Simple and Rapid On-site molecular detection of Mycobacterium tuberculosis

描述(由申请人提供)：这项SBIR第一阶段申请将专注于开发一种简单且廉价的手持电池供电的生物传感器，该传感器集成了微流体和分子生物学，以快速检测人类结核病(TB)的病原体结核分枝杆菌。由于每年新增结核病病例多达900万例，死亡约200万人，而且资源有限的国家在准确诊断结核病方面遇到困难，世界卫生组织敦促开发新的诊断平台，以发现结核分枝杆菌。依赖于百年历史的缺乏敏感性的微观方法，资源有限的国家代表着成功产品的主要市场。此外，通过航空旅行将耐多药结核病传播到工业化国家的风险使其成为一个全球卫生问题。由于每年花费近10亿美元进行结核病检测，易于使用的结核分枝杆菌检测的商业市场潜力立竿见影，并将对改善这一全球健康问题产生广泛影响。 为了开发现代分子生物学检测方法，而不需要昂贵的设备和耗时的分析步骤，我们建议使用两组不同的寡核苷酸探针来补充结核分枝杆菌rRNA序列。一组将被结合到磁珠上，而另一组将被连接到包裹酶底物的脂质体上。由此产生的杂交复合体将通过微流控设备中的磁铁进行固定。然后，脂质体将被裂解，允许现在释放的酶底物驱动电化学反应，该电化学反应将通过集成的交叉式超微电极阵列(IDUA)进行定量。因此，IDUA将把生物信号转换成数字电子信号。这些目标和目的的实现将允许直接检测结核分枝杆菌rRNA，而不需要耗时的浓缩或基因扩增步骤。此外，预计这种易于使用的诊断测试将允许不需要广泛培训和分子生物学背景的个人进行近距离测试。 成功完成拟议的第一阶段工作将产生一种独特的生物传感器，能够快速检测结核病的病原体--结核分枝杆菌。由于世界上三分之二以上的结核病病例是在资源有限的国家发现的，这些国家缺乏财政或技术资源来准确诊断新的结核病病例或监测现有病例的治疗，因此这种生物传感器将对改善生活在这些国家的个人健康产生立竿见影的影响。此外，由于结核病可以通过空气传播有效传播，在世界上感染率高的地区控制结核病的能力也将降低向工业化国家传播结核病的风险，包括结核分枝杆菌耐多药菌株造成的风险。

DESCRIPTION (provided by applicant): This SBIR Phase I application will focus on the development of a simple and inexpensive, handheld, battery-operated biosensor integrating microfluidics and molecular biology to rapidly detect Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) in humans. With as many as 9 million new cases of TB and approximately 2 million deaths per year and the difficulties encountered in resource-limited countries to accurately diagnose TB, the World Health Organization has urged the development of new diagnostic platforms for the detection of M. tuberculosis. Relying upon 100-year old microscopic methods that lack sensitivity, resource-limited countries represent a major market for a successful product. Furthermore, the risk of transmission of multi-drug resistant TB to industrialized nations via the availability of air travel makes this a global health issue. With almost $ 1.0 Billion spent every year to perform TB testing, the commercial market potential for an easy-to-use test for M. tuberculosis is immediate and will have broad implications for improving a worldwide health issue. In order to exploit modern molecular biology detection methods without imposing a need for expensive equipment and time-consuming analytical steps, we propose to utilize two different sets of oligonucleotide probes complementary to a M. tuberculosis rRNA sequence. One set will be bound to magnetic beads while a second set will be conjugated to liposomes that encapsulate enzyme substrates. The resulting hybridization complex will be immobilized via a magnet within a microfluidic device. Liposomes will then be lysed allowing the now released enzyme substrates to drive an electrochemical reaction that will be quantitated by an integrated interdigitated ultramicroelectrode array (IDUA). Thus, the IDUA will transduce the biological signal into a digital electronic signal. The accomplishment of these goals and objectives will permit the direct detection of M. tuberculosis rRNA without the need for time-consuming enrichment or gene amplification steps. Furthermore, it is anticipated that this easy-to-use diagnostic test will permit near-patient testing by individuals who will not require extensive training and background in molecular biology. Successful completion of the proposed Phase I effort will result in a unique biosensor, capable of rapidly detecting Mycobacterium tuberculosis, the causative agent of TB. Inasmuch as more than two-thirds of the world's TB cases are found in resource-limited countries that lack the financial or technical resources to accurately diagnose new TB cases or monitor treatment of existing cases, such a biosensor will have an immediate and dramatic impact on improving the health of individuals living in such countries. Furthermore, since TB can be efficiently spread by airborne transmission, the ability to control TB in those portions of the world with high infection rates will also reduce the risk of spreading TB, including that caused by multi-drug resistant strains of M. tuberculosis, to industrialized nations.