量子点荧光免疫检测牛奶中己烯雌酚、雌二醇

研究牛奶中己烯雌酚、雌二醇的荧光免疫检测技术并对实际样品进行检测.辅助量子点标记技术,结合间接竞争酶联免疫吸附法原理建立检测标准曲线,评价特异性,对实际样品进行检测.己烯雌酚的荧光免疫检测在0.472 ng/mL～66.597 ng/mL内呈线性关系,最低检测限为0.236 ng/mL.雌二醇在0.418 ng/mL～...     

Quantum bead-based fluorescence-linked immunosorbent assay for ultrasensitive detection of aflatoxin M1 in pasteurized milk,yogurt, and milk powder

Herein, we reported a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of aflatoxin M₁ (AFM₁) in pasteurized milk, yogurt, and milk powder using 150-nm quantum dot beads (QB) as the carrier of competing antigen. Large QB were applied to decrease the binding affinity of the competing antigen to antibody and enhance the fluorescent signal intensity. The aflatoxin B₁ molecule was used as the surrogate of AFM₁ to label with BSA on the surface of QB because of its 63% cross reaction to anti-AFM₁ mAb. The binding affinity of the competing antigen to mAb was tuned by changing the labeled molar ratios of aflatoxin B₁ to BSA. Through combining the advantages of QB as the carrier of the competing antigen, including low binding affinity to mAb and highly fluorescent signal output, the proposed dcFLISA exhibited an ultrahigh sensitivity for AFM₁ detection, with a half-maximal inhibitory concentration of 3.15 pg/mL in 0.01 M phosphate-buffered saline solution (pH 7.4), which is substantially lower than that of the traditional horseradish peroxidase-based ELISA. The proposed method also exhibited very low detection limitations of 0.5, 0.6, and 0.72 pg/mL for real pasteurized milk, yogurt, and milk powder, respectively. These values are considerably below the maximum permissible level of the European Commission standard for AFM₁ in dairy products. In summary, the proposed dcFLISA offers a novel strategy with an ultrahigh sensitivity for the routine monitoring of AFM₁ in various dairy products.     

膜种类及前处理方式对原奶中葡萄球菌肠毒素层析试纸条检测的影响

对用于原料奶中葡萄球菌A、B、C型肠毒素现场快速检测的免疫层析试纸条进行了初步研究。研究内容主要包括层析膜和原奶预处理方法的选择及优化,通过实验,最终选择了型号为HF09002的层析膜,采用了2倍稀释的样品预处理方法,研制所得的3种免疫层析试纸条分别检测原奶中SEA的灵敏度为10 ng/mL,SEB的灵敏度为4 ng/mL,SEC的灵敏度为8 ng/mL,与3 MTM TecraTM微孔板法检测结果相比无显著性差异。     

基于酶联适配体快速检测食品中葡萄球菌肠毒素A

该研究基于双适配体夹心原理,建立了一种酶联适配体检测葡萄球菌肠毒素A(Staphylococcal enterotoxin A,SEA)的新方法,并对适配体浓度、链霉亲和素-辣根过氧化物酶浓度、反应体系、封闭条件、反应时间等条件进行了优化.结果显示,在适配体浓度40 nmol/L、牛血清白蛋白(Bovine serum...     

共价有机聚合物固相微萃取结合高效液相色谱-串联质谱法检测牛奶中黄曲霉毒素

目的 建立基于共价有机聚合物固相微萃取前处理结合高效液相色谱-串联质谱法(highperformance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS)快速、高灵敏检测牛奶中6种黄曲霉毒素残留的分析方法。方法 以1,3,5-三(4-氨苯基)苯[1,3,5-tri(4-aminophenyl)benzene,TAPB]和1,3,5-三甲酰基间苯三酚(1,3,5-triformyl-phloroglucinol,Tp)为单体制备了共价有机聚合物材料并固定在木签表面,用于黄曲霉毒素的固相微萃取前处理。在萃取时间为20 min、洗脱溶剂为乙腈、洗脱时间为6 min条件下,对黄曲霉毒素进行萃取和洗脱。结果 6种黄曲霉毒素在一定质量浓度范围内线性关系良好,相关系数均大于0.9995。日内和日间精密度的相对标准偏差(relative standard deviations, RSDs)分别为4.39%～8.21%和4.97%～9.12%。将开发的方法用于实际牛奶样品中黄曲霉毒素残留检测,加标回收率为80.01%～92.71%,R...     

适配体结合量子点技术同时检测金黄色葡萄球菌和大肠埃希氏菌O157:H7方法

目的：基于适配体结合量子点技术,建立一种同时针对金黄色葡萄球菌、大肠埃希氏菌O157:H7两种食源性致病菌快速高效、灵敏的检测方法。方法：利用表面增强拉曼光谱技术筛选出与目标菌结合特异性较好的2?种适配体;利用免疫磁珠与适配体偶联技术特异性捕获2?种目标菌;选取不同发光原理的2?种量子点,并结合量子点荧光标记技术构建“磁珠＋目标菌＋量子点”的“三明治结构”,通过对结构条件优化确定2?种目标菌的检出限,并应用到市售无菌牛乳实际样品中进行加标回收率实验。结果：本研究证明所选取的2?条适配体与目标菌的结合具有高特异性,可实现对2?种目标菌的同时检测。对“三明治结构”优化结果表明：2?种目标菌的最佳捕获条件为免疫捕获时间45?min、磁分离时间2?min;适配体最适浓度为400?nmol/L;金黄色葡萄球菌检出限为101?CFU/mL,线性方程为Y=28.51X＋126.67（R2=0.973）;大肠埃希氏菌O157:H7检出限均为102?CFU/mL,线性方程为Y=92.86X－64.67（R2=0.987）,其中,Y为荧光强度,X为细菌菌落数的对数值,拟合度良好。且在牛乳样品中金黄色葡萄球菌和大肠埃希氏菌O157:H7回收率分别为94.6%～102.8%和93.4%～100.4%。结论：本研究所建立的方法能够实现同时对2?种食源性致病菌快速、高效检测,该方法易操作且灵敏度高,在食品加工生产安全检测方面具有良好的应用前景。     

Comparison of immunochromatographic assays based on fluorescent microsphere and quantum-dot submicrobead for quantitative detection of aflatoxin M1 in milk

The performance of fluorescent microsphere immunochromatographic assay (FM-ICA) and quantum-dot submicrobead immunochromatographic assay (QB-ICA) was systematically and comprehensively compared in quantitative detection of aflatoxin M₁ in milk. Under optimum conditions, the advantages of FM-ICA include lower limit of detection of 42.3 pg/mL with better accuracy, precision, reliability, and practicability. The advantages of QB-ICA include shorter detection time and lower monoclonal antibody consumption. The 2 ICA were consistent with liquid chromatography-tandem mass spectrometry. This study serves as a reference for selecting appropriate fluorescent labels for the immunochromatographic assay of aflatoxin M₁.     

石墨烯量子点荧光“开启”适配体传感器检测卡那霉素

该文开发一种基于结构转换适配体(aptamers)的新型高灵敏度荧光"开启"适配体传感器,用于快速、灵敏检测动物源性食品中卡那霉素(kanamycin,KAN)。适配体的结构转换诱导氮掺杂石墨烯量子点(nitrogen-doped graphene quantum dots,N-GQDs)的聚集/解聚行为,从而引起体系荧光的淬灭/恢复。与之前文献方法相比,该研究方法在效率、灵敏度、选择性和稳定性等方面均表现出优异性能:线性范围为0.1 ng/mL~10.0 ng/mL,检测限低至0.036 ng/mL(S/N=3),远远低于动物源性食品中KAN的最大残留限量。并且整个检测过程(包括样品提取)可在45 min内完成。此外,该方法成功用于5种动物源性食品样品(牛奶、蜂蜜、鱼、蛋、鸡肉)中KAN的检测。     

Real time biosensor analysis of staphylococcal enterotoxin A in food.

Currently there is no 'real-time' detection system to identify food borne toxins. In order to develop such a system, we have used a evanescent wave biosensor for real time detection of staphylococcal enterotoxin A (SEA) in foods. The approach used here is sandwich biosensor, a method utilizing two antibodies. The toxin binds initially to a capturing antibody which is bound covalently on the surface of the biosensor detector. The second antibody binds to the captured toxin. We were able to measure SEA in foods with little or no background interference, demonstrating that biosensor-based measurement of SEA was possible not only with purified SEA but also in complex food matrices such as hot dogs, potato salad, milk and mushrooms. Autoclaved samples of SEA did not evoke a positive response. With both purified SEA and SEA-spiked foods, the assay sensitivity is 10-100 ng/g depending on the material tested and the assay is rapid ( <4 min) when a single antibody is used.     

Rapid detection and characterization of postpasteurization contaminants in pasteurized fluid milk

Microbial spoilage of pasteurized fluid milk is typically due to either (1) postpasteurization contamination (PPC) with psychrotolerant gram-negative bacteria (predominantly Pseudomonas) or (2) growth of psychrotolerant sporeformers (e.g., Paenibacillus) that have the ability to survive pasteurization when present as spores in raw milk, and to subsequently grow at refrigeration temperatures. While fluid milk quality has improved over the last several decades, continued reduction of PPC is hampered by the lack of rapid, sensitive, and specific methods that allow for detection of PPC in fluid milk, with fluid milk processors still often using time-consuming methods (e.g., Moseley keeping quality test). The goal of this project was to utilize a set of commercial fluid milk samples that are characterized by a mixture of samples with PPC due to psychrotolerant gram-negative bacteria and samples with presence and growth of psychrotolerant sporeforming bacteria to evaluate different approaches for rapid detection of PPC. Comprehensive microbiological shelf-life characterization of 105 pasteurized fluid milk samples obtained from 20 dairy processing plants showed that 60/105 samples reached bacterial counts >20,000 cfu/mL over the shelf-life due to PPC with gram-negative bacteria. Among these 60 samples with evidence of gram-negative PPC spoilage over the shelf-life, 100% (60/60) showed evidence of contamination with noncoliform, non-Enterobacteriaceae (EB) gram-negative bacteria (e.g., Pseudomonas), 20% (12/60) showed evidence of contamination with coliforms, and 7% (4/60) showed evidence of contamination with noncoliform EB. Among the remaining 45 samples, 28 showed levels of gram-positive bacteria above 20,000 cfu/mL and the remaining 17 samples did not exceed 20,000 cfu/mL over the shelf-life. Evaluation of the same set of 105 samples using 6 different approaches {all possible combinations of 2 different enrichment protocols (13°C or 21°C for 18 h) and 3 different plating media [crystal violet tetrazolium agar, EB Petrifilm (3M, St. Paul, MN), and Coliform Petrifilm]} showed that enrichment at 21°C for 18 h, followed by plating on crystal violet tetrazolium agar provided for the most sensitive, accelerated detection of samples that reached >20,000 cfu/mL due to PPC with psychrotolerant gram-negatives (70% sensitivity). These results show that tests still required and traditionally used in the dairy industry (e.g., coliform testing) are not suitable for monitoring for PPC. Rather, approaches that allow for detection of all gram-negative bacteria are essential for improved detection of PPC in fluid milk.     

亲水作用色谱-串联质谱法测定婴幼儿乳粉中的牛磺酸

目的:建立婴幼儿乳粉中牛磺酸直接测定的亲水作用色谱-串联质谱(HILIC-MS/MS)检测方法。方法:样品经提取后,采用乙腈和10 mmol/L甲酸铵(0.05%甲酸)作为流动相进行梯度洗脱,质谱在负离子模式下采用多反应监测(MRM),对牛磺酸进行定量测定。结果:牛磺酸在10~200 ng/mL浓度范围线性关系良好,相关系数r为0.9997,检出限为0.04 mg/kg,回收率在81.2~90.5%之间,相对标准偏差小于4.0%。结论:该方法快速、准确、灵敏度高,可用于婴幼儿乳粉中中牛磺酸的直接测定。     

基于金纳米颗粒信号放大ELISA检测食品中恩诺沙星

以金纳米颗粒（gold nanoparticles,AuNPs）为信号放大载体,利用辣根过氧化物酶（horseradish peroxidase,HRP）标记的二抗为信号探针,建立一种基于AuNPs的信号放大酶联免疫检测方法（AuNPs signal amplification enzyme-linked immunosorbent assay,AuNPs-HRP-IgG ic-ELISA）,检测食品中恩诺沙星（enrofloxacin,ENR）。该方法对ENR的检测限（IC15）为5×10－4?ng/mL,半数抑制浓度（IC50）为0.24?ng/mL,检测范围为0.16～500?ng/mL,与建立的传统ELISA方法（IC50=8.76?ng/mL）相比,显著提高了检测灵敏度,并且该方法与环丙沙星、氧氟沙星、诺氟沙星交叉反应率均小于0.1%,具有良好的特异性。该AuNPs-HRP-IgG?ic-ELISA方法的实用性得到了样品添加回收实验和商业化ELISA检测试剂盒的验证,其在牛奶样品中的加标回收率可达80.52%～102.66%,适用于实际牛奶样本中ENR的快速灵敏检测,也为建立其他食品危害物质的精准检测技术开发提供参考。     

基于量子点二抗偶联物的荧光免疫分析法测定牛奶中的氨苄青霉素

目的建立基于量子点二抗偶联物的荧光免疫分析法测定牛奶中的氨苄青霉素。方法采用共价偶联的方法将Qdot 655红色荧光量子点(quantum dots, QDs)与二抗偶联,利用制备好的QDs-二抗偶联物代替传统酶标二抗应用到检测牛奶中氨苄青霉素残留检测的荧光免疫分析方法中,并将该方法与酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)和高效液相色谱法(high performance liquid chromatography, HPLC)进行比较。结果该方法 50%抑制浓度(IC50)为8.3μg/L;检测限为2.5μg/L。空白牛奶加标回收率为94.0%～106.2%,变异系数为2.1%～9.2%。在实际样品的检测中,该方法与ELISA方法和HPLC方法测定的结果相比无显著差异(P0.05)。结论该方法准确、灵敏,适用于牛奶中氨苄青霉素残留的检测。     

Development of Sensitive,Rapid, and Effective Immunoassays for the Detection of Vitamin B<Subscript>12</Subscript> in Fortified Food and Nutritional Supplements

An indirect competitive enzyme-linked immunosorbent assay (Ic-ELISA) method and lateral-flow immunochromatographic (ICA) strip assay method were developed for the detection of vitamin B₁₂ (four major forms, cyanocobalamin, hydroxocobalamin, adenosylcobalamin, and methylcobalamin) in different food products. The limit of detection and 50 % inhibitory concentration for the Ic-ELISA method were 0.065 and 0.43 ng/mL, respectively. The visual limit of detection and cutoff value for the lateral-flow ICA strip assay method were 1 and 4 ng/mL, respectively. For the detection of fortified food and nutritional supplements in vitamin tablets, energy drink, and infant milk powder samples, the recovery rates were in the range of 81 to 122 % for the Ic-ELISA method, and even the lowest content in infant milk powder was identified by the lateral-flow ICA strip assay. Therefore, both of these methods are sensitive, rapid, and effective and are suitable for the on-site detection and rapid screening of mass samples.     

超高效液相色谱–质谱联用法定量测定奶茶中的 3种毒品成分

目的建立超高效液相色谱质谱联用法测定奶茶中苯丙胺、甲基苯丙胺和氯胺酮的分析方法。方法样品经甲醇超声提取处理,利用超高效液相色谱质谱联用法, 4.0 min内完成样品的上机分析。结果方法在0.1～50.0 ng/mL范围内,线性良好,相关系数r大于0.999。仪器检出限在0.008～0.027 ng/mL之间,定量限在0.028～0.089 ng/mL之间。三水平加标回收率在80.7%～112.0%之间。高浓度样品分析后, 3种毒品无明显仪器残留。使用本方法分析市售奶茶样品,未检出3种毒品。结论本方法灵敏、快速、线性范围宽、选择性好、前处理简单,适用于奶茶中3种毒品的准确定量检测。     

Sensitive enzyme-linked immunosorbent assay and gold nanoparticle immunochromatocgraphic strip for rapid detecting chloramphenicol in food

Antibody specific to chloramphenicol (CAP) was produced from rabbit that had been immunized with CAP-keyhole limpet hemocyanin (KLH). Using the antibodies, we established a sensitive direct competitive enzyme-linked immunosorbent assay (dcELISA) and a gold nanoparticle immunochromatographic strip (immunostrip) for detection CAP in food samples. In the dcELISA, CAP at levels of 0.15 ng/ml causes 50% inhibition (IC₅₀) of the binding of CAP-horseradish peroxidase to the antibodies. The overall analytical recoveries of CAP (0.25–100 ng/g) added to the honey or milk samples in the dcELISA were 81.9 and 73.7%, respectively. Onsite determination of CAP was accomplished by immunostrips with a detection limit of 0.5 ng/ml and completed within 10 min. Carefully studying 10 honey and 6 milk samples using the dcELISA and immunostrip indicated that all examined samples were negative for CAP. The presented dcELISA and immunostrip methods are sensitive enough for the rapid determination of CAP in the samples.     

Immunochromatographic assays for ultrasensitive and high specific determination of enrofloxacin in milk,eggs, honey,and chicken meat

Enrofloxacin, a veterinary antibiotic that persists in food, poses a risk to human health. Here, a monoclonal antibody against enrofloxacin, 1H12, was prepared based on the hapten ENR-1, and showed excellent sensitivity with a 50% inhibitory concentration (IC₅₀) of 0.03 ng/mL. Using this antibody, 2 lateral-flow immunochromatographic assays were developed for determination of enrofloxacin in egg, milk, honey, and chicken meat samples. The detection ranges (IC₂₀–IC₈₀) were 0.16–0.82 ng/g, 0.24–1.8 ng/g, 0.25–3.6 ng/g, and 0.61–3.9 ng/g by colloidal gold-immunochromatographic sensor (CG-ICS) analysis, and 0.022–0.42 ng/g, 0.054–0.42 ng/g, 0.069–1.4 ng/g, and 0.19–2.2 ng/g by Eu-fluorescence-immunochromatographic sensor (EF-ICS) analysis. The intraassay and interassay recovery rates were 88.9 to 108.5% with coefficients of variation of 1.3 to 7.0% by CG-ICS analysis, and 88.6 to 113.6% with coefficients of variation of 1.3 to 8.1% by EF-ICS analysis. Thus, our newly developed ICS are sensitive and reliable, providing an option for rapid quantitative detection of enrofloxacin in food samples.     

High throughput detection of antibiotic residues in milk by time-resolved fluorescence immunochromatography based on QR code

ABSTRACT

Herein, we have successfully established a novel, rapid, and simple lateral-flow immunoassay based on time-resolved fluorescence and biotin-streptavidin to detect the residues of various antibiotics in milk. The fluorescence signal and sensitivity of immunochromatography were enhanced through biotinylated antibody coupled with streptavidin europium microspheres. Moreover, due to the use of a QR Code and fluorescent reader, quantitative detection and real-time data uploading can be achieved. Under the optimal conditions, the various antibiotic residues were detected in the milk samples. The results showed that the limits of detection of tylosin, lincomycin and doxycycline were 0.10, 0.06, and 0.27 ng/mL, respectively. The recoveries of the spiked milk samples were 88.9%~127%, with coefficients of variation less than 11%, and the test strip can be stored at room temperature for 12 months. This study shows that the proposed time-resolved fluorescence immunoassay is sensitive, rapid and reliable, and has the potential to be used for detection of veterinary antibiotic residues in food safety fields.     

Rapid propidium monoazide PCR assay for the exclusive detection of viable Enterobacteriaceae cells in pasteurized milk

Pasteurized milk is a complex food and contains numerous PCR inhibitors and can often contain high levels of dead Enterobacteriaceae cells, depending on the condition of food sanitation. Usually, propidium monoazide (PMA) or ethidium monoazide PCR techniques decrease the number of dead bacteria by up to 3.5 log to the associated dead bacteria with no treatment. However, this difference could be insufficient to completely inhibit DNA amplification in the PCR from 10(6) cells of dead Enterobacteriaceae bacteria/mL, potentially contaminated in pasteurized milk. Actually, such potentially high levels of dead Enterobacteriaceae cells in milk has prevented milk researchers from applying PMA- or ethidium monoazide PCR to the assay of viable Enterobacteriaceae cells in milk. We, therefore, developed a rapid PMA real-time PCR whose minimum levels of detection were 1.5 log cfu/PCR for Cronobacter muytjensii and Escherichia coli, and 2.5 log cfu/PCR for Salmonella enteritidis without DNA purification in milk matrices. The PMA real-time PCR allowed us to specifically detect viable Enterobacteriaceae cells (5-10 cfu/mL) in pasteurized milk (20 mL) within 7.5h of total testing time, following the hygienic guidelines for pasteurized milk in the United States and European Union. The long DNA amplification (mainly 2,451 bp) of the 16S-23S rRNA gene was completely suppressed in highly contaminated dead Enterobacteriaceae cells (7.5 log cfu of Cronobacter muytjensii) in 20 mL of pasteurized milk by 23-μM PMA treatment. Although the contamination of the PCR reaction with 5% milk usually causes great inhibition, our method led to the successful elongation of PCR from viable Enterobacteriaceae cells still in the pasteurized milk matrices finally corresponding to 2 to 4 mL of milk PCR inhibitors without a DNA purification step. To comply with current customer demands for chilled pasteurized milk at the most excellent possible quality, our new technique could enable laboratory persons in a factory to conduct rapid milk coliform testing before shipping from a factory.     

高效液相色谱法测定羊乳中的乳铁蛋白

为开发羊乳中乳铁蛋白的高效液相色谱测定方法及报告羊乳中乳铁蛋白的分布规律。本研究采集哈尔滨地区羊乳样品(n=24)和经过巴氏杀菌的羊乳样品(n=24),采用高效液相色谱测定其乳铁蛋白质量浓度。样品脱脂后,调整pH沉淀酪蛋白,采用C8色谱柱分离,二极管阵列检测器210nm检测,以水、乙腈、三氟乙酸为流动相进行线性梯度洗脱。方法回收率94.2%,线性范围50~300μg/mL,检出限12μg/mL,定量限40μg/mL。原料羊乳中乳铁蛋白的质量浓度的平均值122.1μg/mL,巴氏杀菌后羊乳中乳铁蛋白的平均值103.9μg/mL。巴氏杀菌对羊乳中乳铁蛋白浓度无影响。