Immuno-Based Molecular Scaffolding of Glucose Dehydrogenase and Ferrocene Mediator on fd Viral Particles Yields Enhanced Bioelectrocatalysis

Abstract

A virus-based nanostructuring strategy is proposed for improving the catalytic performance of integrated redox enzyme electrodes. Random arrays of adsorbed filamentous fd bacteriophage particles, used as scaffolds, are assembled onto gold electrode surfaces. The viral particles are endowed with functionally coupled enzymatic and redox properties, by the sequential immunological assembly on their protein shell of quinoprotein glucose dehydrogenase conjugated antibodies and ferrocene PEGylated antibodies. The resulting virus-scaffolded enzyme/redox mediator integrated system displays a large enhancement in the catalytic current generated per enzyme molecule (i.e. in enzymatic turnover) as compared to non-scaffolded integrated glucose oxidizing enzyme electrodes. The mechanism underlying the observed scaffolding-induced catalytic enhancement is deciphered. Confinement of the mediator on the viral scaffold enables fast electron transport rate and shifts the enzyme behavior into its most effective cooperative kinetic mode.

Probing the Enzymatic Activity of Individual Biocatalytic fd-Viral Particles by Electrochemical-Atomic Force Microscopy

“…This result is in agreement with our previous finding that confinement of the integrated system on the fd-scaffold turns the enzyme into its fastest kinetic mode. 47 Moreover, it also demonstrates the validity of our Mt/AFM-SECM approach, and its associated theoretical model, for probing and analyzing the catalytic activity of individual nano-scaffolded integrated enzymatic systems.…”

“…The anti-rabbit GDH-IgG conjugate was prepared in-house by conjugating PQQ-GDH to a polyclonal goat anti-rabbit IgG, as previously detailed. 47 The anti-GDH polyclonal antibody was custom produced in rabbit by Genosphere Biotechnologies using apo-GDH monomers as antigens. The goat anti-rabbit antibody to be conjugated with GDH or Fc-PEG chains, and the bovine serum albumin (BSA) (IgG-free grade) blocking agent were from Jackson ImmunoResearch Laboratories.…”

“…Dotted blue curve: Dependence of the experimental k app value measured at the ensemble scale by cyclic voltammetry in our previous work. 47 One can clearly visualize that the data points presented in all of the cross-correlation plots tend to align along straight lines passing through the origin. This result indicates that the catalytic tip current determined for all of the particles are simply proportional to each other.…”

“…This result evidences that confinement of the flexible Fc-PEG chains on the fd-scaffold enables a fast electron transport rate, which, as shown previously, contributes to enhance the catalytic performance of the Fc-PEG/GDH integrated system, as compared to non-scaffolded systems. 47 The question that arises now is how can one quantitatively Examination of the kinetic plots reproduced in Figure 4B reveals that, for all of the particles interrogated, qualitatively similar bell-shaped variations of the catalytic tip current with glucose concentration are obtained. Yet, one also notices that the magnitude of the curves can differ from one particle to the other, and so can possibly the exact shape of the curves ( Figure 4B).…”

Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

“…VLPs have made significant advances in various fields, from vaccinology to industrial uses due to their promising characteristics, including monodispersed particle size distribution, defined geometric surfaces, biosafety, and functional programmability (Mohsen et al, 2017;Janitzek et al, 2019). Additionally, the viral capsids are stable over a wide range of environmental conditions, such as temperatures and pHs, which make them suitable for different applications, including industrial biocatalysis (Stanley, 2014;Koch et al, 2015;Brasch et al, 2017;Torbensen et al, 2019). Nevertheless, VLPs are challenging to be abundantly produced at the industrial scale (Mohsen et al, 2017;Tan and Jiang, 2017).…”