Synthesis of a Ferrocene-Modified Coenzyme for Electrochemical Biosensor Applications

Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme for many dehydrogenases that participate in the oxidation or reduction of metabolities. The NAD⁺ is also consumed in a process called ADP-Ribosylation. The reaction is typically catalyzed by ADP-ribosyltransferases which transfer the ADP-ribose moiety from NAD⁺ to specific proteins. Pseudomonas aeruginosa (Pa) releases two ADP-ribosyltransferases; Exotoxin A and Exoenzyme S. Exo A acts on the eukaryotic elongation factor 2 (eEF-2) while Exo S acts on GTPases. These toxins are implicated as major pathogenic factors in Pa infections. The detection of these toxins could help in the early detection of pathogens. We have synthesized a ferrocene-labeled NAD⁺ cofactor analog (Fc-NAAD) through the reductive amination of the primary amine in the adenine moiety, using FcCOH and NaCNBH₃. Our new product shows chemical reversibility (Ipa/Ipc = 1.087 uA at 200 mV/s) and electrochemical quasi-reversibility. OSWV analysis showed an anodic peak in 292 mV for Fc-NAAD. Compared with the anodic peak of FcCOH in 508 mV the potential decreases for Fc-NAAD. H¹-NMR and C¹³-NMR studies showed the presence of NAAD aromatics in the 8.0-9.5 ppm and 125-160 ppm regions while the ferrocene appeared in the 4.2-5 ppm and 65-90 ppm regions. UV-VIS spectroscopy analysis showed a 259.41 nm absorption peak that was assigned to the adenine moiety absorption in NAAD. We have modified glassy carbon electrode surfaces with Fc-NAAD using several poly(Styrene-co-N-acryloxysuccinimide) copolymers and our work is now focused on testing and optimizing the electrochemical detection of a gram-negative bacteria.