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To identify biologically relevant compounds in basic biology and drug discovery processes, rapid quantitative methods for elucidating protein protein interactions have become necessary. We describe a novel optical technique for monitoring protein-protein interactions in living cells based on complementation of split luciferase fragments from click beetle (Brazilian Pyrearlnus termitilluminans). A new pair of amino-terminal and carboxy-terminal fragments of the luciferase was identified using semirafional library screening, demonstrating achieved markedly higher sensitivity and signal-to-background ratio. The identified fragments were applied to the study of five G-protein coupled receptors (GPCR) that interact with [beta]-arrestin on the plasma membrane. By generating cell lines stably expressing the GPCRs and [beta]-arrestin connected with the luciferase fragments, we demonslrated rapid and sensitive screening of potential chemicals that act on GPCRs using a 96-well microtiter plate format. The screening time was reduced to 5-10 min after ligand stimulation. The maximum response became more than 15-fold higher than the background signal. This luciferase complemenration method also enabled accurate spatial and temporal analyses of interactions in single living cells using bioluminescence microscopy. These GPCR assays will facilitate developments of high-throughput screening systems in a multiwell plate format. Furthermore, using specific proteins of interest, the novel fragments of luciferase will provide different assay methods for the study of many intracellular signals in living cells and animals. 10.1021/ac100104q

Abstract: A novel hydrogen peroxide biosensor has been designed based on the co-immobilization of hemoglobin (Hb), TiO2 nanotubes and methylene blue (MB) onto a glassy carbon (GC) electrode. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; and then detached from the Ti substrate using ultrasonication. A pair of well-defined redox peaks of Hb, enhanced by MB, appeared at an applied potential around −0.425V, revealing that the immobilized Hb exhibits direct electrochemistry. Our electrochemical study shows that the immobilized Hb remains highly bioactive and displays excellent electrocatalytic response toward H2O2. The effect of pH and applied electrode potential in H2O2 sensing has been systemically studied. Besides the advantages of fast response and high stability, under the optimized conditions the linearity of the developed biosensor for the detection of H2O2 was observed from 2×10−7 to 1.6×10−4 M with a detection limit as low as 8×10−8 M (based on S/N =3). [Copyright &y& Elsevier]

Abstract: The formation of horseradish peroxidase-lipid Langmuir–Blodgett film and its applicability as a biosensor have been studied. HRP was spread directly onto the subphase covered with a layer of lipid in LB trough. Our experimental results showed that surface pressure of this film from liquid to solid state ranged between 15 to 25 mN/m. At surface pressure of 25 mN/m, the monolayer was successfully transferred onto the gold surface. In addition, electrochemical properties of this film showed that protein molecules still kept their natural structure and can give a well electrocatalytic activity to H2O2. As an H2O2 biosensor, this LB film was able to detect concentration of H2O2 which were 3×10−7 M. [Copyright &y& Elsevier]