液态悬浮细胞多路复用检测/靶向吸收毒性重金属离子

Water heavy metal pollution have been become an urgent environmental issue in China. In this proposal, by using genetic recombination technique, we present the first description of a liquid suspension approach to construct a molecular-scale multiplexing platform on living Chlorella cells for simultaneous detection of heavy metal ions. It mainly imitates the irreversible binding of Vp130 (a chloroviral surface protein) to the specific receptors on Chlorella cell wall and the anti-parallel assemble model of SPA (staphylococcal protein A) with heavy metal monoclonal antibodies. Meanwhile, inspired by the heavy metal-binding proteins evolved over time from mineral deposit associated microorganisms, a cellular level selective and active accumulation system will be established by inserting the metal-binding protein coding genes into Chlorella. Consequently, the Chlorella with both heavy metal detection and uptake function is obtained, and the integration of simultaneous detection and targeted bioremediation of multiple heavy metal ions can be realized. Flow cytometry, ELISA (enzyme-linked immunosorbent assay), crystal XRD (X-ray diffraction), Western Blot, 2D-DIGE (two-dimensional fluorescence difference in gel electrophoresis), MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), CLSM (confocal laser scanning microscopy) and TIRFM (total internal reflection fluorescence microscopy) are used as tools to analyse multi-layer assembly mechanism of living cells/functional molecules and the interaction processes, spatial orientation of binding sites and the structure-activity relationship with immune recognition/response, time-course variation of heavy metal-binding proteins and its main effect extent in the involved regulatory networks, thereby building up new methods for the comprehensive detection and remediation of toxic heavy metals.

水体重金属污染是我国亟待解决的环境问题。本课题以基因重组为技术手段，仿效藻病毒表面蛋白Vp130与小球藻细胞壁特异受体的不可逆结合，以及葡萄球菌蛋白A与重金属单克隆抗体的反向平行组装模式，首次提出在活体藻细胞表面构建分子尺度的液态悬浮重金属多路复用检测平台；同时，借鉴矿床伴生微生物长期进化而来的重金属结合蛋白，将其编码基因转入小球藻基因组，建立细胞水平的选择性主动吸收体系，获得兼具重金属检测与摄取功能的小球藻，由此实现多种重金属离子同步检测与靶向生物修复的一体化集成。采用流式细胞术、酶联免疫、X射线晶体衍射、蛋白质印迹、双向荧光差异凝胶电泳、基质辅助飞行时间质谱、激光共聚焦扫描及全内反射显微等方法分析活体细胞/功能分子的层层装配机制及相互作用过程，结合位点的空间取向及与识别响应的构效关系，重金属结合蛋白的时程变化及在所参与调控网络中的主效程度，由此建立全方位的毒性重金属检测/治理新方法。

重金属尤其是毒性重金属的开采、冶炼及其在工业领域的广泛应用给生态环境带来了巨大的灾难。重金属污染检测是重金属防治的前提，本项目通过克隆藻病毒表面蛋白Vp130的C端重复序列，表达Vp130-SPA融合蛋白特异识别小球藻并与其细胞壁结合，并通过2分子Vp130-SPA融合蛋白以反向平行的方式与5分子重金属单克隆抗体组装成豆荚结构，采用竞争法同步检测水体中的毒性重金属离子，获得兼具重金属检测与吸附功能的小球藻。同时，项目针对微量重金属检测干扰大、难以实现多靶标并行检测等问题，采用原子掺杂过渡金属硫化物/硒化物以及靶标依赖的比率型探针搭建荧光生物传感器等策略，开展了复杂样品中多种重金属离子高灵敏检测的方法学研究，阐明了依赖特异重金属离子的荧光猝灭机制以及依赖核酸碱基序列的特殊结合模式，设计并制备了一系列靶标诱导的免标记荧光探针，实现了同一体系内多种重金属离子的精准检测。对重金属离子的响应时间短至1 min，检测限低至2.9 nM。项目研究成果已发表第一标注学术论文21篇，第二/三标注论文7篇，申请并授权国家发明专利2项。培养博士及硕士毕业生7名，“新型功能纳米材料的构筑方法及净化废水的作用机理”获湖南省自然科学三等奖。相关工作可为重金属污染监测与防治提供技术支撑，为微量重金属离子的检测与吸收、功能分子/细胞器件的仿生设计、生物有机体与无机离子之间的相互作用、核酸/蛋白质复合体的有序组装及干预等基础研究提供新思路，并具有较好的产业化前景。