胶体金免疫渗滤法检测淡水鱼及牛乳中氯霉素残留

制备了平均粒径20 nm的胶体金,用以标记抗氯霉素多克隆抗体;将氯霉素半抗原与卵清蛋白的偶联物包被在硝酸纤维素膜上作为检测带,羊抗兔二抗作为质控带,依据免疫竞争法原理,建立通过免疫胶体金渗滤式试纸条检测六种淡水鱼及牛乳中氯霉素残留的方法.样品前处理方法简便,方法灵敏度为1 μg/L,只需10～15 min即可目测判断结果,可以作为淡水鱼及牛乳中氯霉素残留现场大量筛选的有效手段.     

胶体金免疫层析法检测食用肉中的氨基脲

制备特异性胶体金免疫层析试纸条,检测肉类食品中氨基脲的残留。用活性酯法将氨基脲(SEM)衍生物CPSEM交联到牛血清白蛋白(BSA)上制备完全抗原CPSEM-BSA,将硫酸铵沉淀法纯化的抗CPSEM单克隆抗体标记到柠檬酸三钠还原法制备的胶体金,通过优化C/T线及金标抗体工作浓度,制备检测氨基脲的胶体金免疫层析试纸条,并对试纸条的灵敏度、特异性、准确性和稳定性进行测定。制备的试纸条检测5min后即可用肉眼观察到结果;对SEM的最低检测限达0.72ng/ml;除与呋喃西林有弱交叉反应外,与其他同类物均无交叉反应;该试纸条对猪肉中添加的SEM检测结果与酶联免疫吸附测定法(ELISA)检测结果呈现了很好的相关性;试纸条在室温下保存7周后不会失效。通过制备针对SEM的试纸条所建立的胶体金免疫层析法(ICA),快速简便、灵敏度高、特异性强、准确性和稳定性好,基本能达到商用检测要求。     

胶体金免疫层析法和气相色谱法测定动物产品中氯霉素残留

对动物产品中氯霉素残留检测的胶体金免疫层析法和气相色谱法进行比较研究。向样品中分别添加0.3、0.5、1.5μg/kg三个添加水平的氯霉素标准品,用两种方法进行检测。胶体金免疫层析法对0.5μg/kg以上均能检出,气相色谱法平均回收率分别为78.1%、85.3%和88.3%,变异系数为5.3%～9.0%,最低检测限为0.3μg/kg。对108个样品用胶体金免疫层析法筛选出3个阳性样品,经气相色谱法验证皆为阳性,105个阴性样品经气相色谱法检测皆为阴性,未发现假阳性和假阴性。结果表明：胶体金免疫层析法具有快速,简便,特异,灵敏度高的特点,适合基层检验机构对动物产品中氯霉素残留现场快速筛选;气相色谱法灵敏度高,适用于阳性样品的精确定量。     

高效液相色谱-电喷雾离子阱质谱测定乳品中氯霉素、甲砜霉素和氟甲砜霉素残留的研究

本实验建立了乳制品中氯霉素、甲砜霉素和氟甲砜霉素残留的高效液相色谱-电喷雾离子阱质谱联用测定方法.该方法采用多反应监测负离子模式,可一次对氯霉素、甲砜霉素和氟甲砜霉素进行定性和定量.该方法的检出限0.3～1.0μg/L,测定低限牛奶0.3～1.1μg/L,乳粉为3.0～11.0μg/kg,线性范围1.0～30.0.g/L,加标回收率77.9%～96.6%,相对标准偏差为7.7%～14.0%.该法具有样品预处理简单,灵敏度高,分析时间短等优点.     

养殖对虾中氯霉素残留的放射免疫分析

采用放射免疫分析法测定了湛江市水产市场3份新鲜养殖对虾及2份出口养殖对虾氯霉素残留量。结果表明,本方法的检测限量为0.15×10-12,3份新鲜养殖对虾体内氯霉素残留量均大于0.15×10-12,2份出口养殖对虾氯霉素残留量均小于0.15×10-12。提示放射免疫分析法可作为水产品中氯霉素残留量的有效检测手段。     

胶体金免疫层析法快速检测食品中金霉素残留

研究食品中金霉素残留的胶体金免疫层析法的快速检测方法。采用戊二醛交联法制备金霉素-BSA 免疫抗原,按照常规的免疫程序免疫新西兰大白兔制备抗金霉素多克隆抗体。纯化后的抗体用ELISA 法检测其特异性和效价。采用柠檬酸三钠还原法制备颗粒大小为15nm 的胶体金,并制备抗体- 胶体金复合物,以组装检测试纸。通过可见的颜色深浅,检测样品中金霉素的残留量。结果表明：试纸条法的金霉素检测限为0.7μg/mL,在四环素质量浓度大于1.0μg/mL 时,与金霉素存在交叉反应性,其结果与高效液相色谱法测定的结果一致。本方法检测时间仅需5min,适用于食品中金霉素残留的快速检测。     

UPLC-MS-MS法测定饲料、原料乳及乳制品中3 种氯霉素类药物残留

建立超高效液相色谱-质谱联用法检测饲料、牛乳及广义乳制品中氯霉素类药物残留的通用检测方法。研究适用于检测饲料、原料乳、液体乳、发酵乳、含乳饮料、乳粉、奶片、冷冻饮品中氯霉素类药物的前处理方法以及适用的超高效液相色谱及质谱条件。样品经低温冷冻沉淀蛋白质后,乙腈提取,饱和氯化钠使乙腈、水分层,取上清液氮气吹干定容后,正己烷除脂,乙腈、水梯度洗脱,电喷雾负离子多反应监测模式进行检测。氯霉素、甲砜霉素、氟甲砜霉素定量限0.1 μg/kg。氯霉素类药物添加范围0.1～2.5 μg/kg时,基质效应为57.9%～139.2%,回收率为64.3%～120.2%,相对标准偏差为2.1%～10.0%。该方法具有简单方便、检测周期短、适用于更广泛样品检测的特点。     

牛奶中氯霉素的纳米纤维固相萃取快速前处理方法

将纳米纤维固相萃取柱应用于氯霉素检测的前处理,建立牛奶中氯霉素检测的快速前处理方法。在样液中加入高氯酸沉淀蛋白,离心后取上清液调节pH值至中性后直接过柱,100μL甲醇洗脱得洗脱液,取20μL注入高效液相色谱仪检测。流动相为甲醇:Na2HPO4-柠檬酸缓冲液(1:2,V/V,pH3.8),流速1.0mL/min,紫外检测波长277nm。优化高氯酸体积分数、上清液的pH值、盐质量浓度、洗脱剂用量。在最优条件下,方法定量线性范围为0.05～10μg/mL。方法回收率为92.3%～105.3%,日内RSD为2.5%～8.9%,日间RSD为11.7%。与C18固相萃取柱相比,本方法操作简便,效率高,且精密度高,回收率好,是一种可靠并且相对高效环保的方法,可应用于牛奶中氯霉素的检测。     

Advantage of Eu3+‐Doped Polystyrene Microspheres Compared with Colloidal Gold Used in Immunochromatographic Assays for the Detection of Melamine in Milk

Colloidal gold and Eu³⁺‐doped fluorescent microspheres were applied as labels to develop the immunochromatographic strips for detecting melamine in milk. Under the optimized condition, the visual detection limit of colloidal gold‐immunochromatographic test strip (ICTS) was 150 μg/L of melamine in phosphate‐buffered saline (PBS), although the visual detection limit of fluorescent nanoparticles (FN)‐ICTS was 75 μg/L in PBS. As thermal acceleration test, FN‐ICTS could be stored at 37 °C for at least 11 d before sample testing, but the color of the lines on colloidal gold‐ICTS faded away after 7‐d storage. The visual result of FN‐ICTS was more stable than that of colloidal gold‐ICTS, and the fluorescence intensity of the line on FN‐ICTS could be maintained up to 30 d at 22 °C after sample testing. Once the immunochromatographic strips were used to detect melamine in milk, no negative effect of milk components on the performance of FN‐ICTS was identified, whereas the performance of colloidal gold‐ICTS was significantly influenced by milk matrix.     

胶体金免疫层析法联检食品中5 种典型沙门氏菌模型的建立和优化

采用胶体金标记抗沙门氏菌单克隆抗体,将沙门氏菌单克隆抗体和驴抗鼠抗体（二抗）喷涂于硝酸纤维膜上分别作为检测线和质控线,研制能联检5 种典型沙门氏菌（甲型副伤寒沙门氏菌、鼠伤寒沙门氏菌、猪霍乱沙门氏菌、肠炎沙门氏菌、鸭沙门氏菌）的胶体金免疫层析试纸条。结果表明,该试纸条能达到同时检测5 种沙门氏菌的目的,其中甲型副伤寒沙门氏菌的检测灵敏度最高,为105 CFU/mL;鼠伤寒沙门氏菌、猪霍乱沙门氏菌、肠炎沙门氏菌和鸭沙门氏菌的检测灵敏度为106 CFU/mL。沙门氏菌加入量为10～100 CFU时,增菌16～20 h,该试纸条能够检测出牛奶、鸡蛋样本中的5 种沙门氏菌。本实验研制的试纸条能快速高效地联检食品中5 种典型的沙门氏菌,特异性高、灵敏度好,在对多种沙门氏菌进行联检方面有很重要的应用价值。     

免疫亲和柱-高效液相色谱法测定牛奶中氯霉素和玉米赤霉醇及其类似物

建立免疫亲和柱同时净化-高效液相色谱法测定牛奶中氯霉素和玉米赤霉醇及其类似物（α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮和玉米赤霉烯酮）残留量的方法。样品经免疫亲和柱净化与富集后,用高效液相色谱-紫外检测器检测。采用Cloversil-C18反相柱分离,流动相为乙腈-甲醇-水溶液,检测波长为265 nm。结果表明,牛奶中添加氯霉素和玉米赤霉醇及其类似物的回收率在74%～101%,且相对标准偏差均小于7%。氯霉素的检出限为0.02 μg/L,玉米赤霉醇及其类似物的检出限分别为α-玉米赤霉醇0.03 μg/L、β-玉米赤霉醇0.03 μg/L、α-玉米赤霉烯醇0.03 μg/L、β-玉米赤霉烯醇0.03 μg/L、玉米赤霉酮0.04 μg/L和玉米赤霉烯酮0.05 μg/L。该方法灵敏度高、重复性好,提高了检测速率,可满足牛奶样品中痕量氯霉素和玉米赤霉醇及其类似物残留的测定。     

氯霉素毛细管电泳免疫分析方法研究

针对目前食品安全领域中氯霉素的检测,建立一种快速灵敏的氯霉素毛细管电泳免疫分析方法。该方法对氯霉素检测的线性范围和最低检测限分别为0.008～5μg/L 和 0.0016μg/L。与相应固相酶联免疫分析ELISA相比,对氯霉素的检测灵敏提高了约20 倍且有效缩短了分析时间。建立的氯霉素毛细管电泳免疫分析方法对动物源性食物(牛奶、鸡肉和鱼肉)进行分析,得到氯霉素在实际样品中的检测限为0.035μg/kg。     

Detection and Quantification of Chloramphenicol in Milk and Honey Using Molecularly Imprinted Polymers: Canadian Penny‐Based SERS Nano‐Biosensor

We integrated molecularly imprinted polymers with surface‐enhanced Raman spectroscopy (MIPs‐SERS) to develop an innovative nano‐biosensor for the determination of chloramphenicol (CAP) in milk and honey products. Template molecule (CAP), functional monomer (acrylamide), cross‐linking agent (ethylene glycol dimethacrylate), initiator (2,2’‐azobis(isobutyronitrile)), and porogen (methanol) were employed to form MIPs via “dummy” precipitation polymerization. Static and kinetic studies validated the specific selectivity of MIPs toward CAP over nonimprinted polymers (imprinting factor >4). Canadian penny‐based silver nano‐structure was synthesized as SERS‐active substrate for determination of CAP in food matrices. Collected spectra were processed by principal component analysis to differentiate various concentrations of CAP in foods. Partial least squares regression models showed good prediction values (R > 0.9) of actual spiked contents (0, 0.1, 0.5, 1, 5 ppm) of CAP in milk and honey. This developed nano‐biosensor is low cost, requires little sample pretreatment, and can provide reliable detection of trace level of chemical hazards in food systems within a total of 15 min.     

液相色谱- 串联质谱法测定八种食品中甲霜灵

为研究建立动物源性食品(鸽肉、鸭肾)、植物源性食品(蔬菜汁、苹果、橙子、葡萄、柠檬、奇异果)中农药甲霜灵残留的液相色谱- 串联质谱测定方法。采用外标法(添加水平分别为0.005、0.010、0.020mg/kg)测定回收率,其中：动物源性食品中平均回收率为95.9%～102.0%,相对标准偏差为2.13%～12.7%(n=5),方法检出限为0.002mg/kg;植物源性食品中平均回收率为116.4%～141.0%,相对标准偏差为0.37%～4.09%(n=5),方法检出限为0.001mg/kg。该方法的测定结果满足农残检测要求。     

毒死蜱残留检测间接竞争ELISA 试剂盒的研制

在多克隆抗体的基础上研制一种适用于检测样品中毒死蜱残留的间接竞争酶联免疫吸附分析(ELISA)试剂盒,并对试剂盒的灵敏度、特异性、精密度、准确度和有效期进行测定。结果表明,研制的试剂盒最低检测限为0.5μg/L,线性检测范围为1.8～1000μg/L,供试样品检测结果的批内、批间变异系数均低于8%,回收率均高于85%。试剂盒在4℃或－ 20℃条件下至少可保存6 个月。     

铬离子单克隆抗体的制备及食品总铬含量检测胶体金免疫层析试纸条的研制

目的:制备三价铬离子螯合物单克隆抗体(Cr³⁺-EDTA m Ab),研制胶体金免疫层析试纸条（Cr-Strip）。方法:用异硫氰酯法合成Cr³⁺-i EDTA-BSA,UV和ICP-AES进行鉴定;细胞融合技术筛选Cr³⁺-EDTA m Ab细胞株,体内诱生腹水法制备Cr³⁺-EDTA m Ab,并对其特性进行分析;应用Cr³⁺-EDTA m Ab研制Cr-Strip,并测定其性能。结果:筛选出3株杂交瘤,最好的是2A11G5,Ka为2.69×109L/mol,对Cr³⁺-EDTA的IC50为9.84μg/L,与其他重金属离子无交叉反应;Cr-Strip的检测时间为10 min,检测限为5μg/L,其检测结果与ICP-AES符合率为100%。结论:制备出了高质量的Cr³⁺-EDTA m Ab,研制出更为灵敏的Cr-Strip。      

悬液芯片与常规酶联免疫吸附法检测庆大霉素残留的比较

目的:建立庆大霉素兽药残留的悬液芯片直接竞争检测法,并同时与常规酶联免疫法进行比较。方法:将庆大霉素单克隆抗体与表面带有羧基的聚苯乙烯微球通过酰胺键偶联形成检测探针,利用碳二亚胺法将庆大霉素与藻红蛋白偶联后形成酶标物,采用直接竞争法,在液相体系中庆大霉素与藻红蛋白酶标物竞争性与单克隆抗体相结合,反应后通过荧光信号获得庆大霉素竞争曲线。同时进行庆大霉素直接竞争酶联免疫法曲线的测定。对两种方法在检出限、检测区间以及加样回收率方面进行比较。结果表明:悬液芯片IC10、IC50分别为0.1、2.8ng/mL,检测区间为0.1～100ng/mL,酶联免疫竞争法IC10、IC50为0.27、1.95ng/mL,检测区间为0.1～10ng/mL。两种方法在牛奶、鸡肉、鸡肝、猪肉和猪肝中的回收率均在80%～120%之间。另外,悬液芯片法与其他氨基糖苷类抗生素基本无交叉反应,特异性良好。结论:悬液芯片系统具有操作简单,灵敏度高以及特异性良好等特点,与传统经典的农兽药残留酶联免疫检测法相比,具有一定的优势,从而为农兽药残留检测提供了新的检测技术。     

ABTS法体外测定果蔬类总抗氧化能力的研究进展

ABTS法作为一种用于体外测定物质总抗氧化能力的新方法,在国内报道甚少,文中就该方法在国外的发展、在果蔬中的应用以及存在的问题进行了概述和分析,为该方法在国内的应用提供理论依据。     

Intra- and inter-laboratory validation of a dipstick immunoassay for the detection of tropane alkaloids hyoscyamine and scopolamine in animal feed

Tropane alkaloids (TAs) are toxic secondary metabolites produced by plants of, inter alia, the genera Datura (thorn apple) and Atropa (deadly nightshade). The most relevant TAs are (–)-L-hyoscyamine and (–)-L-scopolamine, which act as antagonists of acetylcholine muscarinic receptors and can induce a variety of distinct toxic syndromes in mammals (anti-cholinergic poisoning). The European Union has regulated the presence of seeds of Datura sp. in animal feeds, specifying that the content should not exceed 1000 mg kg^–1 (Directive 2002/32/EC). For materials that have not been ground, visual screening methods are often used to comply with these regulations, but these cannot be used for ground materials and compound feeds. Immunological assays, preferably in dipstick format, can be a simple and cost-effective approach to monitor feedstuffs in an HACCP setting in control laboratories. So far no reports have been published on immunoassays that are capable of detecting both hyoscyamine and scopolamine with equal sensitivity and that can be used, preferably in dipstick format, for application as a fast screening tool in feed analysis. This study presents the results obtained for the in-house and inter-laboratory validation of a dipstick immunoassay for the detection of hyoscyamine and scopolamine in animal feed. The target level was set at 800 µg kg^–1 for the sum of both alkaloids. By using a representative set of compound feeds during validation and a robust study design, a reliable impression of the relevant characteristics of the assay could be obtained. The dipstick test displayed similar sensitivity towards the two alkaloids and it could be concluded that the test has a very low probability of producing a false-positive result at blank level or a false-negative result at target level. The assay can be used for monitoring of TAs in feedstuffs, but has also potential as a quick screening tool in food- or feed-related poisonings.