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Nanofluidic Sequencing of Polypeptides


OBJECTIVE: Design, fabrication, and demonstration of an electrophoretic capillary nanofluidic integrated sensor platform effective for sequencing polypeptides. The goal is to rapidly determine the amino acid sequence of a large polypeptide in a non-destructive manner. DESCRIPTION: Standard methods of proteomics, such as mass spectrometry and SDS-PAGE, involve an extensive amount of sample preparation that is usually performed in a well-equipped laboratory. Such methods are very difficult to move to field environments. Also, standard methods of proteomics involve fragmenting a protein into small peptides before analysis. Following analysis of the smaller peptides, the researcher is required to reassemble the data to determine the amino acid sequence of the original large protein. There is a need in the DoD for rapid non-destructive methods of protein analysis that have the potential for use in the field. Nanofluidic analysis has emerged as a method to address this important problem. Recently, methods have been developed to rapidly separate long-strand polymers according to length. The separation mechanism utilizes confinement-induced forces to separate the polymers into different size fractions. Researchers have examined interfaces between regions of vastly different configuration entropy, where small fragments can become trapped in favorable regions, but the larger fragments cannot completely enter due to the large size. Researchers have also examined mechanical and/or field-induced dielectrophoretic trapping due to the surface roughness within nanopores and selective binding of proteins and nucleic acids to silica particles. Nanofluidics holds the promise for rapid, inexpensive, non-destructive analysis of biopolymers. In particular, nanofluidics may address rapid, non-destructive determination of the amino acid sequence of a large polypeptide. A better understanding of the transport behavior of long and short biopolymer strands in nanochannels is required. Better reporter mechanisms are also needed. PHASE I: Examine the transport behavior of large polypeptide strands in fused silica nanochannels with the application of electrical fields of different strengths. Examine mechanical and/or field-induced dielectrophoretic trapping due to the surface roughness within the nanochannels. Examine areas within the nanofluidic channels with induced interfaces between regions of vastly different configuration entropy. Examine methods for utilizing polypeptide transport in nanochannels as a method for the non-destructive determination of the amino acid sequence. Examine methods of amino acid reporting, including optical and electrical methods. PHASE II: During Phase II, the offeror should build and test a functioning nanofluidic proteomics platform. The research and development work should include an assessment of the prototype"s ability to analyze large polypeptides and report the amino acid sequence. The nanofluidic proteomics system should allow for stand-alone operation with fluidic and electrophoretic control that is self-sustained to facilitate a transparent operation by the user, with the ultimate goal of developing a system that can be used in a field environment. The ultimate goal of the effort is to develop a system that is the size of a smart-phone that can operate on available battery power. Analysis time will depend on the size of the polypeptide strand under analysis. Target analysis time should be approximately 30 minutes. PHASE III: Further research and development during Phase III efforts will be directed towards refining a final deployable design, incorporating design modifications based on results from tests conducted during Phase II, and improving engineering/form-factors, equipment hardening, and manufacturability designs to meet U.S. Army CONOPS and end-user requirements to include the Joint Chemical and Biological Defense Program (CBDP). Specifically improved nanofluidic analysis will have relevance to scientific studies on biological materials and structures, to the detection and identification of biological threats, to medical diagnostics of biological induced diseases, to the monitoring of commercial consumables for biological contamination, just to name a few possibilities.
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