Aflatoxin M1 determination in raw milk using a flow-injection immunoassay system

A flow-injection immunoassay (FI-IA) method with amperometric detection for aflatoxin M1 (AFM1) determination in milk has been developed. The first step consists in an incubation of the sample containing AFM1 (Ag) with fixed amounts of anti-AFM1 antibody (Ab) and of the tracer (Ag^*, AFM1 covalently coupled to HRP) until equilibrium is reached. In this mixture a competition occurs between Ag and Ag^* for the Ab. The mixture is then injected into a flow system where the separation of the free tracer (Ag^*) and the antibody-bound tracer (AbAg^*) is performed in a column with immobilized Protein G. The antigen–antibody complexes are retained in the column due to the high affinity of the Protein G for the antibody. The activity of the eluted enzyme label is then amperometrically detected.

The immunoassay was optimised relative to conditions for antibody–antigen incubation (pH, incubation time, ionic strength, temperature) and enzymatic label detection. This method showed a dynamic concentration range between 20 and 500 ppt AFM1, a low detection limit (11 ppt), good reproducibility (RSD < 8%) and a high throughput (six samples per hour in triplicate). Different milk samples were analysed and the results were in good agreement with those obtained by HPLC using the AOAC 2000.08 method.     

Molecular Recognition of Aflatoxin M1 in Water and Milk Samples

Two stochastic microsensors based on diamond paste modified with 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) were proposed for the molecular recognition of aflatoxin M1 in water and milk samples. Two types of diamond were used: a monocrystalline diamond powder (1 μm) (DP) and a nanopowder of monocrystalline diamond (10 nm) (nDP) for the design of the stochastic microsensors. The microsensors were incorporated as combined stochastic microsensors (a microcell containing the stochastic microsensor and the Pt wire, and a Ag/AgCl sensor) in a platform used for monitoring of waste waters and milk samples. The platforms were used for the fast screening of water and milk samples for the qualitative and quantitative analysis of aflatoxin M1 at very low concentration levels (up to 0.001 fg/L). The linear concentration ranges were wide, being able to cover concentrations between 0.001 fg/L to 20 μg/L. The highest sensitive microsensor (sensitivity of 4.74×10¹⁰ s mg^?1 L) was the one based on nDP. The results provided by the platforms were in agreement with those obtained by utilization of standard method (ISO certified methods (HPLC/fluorescence method)), recoveries being higher than 99.00 % and RSD lower than 1.00 % proving that the method can be reliable used for molecular recognition of aflatoxin M1 in water and milk samples.     

反相高效液相色谱法测定乳品及乳制品中的黄曲霉毒素M_1

采用反相高效液相色谱测定乳品及乳制品中黄曲霉毒素M_1(AFM_1)的含量。样品经氯仿提取,过硅胶固相萃取柱净化,用氯仿-丙酮(1+1)混合溶液将黄曲霉毒素M_1从固相萃取柱上洗脱下来。以ZORBAX SB C_(18)色谱柱为分离柱,水和乙腈为流动相梯度淋洗,用荧光检测器检测,外标法定量。黄曲霉毒素M_1在1.0～25μg·L~(-1)质量浓度范围内与其峰面积呈线性关系,检出限(3S/N)为0.05μg·kg~(-1)。应用此法测定了牛奶和乳粉中AFM_1的含量,并测得其平均回收率分别在76.0%～80.0%和76.7%～90.8%之间,相对标准偏差(n=6)均小于7.0%。     

磁性微粒子化学发光免疫分析法测定人血清中雌三醇

利用化学发光免疫分析(chemiluminescence immunoassay, CLIA)高灵敏度和高特异性的特点, 将磁性微粒子应用于化学发光免疫分析中, 用两种不同的方法对人体血清内的雌三醇(E3)含量进行了测定. 磁性微粒子分别作为固相一抗包被材料和二抗分离剂参与反应. 两种方法检测雌三醇浓度的线形范围均为0.6～60 ng/mL. 其中, 固相一抗法的批内变异及批间变异系数分别小于11%和15%, 回收率为90%～116%, 健全性系数为0.9987. 二抗分离法的批内变异及批间变异系数分别小于8%和10%, 回收率为88%～118%, 健全性系数为0.9974. 两种方法分别与经典板式化学发光法对比, 检测人血清样本, 结果相关性较好, 且磁性微粒子法更为省时、简便, 适于推广应用.     

A Strategy for Sample Preparation: Using Egg White Gel to Promote the Determination of Aflatoxin M1 Content in Milk Samples

The analysis of food samples is a challenging task. The high complexity of food matrices hinders the extraction and detection of analytes from them. Therefore, the correct preparation of food samples is a crucial step for their subsequent analysis, as it achieves the proper isolation and preconcentration of analytes and removes the interfering proportion of the food matrix before instrumental analysis. We aimed to develop a method that not only satisfies the requirement of detecting trace compounds in complex matrices but also achieves a “greener” approach by reducing the use of organic solvents and non-degradable materials to minimize the health hazards posed to the operators as well as pollution to the environment. In this study, we prepared egg white as a concentrated gel and used this material for the biological purification of milk samples. After the milk protein was removed by acidification and salting, the residual amount of aflatoxin M1 in milk samples was quantitatively determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the novel egg white purification method possessed advantages over the immunoaffinity technique used as the reference method in extraction recovery, sensitivity, repeatability, and operability. The limit of detection (LOD) was 0.001 μg/kg. In spiked samples containing 0.01 μg/kg to 2 μg/kg of AFM1, the average recovery was 88.3–94.7%, with a precision of 6.1–11.0%. Improved repeatability was obtained by significantly reducing the operation time and resource requirements compared with the immunoaffinity technique currently used internationally. This study provides a reference for the further improvement of the relevant international standards in place for the detection of aflatoxin M1 in milk.     

Occurrence of aflatoxin M1 in milk samples from Italy analysed by online-SPE UHPLC-MS/MS

The occurrence of aflatoxin M1 in 69 milk samples collected in a south region of Italy in 2016 was evaluated. The samples were analysed using an automated method based on online SPE coupled with UHPLC tandem mass spectrometry. After a salt induced liquid–liquid extraction with acetonitrile to remove protein from milk, the extract was diluted with water and analysed using an automated online SPE MS/MS method. Among the analysed samples no one had AFM1 higher than the legally allowable limits whereas 71.4% of the other analysed samples were above the LOD of the method. The highest contamination level of AFM1 was found in pasteurised milk (44.39 ng kg^?1). The results show the worrying and widespread of AFM1 contamination, highlighting the necessity of monitoring studies in order to evaluate the reduction of the maximum legal limit.     

Electrochemical Aptasensor with Layer‐by‐layer Deposited Polyaniline for Aflatoxin M1 Voltammetric Determination

Mycotoxins are highly toxic metabolites of some fungi that frequently contaminate water, food and feed and hence cause several human and animal diseases. In this work, a new approach to the fast and reliable determination of aflatoxin M1 (AFM1) in water and milk has been proposed with reagent free protocol of signal measurement. For this purpose, DNA aptamer selective to AFM1 was entrapped between two thin layers of polyaniline (PANI) electrodeposited on glassy carbon electrode. The incubation of the aptasensor in the AFM1 solution results in remarkable decrease of the PANI intrinsic activity monitored by direct current voltammetry or electrochemical impedance spectroscopy. Appropriate calibration curves were linear in the range from 3 to 90 ng/L with limit of detection (LOD) 1–5 ng/L depending on the measurement mode. Mechanism of signal generation involves shielding electrostatic interactions between the PANI and aptamer in the surface layer and variation of its redox activity attributed to the emeraldine form of PANI. Selectivity of the response was proved by similar experiments with aflatoxin B1 and ochratoxin A and by comparison of the results with those obtained with non‐specific aptamer in the sensing layer. Simple protocol for milk pretreatment has been proposed for reliable detection of AFM1 on the level of its threshold limited values (20 ng/L).     

直接竞争荧光免疫法测定中药材中的黄曲霉毒素B1

本文采用Mannich反应合成黄曲霉毒素B1(AFB1)人工抗原,免疫小鼠制备AFB1单克隆抗体。采用直接搅拌法将异硫氰酸荧光素(FITC)标记AFB1抗体,经Sephadex-50凝胶柱纯化,制得FITC-AFB1荧光标记抗体,分析其免疫学特性,从而建立了一种快速灵敏的直接竞争荧光免疫分析方法(FIA)以用于检测中药材中的AFB1含量。结果表明,AFB1-FITC标记抗体的结合比率为4.19。通过对检测体系多项影响因素的筛选优化,FIA检测方法的标准曲线方程为I=33.45 log C+25.55,R=0.9913,线性检测范围1~100 ng/m L,检测限0.69ng/m L,回收率90.4%~106.6%。该方法具有操作简单、快速灵敏、特异性高等特点,可用于中药材中AFB1的分析测定。     

Micro-Plate Chemiluminescence Enzyme Immunoassay for Determination of Zeranol in Bovine Milk and Urine

Zeranol (α-Zearalanol, α-ZAL), well-known as an anabolic promoter, was officially banned in Europe due to its potential carcinogenic and endocrine-disrupting biological activities. In this study, a method for the determination of zeranol residues in bovine milk and urine was developed based on micro-plate chemiluminescence enzyme immunoassay (CLEIA). The limit of detection (LOD) was 50 ng/L, and the linear range was between 100 ng/L and 4510 ng/L, with a correlation coefficient (R²)0.9982. The recoveries of zeranol in bovine milk and urine were between 84.7% and 123.6%. This study showed that CLEIA was a reliable, convenient, and sensitive method for screening zeranol residues in bovine milk and urine.     

时间分辨荧光免疫分析方法检测烟草中菌核净残留

建立了简便、灵敏地检测烟草中菌核净残留的时间分辨荧光免疫分析方法(TRFIA),及配套的无净化的快速前处理技术。采用镧系元素螯合物Eu-N1标记亲和纯化后的羊抗兔IgG示踪抗体。采用间接竞争TRFIA法建立了菌核净标准抑制曲线,方法的检出限I10为2.0μg/L;抑制终浓度I50为32.00μg/L;检测线性范围为4～128μg/L。考察了丙酮提取后33%,10%和1%的烟草基质对菌核净-TRFIA分析方法的影响,表明在1%烟叶基质条件下,建立的菌核净的抑制曲线的线性范围与标准抑制曲线趋于平行,确定烟草样品的前处理稀释倍数为100倍,计算得到基质影响因子(Im)为17.1。对烟叶样品中添加1,7和24mg/L的菌核净标样,连续3d的添加回收实验表明,方法的回收率为73%～128%,相对标准标准偏差(RSD)在4.3%～13.2%之间;烟草中菌核净的实际最低检出限为1mg/L。本方法可望用于烟草中菌核净残留的快速筛选检测。     

Electrochemical Aptasensor Based on Poly(Neutral Red) and Carboxylated Pillar[5]arene for Sensitive Determination of Aflatoxin M1

Aptasensor for highly sensitive determination of aflatoxin M1 (AFM1) was developed on the base of glassy carbon electrode (GCE) covered with polymeric Neutral red (NR) dye obtained by electropolymerization in the presence of polycarboxylated pillar[5]arene derivative. Aptamer against AFM1 and NR label were then covalently linked to the carboxylic groups of the carrier by carbodiimide binding. At presence of AFM1 the cathodic peak current related to the NR conversion decreases. AFM1 induced also an increase of the charge transfer resistance measured by electrochemical impedance spectroscopy. In optimal conditions, this make it possible to determine from 5 to 120 ng/L AFM1 in standard solutions with limit of detection (LOD) of 0.5 ng/L. The aptasensor was validated on the spiked samples of cow and sheep milk as well as in kefir after their methanol dilution. Reliable detection of the 40–160 ng/kg of mycotoxins was reached. This is below limited threshold value (50 μg/kg) established in EC.     

高效液相色谱法测定牛奶中黄曲霉毒素M1含量的不确定度评定

根据方法GB 5009.24-2016《食品安全国家标准食品中黄曲霉毒素M族的测定》,建立高效液相色谱法测定牛奶中黄曲霉毒素M₁含量不确定度的数学模型,分析检测过程中的不确定度来源,并对不确定度分量进行量化和合成.当牛奶中黄曲霉毒素M₁的质量分数为0.842 μg/kg时,其扩展不确定度为0.048 4 μg/kg（k=2）.方法相对不确定度主要来源于标准品纯度和样品回收率.     

毛细管电泳免疫分析法分析雌三醇

利用一种温敏水凝胶作为毛细管电泳的填充介质，建立了毛细管电泳免疫分析血清中雌三醇的方法。研究了缓冲溶液的浓度和pH值、水凝胶的浓度、电压等因素对分析结果的影响。雌三醇的检出限和线性范围分别为31．6ng／L和50-5000ng／L。     

柱前衍生-高效液相色谱法测定茶叶中的黄曲霉毒素B1

应用柱前衍生-高效液相色谱法测定茶叶中黄曲霉毒素B1的含量。样品采用乙腈(85+15)溶液提取,滤液用MycoSepTM226柱净化,加入正己烷和三氟乙酸衍生,经C18色谱柱分离,荧光检测器检测。黄曲霉毒素B1的质量浓度在0.20~10.0μg·L-1范围内与其峰面积呈线性关系,检出限(3S/N)为0.1μg·kg-1。在0.5,1.0,5.0μg·L-1等3个浓度水平进行加标回收试验,回收率在91.9%~102%之间,测定值的相对标准偏差(n=6)在1.5%~6.9%之间。     

Disposable Electrochemical Aptasensor for Ultrasensitive Determination of Aflatoxin B1 Using Copper Nanoparticles as Probes

An electrochemical aptasensor for aflatoxin B1 (AFB1) detection was constructed based on the copper nanoparticles (CuNPs) and gold nanoflowers modified screen-printed carbon electrodes as electrochemical probes and substrates, respectively. In the range of 100 aM to 100 pM, a good linear relationship between oxidation peak current of CuNPs and concentration of AFB1 was obtained. The high sensitivity could be ascribed to the amplified electrochemical signals by CuNPs. The high affinity of AFB1 with aptamer endowed its high selectivity. The above advantages and disposable traits made this aptasensor as an ideal platform for evaluation of AFB1 level in food samples.     

高灵敏度化学发光磁酶免疫法检测人绒毛膜促性腺激素

将磁性微粒与抗体的偶联,通过优化偶联条件提高了偶联效率,制备了高灵敏度的磁微粒生物探针;采用3-(2-螺旋金刚烷)-4-甲氧基-4-(3-邻氧酰苯基)-1,2-二氧杂环丁烷(AMPPD)--碱性磷酸酶(ALP)化学发光体系,建立了化学发光磁酶免疫检测方法;对检测方法进行了优化和改进,提高了系统的灵敏度和检测速度,并对HCG样品进行相关检测.结果表明,HCG浓度在0.15～150 IU/L范围内,光强随HCG浓度增大而增加,两者之间线性关系良好,相关系数r为0.960;检出限可达0.15 IU/L; 相对标准偏差(RSD)小于5%; 检测总时间少于1 h.本方法可以应用于其它免疫分子的检测,在临床免疫检测方面具有广阔的应用前景.     

Immunofluorescent Analysis with Magnetic Nanoparticles for Simultaneous Determination of Antibiotic Residues in Milk

An analytical system for simultaneous detection of three antibiotic residues in milk (penicillin, tetracycline, and sulfadimethoxine sodium salt) was developed. The method was based on a fluorescence immunoassay. Magnetic nanoparticles (MNPs) were prepared and then coated with 3-(aminopropyl) triethoxysilane (APTES). Antibodies, against penicillin, tetracycline, and sulfadimethoxine, were immobilized on APTES-MNPs. Two immobilization methods were used: a random and an oriented method by protein A. The fluorochrome-antigen conjugates, containing FITC, ATTO 590, and ATTO 630 were obtained. Three separate immunofluorescent analyses were investigated for penicillin, tetracycline, and sulfadimethoxine, first in buffer and then in milk. Linearity values of standard curves in milk were: for penicillin 4–15 ng mL^?1, for tetracycline 50–500 ng mL^?1 (using random immobilization) and 100–500 ng mL^?1(using oriented immobilization), and for sulfadimethoxine 100–500 ng mL^?1. The linearity and sensitivity of calibration curves of determination of penicillin, tetracycline and sulfadimethoxine in milk were very close to the obtained results for separate determination of three antibiotics. It was shown that the multiple immunofluorescence analysis for antibiotic residues in milk without preliminary treatment is effective and rapid.     

Determination of Enrofloxacin in Bovine Milk by a Novel Single-Stranded DNA Aptamer Chemiluminescent Enzyme Immunoassay

Enrofloxacin, a widely used fluoroquinolone antibiotic, may be a cause of bacterial drug resistance and is forbidden in poultry. Consequently, a sensitive and rapid method is required for its determination. Aptamers, which are more stable and easily synthesized than antibodies, may serve as alternatives in the development of methods for rapid detection. Six single-strand DNA aptamers binding to enrofloxacin were selected by in vitro selection. Aptamer number 17 showed the highest affinity for enrofloxacin with a dissociation constant of 188 nM and the highest guanine concentration (35%), which was predicted to be crucial for strong affinity of the aptamer to enrofloxacin, and successfully distinguished enrofloxacin from its structure analogs. Using aptamer number 17, a novel chemiluminescent enzyme immunoassay associating with biotin-streptavidin was developed that allowed the determination of enrofloxacin to 2.26 ng/mL. Due to its capability to determine enrofloxacin in bovine milk, this newly selected aptamer may find broad application in food and environmental monitoring.