BRIGE: Microfabricated Bacterial Environments with Integrated Nanofluidic Electrochemical Sensors for Systems Biology Applications

新工具正在使生物学和医学取得前所未有的进步。该提案描述了一个跨学科的研究项目，旨在创建、测试和传播用于定量生物学研究的微纳米流体技术。具体来说，将使用纳米流体电化学传感器结合传统光学技术来研究单个细菌细胞的行为。最初的实验重点是检测群体感应（或自动诱导）分子，这些分子是由许多细菌细胞分泌的小电活性分子，包括一些与人类疾病有关的细菌细胞。该平台将用于研究外部刺激的影响、静态异质性以及细菌种群中化学通讯的作用。这种方法将利用在大型硅衬底上微制造的纳米流体电化学传感器。这些纳米流体传感器允许低至100 nM的浓度的实时电化学检测，并且可以很容易地与更大的流体系统集成。一个微流体结构，连同控制阀，将被排列在硅衬底上的纳米流体传感器上，以便在传感器附近运输和捕获单个细菌细胞。微流控结构将通过软光刻技术使用光固化透明聚合物（聚二甲基硅氧烷）构建。微流体通道的透明性质将允许对细胞进行光学监测。组装系统将允许同时实时电化学和光学表征单个细胞。特别令人感兴趣的是，当细胞暴露于各种外部因素（如pH值、温度、缓冲液浓度、表面修饰和药物分子）时，细胞将经历的物理和生化变化。最初的电化学测试将使用已知的商业上可用的群体感应分子（例如，自动诱导剂-2，pyocyanin）进行，微流体结构的验证将利用微珠来模拟细菌细胞。在这个项目的最后阶段，细菌细胞（如大肠杆菌、铜绿假单胞菌）将被运输并捕获在纳米流体传感元件旁边，使用微流体平台。细胞产生的群体感应分子将被电化学监测，而细胞对分子的反应将被光学观察。这项研究的长期目标是为系统生物学实验提供一个集成的实验室芯片平台，研究人员将能够同时刺激和监测数百个单个细胞。智力优势：几乎没有其他方法可以监测单个细胞。这项工作将研究电化学检测作为一种技术的可行性和实用性，结合现有的光学技术，以高通量的方式研究单个细胞的行为。该提案还将研究用于处理直径为1-2微米的单个细菌细胞的微流体系统，这仍然是一个困难的技术挑战。这个广泛适用的平台将通过同时为潜在的数千个单个细胞提供无标签化学信息来彻底改变系统生物学领域。目前还没有这样的高通量电子传感器技术。正在研究的基本问题将深入了解细菌细胞的行为和相互作用，这些行为和相互作用可以应用于生物技术、医学和环境研究。更广泛的影响：本研究的跨学科性质将促进工程师和生物学家之间的合作，并为新兴的动态工作环境培训研究人员。所提出的技术可以潜在地用于多种应用，从微生物燃料电池到药物筛选，从生物医学仪器到进化生物学。总的来说，对电化学和微/纳米级系统的更好的基本理解将为生物科学研究人员带来下一代的工具。通过中学到大学的教师和学生的积极参与，这个项目和整个工程的重要性将被传播给公众。PI将访问当地的内城学校，提供有关工程职业机会的信息。将提供现场和虚拟实验室参观。实验室将为来自波士顿市中心学校（如罗克斯伯里预备特许学校）的高中生和教师提供动手培训。参加培训计划的初中和高中教师将与研究小组合作，设计工具包和教学模块，帮助初中和高中科学课程的学生学习纳米生物技术。开发的材料将首先在教皇约翰保罗二世天主教小学进行评估，这是芝加哥的一所以少数民族为主的学校。西南偏南。在大学阶段，研究小组将为本科生提供研究经验，以加强在课堂上学到的工程概念。

PI: GoluchProposal Number: 1125535 New tools are enabling unprecedented advances in biology and medicine. This proposal describes an interdisciplinary research program that aims to create, test, and disseminate micro and nanofluidic technology for quantitative biological studies. Specifically, the behavior of individual bacterial cells will be investigated using nanofluidic electrochemical sensors in combination with traditional optical techniques. Initial experiments are focused on detection of quorum sensing (or auto-inducing) molecules which are small electroactive molecules that are excreted by many bacterial cells, including several involved in human diseases. The platform will be used to investigate effects of external stimuli, static heterogeneity, and the role of chemical communication in bacterial populations. This approach will utilize nanofluidic electrochemical sensors that are microfabricated on large silicon substrates. These nanofluidic sensors allow for real-time electrochemical detection of concentrations as low as 100 nM and can be easily integrated with larger fluidic systems. A microfluidic architecture, complete with control valves, will be aligned over the nanofluidic sensors on the silicon substrate to allow transport and trapping of individual bacterial cells near the sensors. The microfluidic architecture will be constructed using a photo-curable transparent polymer (polydimethylsiloxane) via soft lithography. The transparent nature of the microfluidic channels will allow for optical monitoring of the cells. The assembled system will allow the simultaneous real-time electrochemical and optical characterization of individual cells. Of particular interest are the physical and biochemical changes the cells will undergo when exposed to various external factors, such as pH, temperature, buffer concentration, surface modification, and drug molecules. Initial electrochemical testing will be conducted with known commercially available quorum sensing molecules (e.g. autoinducer-2, pyocyanin) and validation of the microfluidic architecture will utilize microscale beads to simulate bacterial cells. In the final stage of this project, bacterial cells (e.g. E. coli, P. aeruginosa) will be transported and trapped next to the nanofluidic sensing elements using a microfluidic platform. The generation of quorum sensing molecules by the cells will be monitored electrochemically while the cellular response to the molecules will be observed optically. The long term objective for this research is to provide an integrated chip-in-a-lab platform for systems biology experiments where researchers will be able to stimulate and monitor hundreds of individual cells simultaneously. Intellectual Merit: Few alternatives exist for monitoring single cells. The proposed work will investigate the feasibility and practicality of electrochemical detection as a technique for studying the behavior of individual cells in conjunction with existing optical techniques in a high-throughput manner. The proposal will also investigate microfluidic systems for handling of individual bacterial cells with diameters of 1-2 micrometers, which remains a difficult technological challenge. This broadly applicable platform will revolutionize the field of systems biology by providing label-free chemical information for potentially thousands of individual cells simultaneously. No such high-throughput electronic sensor technology is currently available. The fundamental questions being investigated will give insight to the behavior and interactions of bacterial cells that can be applied to biotechnology, medicine, and environmental research. Broader Impacts: The interdisciplinary nature of this research will foster collaborations between engineers and biologists and train researchers for emerging dynamic work environments. The proposed technology can be potentially employed for a large variety applications ranging from microbial fuel cells to drug screening to biomedical instrumentation to evolutionary biology. In general, a better fundamental understanding of the electrochemical and micro/nanoscale systems involved in this project will lead to the next generation of tools for researchers in the biosciences. The importance of this project and engineering in general will be disseminated to the public by actively engaging teachers and students ranging from the middle school to undergraduate levels. The PI will visit local inner city school to provide information about engineering career opportunities. Live and virtual laboratory tours will be offered. Hands on training in the laboratory will be provided for high school students and teachers from Boston inner city schools, such as Roxbury Preparatory Charter School. Middle and high school teachers participating in the training program will be partner with the research group to design kits and teaching modules to help educate students in junior high and high school science classes about nanobiotechonolgy. The developed material will initially be assessed at Pope John Paul II Catholic Grade School, a predominantly minority school on Chicago?s southwest side. At the college level, research experiences for undergraduates will be offered in the research group to reinforce engineering concepts learned in the classroom.