酶纳米胶囊传感器研制及其双酚A高灵敏快速检测

Bisphenol A (BPA) is an emerging toxic pollutant that has affected food safety, environment and health of human. As one of the most important endocrine disrupting chemicals, BPA can adversely interfere with endocrine system and immune system of human, and increase risk of prostate cancer, breast cancer, etc.. BPA mainly comes from polycarbonate (PC) and epoxy resins plastic products, such as feeding bottles, water bottles, food packaging materials and container. Aiming at the rapid determination of BPA in PC products (content and amount of migration), and focusing on solving several common key problems that limit the development of enzyme based biosensors, this project will mimic the principle of cell reactor (the enzymes show the highest activity and stability in cells), synthesize enzyme unimolecular “nanocapsule” with high bioactivity and stability by encapsulating tyrosinase biomolecule within a thin permeable polymer shell. By making use of the biocompatible microenvironment and the interspace confinement effect of nanocapsule, it will significantly improve the long-term storage stablity and long-term bioactivity of tyrosinase biomolecules. These nanocapsules will further be assembled into three dimentional (3D) carbon nanomaterial (graphene, N-doped carbon nanotube) to fabricate nanocapsule-based biosensors with improved sensitivity and detection limit. The biological and chemical reaction, electron transfer, mass transport rules and response characteristics of substrate on the biomimic nano-biosensing interface will be systematically investigated. The research will fabricate ultrasensitive, cost-effective and portable biosensor with excellent performance index (especially long-term storage and use stability), and set up new method to meet the rapid screening and detection of BPA and other important targets. The project will promote more kinds of enzyme-based biosensor for realization of commercial applications. In addition, the researches also have very important meaning for the development of many fields, such as biological fermentation, enzyme chemical engineering, enzyme assisted pollution treatment, biomolecular devices, life science, and material science areas.

双酚A(BPA)是影响食品安全、环境、生命健康的最重要新型污染物之一，主要来自聚碳酸酯（PC）等食品包装材料和日用品，干扰内分泌、免疫等系统，与多种癌症有关。本项目围绕着解决限制酶传感器领域发展的稳定性、灵敏度、检测限性能亟需提高的共性关键科学问题，拟以PC等产品中强制检测的BPA（含量及迁移量）为模式目标物，以酪氨酸酶为模式酶，通过模拟细胞反应器的原理，研发高活性酶单分子纳米胶囊反应器，探究纳米胶囊微环境和空间限制效应对酶活性、稳定性的影响机制，解决酶分子长期存贮和使用稳定性差、易失活的难题。研制基于酶纳米胶囊/三维导电网络(石墨烯等)的高稳定性新型传感器，探究纳米生物仿生传感界面的生物化学反应、电子传递、物质传输规律和响应特征，通过提高电信号交换效率与传质速率并利用纳米放大效应及富集效应，成倍提高传感器灵敏度、检测限等性能，建立双酚A等目标物快速、高灵敏、低成本检测的新方法。

双酚A(BPA)是影响食品安全、环境、生命健康最重要新型污染物之一，主要来自聚碳酸酯（PC）等食品包装材料和日用品，干扰内分泌、免疫等系统，与多种癌症有关。本项目围绕着解决限制酶基生物传感器领域发展的稳定性、检测限、灵敏度有待进一步提高的共性关键科学问题，以PC等产品中强制检测的BPA（含量及迁移量）为模式目标物，以酪氨酸酶为模式酶，按照研究计划开展了酪氨酸酶纳米胶囊制备方法优化、基于酶纳米胶囊的传感器稳定性等性能研究、新型纳米传感增敏材料性能探索、基于三维导电网络的生物传感器研制与性能表征以及双酚A的高灵敏快速检测研究。探究了纳米胶囊生物相容性微环境和“空间限制效应”抑制酶分子去折叠失活提高酶稳定性的影响机制，解决了酶分子长期存贮和使用稳定性差、易失活的难题。动态光散射结果显示，天然游离的酪氨酸酶（Tyr）的粒径大小约为8nm，而酪氨酸酶纳米胶囊（nTyr）的单分散粒径约为13nm，表明在酪氨酸酶表面成功包裹了一薄层聚合物外壳。研制的nTyr和酶纳米胶囊传感器（nTyr-Chi/GC）的热稳定性、有机溶剂耐受性、酸碱稳定性、存储和使用稳定性等性能跟天然酶分子基传感器（Tyr-Chi/GC）相比都有显著的提高，使得传感器可以适用于有机溶剂检测体系、可穿戴设备、宽pH范围等不同的应用场景。热稳定性研究表明在65℃下孵育2h后天然酪氨酸酶剩余相对活性只剩23.2%，而酪氨酸酶纳米胶囊剩余相对活性仍然可以保持52.74%。在有机溶剂（MeOH、DMSO、DMF和EtOH）和磷酸缓冲液的体积比为1:1体系中，酪氨酸酶纳米胶囊在有机溶剂中稳定性都明显更高。如nTyr-Chi/GC经甲醇/磷酸缓冲液(1:1)浸泡1ｈ后仍能保持85.6%活性，而Tyr-Chi/GC的残留活性只剩下56.56%。在未使用任何增敏纳米材料的条件下，制备的nTyr-Chi/GC传感器检测双酚A检出限为8.2nM，可满足传感器对实际样品中双酚A的快检需求。为了进一步提高电化学酪氨酸酶传感器的灵敏度，本项目开发了MXene、石墨炔等新型纳米传感材料，研制的生物传感器在实际样品中双酚A快速检测方面显示了非常优良的检出限、灵敏度等性能，建立了满足双酚A快速、低成本、高灵敏检测的新方法。