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保健成本，快速“护理点”（POC）测试在过去十年中急剧增长。由于其相对容易，免疫学方法经常用于此类测试。分子分析（即，然而，基于DNA和/或RNA）在很大程度上局限于临床实验室环境，其中可以进行固有地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，从而改善诊断数据并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。通过消除酶基因扩增步骤，POC测试可以更容易地在预期的快速时间范围内完成。病原微生物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。一种（“捕获”探针）将与磁珠结合，磁珠将用于结合并固定所提出的装置内的靶序列。第二寡核苷酸探针（“报告”探针）将结合到脂质体纳米囊泡，其进而将包封量子点（QDot）。由于QDots可以根据其大小进行“调整”，以在特定波长下发出荧光，因此这一功能将使我们能够在第二阶段最终设计一种简单的多分析物测试。通过首先预测其二级结构（使用自由能计算），然后评价这些序列的特异性和杂交可用性，选择病原生物体的靶rRNA序列。“捕获”和“报告”探针将被设计为与这些可用序列杂交。将这些技术整合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测定的速度和易用性。最后，成功地开发出一种无需样品操作的“样品输入-结果输出”装置，将大大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.