基于三唑磷残留限量值的多检测线免疫试纸条的制备与应用

建立了基于三唑磷残留限量值(MRL)的免疫层析检测方法,可裸眼观察判断谷物、蔬菜和水果中的三唑磷农药残留水平。以粒径20 nm的胶体金标记三唑磷单克隆抗体于金标垫上,包被不同浓度的三唑磷包被原(检测线T1、T2、T3线)、羊抗小鼠Ig G抗体(质控线C线)于硝酸纤维素(NC膜)上,与吸水垫及PVC底板组合成层析试纸条。以大米、甘蓝和苹果为对象,优化了样品前处理方法、提取试剂等条件。结果表明,基于1/10三唑磷MRL值的试纸条(I)对三唑磷的裸眼观察检测灵敏度可达0.005、0.01和0.02μg/m L,优化调整后获得试纸条Ⅱ灵敏度设定为国标GB2763中的三唑磷MRL值依次为0.05、0.1和0.2μg/m L,并与3条检测线一一对应。结合前处理方法优化结果,样品经乙腈提取后,用PBS稀释10倍或者等体积溶剂置换后检测,此两种试纸条在8~12 min内均可准确判定农产品中三唑磷的残留是否超过MRL值,同时多区间定量范围的设定亦可较为准确地定量其残留值,结果与GC方法一致。本研究采用基于MRL值的3条检测线,对检测结果的判定更为直接、准确,无需换算,为基于MRL农产品安全性的快速判断提供了更为直观、简单、准确的方法。     

拟除虫菊酯类农药多残留直接竞争ELISA 建立及初步应用

以辣根过氧化物酶对兔抗拟除虫菊酯类农药抗体进行标记,通过优化分析条件和样品前处理方法,建立了可同时检测蔬菜中多种拟除虫菊酯类农药的直接竞争抑制ELISA方法(DC-ELISA),并在实际检测中进行了初步应用.该方法可用于同时检测蔬菜中甲氰菊酯、氯氰菊酯、三氟氯氰菊酯、溴氰菊酯4种农药的残留,检出限(I10)分别为0.25、0.30、0.43、0.81 mg·L-1,青菜样品添加含量为0.5～5.0 mg·kg-1,回收率分别为93%～114%,97%～110%,89%～126%,93%～113%.采用该方法对南京市场抽取的107个样品进行检测,并与气相色谱法进行比较,直接竞争ELISA阳性检出率为8.46%,气相色谱法阳性检出率为3.74%.     

粮食中百草枯残留的金标免疫层析检测方法研究

采用柠檬酸三钠还原法制备胶体金,研究胶体金与抗体蛋白的作用过程,确定稳定标记1.0 mL胶体金需8.10μg抗体蛋白,标记体系的最佳pH值为8.5。以金标抗体为分析探针,PQ-h-OVA为竞争抗原,羊抗兔IgG为控制抗体,构建直接竞争胶体金标免疫层析检测体系(GICA)。确定膜上包被抗原的质量浓度为0.50 g/L,点样量为1.0μL/条;金标点样量:5.0μL/条;羊抗兔二抗的最佳包被质量浓度为0.11 g/L,点样量为1.0μL/条。金标试纸条的目测检出限为10μg/L,检测时间约5 min,交叉反应率小于0.10%。方法的重复性和稳定性较好,该试纸条在室温下至少可保存5个月。     

胶体金免疫层析法快速检测牛奶、奶粉、饲料中的三聚氰胺

建立了快速检测牛奶、奶粉、饲料样品中三聚氰胺的胶体金免疫层析分析法。将三聚氰胺进行分子修饰得到两种衍生物,分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)相连制得免疫原和包被抗原。运用杂交瘤抗体制备技术得到抗三聚氰胺的单克隆抗体(mAb)。利用柠檬酸三钠还原法制得平均粒径18 nm的胶体金,将胶体金与抗三聚氰胺单克隆抗体相连,所形成的金标抗体(Au-mAb)包被在胶体金垫上,包被抗原和羊抗鼠二抗分别包被在硝酸纤维素膜(NC)上作为检测线(T线)和质控线(C线)。将胶金垫、NC膜、样品垫和吸水纸组装成免疫层析快速检测试剂条。将标准溶液(或待测液)滴加到样品垫上,10 min后可在NC膜C线和T线位置上用肉眼观察到胶体金的颜色(红色),通过比对颜色的深浅判断样品中三聚氰胺的含量。结果表明,三聚氰胺的检出限为10μg/L;选用了其它10种物质对免疫层析试剂条进行了特异性实验,免疫层析试剂条与2-氯-4,6-二氨基-1,3,5-三嗪和环丙氨嗪的交叉反应率约为1%,与其它8种物质没有交叉反应;加标样品用免疫层析试剂条和酶联免疫吸附分析法(ELISA)同时检查,结果一致。本方法适用于牛奶、奶粉、饲料样品中三聚氰胺的现场快速检测。     

猪肉中1-氨基乙内酰脲的胶体金免疫层析法快速检测

基于免疫竞争胶体金免疫层析原理,研制了检测食用肉中呋喃妥因代谢物1-氨基乙内酰脲(AHD)的免疫试纸条。用柠檬酸钠还原法制备胶体金颗粒,标记抗1-氨基乙内酰脲的衍生物(CPAHD)的单克隆抗体并喷于玻璃纤维上,CPAHD-BSA(Bovine serum albumin)抗原和羊抗鼠IgG分别结合于硝酸纤维膜上,依次将样品垫、胶体金垫、硝酸纤维素膜和吸水纸组装切割成AHD胶体金免疫层析快速检测试纸条。在5 min内肉眼观察结果,该试纸条对AHD的最低检测限为2.33μg/L,除与呋喃妥因有弱交叉反应外,与其他同类物均无交叉反应,用该试纸条和ELISA(Enzyme linked immunosorbent assay)检测猪肉中添加的AHD,结果呈现很好的相关性。该方法灵敏度高,简便快速,无需特殊仪器设备,可作为呋喃妥因残留批量检测的筛选方法。     

氟苯尼考胶体金免疫层析定量检测方法的建立

建立了定量检测氟苯尼考的胶体金免疫层析方法.对胶体金标记抗体时溶液pH和抗体浓度、金标抗体用量、检测线上抗原浓度以及检测时间进行了优化.采用胶体金试纸条读取仪测定试纸条检测线和质控线的信号强度,以标准品的浓度为横坐标,阳性样本和阴性样本的检测线/质控线的信号比值(Bx/B0)为纵坐标建立标准曲线.结果表明,胶体金免疫层析试纸定量检测氟苯尼考的线性范围为0.1~1.5 ng/mL,检出限为0.08 ng/mL,检测时间为15 min.本方法具有简便、快速和可定量等特点,适于大批量样品的现场筛查.     

五氯酚免疫层析检测试纸条的研究

利用胶体金免疫层析技术建立了一种快速检测五氯酚(PCP)残留的方法。采用柠檬酸三钠还原法制备大小一致、分布均匀、粒径为20 nm的胶体金颗粒,以此标记五氯酚抗体,制备金标抗体。将五氯酚包被抗原和羊抗鼠二抗分别结合于硝酸纤维膜上,依次将型号Millipore135硝酸纤维膜、型号VL78金标垫、型号SB06样品垫及吸水纸组装于PVC底板上,组装成胶体金免疫层析检测试纸条。通过试纸条上颜色的深浅,检测样品中PCP的残留量。试纸条检出限为10 ng/mL,检测时间为5 min。该方法检测所需试剂已预先包被在试纸条上,操作简单、重复性好、成本低廉,可用于五氯酚的现场快速检测。     

孔雀石绿的磁性免疫层析快速检测方法研究

开发了一种适用于现场快速检测孔雀石绿(MG)的免疫层析试纸条,在超顺磁性纳米微球上偶联MG单克隆抗体作为检测探针,分别将孔雀石绿完全抗原(MG-B SA)和羊抗鼠IgG喷涂于NC膜的T线和C线.结果 发现,T线最佳喷涂量为0.25 mg/mL,抗体最佳偶联量为20 μg,构建的试纸条可在25 min内实现养殖用水及鱼肉...     

基于上转换发光技术和免疫层析技术对氯霉素定量的检测方法

基于上转换发光技术和免疫层析技术对氯霉素定量的方法,属于纳米生物标记、免疫学及光学检测领域。将200~300 nm的上转换荧光纳米颗粒进行羧基化修饰,使之成为水溶性好、分散性高且易于与生物分子偶联的标记物;将样品垫、上转换标记物处理过的结合垫、抗原抗体点样过的硝酸纤维素膜(NC膜)和吸水纸通过粘性底衬结合起来,抗原为氯霉素–BSA作检测线(T),抗体为羊抗鼠抗体作质控线(C),且相距0.5 cm。用切条机将组装好的试纸切成长6 cm、宽4 mm的试纸条,装备成壳,建立免疫层析试纸;将不同浓度梯度的氯霉素标准抗原加于试纸壳中的样品垫里,经10~15 min静置后,放于上转换检测器里进行检测。本发明制备工艺和操作简单,不需要复杂的仪器设备,快速、灵敏,能准确定量检测食品中氯霉素含量。     

胶体金免疫层析方法快速检测腹泻性贝毒软海绵酸的初步研究

建立了赤潮毒素腹泻性贝毒软海绵酸的快速胶体金免疫层析检测方法。通过细胞融合,制备抗软海绵酸单克隆抗体,胶体金标记抗体,建立快速检测软海绵酸的免疫层析试纸条方法。检出限500 ng/mL(50 ng/条),探讨了影响测试方法的因素和提高灵敏度的可能手段。     

Identification and quantitation of pyrethroid pesticide residues in vegetables by solid-phase extraction and liquid chromatography/electrospray ionization ion trap mass spectrometry

A quantitative method consisting of solid-phase extraction (SPE) followed by liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) analysis was developed for the identification and quantitation of ten pyrethroid pesticides commonly used in vegetables. The best HPLC separation was achieved using a gradient program of methanol/water mixture. For the vegetable samples, an SPE procedure to clean up the matrices was carried out prior to LC/MS analysis. Under the optimum conditions, the limits of quantification of the pyrethroid pesticides (tetramethrin, allethrin, fenpropathrin, lambda-cyhalothrin, cypermethrin, deltamethrin, fenvalerate, bioresmethrin, permethrin and bifenthrin) ranged from 0.03 to 0.1 mg kg-1 with relative standard deviations<20%, and the mean recoveries ranged from 69.5 to 102.5%. The proposed method has been successfully applied to the determination of pyrethroids in six vegetables with satisfactory results.     

Development of colloidal gold-based flow-through and lateral-flow immunoassays for the rapid detection of the insecticide carbaryl

One-step membrane-based competitive colloidal gold-based immunoassays in flow-through and lateral-flow formats for the rapid detection of carbaryl were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and carbaryl hapten-OVA conjugate (test line). Anti-carbaryl antibody labeled with colloidal gold particles was firstly incubated with carbaryl. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for flow-through and lateral flow of 50 and l00 μg/L, respectively. The assay time for both tests was less than 5 min, suitable for rapid testing on-site.     

Gold nanoparticle-based immunochromatographic assay for the detection of 7-aminoclonazepam in urine

An analytical system of immunochromatographic assay based on gold nanoparticles was developed for the detection of 7-aminoclonazepam (7-ACLZ) in human urine. The qualitative assay was based on the competitive immunoassay using anti-7-ACLZ polyclonal antibody (PcAb) and a detector reagent that contains colloidal gold particles coated with anti-7-ACLZ PcAb. Nitrocellulose membrane was separately immobilised with goat anti-rabbit IgG (control line) and 7-ACLZ-OVA conjugate (test line). The sensitivity of the strip was tested for detecting 7-ACLZ spiked in urine and each specimen was independently measured by liquid chromatography tandem mass spectrometry. Good correlation was showed by the recovery results. The limit of detection for the strip test in urine was 100 ng mL^?1. The assay can be applied to the rapid detection of 7-ACLZ with the short testing time.     

基于多壁碳纳米管改进QuEChERS法结合气相色谱-串联质谱检测茶叶中10种拟除虫菊酯类农药残留

利用多壁碳纳米管(MWCNTs)QuEChERS法提取茶叶中拟除虫菊酯类残留农药,采用气相色谱-串联质谱(GC-MS/MS)分析测定,建立了一种灵敏度高、可靠性强的茶叶中农药残留检测方法。比较了单壁碳纳米管(SWCNTs)、MWCNTS、氨基化多壁碳纳米管和石墨烯4种碳纳米材料和其不同用量下的净化效果;采用正交试验设计对前处理最佳实验条件进行筛选,并对实验影响因素进行方差分析。结果表明:提取溶剂、碳纳米材料种类对10种拟除虫菊酯类农药回收率的影响具有极显著统计学差异(p<0.001),提取时间对回收率的影响有统计学差异(p<0.05),碳纳米材料用量对回收率影响不显著(p>0.05);最佳样品前处理条件为以乙腈为提取溶剂,超声提取35 min,净化剂为60 mg MWCNTs、200 mg PSA和200 mg C18。方法学考察表明,10种拟除虫菊酯类农药在0.01~2 mg/L范围内线性良好;检出限(LOD)为0.001~0.01 mg/kg,定量限(LOQ)为0.005~0.04 mg/kg;绿茶样品空白基质加标试验中,10种农药的回收率为91.4%~109.7%,相对标准偏差为0.12%~9.80%(n=6)。对花茶、绿茶、红茶3种茶叶基质进行基质效应(ME)评价,结果发现净化剂中加入MWCNTs在绿茶和红茶基质中能有效降低ME。利用该方法检测了市售120份茶叶中拟除虫菊酯类农药的残留,多个样品中检出目标物,但均未超标。该方法检测灵敏度高,可靠性好,具有良好的回收率和稳定性,能满足茶叶中农药残留快速定量分析的要求。     

Development of colloidal gold-based immumochromatographic assay for the rapid detection of medroxyprogesterone acetate residues in biological materials

A competitive colloidal gold-based immunoassay in lateral-flow format for the rapid detection of medroxyprogesterone acetate (MPA) in biological materials was developed. A nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and MPA hapten-OVA conjugate (test line). Anti-MPA polyclonal antibody labelled with colloidal gold particles was first incubated with MPA. The limit of detection for lateral flow was 5?ng?g^?1 for detecting an MPA standard solution, and the limit of detection was 10?ng?mL^?1 for detecting the MPA spiked in pig urine and 10?ng?g^?1 for spiked in pig liver. The results were confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) and indicated that there was a good agreement between both methods (R ²?=?0.976). The assay time for the test was less than 5?min, suitable for rapid testing on site.     

浊点萃取-异辛烷反萃取-气相色谱测定茶叶中拟除虫菊酯农药残留

建立了浊点萃取-异辛烷反萃取-气相色谱(ECD)检测茶叶中联苯菊酯(Bifenthrin)、甲氰菊酯(Fenpropathrin)、功夫菊酯(Cyhalothrin)、氯菊酯(Permethrin)、氰戊菊酯(Fenvalerate)、溴氰菊酯(Deltamethrin)6种拟除虫菊酯农药残留的方法.对含1.2%(m/V)聚乙二醇6000(PEG6000)表面活性剂和40%(m/V)(NH4)2SO4的6种拟除虫菊酯溶液进行加热萃取,所获得的富集相用异辛烷超声反萃取,并经离心对上层异辛烷溶液进行进一步净化处理,即可获得富集倍数达75倍的6种农药.本方法的检出限(LOD)为:联苯菊酯、甲氰菊酯和功夫菊酯0.4 μg/kg;氰戊菊酯2.1 μg/kg;氯菊酯和溴氰菊酯3.0 μg/kg;用本方法测定了新鲜茶叶中6种拟除虫菊酯农药,含量分别为4.17, 4.15, 4.09, 4.01, 3.93和3.51 μg/kg.在上述茶叶样品中添加20 μg/kg 的6种农药后测定,添加回收率为72.3%～85.6%; 相对标准偏差为2.2%～5.6%.     

Colloidal gold-based immunochromatographic assay for detection of diethylstilbestrol residues

One-step membrane-based competitive colloidal gold-based immunoassays in immunochromatographic formats for the rapid detection of diethylstilbestrol (DES) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and DES hapten-ovalubumin conjugate (test line). Anti-DES polyclonal antibody labeled with colloidal gold particles was first incubated with DES. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for immunochromatographic of 0.5 microg/kg for detecting DES standard solution, and the limit of detection was 5 microg/kg for detecting the DES spiked in swine pork and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.     

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for screening 19-nortestosterone

The lateral flow strip test for 19-nortestosterone is one kind of immunochromatographic assay. Nitrocellulose membrane was separately immobilized with goat anti-rabbit IgG (control line) and 19-NT-OVA conjugate (test line). Anti-19-NT polyclonal antibody labeled with colloidal gold particles acted as the detector reagent. The assay is qualitatively, not quantitatively, judged with positive or negative result. We tested the sensitivity of the strip using spiked swine urine, and each specimen was independently measured by LC/MS/MS. The sensitivity, measure by eye, was determined to be 200 ng/mL. The assay time was less than 15 min, and so suitable for on-site rapid test.     

基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

Electrochemical Metalloimmunoassay Based on Enlargement of Gold Nanoparticle Label Treated with Ag Enhancement System

A novel sensitive electrochemical immunoassay with colloidal gold as the antibody labeling tag and subse-quent signal amplification by silver enhancement is described. Colloidal gold was treated by a light-sensitive silver enhancement system which made silver deposit on the surface of colloidal gold(form Au/Ag core-shell structure), followed by the release of the metallic silver atoms anchored on the antibody by oxidative dissolu-tion of them in an acidic solution and the indirect determination of the dissolved Ag ions by anodic stripping voltamrnetry(ASV) at a carbon fiber microelectrode. The electrochemical signal is directly proportional to the amount of analyte(goat IgG) in the standard or a sample, The method was evaluated by means of a non-competitive heterogeneous immunoassay of immunoglobulin G(IgG) with a concentration as low as 0. 2 ng/mL. The high performance of the method is related to the sensitive ASV determination of silver( I ) at a car-bon fiber microelectrode and to the release of a large number of Ag^ ions from each silver shell anchored on the analyte (goat IgG).