摘要
食品安全直接关系到人类的生命健康,影响人类生活质量,是关乎国计民生的重大问题,一直备受关注。我国食品安全问题面临巨大挑战,最突出的就是食品中农、兽药残留以及微生物污染问题。食品安全检测技术方法,作为解决这一复杂问题的基础条件,因此显得尤为重要。目前农药残留检测方法主要有色谱法,免疫法、传感器方法等。其中色谱方法需要大规模昂贵仪器和专业技术人才,并且要对样品进行预处理,萃取方法包括液-液萃取和固相萃取等,去除干扰杂质,富集痕迹量待测目的物质,其原理是利用物质极性差异达到分离的目的,往往萃取富集得率低;免疫学方法需要制备抗体,制备农药抗体需要合成完全抗原并免疫动物,制备周期长,且生物活性受多种因素影响易于失活,难于工业化生产。目前的生物传感器中多利用生物分子如抗体、酶等作为识别元件,生物分子的稳定性、重复检测性较差;化学传感器往往也是利用待测物极性差异进行检测,其检测缺乏特异性。因此,开发对待测组分有特异识别性能的敏感材料,成为研发新的检测方法的关键。随着科学技术的发展,分子印迹技术产生了,分子印迹技术为制备具有特异吸附性材料提供了技术支持。
分子印迹聚合物(Molecularly imprinted polymers,MIPs),是基于抗原-抗体反应原理而采用化学方法合成的高聚物。作为一种新型的吸附材料,MIPs具有从复杂样品中选择性识别目标分子或与其结构相近的某一类化合物的能力,因而被称为“人工抗体”。由于其化学性能稳定,目前已经广泛应用于样品分离、固相萃取、仿生传感器检测等领域。本文拟以部分农药、毒素为目标物质,制备其分子印迹聚合物,对其吸附性能进行测定并用于样品的富集处理。
首先对传统沉淀聚合制备微球条件进行摸索,探讨聚合溶剂体积、模板、单体各组分比例,通氮气除氧时间和聚合时间对聚合物微球形貌和产量的影响。得出在乙腈中可以制备出纳米级别的印迹聚合物微球。选取部分农药毒死蜱、西维因及呋喃丹分子为模板物质,采用沉淀聚合方法制备了各自分子印迹聚合物微球,扫描电镜观察产物颗粒均匀,分散性较好;光谱法和计算机模拟方法预测模板农药分子与功能单体甲基丙烯酸的相互作用方式,结果显示毒死蜱、西维因及呋喃丹与功能单体之间分别以1∶1、1∶1、1∶2的方式发生相互作用,这在一定程度上阐明了聚合物对模板分子进行识别的理论基础。而后采用静态吸附试验测定了分子印迹聚合物对模板农药的结合能力,Scatchard分析结果表明毒死蜱分子印迹聚合物对模板最大表观结合量可达53.91μmol/g;并将聚合物用于环境介质中农药的吸附,取得较好的效果。西维因分子印迹聚合物对模板的最大表观结合量可达1.95μmol/g,同时以印迹聚合物为识别元件,固定于石英晶体微天平电极表面构建用于农药检测的分子印迹压电石英晶体传感器;对其检测条件进行优化,农药西维因浓度在10 - 1000ng/mL范围内,传感器响应方程为y = 0.139 x + 2.99,最低检测限为12.5 ng/mL,并且具有较好的选择性和重复性。
葡萄球菌肠毒素B(SEB)是引起食物中毒的重要因素,本文尝试以此蛋白毒素为模板物质,制备其人工抗体并用于毒素的分离富集。实验首先采用水相悬浮聚合方法合成其分子印迹聚合物凝胶微球,并对其合成工艺进行了优化。平衡吸附试验对制备的SEB印迹凝胶微球的吸附动力学及等温吸附性能进行考察,确定了其对模板毒素SEB的最大吸附量为8.4 mg/g印迹聚合物微球。存在问题是SEB水凝胶微球干燥容易破裂,导致其吸附能力不稳定,再生性能较差。为了克服这一缺点,实验中以聚苯乙烯微球为基质,采用表面印迹法成功制备了SEB分子印迹聚合物微球。首先在聚苯乙烯微球表面共价修饰模板毒素,采用硅烷化试剂(APTMS, OTMS)聚合,而后洗脱模板毒素,从而获得特异的针对模板分子的印迹位点。试验中对聚合过程中的蛋白用量、硅烷化试剂比例、蛋白洗脱条件等参数进行优化,平衡吸附试验考察了聚合物的吸附动力学以及等温吸附性能,结果表明制备的SEB分子印迹聚合物微球对模板毒素有较好的吸附性能,Langmuir方程计算最大表观吸附量Qmax为3.86 mg/g印迹聚合物微球,明显高于非印迹聚合物微球;选用SEA、SEC以及BSA为竞争蛋白对聚合物吸附选择性进行分析,结果合成的SEB印迹聚合物具有较好的吸附特异性。
Food safety has been much concerned for related to human health and quality of life. The most prominent problems for China are the contamination of pesticide, veterinary drug residues and microbiology factors. As a solution to the complex problem, the determination techniques play an important role in regulation the use of pesticide residues. At present, chromatography and immunity methods have been commonly used for an efficient determination of pesticide residue. However, chromatography methods requires expensive equipment, large-scale professional personnel, and the pretreatment of samples, including liquid extraction - liquid extraction and solid-phase extraction, removal of interference of impurities; General drawbacks of immunoassay techniques include the lack of suitable antibodies and their instability, which is difficult to industrial production. Using of biological molecules such as antibodies and enzyme as recognition elements, the biological sensors are limited by the stability and regeneration of biological molecules. So it is necessary to synthesis a kind of materials which can specificly recognize the target molecules with high sensitivity and stability. With the development of science and technology, molecular imprinting has been developed for preparation of the new materials.
Molecularly imprinted polymers (MIPs) are polymers prepared in presence of a template that serves as a mould for the formation of template-complementary binding sites. Thus, MIPs can be created to recognize a target molecule with affinity and selectivity comparable to natural receptors and were called as“artificial antibody”. And these materials can withstanding much harsher conditions than antibodies, such as high temperature, pressure, extreme pH, and organic solvents. These properties have made them extremely attractive for solving problems in the fields of preparative chemical separations, purification and sensors, or for the removal of specific molecular targets from complex mixtures.
In this article, traditional precipitation polymerization was investigated in order to obtaine desired uniform microspheres. Several pesticides such as chlorpyrifos, carbaryl and cabofuran were used as templates to synthesize the molecularly imprinted polymers. Computer simulation technology was employed to investigate the interaction between them and MAA for elucidating the recognition mechanism. Results showed that the ratio between them and MAA was 1∶1, 1∶1, 1∶2. The saturation rebinding experiments and Scatchard analysis were carried out to evaluate rebinding performance. maximum number (Qmax)of binding site of MIPs is 53.91μmol/g for chlorpyrifos and 1.95μmol/g for carbaryl. Used as the recognition elements, the polymer particles were fixed on the surface of the electrode to construct the MIPs-QCM sensor. Atomic force microscopy (AFM) was employed to evaluate the obtained MIPs sensitive film coated on the electrode. The sensor developed exhibits a liner relationship between the frequency shift and carbaryl concentration in the range of 10ng/mL up to 1000ng/mL (y= 0.139 x + 2.99), and the detection limit was 12.5 ng/mL.
The staphylococcus enterotoxin B (SEB) is one major cause of symptoms of food poisoning in humans. So the SEB-imprinted polyacrylamide gel beads were synthesized using suspension polymerization. Adsorption dynamic and isotherms were investigated by equilibrium adsorption experiment, and the maximum adsorption capacity of 3.86mg SEB/g imprinted beads. The problem is that the material has poor mechanical strength. In order to overcome the issue, the SEB-imprinted beads were prepared using microsphere surface-imprinting method. Two kinds of organic silane (APTMS and OTMS) were polymerized on a surface of polystyrene microspheres after the SEB template was covalently immobilized by forming imine bonds. The resulting imprinted beads were selective for SEB. The Langmuir adsorption models were applied to describe the equilibrium isotherms. The results showed that an equal class of adsorption was formed in the MIP with the maximum adsorption capacity of 3.86mg SEB/g imprinted beads. The MIP has much higher adsorption capacity for SEB than the non-imprinted polymer, and the MIP beads have a higher selectivity for the template molecule.
引文
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