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

Journal article
Authors Jenny Bergman
Y. M. Wang
J. Wigstrom
Ann-Sofie Cans
Published in Analytical and Bioanalytical Chemistry
Volume 410
Issue 6
Pages 1775-1783
ISSN 1618-2642
Publication year 2018
Published at Department of Chemistry and Molecular Biology
Pages 1775-1783
Language en
Keywords Gold nanoparticles, Immobilized enzyme, Enzyme quantification, Microelectrode, Electrochemical, glassy-carbon electrodes, glucose-oxidase, gold nanoparticles, electrochemical dissolution, surface-area, biosensors, microscopy, adsorption, acetylcholine, morphology, Biochemistry & Molecular Biology, Chemistry
Subject categories Biochemistry, Chemistry, Chemical Sciences


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.

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