莱克多巴胺兽药残留初筛检测,对阳性可疑样品采用液相色谱.串联质谱分析方法进行确证检测,并对两种方法的检测结果进行对比分析。结果样品在腐败变质后产生含有可以产生非特异性显色内源性辣根过氧化物酶的细菌,从而造成假阳性,干扰检测结果。结论莱克多巴胺检测中造成酶联免疫吸附(ELISA)方法假阳性结果的主要原因包括莱克多巴胺的代谢速度快,ELISA检测所用抗体与莱克多巴胺的交叉反应性高,以及样品因素的影响。     

SPR技术检测奶粉中生物素

目的利用表面等离子共振(surface plasmon resonance,SPR)技术,建立快速定量测定牛奶中生物素的方法。方法将生物素共价偶联到表面等离子共振芯片CM5表面,并对竞争结合的生物素结合蛋白的结合浓度及芯片的再生条件进行优化,检测芯片的稳定性。在无抗生素牛奶中添加系列质量浓度的生物素,利用免疫竞争抑制原理构建标准曲线,并对市售10个奶粉样品进行检测。结果制备的芯片稳定,50个循环相对标准偏差(relative standarddeviation,RSD)小于10%。日间批内同一样品差异为8.75%,该方法的检测限为0.1μg/100g,回收率为80.4%~91.2%。10个牛奶产品中生物素含量全部在固定的允许范围内。所建立的方法可以在4 h内完成样品的前处理和检测。结论该方法是一种简便、快捷的定量检测方法。     

GC-MS分析法在猪肉氯霉素残留检测中的应用

建立了猪肉中残留氯霉素(CAP)的GC-MS分析方法。样品中CAP用乙酸乙酯提取,脂肪用正己烷去除,BondElut-C18柱纯化样品,BSTFA-TMCS衍生后用NCI源选择m/z为466的特征离子为目标离子,在SIM模式下进行GC-MS分析。被检测CAP质量浓度在0.08~100μg/kg内,方法的线性关系良好,相关系数为0.998。方法的最低检测限达到0.08μg/kg。用此方法对市场上销售的猪肉样品进行测定,结果显示:只有一份样品CAP残留量为(0.236±0.007)μg/kg,另4份猪肉样品中未检出CAP残留。应用本方法测定新鲜猪肉中CAP残留,基本上满足了灵敏、快速、准确可靠的分析方法要求。     

Determination of residual ractopamine concentrations by enzyme immunoassay in treated pig's tissues on days after withdrawal

The objective of this study was to measure residual ractopamine concentrations in tissues of pigs as experimental animals after treatment with dietary ractopamine for 28 consecutive days. Ractopamine was administered orally to the experimental group (n = 9) in a dose of 0.1 mg/kg body mass per day, whereas control animals (n = 3) were left untreated. Treated pigs (60 kg) were sacrificed on days 1, 3 and 8 of treatment discontinuation and residues were determined in kidney, liver, muscle, brain and heart tissues using previously validated enzyme-linked immunosorbent assay (ELISA) as a quantitative screening method. Validation showed good mean recoveries (approx. 70-90%) with acceptable inter- and intra-day relative standard deviations (RSD < 13%), demonstrating the method efficiency in determination of ractopamine tissue concentrations. The highest ractopamine concentration on day 1 (24 h) after the last exposure was recorded in the kidney (12.49 ± 7.96 ng/g), followed by the liver (7.21 ± 2.73 ng/g), heart (1.26 ± 0.12 ng/g) and brain (0.63 ± 0.05 ng/g); at this time of withdrawal, residues were not detected in the muscle. Ractopamine was depleted rapidly from all study tissues, with mostly no detectable residues on day 8 of withdrawal.     

纺织品中双酚A的表面等离子体共振辅助检测方法

鉴于双酚Ａ（BPA）能够干扰生物的内分泌功能,引起各种生殖异常,威胁着婴幼儿的健康,甚至有致癌的危险等生物危害性,采用表面等离子体共振技术（SPR）对BPA 进行定性定量检测。从传感芯片的修饰、BPA-BSA溶液最佳质量浓度的确定、不同质量浓度BPA的响应情况以及纺织品中BPA的萃取等4个方面出发,探索了SPR技术检测纺织品中BPA的实用性。结果表明,BPA的SPR检测法具有检测速度快、对样品要求低、检测针对性强、检测灵敏度高等优点,适合检测领域的广泛应用,可应用于纺织品中双酚A含量的测定。     

表面等离子共振技术快速检测食品中的 玉米赤霉烯酮毒素

目的建立基于表面等离子共振(surface plasmon resonance,SPR)技术快速检测食品中玉米赤霉烯酮毒素(zearalenone,ZEN)的方法。方法采用表面自组装技术(self-assembled monolayer,SAM)在金膜的表面修饰羧基基团,将ZEN抗原与牛血清白蛋白(albumin from bovine serum,BSA)偶联物(ZEN-BSA)通过共价键固定在芯片的表面,采用竞争法检测样品中的玉米赤霉烯酮毒素。结果该方法的检测限为8.2 ng/m L,ZEN单克隆抗体与呕吐毒素、黄曲霉毒素B_1、赭曲霉毒素、伏马毒素等没有交叉反应,与α-玉米赤霉烯醇和β-玉米赤霉烯醇交叉反应率分别为15.3%和11.5%。结论本方法具有简便、快速和高灵敏度等优势,在食品中真菌毒素的快速检测方面具有潜在的应用价值。     

Direct detection of E. Coli O157:H7 in selected food systems by a surface plasmon resonance biosensor

The Spreeta^TM, surface plasmon resonance (SPR)-based biosensor, was used to detect Escherichia coli O157:H7 spiked in milk, apple juice and ground beef extract using specific antibodies. In the Spreeta^TM biosensor light from an LED is reflected off a gold surface, and the angle and intensity corresponding to the SPR minimum is measured and represented as a refractive index (RI) change corresponding to the antigen-antibody coupling at the sensor surface. Milk, apple juice, and ground beef patties spiked with E. coli O157:H7, at varying concentrations, were injected on the sensor surface immobilized with antibodies against the pathogen at a rate of 1 ml/min for a total of 2 min. The change in RI due to the binding of O157:H7 corresponding to each concentration was computed as an average of three replications over a 2 min interaction period. Assays were conducted at near real-time and results obtained after about 30 min of sample injection. Sensitivity of the E. coli O157:H7 assay was 10²-10³ colony forming unit (CFU)/ml. The biosensor assay was also specific to E. coli O157:H7 as other organisms (E. coli K12 and Shigella) did not produce any appreciable change in the sensogram. Further experiments are needed to establish well-defined methods for detecting other food-borne pathogens in more complex and specific food matrices.     

Levels and distribution of PBDEs and PFASs in pork from different European countries

ABSTRACT

Meat and meat products are included in a great number of human diets. However, the great consumption of meat needs to be controlled for the presence of traces of contaminants. The European Commission has not stated maximum limits for some environmental pollutants such as the perfluoroalkyl substances (PFASs) and polybrominated diphenyl ether (PBDE); the European Food Safety Authority (EFSA) Scientific Panel has recommended that more occurrence data for PFASs in food should be collected to improve the accuracy of future exposure calculations. Therefore, the distribution of PFASs and PBDEs trace contaminants from eight EU Member States were investigated through liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and Gas Chromatography-Mass Spectrometry (GC-MS/MS). No PFASs were detected, except perfluorooctanoic acid, in only one Austrian sample at the concentration of 0.531 ng g^?1. PBDEs were detected in 3 out of 77 samples: one from Germany showed the presence of all congeners analysed in the concentration range 0.53–0.77 ng g^?1, the others, from Netherland and Italy, respectively contained PBDE 153 (0.53 ng g^?1) and PBDE 100 (0.62 ng g^?1). The results show that the analysed samples do not pose a risk for human beings in regard to PFASs and PBDEs. Further studies are needed to keep monitoring their presence in foodstuff, as it has been suggested by European Commission.     

酶联免疫法与液相色谱-串联质谱法检测猪肉中沙丁胺醇

运用酶联免疫法（ELISA）与液相色谱-串联质谱法（LC-MS/MS）对猪肉中沙丁胺醇残留进行测定,并比较了二者在灵敏度、准确性和重现性等方面的差异。结果显示,ELISA法和LC-MS/MS法在猪肉样品中的检测限可达到0.5 μg/kg和0.25 μg/kg,分别向猪肉样品中添加3个质量浓度水平（1.0 μg/kg、2.0 μg/kg、4.0 μg/kg）的沙丁胺醇时,回收率分别为83.7%～90.9%和86.6%～93.5%,变异系数（CV）分别为5.4%～10.3%和6.0%～8.3%。用酶联免疫法实际样品进行检测,筛选出2个阳性样品,经液相色谱-串联质谱法确证亦为阳性,测定结果一致。研究表明,酶联免疫法重复性较好、准确度较高,适用于进行猪肉样品中沙丁胺醇的快速筛选,液相色谱-串联质谱法适用于阳性样品的确证和精确定量。     

Determination of concentration and binding affinity of antibody fragments by use of surface plasmon resonance

An assay method using a surface plasmon resonance (SPR) biosensor has been developed that allows quantitative measurement of the specific antibody concentration in crude materials. By injecting non-labeled antibody samples onto a biosensor surface on which antigen was immobilized at high densities, the concentration of active antibodies can be accurately measured. To clarify applicability of this method to pharmacokinetic studies, the concentration of active antibodies in mouse plasma was measured for 4 h after injection of antibodies in mice. Although this period of measurement might be insufficient for determining the pharmacokinetics of blood pool clearance, this method has some advantages over conventional methods in measurement of single-chain antibody fragment (scFv) concentrations. Using the SPR biosensor, scFv and antibodies without epitope tag peptides were easily detected in real time, requiring as little as 20 mul of blood sample. Moreover, from the apparent dissociation rate in the dissociation phase of the sensorgrams, we could identify whether the antibody fragments existed as bivalent or monovalent in animal blood. We also evaluated the antigen binding activity of the scFvs against human CD47 and found scFvs had slightly weak affinity to their antigen (K(D), about 10 nM) compared with F(ab')2 and Fab' fragments (K(D), about 3-4 nM). This assay method promises to be a convenient tool for quality control, screening, and simple pharmacokinetic analysis of antibody fragments and other recombinant proteins not having epitope tags.     

HPLC-MS/MS测定猪肉和猪肝中氯霉素残留量的研究

建立高效液相色谱-串联质谱法(HPLC-MS/MS)检测猪肉和猪肝中氯霉素药物残留的分析方法。样品经4%的氯化钠溶液和乙腈提取并沉淀蛋白质,正己烷脱脂净化,乙酸乙酯液液萃取,再浓缩定容,上HPLC-MS/MS仪器,用Syncronis C18柱分离,以乙腈-水(40∶60)作为流动相,采用电喷雾离子源(ESI),负离子模式,多反应监测(MRM)方式检测,用氯霉素-D5作内标物来定量。在该试验条件下,氯霉素在1～15ng/mL浓度呈良好的线性关系,r=0.999 2,最低检出浓度为0.1μg/kg,样品的加样回收率在90.0%～110.0%,重复性好RSD<3.0%。该方法能对猪肉和猪肝中氯霉素残留进行定性及定量分析,方法精密度较好、准确度较高、选择性好,结果满意。     

利用捕获法建立筛选鸭坦布苏病毒适配单抗的 表面离子共振方法

目的以表面离子共振(surface plasmon resonance,SPR)系统为技术平台,利用捕获法建立检测鸭坦布苏病毒适配单抗的SPR方法,并对抗坦布苏病毒单克隆抗体的亲和力及抗原抗体结合的动力学进行研究。方法将鼠抗抗体偶联于CM5芯片上,采用Kinetics程序将HBS缓冲液梯度稀释的不同浓度的单克隆抗体进行捕获反应,拟合解离曲线计算不同单抗的K_D值并确立检测灵敏度低限并对其他不同亚型及5种常见禽类病毒抗体进行特异性检测试验。结果测得3株单抗的K_D值分别为1.50×10~(-13)、1.16×10~(-11)、7.85×10~(-12),建立的表面离子共振方法在检测其他禽源病毒时无阳性响应信号,坦布苏重组E蛋白的检测极限达0.625nmol/L。结论该方法可用于快速筛选检测坦布苏病毒的稳定适配单抗,并可有效获知其亲和力数据,为大规模推广建立免疫筛查方法奠定基础。     

高效液相色谱-串联质谱法测定猪肉中氯霉素和五种磺胺类药物的残留量

目的:建立超高效液相色谱-串联质谱（UPLC-MS/MS）同时快速测定猪肉中氯霉素、磺胺甲嘧啶、磺胺二甲嘧啶、磺胺噻唑、甲氧苄啶和磺胺嘧啶残留量的分析方法。方法:猪肉加入乙酸乙酯后匀浆,转移有机层N₂吹干,残渣用10%乙腈水溶液溶解,正己烷除脂,采用BEH C₁8（2.1 mm×50 mm,1.7μm）色谱柱分离,以乙腈和水为流动相进行梯度洗脱,采用电喷雾离子源（ESI）,以多反应监测（MRM）方式检测。结果:在该实验条件下,氯霉素和5种磺胺类药物分别在9.6³84 ng/kg和0.51²0.8μg/kg浓度范围呈良好的线性关系,R²均大于0.99;氯霉素检出限为3.2 ng/kg,定量限为9.6 ng/kg,5种磺胺类药物检出限为0.158⁰.173μg/kg,定量限为0.475⁰.52μg/kg;方法的平均回收率范围为70.84%¹03.99%,相对标准偏差为1.98%⁸.01%。结论:此方法具有简便快捷、灵敏度高、定量准确等特点。      

微波辅助萃取-液相色谱-串联质谱法检测猪肉中四种氟喹诺酮类药物残留

建立了微波辅助萃取-液相色谱-串联质谱法检测猪肉中氧氟沙星、诺氟沙星、环丙沙星、恩诺沙星四种氟喹诺酮类药物残留的方法。样品经微波辅助萃取,正己烷脱脂,无水硫酸钠除水,浓缩提取物至干,用1.0mL0.1%甲酸溶液溶解后上机检测。采用Hypersil GOLD-1.9μm,50 mm×2.1 mm(i.d)色谱柱分离,在多反应监测模式下检测,外标法定量。结果表明:四种氟喹诺酮类药物在浓度10～200μg/L范围内线性关系良好,相关系数r=0.9956～0.9991,定量限为10μg/kg。在不同添加水平下,其平均回收率为88%～101%,变异系数为1.4%～4.1%。     

Plant pectin methylesterase and its inhibitor from kiwi fruit: Interaction analysis by surface plasmon resonance

Two surface plasmon resonance (SPR)-based interaction analysis methods were successfully implemented to explore the binding between plant PME and kiwi PMEI. In a first method, plant PMEs were immobilised on a chip surface via amine coupling. This experimental setup allowed studying the effect of pH and ionic strength on the PME–PMEI interaction kinetics. Strong binding was obtained at pH < 7 and at low salt concentrations, whereas both pH ? 8 and [NaCl] of ca. 1.0 M effectively caused dissociation. In a second method, kiwi PMEI was immobilised on a chip surface to which streptavidin had been covalently attached. Hereto, PMEI was biotinylated by means of a NHS-biotin reagent. With this immobilisation strategy, the effect of (partial) thermal or high pressure-induced denaturation of PME on its affinity towards PMEI was investigated. A notable degree of enzyme inactivation was required before interaction characteristics were significantly altered. Any incomplete inactivation of PME resulted in binding to the PMEI surface.     

液相色谱—质谱/质谱法测定猪肉中4种磺胺类药物残留量的不确定度评定

目的：对采用液相色谱—质谱/质谱法测定猪肉中磺胺间甲氧嘧啶、磺胺二甲氧嘧啶、磺胺甲噁唑和磺胺二甲嘧啶残留量的不确定度进行评定。方法：分析猪肉中磺胺类药物残留量测定过程中的各种影响因素,包括称量、标准溶液的配制、样品提取净化、仪器测量、回收率等进行分析评定。结果：该方法测定磺胺间甲氧嘧啶、磺胺二甲氧嘧啶、磺胺甲噁唑和磺胺二甲嘧啶残留量的扩展不确定度分别为9.4,18.1,12.0,15.6 μg/kg。结论：试验的不确定度主要由测量重复性、标准溶液的配制和标准曲线拟合引入。     

SPR技术对奶粉中维生素B12检测方法的建立

目的 利用表面等离子共振(surface plasmon resonance, SPR)技术, 建立快速定量测定牛奶中维生素B12的方法。方法 将钴胺素共价偶联到表面等离子共振芯片CM5表面, 并对竞争结合的维生素B12结合蛋白的结合浓度及芯片的再生条件进行优化, 检测芯片的稳定性。在无抗生素牛奶中添加系列质量浓度的维生素B12, 利用免疫竞争抑制原理构建标准曲线, 并对市售10个奶粉样品进行检测。结果 制备的芯片稳定, 50个循环相对标准偏差(relative standard deviation, RSD)小于10%。日间批内同一样品差异为0.54%, 该方法的检测限为0.006 μg/100 g, 回收率为92.1%~104.1%。测得的10个牛奶产品中维生素B12含量与对应商品标签值全部在标准规定的范围内。结论 所建立的方法可以在6 h内完成样品的前处理和检测, 是一种简便、快捷的定量检测方法。