甲基对硫磷压电免疫传感技术的初步探讨

分别利用蛋白A法和巯基自组装膜法将抗体固定在压电石英晶体金电极上,结果表明巯基自组装膜法效果更好。在利用巯基自组装膜法固定甲基对硫磷抗体的基础上,初步建立了基于直接法和竞争法的甲基对硫磷压电免疫传感技术并对两种技术作了比较,结果表明,基于竞争法的压电免疫传感技术对对硫磷抗原抗体反应的响应频移值较大,可提高检测灵敏度,降低检测限。     

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。     

二氟沙星残留检测直接竞争ELISA试剂盒的研制及其性能测定

应用抗二氟沙星单克隆抗体（DIFmAb）杂交瘤细胞株建立直接竞争ELISA分析方法,研制出DIF残留快速检测试剂盒（DIF-Kit）并对其性能进行了测定。特异性试验结果表明与达氟沙星的交叉反应率为0.32％,与其它氟喹诺酮类药物和磺胺类药物无交叉反应,该方法对DIF的检测限可达到0.1µg/L,半数抑制浓度IC50为2.2µg/L,线性范围0.1～128µg/L, 平均批内和批间变异系数小于<15%。对牛奶、鸡肉分别添加0.4、2.0、10.0、50.0µg/LDIF标准品,平均回收率分别为89.3%和83.9%。DIF-Kit性能稳定,在4℃可保存6个月。DIF-Kit准确度高、重复性好、特异性强,适用于DIF残留快速检测的推广应用。     

用于农药残留快速检测的压电免疫生物传感器的研究

该文建立了一种压电免疫生物传感器结合流动注射的方法检测样品中的农药残留.为了有效地捕获有机磷农药抗原,比较了三种在石英晶体金电极表面上固定有机磷单克隆抗体的方法.在0.005～10μg/mL范围内,有机磷浓度与晶体频率的变化之间呈较好的相关关系.回归方程:y=5.9111Ln(x) 51.979,决定系数为:0.93.该传感器的最低检测限为2.16×103μg/mL,选择性好,可以重复使用.     

电子舌检测奶粉中抗生素残留

为了找到能快速检测乳制品中抗生素残留的方法,该文利用电子舌对奶粉中相同质量浓度的6种抗生素进行了辨识,并对新霉素检测质量浓度进行了初步研究。采用铂、金、钯、钨、钛和银6个电极组成的传感器阵列和1、10和100 Hz 3个脉冲频率进行检测,并通过主成分分析、线性判别分析和偏最小二乘法进行数据分析。结果显示：电子舌对不同种抗生素和不同质量浓度的新霉素具有较好的辨识能力,定性分析能够达到国家最高残留限量标准;利用偏最小二乘法（PLS）建立模型定量分析,新霉素最适检测质量浓度范围在300～1 100 μg/L附近。电子舌依据其独特的优点,为食品掺杂掺假的检测提供了新的思路和方法。     

CuO-CG纳米复合材料修饰电极的制备及其在日落黄检测中应用

为预防和避免由滥用日落黄引发的食品安全隐患,建立快速、灵敏、准确测定日落黄的分析方法。该研究基于氧化铜-羧基化石墨烯（Copper Oxide-Carboxylated Graphene,CuO-CG）纳米复合材料修饰电极构建了一种用于日落黄检测的电化学传感器。采用电沉积法制备CuO-CG纳米复合材料修饰电极,利用循环伏安法和计时电流法研究了日落黄在CuO-CG修饰电极上的电化学氧化行为。结果表明：对影响日落黄检测的条件优化后,得出较佳的试验条件为先沉积CG后沉积CuO、CG的沉积时间和沉积电压分别为900 s和-1.4 V、Cu的沉积时间和沉积电压为120 s和-1.1 V、氢氧化钠浓度为0.10 mol/L、计时电流法测定日落黄的施加电压为0.55 V。在优选试验条件下,该电化学传感器对日落黄的响应时间在5 s以内、线性范围为0.20μg/mL～4.07 mg/mL、检出限为79.36 ng/mL。进一步将此传感器用于饮料样品检测,无需复杂的样品处理步骤,测定回收率为99.35%～105.88%。该研究建立的电化学传感器具有响应时间短、线性范围宽、检出限低、重现性好、稳定性佳、选择...     

智舌对奶粉中抗生素残留的检测研究

本文利用智舌对奶粉中相同浓度的六种抗生素进行了辨识,并对新霉素检测浓度进行了初步研究。采用铂、金、钯、钨、钛和银6个电极组成的传感器阵列和1、10和100Hz三个脉冲频率进行检测,并通过主成分分析、线性判别分析和偏最小二乘法进行数据分析。结果显示：智舌对不同种抗生素和不同浓度的新霉素具有较好的辨识能力,定性分析能够达到国家最高残留限量标准;利用PLS建立模型定量分析,新霉素最适检测浓度范围在200-1000ug/kg附近。     

日本血吸虫SjIR3基因的克隆、表达和免疫保护力研究

鉴定SjIR3诱导小鼠抗攻击感染的免疫保护力。根据GenBank中的SjIR3（AY251481）序列设计特异性引物一对，以日本血吸虫cDNA文库为模板PCR扩增SjIR3基因，利用网上生物信息资源对SjIR3基因进行生物信息学分析。常规方法构建重组质粒pET-32a(+)/SjIR3，转化E.coliLB21，IPTG诱导表达重组蛋白SjIR3（rSjIR3），并利用SDS-PAGE及western-blot进行鉴定分析。动物保护试验用动物为昆明小鼠（雌性，20g），44只，随机分4组，每组11只：A组和B组为对照组，分别接种PBS和FCA；C组和D组为试验组，分别于背部多点注射接种50µg rSjIR3和50µg rSjIR3+FCA，免疫程序为0-2-4-8周。末次免疫后2 wK，每只小鼠攻击40±1条尾蚴，45天后剖杀，冲虫，计数减虫率和减卵率。免疫前和感染前尾静脉采血，ELISA检测IgG抗体水平。结果表明SjIR3 PCR扩增产物约为900bp，与GenBank 中的SjIR3（AY251481）100%的同源，为一跨膜蛋白，具有PS50204和SSF69572两个模体（Domain），为泛素样蛋白激酶家族。动物保护试验表明rSjIR3及rSjIR3+FCA免疫小鼠，可诱导产生26.63%、36.38%的减虫率和34.79%、44.09%的减卵率。结论：rSjIR3可诱导小鼠产生部分抗攻击感染的免疫保护力。     

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。     

苏云金芽孢杆菌cry1Ia基因的克隆、表达与活性研究

根据cry1Ia类基因的全长序列设计引物，以苏云金芽孢杆菌（Bacillus thuringiensis）菌Btc008的总DNA为模板扩增出片段长为2.1kb的cry1Ia的全长基因，插入大肠杆菌（Escherichia coli）表达载体pET-21b，转化大肠杆菌BL21（DE3）菌株，诱导表达出81kD的蛋白。该蛋白由719个氨基酸组成，推导的分子量为81.2kDa。该蛋白的氨基酸序列不同于已知的12种Cry1Ia蛋白，是一种新的Cry1Ia蛋白，该基因已被国际基因命名委员会正式命名为cry1Ia8。杀虫活性测定结果表明：Cry1Ia8对亚洲玉米螟（Ostrinia furnacalis）、小菜蛾（Plutella xylostella）有很强的杀虫活性，LC50分别为0.268 µg/g、2.227 µg/ml，其杀虫效果与Cry1Ab、Cry1Ac相当。对大豆食心虫（Leguminivora glycinivorella）也有较好的活性，但对鞘翅目叶甲科害虫榆兰叶甲（Pyrrhalta aenescens）没有活性。该基因的获得将为我国抗虫转基因作物和工程菌的研制提供新的基因来源，为筛选延缓昆虫抗性产生的基因组合提供了极为重要的依据。     

Monitoring and estimation of the kinetics parameters in the binding process of tannic acid to bovine serum albumin with electrochemical quartz crystal impedance system

Combined measurements of piezoelectric quartz crystal impedance (PQCI) and electrochemical impedance (EI) were utilized in situ to monitor the adsorption of bovine serum albumin (BSA) onto the newly prepared Au colloid-modified electrode and study the binding process of tannic acid (TA) to BSA on the BSA-modified electrode surface. The time courses of the resonant frequency and the equivalent parameters of the sensor were simultaneously obtained during BSA adsorption and TA-BSA binding. Compared with the bare gold electrode, the Au colloid-modified gold electrode showed better biocompatibility, and the absorption capacity for BSA was increased by approximately 2.4 times. The observed frequency decrease was ascribed to the mass increase of the sensor surface resulting from the TA-BSA binding, which is believed to result mainly from the hydrogen bonding from FT-IR characterization. The maximal molar binding ratio of TA binding to immobilized BSA obtained from the frequency shift of the adsorbed BSA and TA was estimated to be 10.3:1. On the basis of the frequency decrease with time, the kinetics of the binding was quantitatively studied. By way of fitting the experimental data, the kinetics parameters, that is, binding and dissociation constant (k1, k(-1)), and the binding equilibrium constant (ka) were determined, giving values of 9.51 x 10(4) M(-1) s(-1), 3.15 s(-1), and 3.1 x 10(4) M(-1), respectively.     

Detection of gliadin in foods using a quartz crystal microbalance biosensor that incorporates gold nanoparticles

This work develops a label-free gliadin immunosensor that is based on changes in the frequency of a quartz crystal microbalance (QCM) chip. A higher sensitivity was obtained by applying 25 nm gold nanoparticles (AuNPs) to the surface of a bare QCM electrode. Subsequently, chicken anti-gliadin antibodies (IgY) were immobilized directly on the AuNP-modified surface by cross-linking amine groups in IgY with glutaraldehyde. Experimental results revealed that the change in frequency exhibited when 2 ppm gliadin was bound to the AuNP-modified electrode was 35 Hz (48%) greater than that of the bare gold electrode. The linear dynamic range in 60% ethanol was from 1 × 10(1) to 2 × 10(5) ppb gliadin, and the calculated limit of detection (LOD) was 8 ppb. The entire detection process was completed in 40 min and was highly repeatable. Additionally, the AuNP-modified QCM system generated results in the detection of gliadin in 10 commercial food products that were consistent with those obtained using an AOAC-approved gliadin kit. In conclusion, the QCM platform provides a potential alternative means of ensuring that people with wheat allergies and celiac patients have access to gliadin-free food.     

Adsorption of chloride and nitrate by variable charge soils in relation to the electric charge of the soil

A cell consisting of a chloride-selective electrode and a nitrate-selective electrode was directly put in the soil suspension to determine the concentration ratio NO₃^?/Cl^? for studying the adsorption of these two ions by three soil samples from variable charge soils. It was found that such factors as the iron oxide content of the soil, the pH of the suspension, the concentration of the respective anion, the kind of accompanying cations, and the dielectric constant of solvent etc. can all affect the amounts and the ratio of the two anions adsorbed. The adsorption was chiefly caused by coulombic force, but another mechanism, presumably a covalent force between the anion and the metal atom on the surface of soil particles, may also be involved, at least for chloride ions.     

Metabolism and lack of DNA reactivity of the mycotoxin ochratoxin a in cultured rat and human primary hepatocytes

It is still unclear whether the carcinogenic mycotoxin ochratoxin A (OTA) is bioactivated to DNA-binding metabolites in rodents and humans. Therefore, we have incubated cultured rat and human primary hepatocytes with noncytotoxic concentrations of (3)H-OTA ranging from 10(-7) to 10(-5) M for 8 h and determined its metabolism and covalent DNA binding. In rat hepatocytes, OTA was metabolized to small amounts of three products, which were further studied by electrospray ionization (ESI)-MS/MS techniques. In addition to 4-hydroxy-OTA, which is a known product of OTA biotransformation, two novel metabolites were detected and tentatively identified as hexose and pentose conjugates of OTA. The in vitro induction with 3-methylcholanthrene (3MC) increased the formation of 4-hydroxy-OTA but did not alter the formation of the conjugated metabolites. No covalent binding of (3)H-OTA or its metabolites to DNA was observed in rat hepatocytes with or without 3MC induction with a limit of detection of 2 adducts per 10(9) nucleotides. However, the cellular ratio of reduced glutathione to oxidized glutathione was significantly decreased by treatment with OTA. In cultured human hepatocytes, (3)H-OTA was only very poorly metabolized, and no covalent DNA binding was observed. In conclusion, the results of this in vitro study do not support the notion that OTA has the potential to undergo metabolic activation and form covalent DNA adducts in rodents and humans.     

Integrated electrochemical gluconic acid biosensor based on self-assembled monolayer-modified gold electrodes. Application to the analysis of gluconic acid in musts and wines

An integrated amperometric gluconic acid biosensor constructed using a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which gluconate dehydrogenase (GADH, 0.84 U) and the mediator tetrathiafulvalene (TTF, 1.5 micromol) were coimmobilized by covering the electrode surface with a dialysis membrane is reported. The working conditions selected were Eapp=+0.15 V and 25+/-1 degrees C. The useful lifetime of one single TTF-GADH-MPA-AuE was surprisingly long. After 53 days of continuous use, the biosensor exhibited 86% of the original sensitivity. A linear calibration plot was obtained for gluconic acid over the 6.0x10(-7) to 2.0x10(-5) M concentration range, with a limit of detection of 1.9x10(-7) M. The effect of potential interferents (glucose, fructose, galactose, arabinose, and tartaric, citric, malic, ascorbic, gallic, and caffeic acids) on the biosensor response was evaluated. The behavior of the biosensor in a flow-injection system in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining gluconic acid in wine and must samples, and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.     

Nanocolloidal gold-based immunoassay for the detection of the N-methylcarbamate pesticide carbofuran

Nanocolloidal gold particles were prepared and labeled to an anti-carbofuran monoclonal antibody (Mab). This conjugate was dispensed on the conjugated pad of a porous glass fiber. Ovalbumin (OVA)-carbofuran and goat anti-mouse IgG were dispensed on the nitrocellulose (NC) membrane and served as the test line and control line, respectively. The carbofuran-containing sample migrated to the NC membrane and reacted with the anti-carbofuran Mab labeled with the colloidal gold. The mixture diffused along the membrane and passed through the OVA-carbofuran in the test line via capillary action. The more analyte present in the sample, the more effectively it will compete with the carbofuran immobilized on the test line for binding to the limited amount of antibody labeled with colloidal gold. An adequate amount of carbofuran could prevent attachment of the colored conjugate to the test line. The presence or absence of a colored band on the test line could indicate a negative or positive result, respectively. When measured to the water sample spiked with carbofuran, this was obtained at or above 0.25 mg/L of carbofuran. The major advantages of the one-step strip test are that the detection time needed was <10 min and all of the reagents are included in the test device.     

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.     

First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.     

Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip

A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples.     

Immunochromatography using colloidal gold-antibody probe for the detection of atrazine in water samples

An immunochromatography (ICG) strip test for rapid detection of atrazine in water samples was developed. A monoclonal antibody (MAb) specific to atrazine was produced from the cloned hybridoma cell (AT-1-M3) and used to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and ICG strip. MAb conjugated to colloidal gold, and that was applied to the conjugate pad of the ICG strip. The visual detection limit for the ICG strip was 3 ng/mL. This test required only 10 min to get results and one step of sample to perform the assay. The results of water samples spiked with 5, 10, 20, and 50 ng/mL of atrazine by ICG strip were in good agreement with those obtained by DC-ELISA. The ICG strip was sufficiently sensitive and accurate to be useful for rapid screening of atrazine in various water samples.