Engineered Self-Assembling Fvs for Piezoimmunosensors

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DESCRIPTION (provided by applicant): Piezoimmunosensors (PZs) are biosensors in which antibodies are placed on a quartz crystal microbalance (QCM) to detect minute changes in mass as the antibodies bind with antigens. However, use of PZ technology in biosensors is problematic due to the complex nature of whole antibodies, which cause problems with non-specific interactions and molecule trapping. To overcome these problems, an approach in which recombinant antibody variable fragments (Fv) in self assembling monolayers (SAM) serve as the biorecognition agent is proposed. Engineered Fvs will form a "lawn" of antigen-binding sites coating the surface of the QCM, providing a sensing layer which is chemically stable, contains high numbers of binding sites, and forms a thin uniform surface. The initial study will focus on developing an a-human/goat IgG Fv- SAM to test the underlying principle. Four bioengineering methods will be used to create an Fv-SAM that possesses the necessary attributes. Electrochemistry, network impedance analysis, and Atomic Force Microscope (AFM) will be used in parallel to characterize the immobilized Fv-SAM layers for defects/pin hole structure, rigidity, orientation, stability, and surface coverage. The Fv-SAM piezoimmunosensor will be evaluated and validated for sensitivity, selectivity, linearity, stability, detection limits, and reliability in comparison to other immunoassays and/or traditional methods (GC/LC or GC/MS, LC/MS). An array of sensors specific for various antigens/toxins could be automated or combined with flow injection systems to assay different analytes in complex samples with on-line display of results. The project could lead to production of an inexpensive, easy-to-use biosensor which provides fast, precise identification of biological agents