Direct Electrochemistry of Phanerochaete chrysosporium Cellobiose Dehydrogenase Covalently Attached onto Gold Nanoparticle Modified Solid Gold Electrodes

Abstract

Achieving efficient electrochemical communication between redox enzymes and various electrode materials is one of the main challenges in bioelectrochemistry and is of great importance for developing electronic applications. Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome composed of a catalytic FAD containing dehydrogenase domain (DH(CDH)), a heme b containing cytochrome domain (CYT(CDH)), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (PcCDH) covalently attached to mixed self-assembled monolayer (SAM) modified gold nanoparticle (AuNP) electrode is presented. The thiols used were as follows: 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MP), 11-mercapto-1-undecanamine (MUNH(2)), 11-mercapto-1-undecanoic acid (MUCOOH), and 11-mercapto-1-undecanol (MUOH). A covalent linkage between PcCDH and 4-ATP or MUNH(2) in the mixed SAMs was formed using glutaraldehyde as cross-linker. The covalent immobilization and the surface coverage of PcCDH were confirmed with surface plasmon resonance (SPR). To improve current density, AuNPs were cast on the top of polycrystalline gold electrodes. For all the immobilized PcCDH modified AuNPs electrodes, cyclic voltammetry exhibited clear electrochemical responses of the CYT(CDH) with fast electron transfer (ET) rates in the absence of substrate (lactose), and the formal potential was evaluated to be +162 mV vs NHE at pH 4.50. The standard ET rate constant (k(s)) was estimated for the first time for CDH and was found to be 52.1, 59.8, 112, and 154 s(-1) for 4-ATP/4-MBA, 4-ATP/4-MP, MUNH(2)/MUCOOH, and MUNH(2)/MUOH modified electrodes, respectively. At all the mixed SAM modified AuNP electrodes, PcCDH showed DET only via the CYT(CDH). No DET communication between the DH(CDH) domain and the electrode was found. The current density for lactose oxidation was remarkably increased by introduction of the AuNPs. The 4-ATP/4-MBA modified AuNPs exhibited a current density up to 30 μA cm(-2), which is ∼70 times higher than that obtained for a 4-ATP/4-MBA modified polycrystalline gold electrode. The results provide insight into fundamental electrochemical properties of CDH covalently immobilized on gold electrodes and promote further applications of CDHs for biosensors, biofuel cells, and bioelectrocatalysis.

Cellobiose dehydrogenase modified electrodes: advances by materials science and biochemical engineering

“…Both the use of nanostructures based on the drop-casting deposition technique of nanomaterials such as single-walled carbon nanotubes (SWCNTs) [47, 89–91] (Ortiz et al submitted) [93] or gold nanoparticles (AuNPs) [48, 49, 51] onto the electrode surface of Au, glassy carbon or graphite electrodes, as well as prefabricated carbon nanotube (CNT)-modified screen-printed electrodes [55, 57, 58] has been used and in all instances the use of nanomaterials resulted in an improved current density. An overview of the nanostructured materials used is presented in Table 3.…”

“…Drop-casting of citrate-stabilised AuNPs with a diameter of 19 nm onto the surface of Au disk electrodes [95] was used in combination with mixed self-assembled thiol monolayers (SAMs), onto which P. chrysosporium CDH [48] or C. thermophilus CDH [49, 51] was covalently attached. The mixed SAM featured different thiols with amino, carboxy or hydroxyl head groups.…”

“…Furthermore a series of genetic work has been done to improve the glucose-oxidising properties of CDH (Ortiz et al submitted) [46], in particular, as well as nanostructuring of both carbon [47] and gold-based electrodes [48, 49] to improve the loading and orientation of CDH on the electrode surface and thus also current densities. Especially in the field of biofuel cells [49–51] great progress has been made on the spatial arrangement of CDH on electrodes (Ortiz et al submitted) [47–49, 52, 53] and CDH-based biosensors [54–58].…”

Development of a fast and simple test system for the semiquantitative protein detection in cerebrospinal liquids based on gold nanoparticles

“…Furthermore nanocrystalline materials can be applied advantageously for sensorial bioelectrochemistry as antibody conjugates in front of the gate of field-effect transistors [29], for amperometric detection of enzyme produced hydrogen peroxide [30], for signal amplification in impedance spectroscopy [31] or for direct electron transfer within protein electrode structures [32,33]. In contact with proteins gold nanoparticles are also able to facilitate the efficient inter protein electron transfer [34].…”

Immobilization of enzymes to silver island films for enhanced enzymatic activity

“…[2], [3], [4] In this regard, enzymes are immobilized on to surfaces through covalent binding [4], direct crosslinking, [5] and encapsulation [6] of enzymes on different platforms such as alumina, [7] silica, [8] electrode [4] and nanoparticles. [9] The extent of enzymatic activity after surface immobilization depends on the binding procedure and on the availability of enzymes to substrates.…”