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

描述(由申请人提供)：启用结合使用纳米级组件和微型探测器系统的诊断技术可能会对医疗保健点诊断产生重大影响。特别是，灵敏、坚固、便携、低成本、能够同时准确量化多个分子靶点的诊断系统，将对生物医学研究和分子诊断产生巨大影响。在这一应用中，我们建议将磁性纳米传感器技术与便携式磁弛豫仪相结合，开发一种对临床样本中的细胞内病原体和毒素进行灵敏诊断的测试方法。细菌感染在美国正在上升，它们对医疗保健部门的经济影响是显著的。受污染的农产品中存在肠出血性大肠杆菌O157：H7,2001年的炭疽杆菌袭击事件，以及最近发现的耐药结核分枝杆菌，都引起了公众的关注，并强调了开发敏感和便携式诊断技术的必要性，以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，根据特定的分子靶相互作用选择性地改变周围水分子的自旋-自旋弛豫时间(T2)(核磁共振信号)。这项赠款申请的主要研究人员此前已经证明，这项技术可以检测到具有极高分子特异性的Femtomolemole范围内的寡核苷酸[自然生物技术]。2002；20：816-820]，以及其他分子靶标，如蛋白质和病毒，不需要靶标扩增。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[Nanoletters 2007；7,380]。此外，与陆军埃奇伍德化学和生物中心的合作者一起，我们已经收集了使用磁性纳米传感器检测蓖麻毒素的初步数据。作为一种探测器，我们使用了一种新开发的小型化便携式核磁共振弛豫仪，它可以显著提高检测阈值和速度。这项应用的总体目标是扩大对磁性纳米传感器的研究，努力开发高灵敏度、微型化和便携的检测系统，以筛选临床样本中特定细菌和毒素的存在。我们假设我们的磁性纳米颗粒系统可以提供一种单一的均一检测方法，可以用于检测血液中细胞内病原体和毒素的存在，而不需要目标扩增。作为细胞内细菌致病的模型，我们将使用鸟型分枝杆菌。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炭疽杆菌产生的。在这个项目中，我们将开发一种诊断技术，将磁性纳米颗粒和微型探测器的使用相结合，以监测临床样本中细胞内病原体和毒素的存在。这项技术可以促进快速、选择性和灵敏的细菌检测，为临床决策提供支持。

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.