Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor

[Display omitted] •A sensor based on a 4-mercaptobenzoic acid SAM on gold nano-particles was created.•Horseradish peroxidase (HRP) was immobilized onto the SAM as sensing element.•We employed two forms of HRP: native and chemically glycosylated with lactose.•Modification of HRP with lactose improved operational and storage stability. We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose (414nAμM−1) compared to 378nAμM−1 for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74μM) than the HRP biosensor (83μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors.