EAGER - Development of a Flagellar Motor Biosensor Prototype for Trace Level TNT Detection

恐怖主义袭击的持续威胁使探测爆炸物成为国土安全的一个重要组成部分。本项目的主要目标是设计，制造和测试鞭毛电机生物传感器原型，将提供灵敏，快速和具体的痕量级检测三硝基甲苯（TNT），最广泛使用的爆炸材料之一。鞭毛马达是纳米级的生物马达，常见于许多细菌物种，如大肠杆菌和沙门氏菌。它们的旋转行为对环境化合物水平的轻微变化非常敏感，如硝酸盐和亚硝酸盐，这是爆炸材料的两个关键成分。该生物传感器集成了E.大肠杆菌鞭毛马达与微流控系统检测TNT的存在下，通过监测的旋转行为的变化，一个单一的鞭毛细丝连接到一个固定的细胞体。基于内部的初步研究，非优化的鞭毛马达生物传感器的灵敏度水平与现有的爆炸物检测系统大致相同，包括传统的电化学和新的基于生物的设计。然而，由于传感机制是由细胞感觉系统的快速趋化过程控制的，因此所提出的生物传感器具有短得多的检测时间。拟议的生物传感器将制造的基础上的核心鞭毛电机组装技术开发的PI的研究小组。PI将利用为期一年的EAGER支持来开发鞭毛马达TNT生物传感器的完整原型。以下是拟议项目的具体目标。优化TNT趋化性信号序列.开发使用PDMS微筛的单细胞系留技术.制作生物感测器原型并评估其效能。 拟议活动的智力价值拟议的鞭毛马达生物传感器是一种非传统的爆炸物检测技术。它独特地将分子生物学与MEMS微加工相结合，实现了一种混合微系统，可以在紧凑的封装中提供快速和灵敏的TNT检测。该项目的成功开发不仅对爆炸物检测领域具有重要意义，而且也是将鞭毛马达应用于多种化学品痕量检测的重要一步。该项目将面临许多科学和工程挑战，包括优化TNT传感细菌趋化性的信号传导过程，微流体系统中的单细胞系留，以及开发将TNT样品输送到生物传感器中的有效接口。该项目的结果预计将提供丰富的信息，为未来的生物和生物医学微系统的设计。拟议的研究小组在鞭毛马达的处理和加工方面具有丰富的经验。他们的工作将在阿肯色州大学设备齐全的生物处理实验室和大型微加工设施中进行。 检测痕量TNT的能力对国土安全、法医科学、环境监测和地雷探测至关重要。拟议的生物传感器的成功开发将导致现场TNT检测的手持式工具的开发，该工具可用于实时样品分析，以便在必要时及时进行后续补救。拟议中的研究将为阿肯色州大学的学生提供重要的学习机会。在这项工作下支持的学生将有一个难得的机会在MEMS微加工和生物处理交叉培训。该项目开发的制造技术和配方将大大提高高密度电子中心（HiDEC）的技术能力，该中心是UA的一个多用户微制造设施，在MEMS，BioMEMS和NEMS领域。

Continuous threat of terrorist attacks has rendered the detection of explosive compounds a crucial component of homeland security. The primary goal of the present project is to design, fabricate, and test a flagellar motor biosensor prototype that will provide sensitive, fast, and specific trace-level detection of trinitrotoluene (TNT), one of the most widely used explosive materials. Flagellar motors are nanoscale biological motors commonly found in many bacterial species such as Escherichia coli and Salmonella. Their rotational behavior is extremely sensitive to slight changes in the level of environmental chemical compounds such as nitrate and nitrite, two key components of explosive materials. The proposed biosensor integrates E. coli flagellar motors with a microfluidic system to detect the presence of TNT by monitoring changes in the rotational behavior of a single flagellar filament attached to an immobilized cell body. Based on an in-house preliminary study, the sensitivity level of a non-optimized flagellar motor biosensor is about the same as the existing explosive detection systems, including both the conventional electrochemical and the new biological based designs. However, the proposed biosensor has a much shorter detection time due to the fact that the sensing mechanism is controlled by the fast chemotactic process of the cell' sensory system. The proposed biosensor will be fabricated based on the core flagellar motor assembly techniques developed by the PIs' research groups. The PIs will utilize the one-year EAGER support to develop a complete prototype of the flagellar motor TNT biosensor. The following are the specific objectives of the proposed project.1. Optimize chemotaxis signaling sequence for TNT sensing.2. Develop a single-cell tethering technique using PDMS micro sieves.3. Fabricate biosensor prototype and evaluate performance. Intellectual Merit of Proposed Activity The proposed flagellar motor biosensor is an unconventional technique for explosive detection. It uniquely combines molecular biology with MEMS microfabrication to realize a hybrid microsystem that can provide fast and sensitive TNT detection in a compact package. Successful development of this project is not only important to the field of explosive detection; it also represents a significant step towards the application of flagellar motors for trace-level detection of a wide variety of chemicals. The proposed project will face many science and engineering challenges, including optimizing the signaling process of bacterial chemotaxis for TNT sensing, single-cell tethering in a microfluidic system, and developing an effective interface for delivering TNT samples into the biosensor. Results of the project are expected to provide a wealth of information for future designs of biological and biomedical microsystems. The proposed research team has extensive experience in the handling and processing of flagellar motors. Their work will be carried out in well-equipped biological processing laboratories and large-scale microfabrication facilities at the University of Arkansas. Broader Impacts of Proposed Activity The ability to detect trace level TNT is critical to homeland security, as well as forensics science, environmental monitoring, and landmine detection. Successful development of the proposed biosensor will lead to the development of a hand-held tool for on-site TNT detection which can be used in real-time sample analysis so that subsequent remediation, when necessary, can be carried out in a timely manner. The proposed research will generate significant learning opportunities for students at the University of Arkansas. Students supported under this effort will have a rare opportunity to be cross-trained in MEMS microfabrication and biological processing. The fabrication techniques and recipes developed by this project will greatly enhance the technical capability of the High Density Electronics Center (HiDEC), a multi-user microfabrication facility at UA, in the areas of MEMS, BioMEMS, and NEMS.