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. ? ?
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