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。：Libera，Matthew R.Title：基于微阵列的综合RNA扩增和检测意义的微凝胶束缚：当患者进入症状的患者进入医院表明某种形式的感染，确定感染是否需要长达24小时，并确定特定的感染物种可能需要72小时或识别72个小时或更多。 在此期间，患者可能会严重遭受痛苦，因为缺乏清晰，快速的诊断意味着患者可能无法接受最适当的治疗，例如数小时甚至几天的治疗方法，例如使用正确的抗生素。 尽管基于分子诊断的新技术通过迅速识别感染物种的DNA开始缓解此问题，但这些新方法无法跟上大型医院所需的吞吐量，这些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，该技术不仅有可能快速诊断，而且还为许多不同的患者做出了许多这样的诊断。 该技术使用水凝胶 - 与软接触透镜和处置尿布中的材料相似 - 大小是微观的，因此每次测试只需要少量的目标DNA。 必须首先要解决如何制造这些微观水凝胶以及如何修改它们的工程和科学问题，因此DNA检测所需的化学反应可以准确，快速进行。技术项目描述：分子诊断（MDX）技术可以检测感染，并确定感染物种在短达一小时内的单个菌株水平。 但是，当前MDX分析的基本复杂性阻碍了它们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，经典，采样，培养和表型的方法。对当前MDX测试开发范式的根本变化将需要一种新的和不同的平台技术。我们假设表面图案的微凝胶可以在单个微流体室内整合检测，等温扩增和广泛的多路复用，因此最终简化了整体分子诊断过程。 具体而言，该研究项目：（i）利用基于分子信标（MB）杂交探针将创新的，实时的，自我报告的基于微阵列的检测方法探测到高度水合的电子束图层的聚（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，该方法将放大引物立即靠近MB检测探针，因此可以实现高度多重的测定； （iii）利用电子束模式和微流体组件的固有显微镜特性来控制样品体积尺寸，并最终促进目标 - 培养子杂交。 重要的是，该项目探讨了基于核相核酸序列的扩增（SP-NASBA）的新概念，其中引物设置（例如分子信标探针）被束缚在表面图案的微凝胶上。 固定底漆与当前的实践有很大的不同。 成功将需要仔细了解化学功能位点在单个微凝胶表面的性质和空间分布，以及这些位点的分区以缠绕各种扩增引物和分子信标检测探针。