Our lack of rapid detection protocols for bioterrorism agents, pathogenic agents. and affordable, easy to operate devices suitable for deployment in field environments has been demonstrated by the confusion and the number of deaths due to exposure to anthrax spores in the past several months. It is chilling to envision the potential chaos that results from simultaneous exposure to multiple pathogenic agents. Effective biosensors require a robust, flexible bioactive signal transduction system, based on the attachment of specific antibodies or DNA probes to a biosensor surface, in a microarray or chromatographic formats. In the long term we propose to synthesize monodisperse macromolecular dendrimers that offer unique design capabilities which allow positioning of recombinant antibodies, multivalent functionalities varied depending on the sensor surface, and a variety of reporter molecules, all of which can be varied independent of one another and spatially separated by synthetic design. Recombinant and monoclonal antibodies must be suitably derivatized with a unique functional group for covalent attachment to, and orientation on, the dendritic tether. In this proposal we describe a highly effective and robust design approach consisting of monodisperse, heterobifunctional two-arm dendrimers that can covalently bind to a variety of derivatized antibodies and also possess multiple thiol functionalities to greatly enhance their attachment to gold, for use in surface plasmon resonance devices, quartz crystal microbalance, and colloidal gold biosensors. To conjugate an antibody to the dendrimer tether we provide the tether with a hydrazide functionalized arm and add an aldehyde functional group to serine residues in the peptide tether of a recombinant antibody directed against anthrax protective antigen. To accomplish derivatization of the antibody, a recombinant 0-glycosyltransferase enzyme will be modified by recombinant techniques such that it will transfer a sugar molecule to the serine residues in the peptide linker of the antibody. The proposal is thus two-pronged:(1) to synthesize dendritic tethers having one arm derivatized for attachment to a gold surface and one arm with a hydrazide functionality (2) to functionalize a previously unglycosylated antibody with a reactive aldehyde group. The result is an antigen-specific capture agent tethered to a dendrimer functionalized for attachment to a biosensor surface.
