Electron Transfer Reactions and Stability of Bio-Anodes with PQQ-Dependent Dehydrogenases

Tuesday, May 13, 2014: 10:40
Floridian Ballroom G, Lobby Level (Hilton Orlando Bonnet Creek)
R. J. Lopez, S. Babanova, and P. Atanassov (University of New Mexico, Center for Emerging Energy Technologies)
The performance of enzymatic electrodes for biofuel cells or bio-sensors is subject among other factors to the rate of interfacial electron transfer between the enzyme and the surface of the electrode as well as decrease in enzyme activity due to enzyme immobilization and operation. Therefore, one approach towards improving generated current, stability, and consistent reproducibility of enzymatic electrodes is to optimize the enzyme-electrode interactions. Towards this goal it has been demonstrated previously by our group that covalent attachment of multi-copper oxidase enzymes to carbonaceous electrodes through a bifunctional cross-linker, 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBSE), resulted in improved interfacial electron transfer and subsequent higher generated current relative to physisorbed enzymes on bare multi-walled carbon nanotubes (MWNT)[1].

In the current work, a similar technique is applied for the immobilization of Pyrroloquinoline quinone (PQQ) dependent dehydrogenases using three cross-linkers (PBSE, N-(1-Pyrenyl) maleimide (PYMAL) and 1-Pyrenecarboxylic acid (PCA)) selected based on their similar structures to that of PBSE. PQQ-dependent dehydrogenases are of interest to the application of biofuel cells and sensors due to their high catalytic activity, insensitivity to oxygen and broader substrate specificity[2] relative to NAD+/FAD+ dependent proteins. Electrochemical characterization using linear voltammetry and potentiostatic polarization of anodes developed using soluble Pyrroloquinoline quinone-dependent Glucose dehydrogenase (sGDH-PQQ) indicate that incorporation of a cross-linker onto the anode surface results in a significant increase in generated current (Fig. 1).  This result implies that the length of the linker “tail” determines the distance between the attached enzyme and the electrode; shorter tail – shorter hoping distance for the electrons, resulting in higher electron transfer efficiency. Further chronoamperometry studies indicate an improvement in electrode stability and resistance to enzyme activity degradation when compared to physisorbed sGDH-PQQ on MWNT.  Expanding on the close interaction of the enzyme to the electrode substrate provided by the cross-linker, this work further characterizes enzyme activity of sGDH-PQQ as well as various quinohaemoproteins in situ immobilized on MWNT-paper to determine the most effective cross-linker for improving interfacial electron transport and preserving enzyme activity when immobilized. 

Subsequent to the selection and incorporation of a cross-linker, heme and heme analogues are covalently attached to the surfaces of gold and MWNT electrodes via techniques including electrochemical grafting of diazonium salts possessing amide functional groups which can be used to covalently attach hemes and their analogues to the electrode (Fig. 2).  The heme is a natural electron “mediator” that carries out the internal electron transfer within quinohaemoproteins.  Therefore, incorporating hemes or other selected porphyrins possessing appropriate redox potential in close proximity to the enzyme could improve the electron transfer from the enzyme to the electrode.  Enzymatic anodes containing immobilized hemes or heme analogues in combination with cross-linking agents are characterized for generated current, enzyme activity, and stability using various electrochemical techniques. 

[1]           S. Brocato, C. Lau, P. Atanassov, Electrochimica Acta 2012, 61, 44-49.

[2]           C. Anthony, Antioxidants & Redox Signaling 2001, 3, 757-774.