Conformation and Activity of Glucose Oxidase on Homogeneously Coated and Nanostructured Surfaces

Abstract

Protein unfolding and loss of protein function upon surface contact is a major problem in biotechnology and biomedicine. Using glucose oxidase (GOx) as a model protein, we investigated the impact of surface chemistry, topography, and confinement on enzyme activity, conformation, and affinity. A particular focus lay on the question whether the conformation of surface-bound proteins can be stabilized by embedding nanoscale adsorption sites, here in the form of monodisperse gold nanoparticles (AuNPs), into a protein-repelling matrix material. It was found that on homogeneous surfaces, GOx activity is generally lower than that in its native state and strongly affected by surface chemistry. Loss of activity is related to an increasing amount of β-sheets in the GOx secondary structure and a corresponding reduction of α-helical elements. In contrast, on AuNP surfaces, the effect of surface chemistry is negligible, and the amount of adsorbed protein only depends on particle size. The low activity of GOx on all nanostructures studied is again accompanied by an increase of β-sheet and a reduction of α-helical secondary structure. The major cause for protein unfolding on AuNPs thus seems to be the curvature of the surface. In addition, the data suggest that unfavorable orientation of the adsorbed enzyme also contributes to the loss of activity.

Recent advances in electrochemical biosensors based on graphene two-dimensional nanomaterials

“…Moreover, GOx adsorbed on a graphene surface was hardly able to maintain the enzyme activity in a biosensor, which indicated that graphene would significantly decrease the average enzymatic activity of GOx ( Figure 3). A similar loss of enzyme activity of GOx adsorbed on nanomaterial-modified surfaces, such as CNT (Wooten et al, 2014, Goran et al, 2013 and Au NPs (Seehuber and Dahint, 2013), have also been reported previously (Table 1). …”

Transforming the blood glucose meter into a general healthcare meter for in vitro diagnostics in mobile health

“…The efficiency of DET depends not only on the distance between the active site of the enzyme and the electrode but also on the properties of the electrode material and on the immobilization technique (Tellechea et al, 2012, Ludwig et al, 2013). For the limited number of GOx and GDH that are capable of DET, the efficient electron transfer still requires conformational change of the enzyme, which may result in an obvious loss of activity (Felice et al, 2013, Seehuber and Dahint, 2013). An appropriate balance between the enzymatic and electrochemical activities is vital for DET glucose biosensors, and more innovation is still necessary to reach that balance and a commercially viable product.…”

Improvement of immunoassay detection sensitivity by using well-defined raspberry-like magnetic microbeads as carriers

“…3(d), for a sample mixed with low concentration biotin, its reactive sites on SA are mostly occupied by biotin-HRP which The saturation amount of biotin is determined by the inflection point on absorbance curve. Theoretically, an SA can bond 4 biotins, but actually an SA molecule on a CMMB bonds only 0.6 biotin while an SA on an RMMB bonds 1.6 biotin in our experiments, which may be due to SA deformation on solid surface [19][20] . Nevertheless, RMMBs show a more efficient and adequate affinity between SA and biotin than CMMBs.…”