Quantifying Protein Adsorption and Function at Nanostructured Materials: Enzymatic Activity of Glucose Oxidase at GLAD Structured Electrodes

Abstract

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.

Perspectives on oblique angle deposition of thin films: From fundamentals to devices

“…As with conventional gas sensors, multisensing is also a challenge in the field of biosensor devices. In this context, Sutherland et al [600] have reported a new method that combines protein adsorption on the surface of a nanostructured quartz crystal microbalance (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical detection (cyclic voltammetry CV). The procedure allows for the quantification of both the amount and activity of bound proteins.…”

Hydrogen bioelectrooxidation on gold nanoparticle-based electrodes modified by Aquifex aeolicus hydrogenase: Application to hydrogen/oxygen enzymatic biofuel cells

“…In bioelectrochemistry, AuNP variable size and electronic properties are expected to provide versatile building blocks as well as large surface area-to-volume ratios suitable for high enzyme loading. The activity, stability and electron transfer properties may be altered at nanostructured interfaces compared to flat surfaces, especially when the curvature of the nanoparticle is comparable to the size of the enzyme [11][12][13]. Because gold nanoparticles (AuNPs) with controlled sizes can be quite easily prepared and functionalized by versatile thiol chemistry, AuNP films on electrochemical interfaces have been targeted.…”

“…It was demonstrated that AuNPs can act as conductive wires between the enzymes and the electrode. Long range electron transfer and efficient catalysis were highlighted for various proteins and enzymes immobilized on AuNP films, such as heme proteins including membrane cytochrome oxidases [14][15][16][17], azurin, a blue copper protein [18], glucose oxidase [19,20], and sulfite oxidase [21]. Porous 3D-networks of AuNPs obtained by drop casting of concentrated gold colloids were shown to enhance electrocatalysis by bilirubin oxidase (BOD) [22,23], cellobiose dehydrogenase [24], and laccase [25][26][27][28].…”

Graphene-based Materials and their Nanocomposites with Metal Oxides: Biosynthesis, Electrochemical, Photocatalytic and Antimicrobial Applications

“…Carbon nanomaterials are the most promising materials in the field of smart nanotechnology. Carbon nanomaterials and their composites or metal-doped carbon nanomaterials have interesting optical and electrical properties that are important for smart devices [3][4][5]2]. In addition, carbon nanomaterials-based composites exhibit excellent mechanical, electrical and thermal properties making them potential for various purposes.…”