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Immunosensors for the detection of endocrine disrupting chemicals in environment and foods: designing a new biorecognition interface

机译:用于检测环境和食品中内分泌干扰化学物质的免疫传感器:设计新的生物识别界面

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摘要

In this thesis, for fabricating immmunosensors for bisphenol-A (BPA) and 4-tert-octylphenol (4-tert-OP), the polyclonal antibodies as recognition elements and the BPA epitope functionalised gold nanoparticles as a labelled antigen were synthesised and characterised using enzyme-linked immunosorbent assay (ELISA). BPA antibodies were generated in vivo using BPA-butyrate-protein and BPA-valerate-protein conjugates. Their binding characteristics were evaluated via the competitive assays with five competing haptens (BPA-butyrate, BPA-valerate, BPA-crotonate, BPA-acetate and BPA-2-valerate). Two indirect ELISAs and one direct ELISA were developed. The 50 % inhibition of antibody binding (IC50) values were 0.78 ± 0.01 – 1.50 ± 0.30 µg L-1 and the limits of detection (LOD) as measured by the IC20 values were 0.10 ± 0.03 – 0.20 ± 0.04 µg L-1. The assays were optimised for the analysis of BPA in, bottled water, carbonated drinks and canned vegetables. The LOD for these three evaluated sample matrices were 0.10 ± 0.03, 0.60 ± 0.10 and 8.4 ± 2.2 µg L-1 respectively. The pilot survey of the three types of food and beverage products for the presence of BPA residues was conducted using three developed ELISAs. For generating 4-tert-OP antibodies, 6 haptens based on OP and NP structures (OPA, OPB, OP1, NPA, NPB and NP1) were synthesised. Specific antibodies against OPA-protein conjugate, OPB-protein conjugate and OP1-protein conjugate were generated and characterised using the direct and indirect ELISAs in 32 antibody/enzyme conjugate combinations. The most sensitive assay for 4-tert-OP was based on the antibody against OP1 hapten with the NP1-BSA conjugate (AbOP1#3/NP1-BSA). The assay exhibited an IC50 of 100 µg L-1, and a LOD of 10 µg L-1. In the end, thiolated BPA modified spherical gold nanoparticles with different epitope coverage were synthesised and tested in competitive and displacement ELISA to measure functionality. The minimum epitope coverage required for antibody binding in this study was determined to be 4.4 × 10-10 mol cm-2. Comparing to hapten-protein conjugates in a conventional ELISA, in which epitope coverage is difficult to control, nanoparticles offer greater control of nanoparticle coverage and hence valency. Multivalent conjugation of small molecule epitope on the surface of nanoparticles can act as protein conjugate to bind to the specific antibodies.
机译:在本文中,为制备双酚A(BPA)和4-叔辛基苯酚(4-tert-OP)的免疫传感器,合成了多克隆抗体作为识别元件,并将BPA表位功能化的金纳米颗粒作为标记抗原,并进行了表征。酶联免疫吸附测定(ELISA)。使用BPA丁酸酯蛋白和BPA戊酸酯蛋白结合物在体内生成BPA抗体。通过竞争测定法用五种竞争性半抗原(BPA丁酸酯,BPA戊酸酯,BPA丁烯酸酯,BPA乙酸酯和BPA-2-戊酸酯)评估它们的结合特性。开发了两种间接ELISA和一种直接ELISA。抗体结合的50%抑制(IC50)值为0.78±0.01 – 1.50±0.30 µg L-1,通过IC20值测得的检出限(LOD)为0.10±0.03 – 0.20±0.04 µg L-1。优化了这些分析方法,用于分析瓶装水,碳酸饮料和罐装蔬菜中的BPA。这三个评估样品矩阵的LOD分别为0.10±0.03、0.60±0.10和8.4±2.2 µg L-1。使用三种开发的ELISA对三种类型的食品和饮料产品中是否存在BPA残留进行了初步调查。为了产生4-ter-OP抗体,合成了基于OP和NP结构(OPA,OPB,OP1,NPA,NPB和NP1)的6个半抗原。针对OPA-蛋白结合物,OPB-蛋白结合物和OP1-蛋白结合物的特异性抗体已生成,并使用直接和间接ELISA在32种抗体/酶结合物组合中进行了表征。对4-tert-OP的最灵敏测定是基于带有NP1-BSA缀合物的抗OP1半抗原的抗体(Ab = OP1#3 / NP1-BSA)。该分析的IC50为100 µg L-1,LOD为10 µg L-1。最后,合成了具有不同表位覆盖率的硫醇化BPA修饰的球形金纳米颗粒,并在竞争和置换ELISA中进行了测试,以测量其功能。在这项研究中,抗体结合所需的最小表位覆盖率确定为4.4×10-10 mol cm-2。与难以控制表位覆盖率的常规ELISA中的半抗原-蛋白缀合物相比,纳米颗粒提供了对纳米颗粒覆盖率和价数的更大控制。纳米颗粒表面上的小分子表位的多价缀合可以充当蛋白质缀合物以结合特定抗体。

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