用于细菌趋化性定量检测和筛选的液滴微流控平台及其应用

Bacterial chemotaxis plays a fundamental role in a broad range of processes, including disease pathogenesis, bio?lm formation and bioremediation. Recently, microfluidic approachs have been developed to generate steady or dynamic chemoeffector gradient for studying the mechanism and consequences of bacteira chemotaxis, in which aggregative behavior of bacteira chemotaxis can be recognized and evaluated. However, current technologies are not favorable for studying chemotaxis and bioremediation of individual bacteria. In addition, chemotaxis behavior of enviromental bacteria community with complex bacteria species can not been exploited. In this project, we will integrate continuous flow gradient method with droplet-based microfluidic technology for studying chemotaxis and bioremediation of aromatic hydrocarbons by soil microbes. First, we attempt to develop a new microfluidc system with two parts: a continuous co-flow gradient part to sepearte chemotatic soil bacteria, and a subsequent droplet generation part to isolate individual bacteria into nanoliter droplets for isolated cultivation and study of the bioremediation efficiency in the large scale. Second, we attempt to apply the system we developed to study chemotaxis and bioremediation potential of a specific soil bacteria, Pseudomonas putida, which is recognized as an effective soil inoculant in contaminated soil. In this respect, we will characterize chemotaxis of P. putida towards chloronitrobenzene, benzoic acid, phenol, etc., and screen for high effective mutants using high speed droplet screening. Such a process is going to be repeated with increasing of the chemoeffector concentration, or by switching to multiple compounds for directed evolution of the bacteria. It is envisioned that our system can provide a low consumption, high throughput, and automatic approach for directed evolution of high performance bioremediative bacteria, and can be utilized to separate and identify chemotactic species from environmental samples. The microfluidic systems that we plan to develop in this project can also be extended to study chemotaxis of mammalian cells, such as neutrephils, cancer cells at the single cell level with high throughput.

细菌的趋化性在细菌感染发病机理研究、生物膜形成、微生物污染物降解等方面具有重要研究价值。微流控芯片技术通过动态或者静态浓度梯度的生成和调控，可以实现微生物趋化性的研究。然而，现有方法在研究群体性趋化过程时，往往忽略了微生物个体的差异，无法实现对环境复杂微生物群落趋化的研究。本项目将融合微流控浓度梯度技术和液滴技术，建立集成化的平台，实现趋化细菌的收集、单细胞液滴间隔培养,和细菌对效应物吸收和分解能力的定量分析；利用建立的微流控平台，对恶臭假单胞菌的芳烃类化合物趋化性和降解作用进行研究，测试其对氯代硝基苯、苯甲酸、苯酚等化合物的趋化特征；筛选对这些污染物具有强降解能力的突变个体；实现细菌的定向进化。本项目提出的方法具有低消耗，自动化，高通量等优点，有望加快趋化细菌的发现和挖掘，为发现和开发对污染土壤治理有价值的微生物资源提供基础，此外对研究动物细胞，如白细胞，癌细胞等的趋化也具有指导意义。

趋化性是指运动细菌趋向某些化学吸引剂(正向趋化反应)或避开某些化学驱斥剂(负向趋化反应)的迁移行为。趋化性是微生物的重要生理特征，也是微生物环境适应的重要的机制，在细菌感染发病机理研究、生物膜形成、微生物污染物降解等方面具有重要意义。与传统方法相比，微流控芯片技术通过动态或者静态精确浓度梯度的生成和调控，可以实现更为定量的微生物趋化研究。然而，现有方法在芯片加工、浓度梯度可重现性、趋化菌的回收、以及环境复杂微生物群落趋化分离方面还有待进一步研究。本自然科学基金青年课题提出了两种芯片构型，以解决以上问题：（1）我们开发了一种简易的滑动芯片趋化定量分析芯片，利用芯片的滑动切换，实现了趋化定时启动和终止，利用无扰动的扩散浓度梯度，实现了高度重新的趋化分析。此外该芯片可实现趋化菌的提取。利用这一芯片，我们对睾丸酮丛毛单胞菌CNB-1菌株为实验对象，对其感应苯甲酸这一芳香化合物的20个趋化受体进行了鉴定，初步鉴定了两个甲基化趋化受体蛋白。（2）我们开发了一种融合微流控浓度梯度技术和液滴技术的连续趋化分离装置，建立集成化的平台，实现趋化细菌的收集、单细胞液滴间隔培养。这一芯片通过液滴单细胞培养实现精确的细菌细胞计数和多菌株区分，可不依赖于荧光标记实现趋化的定量分析，可实现复杂环境菌群的的定向分离。本项目开发的趋化分离微流控芯片技术在趋化性定量研究，趋化微生物分离，重要污染土壤治理菌株发现等方面具有重要意义。