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.

EB00000002882K

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