An integrated NMR/magnetic nanosensor system for detection of bacteria and toxins

描述（由申请人提供）：将纳米级组件和小型化检测器系统的使用合并在一起的诊断技术可能对即时诊断产生重大影响。特别是，敏感、坚固、便携、低成本，并能同时精确量化多个分子靶标的诊断系统，将对生物医学研究和分子诊断产生巨大影响。在这个应用中，我们建议使用磁性纳米传感器技术，结合便携式磁弛豫仪，开发一种对临床样本中细胞内病原体和毒素的敏感诊断测试。细菌感染在美国呈上升趋势，其对医疗保健部门的经济影响是显著的。受污染农产品中存在肠出血性大肠杆菌O157:H7、2001年的炭疽杆菌袭击以及最近发现的耐药结核分枝杆菌引发了公众的关注，并强调需要开发敏感和便携式诊断技术，以便快速准确地检测食品和临床样本中的细菌和毒素。我们的方法利用“磁弛豫纳米开关”（MRnS），即磁性纳米颗粒在特定分子靶相互作用时选择性地改变周围水分子（核磁共振信号）的自旋-自旋弛豫时间（T2）。这项拨款申请的首席研究员先前已经证明，这项技术可以在飞分子范围内以极高的分子特异性检测寡核苷酸[自然生物技术，2002；[20:816-820]，以及其他分子目标，如蛋白质和病毒，而不需要目标扩增。最近，我们开发了磁性纳米传感器，用于检测血液和牛奶中的特定细菌[nanoletletters 2007；7, 380]。此外，我们还与陆军埃奇伍德化学与生物中心的合作者一起，利用磁性纳米传感器收集了检测蓖麻毒素的初步数据。作为检测器，我们使用了一种新开发的小型化和便携式核磁共振弛豫仪，可以大幅提高检测阈值和速度。本申请的总体目标是扩展磁性纳米传感器的研究，努力开发高灵敏度，小型化和便携式检测系统，以筛选临床样品中特定细菌和毒素的存在。我们假设我们的磁性纳米粒子系统可以提供一种单一的均匀分析，可用于检测血液中细胞内病原体和毒素的存在，而无需靶扩增。作为细胞内细菌病原体模型，我们将使用鸟分枝杆菌副结核（MAP）。作为毒素模型，我们将使用炭疽毒素（AT），它是由另一种细胞内病原体产生的；炭疽杆菌。在这个项目中，我们将开发一种诊断技术，将磁性纳米颗粒的使用与小型化检测器相结合，以监测临床样本中细胞内病原体和毒素的存在。该技术可以促进快速，选择性和敏感的细菌检测，以支持临床决策。

DESCRIPTION (provided by applicant): Enabling diagnostic technologies that merge the use of nanoscale components and miniaturized detector systems could have a great impact in point-of -care diagnostics. In particular, diagnostic systems that are sensitive, robust, portable, low-cost, and can accurately quantify multiple molecular targets in parallel, would have a tremendous impact in biomedical research and molecular diagnostics. In this application, we propose to use a magnetic nanosensor technology, in conjunction with a portable magnetic relaxometer, to develop a sensitive diagnostic test for intracellular pathogens and toxins in clinical samples. Bacterial infections are on the rise in the United States and their economic impact on the healthcare sector is significant. The presence of enterohemorrhagic E. coli O157:H7 in tainted produce, the B. anthracis attacks in 2001, and the recent identification of drug resistant Mycobacterium tuberculosis have sparked public concern and underscore the need to develop sensitive and portable diagnostic technologies for fast and accurate detection of bacteria and toxins in food and clinical samples. Our approach utilizes "magnetic relaxation nanoswitches" (MRnS), i.e. magnetic nanoparticles that selectively change the spin-spin relaxation times (T2) of surrounding water molecules (NMR signal) upon specific molecular target interaction. The principal investigator of this grant application has previously shown that this technology can detect oligonucleotides in the femtomolemole range with extremely high molecular specificity [Nature Biotech. 2002;20:816-820], as well as other molecular targets such as proteins and viruses, without the need of target amplification. Most recently, we have developed magnetic nanosensors for the detection of a particular bacterium in blood and milk [Nanoletters 2007; 7, 380]. Also, with collaborators at the Army's Edgewood Chemical and Biological Center, we have gathered preliminary data to detect ricin toxin, using magnetic nanosensors. As a detector, we have used a newly developed minituarized and portable NMR relaxometer that allows substantial improvement in detection threshold and speed. The overall goal of this application is to extend research on magnetic nanosensors in an effort to develop highly sensitive, minituarized and portable detection systems to screen for the presence of a particular bacterium and toxin in clinical samples. We hypothesize that our magnetic nanoparticle system can provide a single homogeneous assay that can be used to detect the presence of intracellular pathogens and toxins in blood, without the need for target amplification. As an intracellular bacterial pathogen model, we will use Mycobacterium avium spp. paratuberculosis (MAP). As a toxin model, we will use anthrax toxin (AT), which is produced by another intracellular pathogen; B. anthracis. PUBLIC HEALTH RELEVANCE In this project, we will develop a diagnostic technology that merges the use of magnetic nanoparticles and a miniaturized detector to monitor the presence of an intracellular pathogen and toxin in clinical samples. This technology could facilitate quick, selective and sensitive bacterial detection to support clinical decision making.