Microgel Tethering for Integrated Microarray-Based RNA Amplification and Detection

用于基于微阵列的集成 RNA 扩增和检测的微凝胶束缚

• 批准号: 1402706
• 负责人: Matthew Libera
• 金额: $ 32.03万
• 依托单位: Stevens Institute of Technology
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1402706&HistoricalAwards=false
• 关键词: Microgel; Tethering; Integrated; Microarray; Based

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.

提案编号：1402706P.I.：利伯拉，马修R.标题：微凝胶拴系用于集成的基于微阵列的RNA扩增和检测意义：当患者进入医院时，症状表明某种形式的感染，确定是否存在感染可能需要长达24小时，并确定特定的传染性物种可能需要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在数小时甚至数天内无法接受最适当的治疗，例如给予正确的抗生素。 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，该技术不仅有可能进行快速诊断，而且还可以为许多不同的患者进行许多此类诊断。 这项技术使用水凝胶-类似于软性隐形眼镜和一次性尿布中的材料-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首先是如何制造这些微观水凝胶，其次是如何修改它们，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1小时的时间内检测感染并将感染物种确定到单个菌株的水平。 然而，目前MDx测定的基本复杂性阻碍了其在临床环境中的广泛使用。 因此，许多医院选择性地使用MDx方法，并且仍然严重依赖非常缓慢的经典采样、培养和表型分析方法。要从根本上改变MDx测试开发的当前模式，需要一种新的、不同的平台技术。我们假设，表面图案化的微凝胶可以集成检测，等温扩增，并在一个单一的微流控室广泛的多路复用，从而最终简化整个分子诊断过程。 具体而言，本研究项目：（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，该探针连接到高度水合的电子束图案化的聚（乙二醇）[PEG]微凝胶;（ii）正在建立一种新的基于微阵列的等温，RNA扩增方法，将扩增引物放置在紧邻MB检测探针的位置，从而实现高度多重的测定;和（iii）利用电子束图案化和微流体组件的固有微尺度特性来控制样品体积大小并最终促进靶-引物杂交。 重要的是，该项目探索了基于固相核酸序列的扩增（SP-NASBA）的新概念，其中引物集，如分子信标探针，被拴在表面图案化的微凝胶上。 固定引物与目前的做法有很大的不同。 成功将需要仔细了解的性质和空间分布的化学功能位点的表面上的一个单独的微凝胶，以及分区的这些网站拴各种扩增引物和分子信标检测探针。