基于表面增强拉曼光谱技术在线快速定量分析细胞内能量辅因子

Energy cofactors are the transfer unit of the signal, energy and the ability of redox in the process of cell growth, and they are also the key parameters which can represent the energy levels and the growing status of the cells. The online, rapid and quantitative detection of the energy cofactors is the foundation of the intelligent control of the fermentation process. In this project we intend to adopt the surface-enhance Raman scattering (SERS) technique to achieve the online, rapid, quantitative and micro trace detection of the in vivo energy cofactors (such as ATP/NAD(P)H, and so on) in the fermentation broth. Firstly, we intend to adopt the metal assisted chemical etching technique to obtain the SERS substrate which may possess a large area of three-dimensional micro/nano composite structure, and then a multi enzyme catalysis system will be constructed on the surface of the substrate, so that the highly sensitive, online, rapid and quantitative detection and recognition of the in vivo energy cofactors in the fermentation broth can be achieved based on the characteristic Raman spectra generated during the biochemical reaction process. At the same time, the dynamic analysis of the energy cofactors will be done in the process of the microbial metabolism, which can provide some basic data to achieve the dynamic matching between the material and energy metabolism and to build the gene-protein-metabolism-cofactors model. This project involves much interdisciplinary such as biochemical engineering, physics, and near-field optics, and the research results are expected to aid the ultimate realization of the intelligent control of the fermentation process, which can improve the efficiency of the cell synthesizing target products and further realize green bio-manufacturing.

能量辅因子是细胞生长中信号、能量和氧化还原力的传递单元，是表征细胞能量水平和生长状态的关键参数，细胞内能量辅因子（如ATP及NAD(P)/H等）的在线、快速、定量检测是智能调控发酵过程的重要基础。课题预采用表面增强拉曼光谱(SERS)技术耦合溶菌酶多酶催化体系来在线、快速、定量检测和识别发酵液细胞内能量辅因子。首先采用金属辅助化学腐蚀法制备具有三维微/纳阵列结构的SERS基底，然后在其表面构建溶菌酶催化体系，基于生化反应过程中产生的拉曼“指纹”谱来实现能量辅因子的高灵敏度、高分辨能力及在线、快速、定量与微痕量检测。同时通过动态分析微生物代谢过程中能量辅因子变化，为实现物质和能量代谢的动力学匹配及建立基因-蛋白质-代谢-辅因子之间相互作用模型提供大数据。项目涉及生物化工、物理学、近场光学等交叉学科前沿，研究结果有望实现发酵过程的智能化调控，进而提高细胞合成目标产品的效率并实现绿色生物制造。

微生物发酵是复杂过程，对其智能化调控有利于精准提高发酵效率和目标产品的产率。在发酵体系中，细胞内的能量辅因子（ADP、ATP、NAD，NADH 等）及发酵目标产品作为细胞内能量水平的主要指标可反映细胞的生理状态及代谢水平，因而对发酵液中的关键生物样进行快速、定量检测是生物发酵智能化调控的重要基础。本项目以SERS灵敏度高且稳定性好的大面积三维Ag@SiO2核壳结构硅纳米线阵列（3D-Ag@SiO2/SiNWA）为基础，通过在其表面修饰多酶催化体系和特征色原分子而构筑了生物传感芯片并利用它开展后续研究。该芯片对R6G的检测限达到单分子水平的0.0001 pmol/L，且SiO2纳米层对Ag粒子的包覆使其可抗氧化而具有很好的时间稳定性。实验发现，在对生物能量辅因子进行SERS检测时，以邻联甲苯胺OT代替传统上使用的3,3',5,5'-四甲基联苯胺TMB作为对目标分子进行有效表达的色原可具有更高的信号灵敏度及更好的抗背景干扰性；该芯片对ATP和ADP进行SERS检测的极限低至0.1μM，对NADH的检测限低至0.4μM；利用溶菌酶和特异性反应酶多酶催化体系对Ag@SiO2/SiNWA表面进行分区域功能修饰后，获得了可对发酵液细胞能量辅因子进行快速“捕捉”与“侦测”的多功能生物传感芯片，进而利用它实现了对甘油生成1,3-丙二醇的实际生物发酵过程的实时、快速、定量分析，检测限低至1,3-丙二醇和乳酸的1μg/L以及甘油的4μg/L。本项目探究了生物化学与拉曼光谱学耦合的规律，针对典型工业微生物发酵过程，在复杂背景干扰下实现了目标分子的特异性定量分析，准确建立了 SERS 信号与物质浓度之间的定量关系，发展了一种对微生物发酵过程中的微痕量目标分子进行实时、在线检测的新技术，可用于反馈指导对发酵液中的物质流和能量流进行协同改造以优化发酵过程，并为微生物发酵的智能化调控提供数据支持。