Microfluidic Controlled Gel-Drop Microarrays for Biothreat Diagnostic Devices

描述(申请人提供)：无论是在临床还是流行病学环境中，炭疽杆菌的鉴定和与近亲和具有相似特征的生物体的明确区别仍然是必要的。基因组分析提供了与间接表型分析相反的关于被测生物体的直接信息，但目前通过复杂的(如16S rRNA基因测序)或不可靠的(与聚合酶链式反应相关的)测试表示的是目前认证的炭疽测试，现在只能在高级检测实验室进行。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的临床导向的诊断方法，通过对实验室生物恐怖反应网络(LRN)样品测试所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杆菌和来自蜡样杆菌群的相关病原体。简单、快速、可靠和低成本的Akonni诊断将带来复杂和最终的测试，从C-D级先进的高科技实验室到A级社区医院的典型实验室。Akonni将把3D凝胶滴微阵列技术的优点与微流控平台、最新的1分钟DNA/RNA标记-片段化学以及炭疽杆菌及其近亲系统发育分类的最新进展统一起来。第一阶段的研究将集中在凝胶滴微阵列微流控卡组件的方法开发和验证上。具体目标包括将样品制备技术集成到微流控卡片中，设计、制造和测试玻片上用于炭疽杆菌鉴定的凝胶滴微阵列，以及针对微流控卡片上制备的指定菌株的样品进行凝胶滴微阵列测试。最终卡片将接受~50uL的纯细菌培养悬液；卡片将琼脂平板上生长的1个细菌菌落代表的样本与缓冲液混合，提供rRNA的分离、标记、片段和纯化。Peltier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，并在项目第二阶段将微阵列集成到卡中后，将在光学上兼容荧光检测和数据分析。我们将在我们位于阿贡国家实验室的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 minute 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 1 bacterial colony grown on agar plate with the buffers and provide rRNA isolation, labeling, fragmentation and purification. A peltier 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.