自组装膜电化学免疫传感器测定甲基对硫磷

利用自组装技术将三巯基丙酸固定在金电极上,以1-乙基-3-（3-二甲氨基）碳二亚胺盐酸盐（EDC）和N-羟基硫代琥珀亚胺（NHS）作为活化剂,将甲基对硫磷抗体以共价键形式固定在三巯基丙酸自组装单分子膜修饰的金电极上,制备了一种免疫传感器。根据抗体与抗原之间特异反应前后电极电位的变化对甲基对硫磷进行定量测定,该传感器的检测下限为0.085 µg/ml,检测范围为0.5~15µg/ml,并可以再生、重复利用。     

电化学免疫传感器快速检测农产品中的毒死蜱

研究了一种无标记的电化学免疫传感器,用于农产品中的毒死蜱农药残留的快速检测。将毒死蜱人工抗原作为生物识别元件固定在金电极的表面,采用间接竞争法原理,样品中的被测组分与电极上的固定化包被抗原竞争性结合溶液中的抗体。抗体抗原结合反应通过电化学阻抗谱和石英晶体微天平进行表征。将该免疫传感器用于检测青菜、苹果等农产品中的毒死蜱农药残留。结果表明,此免疫传感器灵敏度好、准确度高;对毒死蜱农药的检测限为0.01μg/mL,回收率大于85%,检测时间小于1 h,变异系数小于5%,传感器经过再生处理后能重复使用,经济性较好。该研究可为实现快速检测农产品中农药残留传感器的商品化提供参考。     

节杆菌属甲基对硫磷的降解菌株L-W的分离及降解特性

从生产甲基对硫磷的华阳农药厂污水曝气池中,分离到1株能以甲基对硫磷及其降解中间产物对硝基苯酚为唯一碳源生长,且能够将其彻底降解为CO2和H2O的细菌L-W,经鉴定,为节杆菌属(Arthrobacter sp)。用气相色谱法和分光光度计法深入研究了L-W的降解能力,其7h内对50mg/L甲基对硫磷的降解率为85%,对50mg/L对硝基苯酚的降解率为99%。     

Bt晶体蛋白Cry1Ac放射免疫检测技术研究

通过苏云金芽孢杆菌(Bt)HD-73培养,提取Bt晶体蛋白Cry1Ac,经酶解后获得高抗虫活性蛋白。免疫新西兰白兔得到高纯度的Bt晶体蛋白1Ac抗体,用125I标记抗原,研究和建立了Bt晶体蛋白Cry1Ac的放射免疫检测技术,该试剂盒用磁性微粒作分离剂,不需离心即可分离,简化了测定步骤,检测结果达到国外同类产品(酶联免疫试剂盒)的水平。     

油菜精量排种器种子流传感装置设计与试验

针对油菜精量播种过程中缺乏小粒径种子流传感而导致播量监测困难的问题,设计了一种油菜精量排种器种子流传感装置。运用高速摄影技术及碰撞动力学模型,记录并分析油菜种子与聚偏氟乙烯压电薄膜的碰撞轨迹,为传感装置的导管、压电薄膜倾角、出种口位置等关键结构参数提供依据。基于油菜种子与压电薄膜的碰撞信号特征分析,设计了沉槽基板-压电薄膜感应结构,将碰撞信号的衰减时间从9缩短至1 ms,提高了对高频种子流检测的时间分辨率,同时能够有效抵抗机械振动带来的干扰影响。对微弱碰撞信号进行放大、半波整流、电压比较、单稳态触发转化为单脉冲信号,通过单片机定时计数采集处理,实现油菜种子流排种频率与排种总量的实时检测,并利用无线收发模块定时发送给监测显示终端,实现播量数据的实时显示与保存。油菜精量排种器台架及数粒仪高频排种试验表明:在排种频率8.1~32.9 Hz范围内,检测准确率不低于99.5%。田间播种试验表明传感装置能够实时检测精量排种器的排种频率与排种总量,在无排种时计数为零,正常播种状态时检测准确率不低于99.1%,机械振动及粉尘对传感装置没有影响。该传感装置为油菜精量播种过程播量监测、漏播检测以及补种提供有效支撑。     

抗氯霉素多克隆抗体的制备

用重氮化和CDI两种方法合成了CAP全抗原 ,紫外分析和商业试剂盒ELISA鉴定表明合成成功 ;动物免疫 4次后采血检测 ,多克隆抗体 50 %抑制率探测限可达1 0ng/ml,效价可达 1 0万以上稀释倍数 ,特异性较好。     

检测睾酮、孕酮、雌酮与雌三醇的双抗体酶免疫分析法

本文报道了自制的试剂建立了检测睾酮、孕酮、雌酮和雌三醇的双抗体酶免疫分析法。实验表明 ,这些测定具有很高灵敏度 (最小可测量为 0 1～ 1pg 孔 ) ,标准曲线范围为 0～ 40pg 孔 ;同相关类固醇激素 (孕酮、孕烯醇酮、睾酮、雄酮、雌酮、雌三醇和皮质酮等 )的交叉反应率一般在 0 1 %以下。测定的批内和批间变异系数分别在5 8%～ 7 2 %与 9 5%～ 1 0 5% (n =8)范围内。向血中添加激素标准品后的回收率在 94%～ 1 1 0 %范围内。用自制防腐剂处理后 ,液态抗体 (包括第二抗体 )、酶标记物和已包被第二抗体的滴定板可保持活性 5～ 1 2个月或更久 ( 4℃～ -2 0℃ )。此测定可节约激素特异抗体 4～ 1 0倍。初测人和动物的血、奶、尿样品表明所建立的 4种测定方法可分别用于人和动物体液中睾酮、孕酮、雌酮和雌三醇的定量检测     

克百威残留放射免疫分析方法研究

通过活化酯法与牛血清蛋白联接 ,制备出较高活性的人工抗原 ,以此来免疫兔子制备抗克百威的多克隆抗体。选择适宜的反应条件 ,以14 C 克百威建立了克百威放射免疫测定法。该方法对克百威标准品的最低检测量为 0 1 75ng ml,线性检测范围为 0 2 56～ 40 0 0 0ng ml,I50 值为 650 0ng ml。在线性检测范围内 ,添加不同浓度克百威的蔬菜样品检测的批内、批间变异系数均小于 1 0 %。在甘蓝菜样品中的添加回收率试验回收率为 93 0 %～ 1 0 4 0 % ,变异系数为 4 3 %～ 1 1 5%。所得克百威抗体特异性强 ,与丁硫克百威的交叉反应率为 9 1 5% ,与呋喃酚的交叉反应率为 5 2 9% ,与供试的其它 3种氨基甲酸酯类的交叉反应率均小于 0 5%。     

水果中毒死蜱农药残留生物条形码免疫分析方法研究

为了更加灵敏地检测水果中毒死蜱农药残留,研究建立基于实时荧光定量PCR (qPCR)的高灵敏生物条形码免疫分析方法。研究将毒死蜱半抗原与鸡卵清白蛋白(OVA)结合,再将此偶联物连接到磁性纳米颗粒上,另将毒死蜱特异性抗体、生物条形码以及经巯基修饰的与生物条形码互补DNA链连接到胶体金纳米颗粒上,随后待检毒死蜱分子会与固定在磁性纳米颗粒上的半抗原竞争性结合胶体金纳米颗粒上的抗体,最后将生物条形码解离下来,通过qPCR检测, DNA相对量与待检农药的含量成负相关。研究结果表明,该方法的最低检测限是0.27μg/L, IC_(50)为19.3μg/L,线性范围是1～1 000μg/L。在苹果和梨两种基质中平均添加回收率为79%～90%,相对标准偏差(RSD)为11%～16%。因此,该方法的建立对于实际样品的检测具有重要意义。     

仿刺参体腔液补体类似物化学发光免疫检测

首次应用酶联化学发光免疫检测(Chemiluminesent Immunoassay,CLIA)技术测定仿刺参体腔液补体类似物AjC3和AjC4。羊抗人C3、C4抗体吸附到经过紫外线处理的聚苯乙烯管内,采用辣根过氧化物酶(HRP)标记抗体。过氧化氢和鲁米诺为辣根过氧化物酶的底物。捕获抗体包被最适浓度为1μg/ml,免疫反应20℃孵育2h达到平衡。HRP-IgC3、IgC4抗体复合物适宜稀释度为1:2000,HRP-IgC3I、gC4抗体复合物4℃下保存8d性能稳定,室温下5d内性能稳定。标准品浓度在0.1～10ng/ml范围内时与化学发光值之间具有良好的线性相关性,检测灵敏度为0.1ng/ml。结果表明应用酶联化学发光免疫检测技术能够检测到仿刺参体腔液中含有补体类似物,AjC3含量为6.58±1.4μg/ml,AjC4含量为0.67±0.3μg/ml。     

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.     

Development of ELISA and immunochromatographic assay for the detection of gentamicin

Competitive direct enzyme-linked immunosorbent assay (ELISA) and the immunochromatographic assay were developed using a monoclonal antibody to detect gentamicin in the animal plasma and milk. No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA, indicating that the antibody is highly specific for gentamicin. On the basis of the standard curves, the detection limits were determined to be 0.9 ng/mL in phosphate-buffered saline (PBS), 1.0 ng/mL in plasma, and 0.5 ng/mL in milk, respectively. Recoveries of gentamicin from spiked plasma and milk at levels of 25-100 ng/mL ranged from 85 to 112%. The concentration of intramuscularly injected gentamicin was successfully monitored in the rabbit plasma through competitive direct ELISA. The detection limits were estimated to be about 6 ng/mL of gentamicin in PBS, plasma, and milk using the colloidal gold-based immunochromatographic assay, which is suitable for the simple screening of gentamicin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could complement each other as well as veterinary field and laboratory findings.     

The Influence of Surfactants and Metals on the Mineralization of 14C-Labeled Methyl Parathion in a Mediterranean Soil under Aerobic Conditions

The mineralization of ¹⁴C-labeled methyl parathion, in a soil from a semiarid Mediterranean area, was followed by a simple method. This method consisted in trapping evolved CO₂ in a NaOH trap, using common laboratory glassware and low quantities of reagents. Volatiles were trapped in polyurethane foams, which are cheap and easy to handle. The influence of soil water content (10?C20%) and the addition of surfactants [Aerosol 22 and hexadecyl trimethyl ammonium bromide (HD)] and metals (Cu and Zn) were assessed. Different models were applied to evaluate parathion mineralization kinetics. Mineralization kinetics was initially faster for 20% soil water content, although the final mineralization extent was similar in the range of tested soil water content. Surfactants behaved differently: HD decreased mineralization rate while Aerosol 22 did not affect parathion mineralization. A bimetal Cu and Zn solution led to a significant decrease in methyl parathion mineralization, which persisted when applied together with HD. Results suggest a possible reduction of pesticide availability in soil or a direct effect of the exogenous chemicals on soil microbial community.     

基于构建微生物传感器的甲基对硫磷降解菌的分离鉴定及其降解特性研究

有机磷农药是目前环境中残留量最多的农药之一,对其残留量的检测及降解机制的研究对于环境污染及生态修复具有重要意义。微生物传感器由生物学元件与换能器构成,因具有成本低廉、易于微型化及选择性高等特点而被广泛应用于各种生化物质的分析和检测。本文从长期受农药污染的土壤中分离出4株能以甲基对硫磷为碳源生长的菌株,根据形态特征和16S r RNA基因序列同源性分析,对4株降解菌进行鉴定,利用高效液相色谱测定降解率,选取降解率最高的1株菌进行降解机制研究,以期将其应用于测定环境中甲基对硫磷残留的电位型微生物传感器的构建。结果表明,在甲基对硫磷初始浓度50 mg·L-1、30℃、p H 7.0的培养条件下培养7 d,4株菌对甲基对硫磷的降解率均在78%以上,其中1株菌的降解效率可达100%。16S r RNA基因序列测定表明,该菌株属于克雷伯氏菌属,命名为Klebsiella sp.MP-6。利用液相色谱-质谱联用对其降解产物的研究表明,菌株MP-6水解甲基对硫磷主要产生二甲基硫代磷酸(dimethyl thiophosphoric acid,DMTP)和对硝基苯酚(p-nitrophenol,PNP),极少部分PNP通过产生4-硝基邻苯二酚(4-nitrocatechol,4-NC)和1,2,4-苯三酚(1,2,4-BT)进一步代谢。结果表明,基于测定中间产物对硝基苯酚(p-nitrophenol,PNP)的电位响应信号,该菌株适用于构建测定海水及土壤等环境中有机磷农药的微生物传感器。     

Rapid and sensitive determination of 4-nitrophenol, 3-methyl-4-nitrophenol, 4,6-dinitro-o-cresol, parathion-methyl, fenitrothion, and parathion-ethyl by liquid chromatography with electrochemical detection

Liquid chromatography with electrochemical detection has been used to determine various nitropesticides, DNOC, fenitrothion, and parathion (methyl and ethyl), and some of their main metabolites, 4-nitrophenol for parathion (methyl and ethyl) and 3-methyl-4-nitrophenol for fenitrothion, by using indirect detection. Analysis of them in river water samples has been performed without a preconcentration step. The recovery efficiencies of the tested compounds yielded values between 96 and 112% at the fortification level of 0.5 ppb in a river water sample, and their relative standard deviations were between 1 and 15%. The detection limits of these compounds ranged between 0.05 and 0.14 ppb.     

Evaluation of residual levels of benomyl, methyl parathion, diuron, and vamidothion in pineapple pulp and bagasse (Smooth cayenne)

The objective of this research was to study the residual levels of benomyl, methyl parathion, diuron, and vamidothion in pineapple bagasse and pulp. Benomyl (benlate), methyl parathion (Folidol 600), diuron (Krovar), and Vamidothion (Kilval 300) were applied pre-harvest to pineapples (smooth cayenne). After harvesting, the fruits were washed (100 ppm sodium hypochlorite) and the pulp was separated from the sub-products (peel, core, tops, and tails). The pulp was not submitted to any heat treatment. The sub-products and the juice expressed from them, were submitted to a blanching process (95 degrees C for 1 min). After separating the juice, the bagasse and pulp were analyzed for residues of diuron and benomyl by high performance liquid chromatography, and for residues of vamidothion and methyl parathion by gas chromatography using a TSD detector. No residues of benomyl, diuron, vamidothion, or methyl parathion were detected in the pulp within the quantification limits of the methods (0.1 mg/kg, 0.1 mg/kg, 0.005 mg/kg, and 0.005 mg/kg, respectively). Only methyl parathion (0.052 mg/kg) and vamidothion (0.021 mg/kg) were detected in the bagasse. The presence of these residues in the bagasse was probably due to the action of the wax found in the peel, which prevented the methyl parathion and vamidothion from dissolving in the juice. According to these results, the pulp was fit for human consumption, as far as pesticide residues were concerned, and the bagasse was fit for animal feed and similar applications, because the residual levels found were below the limits established for these compounds.     

SBSE-GC-ECD/FPD in the analysis of pesticide residues in Passiflora alata Dryander herbal teas

Stir bar sorptive extraction (SBSE) in combination with GC-ECD/FPD analysis is here applied to the determination of the residues of 11 pesticides (hexachlorobenzene, lindane, chlorothalonil, parathion methyl, parathion ethyl, fenitrothion, malathion, dieldrin, alpha- and beta-endosulfan, and tetradifon) in herbal teas prepared with Passiflora alata Dryander spiked leaves. The method was optimized using spiked herbal teas in a range from 0.05 to 1 pg/microL for organochlorine pesticides and from 0.15 to 3 pg/microL for organophosphorus pesticides. The method is reproducible and repeatable with recoveries calculated from herbal teas prepared with spiked plant material versus spiked herbal teas, varying from about 30% for tetradifon to about 90% for parathion methyl and malathion. The limits of quantitation (LOQs) ranged from 0.017 pg/microL for lindane to 0.117 pg/microL for malathion.     

Analysis of zearalenone in cereal and Swine feed samples using an automated flow-through immunosensor

The development of a sensitive flow-though immunosensor for the analysis of the mycotoxin zearalenone in cereal samples is described. The sensor was completely automated and was based on a direct competitive immunosorbent assay and fluorescence detection. The mycotoxin competes with a horseradish-peroxidase-labeled derivative for the binding sites of a rabbit polyclonal antibody. Control pore glass covalently bound to Prot A was used for the oriented immobilization of the antibody-antigen immunocomplexes. The immunosensor shows an IC(50) value of 0.087 ng mL(-1) (RSD = 2.8%, n = 6) and a dynamic range from 0.019 to 0.422 ng mL(-1). The limit of detection (90% of blank signal) of 0.007 ng mL(-1) (RSD = 3.9%, n = 3) is lower than previously published methods. Corn, wheat, and swine feed samples have been analyzed with the device after extraction of the analyte using accelerated solvent extraction (ASE). The immunosensor has been validated using a corn certificate reference material and HPLC with fluorescence detection.     

Functional assembly of a microbial consortium with autofluorescent and mineralizing activity for the biodegradation of organophosphates

Organophosphorus pesticides (OPs) cause serious environmental problems, and bioremediation using bacterial enzymes may provide an efficient and economical method for their detoxification. Green fluorescent protein (GFP) is a stable and easily detectable marker in monitoring genetically engineered microorganisms (GEMs) in the environment. In our research, the methyl parathion hydrolase gene (mpd) and enhanced green fluorescent protein gene (egfp) were successfully coexpressed using pETDuet vector in E. coli BL21 (DE3). The coexpression of methyl parathion hydrolase (MPH) and enhanced green fluorescent protein (EGFP) were confirmed by determining MPH activity and fluorescence intensity. The recombinant protein MPH showed high enzymatic degradative activity of several widely used OP residues on vegetables determined by GC analysis. Subsequently, a dual-species consortium comprising engineered E. coli and a natural p-nitrophenol (PNP) degrader Ochrobactrum sp. strain LL-1 for complete mineralization of dimethyl OPs was studied. It could completely mineralize methyl parathion (MP) via MP through PNP and hydroquinone and eventually through the TCA cycle as determined by HPLC analysis. The accumulation of PNP in suspended culture was prevented. The consortium could be further utilized for complete mineralization of PNP-substituted OPs in a laboratory-scale bioreactor and easily monitored by fluorescence of EGFP for its activity and fate.     

Development of a capillary electrophoresis-based immunoassay with laser-induced fluorescence for the detection of carbaryl in rice samples

A capillary electrophoresis-based competitive immunoassay (CEIA) with a laser-induced fluorescence (LIF) detector for the determination of carbaryl was developed. The method was based on the competitive reactions between fluorescently labeled carbaryl tracer (Ag*) and free carbaryl (Ag) with a limited amount of anticarbaryl antibody (Ab), and the relative amounts of each were separated and determined by capillary electrophoresis (CE) with an LIF detector. Using CEIA, equilibrium was reached in 30 min, and the analytical results were obtained within a further 8 min. The linear range and the detection limit for carbaryl were 0.16-50 ng/mL and 0.05 ng/mL, respectively. The sensitivity of this CEIA with an LIF detector was almost 14 times greater than that of ELISA, which used the same immuno-reagents. The method was also applied to the analysis of carbaryl in rice with rapid and simple sample pretreatment. The method is thus proposed as a fast and sensitive assay for the detection of carbaryl.