Microgel Tethering for Integrated Microarray-Based RNA Amplification and Detection

提案号：1402706pi。题目：基于集成微阵列的RNA扩增和检测的微凝胶系留技术意义：当患者进入医院时，出现某种感染的症状，确定是否存在感染可能需要长达24小时，而确定特定的感染物种可能需要72小时或更长时间。在此期间，患者可能会遭受严重的痛苦，因为缺乏明确和快速的诊断意味着患者可能会在数小时甚至数天内得不到最适当的治疗，例如使用正确的抗生素。虽然基于分子诊断的新技术开始通过快速识别感染物种的DNA来缓解这一问题，但这些新方法无法跟上大医院所需的吞吐量，因为大医院每天必须进行数十次此类测试。因此，该研究项目正在研究一种新技术，该技术不仅有可能做出快速诊断，而且还可以为许多不同的患者做出许多这样的诊断。这项技术使用水凝胶——类似于软性隐形眼镜和一次性尿布的材料——其尺寸非常微小，因此每次测试只需要非常少量的目标DNA。必须解决的工程和科学问题集中在，首先，如何制造这些微观水凝胶，其次，如何修改它们，以便DNA检测所需的化学反应能够准确快速地进行。技术项目描述：分子诊断（MDx）技术可以在短至一小时的时间内检测感染并确定感染物种到单个菌株的水平。然而，当前MDx检测的基本复杂性阻碍了它们在临床环境中的广泛应用。因此，许多医院有选择地使用MDx方法，但仍然严重依赖非常缓慢、经典的取样、培养和分型方法。对当前MDx测试开发范式的根本改变将需要一种新的、不同的平台技术。我们假设表面图案微凝胶可以在单个微流控室中集成检测，等温扩增和广泛的多路复用，从而最终简化整个分子诊断过程。具体来说，该研究项目：(i)利用一种创新的、实时的、自我报告的、基于微阵列的检测方法，该方法基于分子信标（MB）杂交探针，该探针与高度水合的电子束图聚乙二醇（PEG）微凝胶相连；（ii）正在建立一种新的基于微阵列的等温RNA扩增方法，该方法将扩增引物置于MB检测探针附近，从而实现高度复用分析；（iii）利用电子束成像和微流控组件固有的微尺度特性来控制样品体积大小，最终促进目标-引物杂交。重要的是，该项目探索了固相核酸序列扩增（SP-NASBA）的新概念，其中引物集就像分子信标探针一样被拴在表面图案微凝胶上。固定引物是对当前实践的重大偏离。成功将需要仔细了解单个微凝胶表面化学功能位点的性质和空间分布，以及这些位点的划分，以连接各种扩增引物和分子信标检测探针。

Proposal Number: 1402706P.I.: Libera, Matthew R.Title: Microgel Tethering for Integrated Microarray-Based RNA Amplification and DetectionSignificance:When a patient enters a hospital with symptoms suggesting some form of infection, determining whether an infection is present can take as long as 24 hours, and identifying the specific infectious species can take 72 hours or more. During that period, patients can suffer severely, because the lack of a clear and rapid diagnosis means that a patient may not receive the most appropriate treatment, such as the administration of the correct antibiotic, for hours or even days. While new technologies based on molecular diagnostics are beginning to mitigate this problem by rapidly identifying the DNA of infecting species, these new approaches are unable to keep up with the throughput required by major hospitals where dozens of such tests must be run every day. This research project is thus studying a new technology that has the potential to not only make a rapid diagnosis but also make many such diagnoses for many different patients. This technology uses hydrogels - similar to the materials in soft contact lenses and in disposal diapers - that are microscopic in size, so only a very small amount of target DNA is required for each test. The engineering and science questions that must be addressed center, first, on how to make these microscopic hydrogels and, second, how to modify them, so the chemical reactions needed for DNA detection can proceed accurately and quickly. Technical Project Description: Molecular diagnostic (MDx) technologies can detect infection and determine the infecting species to the level of an individual strain in times as short as one hour. However, the fundamental complexity of current MDx assays has hindered their widespread use in clinical settings. Many hospitals thus use MDx methods selectively and still rely heavily on the very slow, classical, method of sampling, culturing and phenotyping. A fundamental change to the current paradigm of MDx test development will require a new and different platform technology. We hypothesize that surface-patterned microgels can integrate detection, isothermal amplification, and extensive multiplexing in a single microfluidic chamber and thus, ultimately, simplify the overall molecular-diagnostic process. Specifically, this research project: (i) exploits an innovative, real-time, self-reporting, microarray-based detection method based on molecular beacon (MB) hybridization probes tethered to highly hydrated electron-beam-patterned poly(ethylene glycol) [PEG] microgels; (ii) is establishing a new microarray-based, isothermal, RNA amplification approach that places the amplification primers in immediate proximity to the MB detection probes and thus enables highly multiplexed assays; and (iii) takes advantage of the inherent microscaling properties of e-beam patterning and microfluidic assembly to control sample volume sizes and ultimately promote target-primer hybridization. Importantly, this project explores the new concept of solid-phase Nucleic Acid Sequence-Based Amplification (SP-NASBA) where the primers sets, like the molecular beacon probes, are tethered to surface-patterned microgels. Immobilizing the primers is a significant departure from current practice. Success will require a careful understanding of the nature and spatial distribution of chemically functional sites on the surface of an individual microgel as well as the partitioning of those sites to tether various amplification primers and molecular beacon detection probes.