Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins.

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Protein Science (Impact Factor: 2.86). 09/2006; 15(8):1936-44. DOI: 10.1110/ps.062261606
Source: PubMed

ABSTRACT Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.

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    • "In the field of GBPs, there has been work in engineering of polypeptide sequences to enhance performance and accept various signal transduction markers (i.e., optical and electrochemical labels) [33] [34] [35] [36] [37] [38]. There have also been some reports in the literature on the immobilization of GBPs towards a glucose-sensing system [39]. However, to the best of the authors' knowledge, there have been no reports to date on the successful immobilization of GBPs or GBP-like sensing elements within a fixed matrix such that a continuous glucose-sensing device is realized. "
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    Sensors and Actuators B Chemical 08/2010; 149(1-149):51-58. DOI:10.1016/j.snb.2010.06.031 · 3.84 Impact Factor
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    • "This can be used to link and spatially orient the biomolecule on the gold surface [15] [16] [17] [18] [19] [20] [21] [22]. When a surface-exposed cysteine residue on a protein is not feasible, thiol linkers to other residues on a protein allow semispecifically oriented layers on the surface [23]. Although there has been some discussion in the literature pointing to protein deposition without exposed cysteines, a number of studies from our laboratories have indicated that native glucose/galactose receptors (GGRs) without a genetically engineered cysteine residue do not bind to the gold surface, whereas receptors with a surface-exposed cysteine form a stable affinity bond to gold surfaces [15] [16] [17] [18] [19] [20] [21] [22]. "
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    ABSTRACT: Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur-gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV-Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 microM.
    Analytical Biochemistry 05/2008; 375(2):282-90. DOI:10.1016/j.ab.2007.12.035 · 2.31 Impact Factor
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    • "dependent emission presumably because of reduced solvent accessibility or loss of conformational flexibility. While a few groups have investigated surface attachment [16] [17] [18], these difficulties with immobilization need to be addressed prior to the production of a practical, implantable fluorescent protein biosensor. "
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