Efficiency of Site‐Specific Clicked Laccase–Carbon Nanotubes Biocathodes towards O₂ Reduction

Abstract

A maximization of a direct electron transfer (DET) between redox enzymes and electrodes can be obtained through the oriented immobilization of enzymes onto an electroactive surface. Here, a strategy for obtaining carbon nanotube (CNTs) based electrodes covalently modified with perfectly control‐oriented fungal laccases is presented. Modelizations of the laccase‐CNT interaction and of electron conduction pathways serve as a guide in choosing grafting positions. Homogeneous populations of alkyne‐modified laccases are obtained through the reductive amination of a unique surface‐accessible lysine residue selectively engineered near either one or the other of the two copper centers in enzyme variants. Immobilization of the site‐specific alkynated enzymes is achieved by copper‐catalyzed click reaction on azido‐modified CNTs. A highly efficient reduction of O2 at low overpotential and catalytic current densities over −3 mA cm−2 are obtained by minimizing the distance from the electrode surface to the trinuclear cluster.

Site directed confinement of laccases in a porous scaffold towards robustness and selectivity

“…Redox reactions occur at two copper-containing centers: a surface located T1 Cu II binding center (blue copper site) responsible for the oxidation of substrate and a burried trinuclear copper T2/T3 center (TNC) responsible for the reduction of O 2 into water. In the past decades, a significant number of reports have focused on the application of this enzyme in technological and bioremediation processes [ 35 ] in addition to their use in organic synthesis [ 36 , 37 ] and as cathodic catalyst [ [38] , [39] , [40] , [41] , [42] ] reducing dioxygen into water in biofuel cells. Laccase has been successfully immobilized on many different types of support attesting for its high stability and robustness [ 3 ].…”

A post-functionalization toolbox for diazonium (electro)-grafted surfaces: review of the coupling methods

“…When the latter are biological application-oriented, chemical coupling with a redox entity constitutes the preliminary step to optimize the reaction, assess the robustness of the linkage and estimate the maximum coupling yield, since the biomolecules to be grafted are biggest than the redox species. 114,129,136,141,156 In these studies, ferrocene, which gives a reversible and stable redox signal, is commercially available, inexpensive and easily chemically functionalizable, was chosen as model molecule. These reasons also motivated the use of this redox probe to validate and better understand different concepts including functionalization of non-conventional electrodes, 132,135,140,157,158 electron transfer, 125,127,128,159 formation of more or less compact monolayers, 105,115,123,124,130,137,139 fabrication of mixed (mono)layers, 105,116,120,122,138 surface structuring, 117,126,131,133 building of dendrimer modified surfaces.…”

“…147 The mild conditions required for the anchoring of the entities by click chemistry make it a very convenient method for the immobilization of biological material, mainly for the development of biosensors. This way has led to the elaboration of DNA (or aptamer), 129,[161][162][163][164] enzyme, 114,141,165,166 non enzymatic protein, 167 peptide 136 or amino acid 168 -modified surfaces.…”

“…The first one, the most usual one, involves the chemical reduction (by L-ascorbic acid or L-sodium ascorbate) of various Cu(II) salts (generally sulfate or acetate) to form Cu(I). 114,117,118,120,[129][130][131]133,136,141,152,153,156,159,161,170,171,182 Large excess in reducing agent (molar ratios [reducing agent/Cu(II)] varying from 1 114,133 to 50 171 ) guarantees a sufficient Cu(I) concentration in the medium, even when the reaction mixture is not kept under inert atmosphere. Cu(I) can also be generated electrochemically by reduction of Cu(II) 126,167,169 or oxidation of Cu(0) (Fig.…”

“…The left columns allow to target the entity to be grafted and the various reactions identified to immobilize these structures. The reactive platforms are drawn 105,115,116,119,[121][122][123][124][125]127,128,134,139,184 graphene, 132 carbon fibers, 157,158 amorphous carbon nitride, 135 BDD, 137 graphite thermoplastic, 140 MWCNT 141 Anion sensing 119 Electronic junctions 127 Catalytic switches 134 Carbon 156 Biological catalysts 156 Post-diazotization GC, 191,196 gold, 191,196 Fluorescent dyes 150 Nitrobenzene Amide coupling Carbon 88,323 Microcontact printing 88 Trace analysis of copper 323 CNT, 102 gold 324 -Click chemistry GC 138 -…”