Microfluidic Controlled Gel-Drop Microarrays for Biothreat Diagnostic Devices

描述（由申请人提供）：无论是在临床还是流行病学环境中，都需要对炭疽芽孢杆菌进行鉴定，并将其与近亲和具有相似特征的生物体明确区分开来。与间接的表型分析相反，基因组分析提供了关于被检测生物的直接信息，然而，在目前经过认证的炭疽检测中，基因组分析是通过复杂的（例如16S rRNA基因测序）或不可靠的（pcr相关的）检测来实现的，并且现在只能在先进的检测实验室进行。Akonni提出的微阵列平台将填补传统临床或疾病诊断与新的生物防御和相关流行病学研究之间的基本技术空白。本应用程序的目的是开发一种便携式临床导向的诊断方法，通过对生物恐怖主义实验室反应网络（LRN）样品测试获得的纯培养物进行微阵列分析，帮助快速可靠地鉴定炭疽芽孢杆菌和蜡样芽孢杆菌群的相关病原体。简单、快速、可靠和低成本的Akonni诊断将从先进的高科技C-D级实验室到典型的社区医院A级实验室带来精密和最终的检测。Akonni将把3D凝胶滴微阵列技术的优势与微流控平台、最新的1分钟DNA/RNA标记-片段化化学以及炭疽芽孢杆菌及其近亲系统发育分类的最新进展结合起来。第一阶段的研究将集中于凝胶滴微阵列微流控卡组件的方法开发和验证。具体目标包括整合微流控卡上的样品制备方案，设计、生产和测试玻璃载玻片上的凝胶滴微阵列，用于鉴定炭疽芽胞杆菌，以及测试凝胶滴微阵列与微流控卡上制备的特定分离株样品的对比。最终卡将接受~50uL纯细菌培养悬浮液；该卡将琼脂板上生长的细菌菌落代表的样品与缓冲液混合，并提供rRNA的分离、标记、碎片化和纯化。一个小型加热器将控制卡内的温度条件。微流控卡将处理所有混合、计量和洗涤步骤，并且在项目第二期将微阵列集成到卡中后，将光学兼容于荧光检测和数据分析。我们将在阿贡国家实验室的BSL-2设施中验证我们的诊断。

DESCRIPTION (provided by applicant): Whether in clinical or epidemiological settings, there is a continued need in identification of B. anthracis and unambiguous discrimination it from close relatives and organisms with similar characteristics. Genomic analysis provides direct information about tested organism in opposite to indirect, phenotype analysis, however represents in current certified Anthrax tests by complicated (e.g. 16S rRNA gene sequencing), or unreliable (PCR-related) tests and may be performed now at advanced detection laboratories only. Akonni proposes microarray platform that will fill a fundamental technology gap between traditional clinical or disease diagnostics and new biodefense and associated epidemiology research. The objective of this application is to develop a portable clinical oriented diagnostic to assist in the rapid and reliable identification of B. anthracis and relative pathogens from B. cereus group by microarray analysis of pure cultures obtained for sample testing at Laboratory Response Network for Bioterrorism (LRN). Simple, rapid, reliable and low- cost Akonni diagnostic will bring sophisticated and final testing from Level C-D, advanced high-tech laboratories, to Level A, laboratories typical for community hospitals. Akonni will unify benefits of 3D gel drop microarray technology with microfluidic platform, newest 1 min DNA/RNA labeling-fragmentation chemistry and last developments in phylogenetic classification of B. anthracis and its close relatives. The research of Phase 1 will focus on the method development and validation of the gel-drop microarray microfluidic card components. Specific aims include integration of sample preparation protocol in microfluidic card, design, produce and test a gel-drop microarray on a glass slide for identification of B. anthracis and testing of gel- drop microarray against samples prepared on microfluidic card from specified isolates. Final card will accept ~50uL of pure bacterial culture suspension; the card will mix the sample represented by one bacterial colony grown on agar plate with the buffers and provide rRNA isolation, labeling, fragmentation and purification. A pettier heater will control the temperature conditions within the card. The microfluidic card will handle all mixing, metering and washing steps and will be optically compatible for fluorescence detection and data analysis after integration of microarray into the card on Phase 2 of the project. We will validate our diagnostic in our BSL-2 facilities in Argonne National Laboratory.