Nanopores for trans-membrane bio-molecule detection

用于跨膜生物分子检测的纳米孔

• 批准号: 7119686
• 负责人: ANDRE MARZIALI
• 金额: $ 20.94万
• 依托单位: UNIVERSITY OF BRITISH COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7119686
• 关键词: bioengineering /biomedical engineering; cell membrane; molecular probes; nanotechnology; nucleic acid quantitation /detection; oligonucleotides; small molecule

DESCRIPTION (provided by applicant): Single-molecule approaches to the collection of biological data can reveal temporal dynamics of processes that would otherwise be unavailable through measurements of ensembles of molecules or cells. The complete elucidation of regulatory networks in cells will require time-resolved gene expression data obtained from a single cell to determine the time constants of the network feedback loops. It has been shown that there is a strong analogy between networks in cell biology and electronic circuits - present tools available to cell biologist are the equivalent of a voltmeter in electronics, yielding information only on slowly varying averages. Cell biologists will eventually need the biological equivalent of an oscilloscope to perform minimally invasive measurements of bio-molecule levels in live cells in real time. Single molecule techniques are the most promising candidate at this time for such a tool. Furthermore, single molecule approaches may lead to highly sensitive assays with broad applications including genotyping, gene expression studies, and protein detection. It is conceivable that arrays of single-molecule nanosensors would provide data similar to microarrays for gene expression or SNP determination, but with increased data quality and higher sensitivity. In preliminary work, we have developed an organic nanosensor capable of detecting and distinguishing between similar nucleic acid strands across a lipid membrane. The sensor is based on a 2 nm wide protein channel that self-assembles into a lipid membrane, with an engineered nucleic acid and protein construct inserted into the pore under an applied electric field. This nanosensor assembly results in a nucleic acid tail protruding through the lipid bilayer the pore is inserted in. This tail is engineered to bind to specific analytes, such that when an analyte is bound and an attempt is made to withdraw the tail from the pore, resistance is encountered - the whole operation resulting in something analogous to ice-fishing. We have successfully used this nanosensor to detect and characterize binding of single DNA strands. In this application, we propose an expansion of this work to determine the operating limitations of this prototype nanosensor, and to develop additional nanosensor prototypes for improved detection of both nucleic acids and other bio-molecules. Though beyond the scope of this initial application, this research is intended to eventually provide a powerful tool for in-vivo sensing of bio-molecules for the study of cellular function and complex cellular diseases (such as cancer), as well as novel synthetic nanosensor arrays for highly accurate quantitation of gene expression and improved, low cost genotyping.

描述（由申请人提供）：收集生物数据的单分子方法可以揭示过程的时间动态，否则通过测量分子或细胞的集合将无法获得这些过程。细胞调控网络的完整阐明将需要从单个细胞获得的时间分辨的基因表达数据，以确定网络反馈回路的时间常数。研究表明，细胞生物学中的网络和电子电路之间有很强的相似性-细胞生物学家可用的现有工具相当于电子学中的电压表，只能产生缓慢变化的平均值的信息。细胞生物学家最终将需要相当于示波器的生物学设备，以便对活细胞中的生物分子水平进行真实的实时微创测量。单分子技术是最有前途的候选人在这个时候这样的工具。此外，单分子方法可导致具有广泛应用的高灵敏度测定，包括基因分型、基因表达研究和蛋白质检测。可以想象的是，单分子纳米传感器阵列将提供类似于用于基因表达或SNP测定的微阵列的数据，但具有提高的数据质量和更高的灵敏度。在初步工作中，我们已经开发出一种有机纳米传感器，能够检测和区分脂质膜上的相似核酸链。 该传感器基于2 nm宽的蛋白质通道，该通道自组装成脂质膜，在施加的电场下将工程核酸和蛋白质构建体插入孔中。这种纳米传感器组件导致核酸尾突出穿过孔插入其中的脂质双层。这条尾巴被设计成与特定的分析物结合，使得当分析物被结合并试图从孔中取出尾巴时，会遇到阻力-整个操作导致类似于冰钓的东西。我们已经成功地使用这种纳米传感器来检测和表征单链DNA的结合。在此应用中，我们提出了这项工作的扩展，以确定该原型纳米传感器的操作限制，并开发额外的纳米传感器原型，用于改进核酸和其他生物分子的检测。虽然超出了最初的应用范围，但这项研究旨在最终为生物分子的体内传感提供一个强大的工具，用于研究细胞功能和复杂的细胞疾病（如癌症），以及新型合成纳米传感器阵列，用于基因表达的高度准确定量和改进的低成本基因分型。