Enzyme immobilization on gold surfaces : effects of surface chemistry and attachment strategies on binding and activity




Correira, Joshua Manuel

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Functional enzymes are the basis for many biotechnological systems, including biosensors, bio-fuel cells, and heterogeneous biocatalysts. In these systems, enzymes are often immobilized on a solid support or surface to capture their catalytic activity. Immobilization has the advantage of improving enzyme stability and reusability but often results in a significant loss of catalytic activity (1-2 orders of magnitude) when compared to the native enzyme. This inactivation is due to direct interactions between the enzyme and the solid support. Here, we developed methods to study enzyme immobilization and resulting inactivation. The aim of this work was to identify optimal surface chemistries and attachment strategies that promote high binding efficiencies while minimizing activity losses. Subsequently, we studied this using three enzymes (acetylcholinesterase (AChE), β-galactosidase (β-gal), horseradish peroxidase (HRP)) immobilized on gold surfaces by direct adsorption, covalent attachment, and DNA-directed attachment. First, AChE was directly adsorbed onto a variety of gold surfaces modified with self-assembled monolayers (SAMs) terminated with -COO⁻, -NH₃⁺, -OH, and -CH₃ functional groups at varying mole % to study the effect of surface hydrophobicity and charge on binding and activity. We found that binding was directly proportional to surface hydrophobicity (r = 0.75) and activity was inversely proportional to surface hydrophobicity (r = -0.62). The highest binding observed was ~40% of a monolayer on the most hydrophobic surfaces and the lowest binding observed was ~10% of a monolayer on the most hydrophilic surfaces. Conversely, on the most hydrophobic surfaces AChE retained <10% of its native activity, and on the most hydrophilic surfaces AChE retained ~40% of its native activity. This illuminated an inherent problem with direct adsorption: high binding and high activity are mutually exclusive. Due to these findings, we next immobilized β-gal and HRP on DNA-functionalized gold surfaces using DNA-DNA interactions, to avoid direct interactions between the enzyme and surface. We found that β-gal retained 62% of its native activity following immobilization, a significant improvement over previous direct adsorption strategies.



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