Use of nanotechnology to rapidly detect human pathogens

描述(由申请人提供)：为了降低不断上升的医疗费用，快速的“护理点”(POC)检测在过去十年中急剧增长。由于相对容易，免疫学方法经常用于此类测试。然而，分子分析(即基于DNA和/或RNA)在很大程度上局限于临床实验室环境，在那里可以执行本质上更复杂的样品制备和分析程序。纳米技术提供了一个独特的机会，将分子技术带给患者，从而改善诊断数据，进一步降低医疗成本。我们建议开发一种纳米医学设备，用于直接检测人类病原体中的特定核酸序列。通过取消酶基因扩增步骤，POC测试可以更容易地在预期的快速时间框架内完成。病原体的检测将通过检测丰富的rRNA序列来完成。将设计和使用两种类型的寡核苷酸探针。一个(“捕获”探针)将被结合到磁珠上，该磁珠将用于结合和固定所提议的装置内的靶序列。第二个寡核苷酸探针(记者探针)将与脂质体纳米微囊结合，后者又将包裹量子点(QDot)。由于QDot可以根据其大小在特定波长发出荧光，这一特性将使我们最终能够在第二阶段设计一个简单的多分析测试。病原体的目标rRNA序列将通过首先预测其二级结构(使用自由能计算)，然后评估这些序列的特异性和杂交可用性来选择。“捕获”和“报告”探针将被设计成与这些可用序列杂交。将这些技术集成到纳米医学诊断设备中将允许快速检测特定的核酸序列，而不需要事先进行基因扩增，从而提高POC和其他人类体外诊断市场中分子诊断分析的速度和易用性。最后，“样品输入-结果输出”装置的成功开发将极大地简化此类测试的性能。

DESCRIPTION (provided by applicant): In order to reduce escalating health care costs, rapid "point-of-care" (POC) testing has dramatically grown over the last decade. Due to their relative ease, immunologic approaches are frequently used for such tests. Molecular analysis (i.e., based upon DNA and/or RNA), however, has largely been limited to the clinical laboratory setting, where the inherently more complicated sample preparation and analysis procedures can be performed. Nanotechnology provides a unique opportunity to bring molecular technology to the patient, thus improving diagnostic data and further reducing healthcare costs. We propose to develop a nanomedical device for the direct detection of specific nucleic acid sequences in human pathogens. By eliminating enzymatic gene amplification steps, POC testing can be more easily accomplished within the rapid time frame expected for such tests. The detection of pathogenic organisms will be accomplished by detecting abundant rRNA sequences. Two types of oligonucleotide probes will be designed and utilized. One ("capture" probe) will be bound to a magnetic bead that will serve to bind to and immobilize the target sequences within the proposed device. The second oligonucleotide probe ("reporter" probe) will be bound to liposome nanovesicles, which in turn, will encapsulate quantum dots (QDots). Because QDots can be "tuned" to fluoresce at specific wavelengths depending on their size, this feature will permit us to ultimately design a simple multianalyte test during Phase II. The target rRNA sequences of the pathogenic organism will be selected by first predicting their secondary structure (using free energy calculations) and then evaluating those sequences for specificity and availability for hybridization. The "capture" and "reporter" probes will be designed to hybridize to these available sequences. Integration of these technologies into a nanomedical diagnostic device will permit the rapid detection of specific nucleic acid sequences without the need for prior gene amplification, thus improving the speed and ease-of-use of molecular diagnostic assays in the POC and other human in vitro diagnostic marketplaces. Finally, the successful development of a "sample in - results out" device whereby no sample manipulation is required, will dramatically simplify the performance of such tests.