Highly Efficient Membraneless Glucose Bioanode Based on Corynascus thermophilus Cellobiose Dehydrogenase on Aryl Diazonium‐Activated Single‐Walled Carbon Nanotubes

Abstract

We present an approach for electrode modification by using the oxidoreductase cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH). CtCDH is a two‐domain enzyme, in which the catalytic dehydrogenase domain (DHCDH) hosts flavin adenine dinucleotide (FAD) as cofactor and is connected through a flexible linker to a small cytochrome domain with a heme b cofactor (CYTCDH). This domain is responsible for the electron transfer from DHCDH to macromolecular electron acceptors, and is capable of direct electron transfer (DET) with electrode surfaces. CtCDH is optimal at pH values between 7 and 9 and exhibits one of the lowest apparent KM values for glucose (2.4×104 μM) in contrast to the majority of other CDHs, which have acidic optimal pH values and have very low or no activity for glucose. Glassy carbon (GC) electrodes were modified by drop‐casting single‐walled carbon nanotubes (SWCTs) and further modifying with aryl diazonium salts (DS). When adsorbed on such GC‐SWCT‐DS electrodes, CYTCDH showed efficient DET in the presence of a substrate. Six different functional groups for the aryl amines were studied and compared to non‐DS modified GC‐SWCT electrodes. The charge of the grafted aryl amines was investigated and it was found that surfaces modified with aniline, 4‐aminophenol, and 4‐aminobenzoic acid (ABA) contain negative charges at pH 7.4. On the other hand, surfaces modified with p‐phenylenediamine (PD), N,N‐dimethyl‐p‐phenylenediamine, and N,N‐diethyl‐p‐phenylenediamine remain uncharged. To date, the highest DET current density (JMAX=32.5 μA cm−2 for lactose and JMAX=16.2 μA cm−2 for glucose) for a CDH‐modified electrode at human physiological pH can be obtained by using a GC‐SWCT‐ABA CtCDH modified electrode. The use of glutaraldehyde (GA) in GC‐SWCT‐PD‐modified electrodes increases the JMAX twofold. The modified electrodes with DS showed a more negative onset potential for the catalytic current. For GC‐SWCT‐ABA modified electrodes and GC‐SWCT‐PD with GA, a change in the onset potential of −62 and −66 mV, respectively, was found compared with non‐DS modified electrodes. The prepared bioanodes show a loss in response current of 15 % after 9 days of continuous measurements from the original signal in 5 mM glucose, using 50 mM phosphate buffer solution at pH 7.40 at 37 °C.

Studying Direct Electron Transfer by Site‐Directed Immobilization of Cellobiose Dehydrogenase

“…In biofuel cells it has been used as an anodic biocatalyst in glucose, lactose or cellobiose biofuel cells . Electrochemical communication between CDH and electrodes has made use of direct electron transfer or mediated electron transfer using different redox mediators or redox polymers and, in recent years, various nanostructured electrodes have been used to increase the efficiency of DET ,,,…”

“…Under suitable conditions, the immobilization of enzymes at electrode surfaces can enhance enzyme stability, activity, selectivity and resistance to inhibition . CDH has been covalently immobilized on electrodes using glutaraldehyde cross‐linking at gold electrodes modified with self‐assembled monolayers (SAMs), or at carbon electrodes modified with diazonium salts . Covalent immobilization of CDH has also been achieved by coupling between glutamic or aspartic acid residues on the enzyme surface to amino‐modified ITO electrodes .…”

Engineering of Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

“…Catalytic waves using CV in the presence of lactose were observed for all the variants and its usage proven for future uses with this electrode material. The presented work combined with the low E values inherent to CDH shows a new methodology to improve the usage of Class II CDHs towards mediator‐less glucose biosensors and BFCs previously reported ,,…”

“…This single Cys to Tyr mutation on the active site of CDH was applied to the Class II CDHs Corynascus thermophilus ( Ct CDH) and Humicola insolens ( Hi CDH) in this work. These enzymes show optimal electrocatalytic properties for DET at neutral pH, a characteristic which has already been explored in biosensors and enzymatic fuel cells (EFC) …”

Carbon Aerogel as Electrode Material for Improved Direct Electron Transfer in Biosensors Incorporating Cellobiose Dehydrogenase

“…Additionally, DET requires a close proximity between the electrode surface and redox active cofactors in or connected to the active site of the redox enzyme . Under these circumstances, taking advantage of the peculiarity of CDH, different types of CDHs were immobilized on various electrode materials such as graphite and diazonium activated carbon nanotubes deposited on glassy carbon , as well as on functionalized electrode surfaces such as alkanethiol functionalized solid gold and gold nanoparticles .…”