Counting the number of enzymes immobilized onto a nanoparticle-coated electrode

Abstract

To immobilize enzymes at the surface of a nanoparticle-based electrochemical sensor is a common method to construct biosensors for non-electroactive analytes. Studying the interactions between the enzymes and nanoparticle support is of great importance in optimizing the conditions for biosensor design. This can be achieved by using a combination of analytical methods to carefully characterize the enzyme nanoparticle coating at the sensor surface while studying the optimal conditions for enzyme immobilization. From this analytical approach, it was found that controlling the enzyme coverage to a monolayer was a key factor to significantly improve the temporal resolution of biosensors. However, these characterization methods involve both tedious methodologies and working with toxic cyanide solutions. Here we introduce a new analytical method that allows direct quantification of the number of immobilized enzymes (glucose oxidase) at the surface of a gold nanoparticle coated glassy carbon electrode. This was achieved by exploiting an electrochemical stripping method for the direct quantification of the density and size of gold nanoparticles coating the electrode surface and combining this information with quantification of fluorophore-labeled enzymes bound to the sensor surface after stripping off their nanoparticle support. This method is both significantly much faster compared to previously reported methods and with the advantage that this method presented is non-toxic. Graphical abstractA new analytical method for direct quantification of the number of enzymes immobilized at the surface of gold nanoparticles covering a glassy carbon electrode using anodic stripping and fluorimetry

Interactions of Catalytic Enzymes with n-Type Polymers for High-Performance Metabolite Sensors

“…It has the lowest amount of enzyme with the largest footprint (after P-0), suggesting that the enzyme has an expanded conformation on this surface which we found to be highly negatively charged. Overall, these calculated footprints are generally larger than the theoretical footprint of GOx predicted from its crystal structure (21, 67, or 195 nm 2 ) ,, but are in line with other reported values on Au (between 528 and 823 nm 2 ) and Au nanoparticles (between 250 and 300 nm 2 ), suggesting that GOx flattens out to some degree during adsorption. , Note that the native GOx dimensions are those of a deglycosylated GOx molecule, thus implying a larger structure for the protein in solution . We also stress the qualitative aspect of these calculations due to our assumptions.…”

Insights into Sustainable Glucose Oxidation Using Magnetically Recoverable Biocatalysts

“…The opposite trend was observed for cytosolic phospholipase also adsorbed on vesicles . For NP based surfaces, it was demonstrated that the enzyme immobilization on a high curvature surface is beneficial to retain enzymatic activity by preventing denaturing and minimizing changes in the enzyme tertiary structure. − Alternatively, in ref the activity of GOx on the gold flat surface was higher than that on NPs. The lack of consensus regarding the surface curvature influence on the enzyme catalytic activity is probably due to multiple factors influencing the activity, thus making it difficult to fully decouple the curvature factor.…”

Optimization of Nanohybrid Biosensors Based on Electro-Crosslinked Tannic Acid Capped Nanoparticles/Enzyme

“…In comparison, the aggregation of NP40 is prevented for R / R th = 2, suggesting the adsorption of more than one layer of the enzyme. A consequent change of GOx conformation could occur on large NPs leading to a larger footprint of the enzyme on a flat surface [ 33 ] than on small NPs, with a high curvature [ 22 ]. Upon adsorption, the enzymes could also unfold leading to the formation of two layers.…”