Coordinated Nanobiosensors For Enhanced Detection: Integration Of Three Dimensional Structures To Toxicological Applications Joanne I. Yeh †%* , Shoucheng Du † , Tian Xia ‡ , Adam Lazareck # , Jin-Ho Kim # , Jimmy Xu # , and Andre E. Nel ‡ † Department of Structural Biology, University of Pittsburgh Medical School; Pittsburgh, PA 15260; ‡ Division of Clinical Immunology and Allergy, Department of Medicine, University of California, Los Angeles, CA 90095; # Division of Engineering, Brown University, Providence, RI 02912; % Department of Bioengineering; 3501 5 th Avenue, Pittsburgh, PA 15260. ‡ . *Corresponding author: jiyeh@pitt.edu A strategy of metallizing peptides to serve as conduits of electronic signals that bridge between a redox enzyme and a carbon-nanotube electrode, has been utilized with enhanced results. In conjunction, a protocol to link the biological elements to the tips of carbon nanotubes has been developed to optimize contact and geometry between the redox enzyme and the carbon nanotube electrode array. These approaches enable direct means of conformationally stabilizing the state of the enzyme relative to the electrode surface. A peptide of 33 amino acids, comprised of a leucine zipper motif, was mutated to bind divalent metals, conferring conductivity into the peptide. Reaction between a thiolate of the peptide with the sulfenic acid of the NADH peroxidase enzyme formed a peptide- enzyme complex that are fully primed to transduce electrons out of the enzyme active site to an electrode upon binding of its endogenous cofactor, NADH. Scanning electron microscopy shows immobilization and linking of the assembly specifically to the tips of carbon nanotube electrodes, as designed. Isothermal titration calorimetry and mass spectrometry indicate a binding stoichiometry of at least three metals bound per peptide strand. The crystal structure of the peptide, solved by X-ray crystallography to 1.6 Å resolution, reveals high structural integrity. The peptide is comprised of linear helices, with an interhelical angle of 35 degrees, forming an “X” shape that coordinates metals at its outer faces. These results show that such peptide bridges can efficiently serve as spacers and as conduits of electronic signals, and are especially useful for coordinated and specific linkage to biomolecular sources. Sensitivity of this system to reactive oxygen species generated in-situ was recently demonstrated. Overall, these results highlight the gain that can be achieved when the signal tranducing units of a biosensor are aligned through directed peptide chemistry. In conjunction with specific linkage to CNT electrodes, arrays of amperometric nanobiosensors can be fabricated to detect multiple signals. ECS Transactions, 3 (29) 115-126 (2007) 10.1149/1.2753296, copyright The Electrochemical Society 115