An integrated micro-sensing system for diagnostic testing of botulinum neurotoxins

该提案的重点是开发1)一个集成的微传感系统，能够检测临床样本（小鼠血清）中肉毒杆菌神经毒素（BoNT）的毫摩尔（fM）浓度，这是一种对国土安全和生物防御具有严重威胁和最重要的生物制剂；2)一个整合高质量教学和推广活动与研究的跨学科项目。BoNT的检测利用与液晶（LC）连接的酶裂解膜，利用BoNT固有的蛋白酶活性，从而利用与酶靶分析相关的固有扩增。由BoNT引起的膜劈裂被LC的协同重新定向放大，这可以通过微传感器的阻抗传感来测量。预测的灵敏度是10微升样品中BoNT的fM浓度，对应于BoNT的飞图（fg）量。这项研究的最终目标是创造一种低成本、易于使用的工具，使肉毒杆菌中毒的快速临床诊断具有高灵敏度和特异性。知识价值：本研究将对肉毒杆菌中毒这一生物防御领域的关键问题的临床诊断做出重大贡献。所提出的微系统还可以应用于许多其他毒素，因此在生物防御，环境监测，食品安全和公共卫生方面具有广泛的应用。此外，这项研究将推动生物活性化学、混合微制造技术、生物制剂传感、微流体和流体/电子集成方案。更广泛的影响：拟议的项目将为大学和K-12级别的学生提供与生物防御相关的前沿研究的重要教育机会。该提案的教育和推广部分包括改进现有课程，开发新的跨学科课程，指导本科生和研究生，特别是代表性不足的学生，以及向当地社区和公众传播和推广科学，工程和国土安全。

This proposal focuses on the development of 1) an integrated micro-sensing system capable of detecting in clinical samples (mouse sera) femtomolar (fM) concentrations of botulinum neurotoxin (BoNT), a bioagent of grave threat and utmost importance in homeland security and biodefense, and 2) a coherent interdisciplinary program integrating high quality teaching and outreach activities with research. The detection of BoNT utilizes an enzymatically cleavable membrane that is interfaced to a liquid crystal (LC), exploiting the intrinsic protease activity of BoNT and thereby leveraging the inherent amplification associated with assaying for an enzyme target. The cleavage of the membrane due to BoNT is amplified by the cooperative reorientation of the LC, which can in turn be measured via impedance sensing with the microsensor. The predicted sensitivity is fM concentrations of BoNT in ten microliter of a sample, corresponding to femtogram (fg) quantities of BoNT. The ultimate goal of this research is the creation of a low-cost, easy-to-use tool that allows rapid clinical diagnosis of botulism with high sensitivity and specificity. Intellectual Merit: This research will make significant contribution to the clinical diagnosis of botulism, a key issue in biodefense. The proposed microsystem can also be applied to many other toxins, thereby having broad range of applications related to biodenfense, environment monitoring, food safety and public health. In addition, this research will advance bioactive chemistries, hybrid microfabrication techniques, bioagent sensing, microfluidics, and fluidic/electronics integration schemes. Broader Impacts: The proposed project will generate significant educational opportunities for students at both college and K-12 levels in frontier research related to biodefense. The education and outreach components of this proposal include improving existing curriculum, developing new interdisciplinary courses, mentoring undergraduate and graduate students, especially underrepresented students, and dissemination and outreach to the local community and general public on science, engineering, and homeland security.