Peptide nanowires for coordination and signal transduction of peroxidase biosensors to carbon nanotube electrode arrays

Abstract

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 developed 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. A peptide nanowire 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 assembly that are fully primed to transduce electrons out of the enzyme active site to an electrode. 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. Overall, these results highlight the gain that can be achieved when the signal tranducing units of a biosensor are aligned through directed peptide chemistry.

Figure 1

SEM image taken at a 30 degree tilt angle; scale bar represents 100 nm, of the bare CNT electrode array.

Figure 2

SEM of CNT Electrode Arrays. A: immobilized bioassembly, localized to CNT tips. B: Controlled deposition of bioassembly can be achieved to attenuate or amplify output.

Figure 3

Formation of a CNT-Npx electrode through subsequent EDC and NHS conjugation. represents the peptide nanowire.

Figure 4

MALDI-TOF-MS spectra of peptide MHP binding complex with Co²⁺ in 10 mM Tris/HCl, pH 9.0. MH⁺ = 4143.11.

Figure 5

of CNT electrode immobilized with Npx-MHP-Co²⁺-AuNP (solid line) and with Npx-AuNP (dash line) bioassemblies in 0.1 M potassium acetate buffer, pH 5.4 at a scan rate of 100 mVs⁻¹.

Figure 6

Plot of peak currents of Cyclic voltammetrys (CVs) of a CNT electrode immobilized with Npx-MHP-Co²⁺-AuNP vs scan rates. The CVs were measured at scan rates of 50, 100, 200, 300, 400, 500 mV/s respectively in 0.1 M potassium acetate buffer, pH 5.4.