Nanopores for trans-membrane bio-molecule detection

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力学，否则无法通过测量分子或细胞的整体来获得。细胞内调控网络的完整阐明将需要从单个细胞获得的时间分辨基因表达数据来确定网络反馈环的时间常数。已经证明，细胞生物学中的网络和电子电路之间有很强的相似性--细胞生物学家目前可用的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上的信息。细胞生物学家最终将需要生物学上的示波器来实时对活细胞中的生物分子水平进行微创测量。单分子技术是目前这种工具最有希望的候选者。此外，单分子方法可能导致高灵敏度的分析，具有广泛的应用，包括基因分型、基因表达研究和蛋白质检测。可以想象，单分子纳米传感器阵列将提供类似于微阵列的数据，用于基因表达或SNP测定，但具有更高的数据质量和更高的灵敏度。在初步工作中，我们开发了一种有机纳米传感器，能够检测和区分跨越脂质膜的相似核酸链。 该传感器基于2纳米宽的蛋白质通道，该通道自组装成脂膜，在外加电场的作用下，工程核酸和蛋白质结构插入孔中。这种纳米传感器的组装导致一条核酸尾巴通过插入小孔的脂质双层突出。这种尾巴被设计成与特定的分析物结合，这样当结合分析物时，试图从毛孔中取出尾巴时，就会遇到阻力-整个操作导致了类似于冰钓的事情。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。在这一应用中，我们建议扩展这项工作，以确定这种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，以改进对核酸和其他生物分子的检测。虽然这项研究超出了最初的应用范围，但这项研究的最终目的是为研究细胞功能和复杂的细胞疾病(如癌症)提供一个强大的生物分子体内传感工具，以及用于高精度基因表达定量和改进的低成本基因分型的新型合成纳米传感器阵列。

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.