title: Coimmobilization of acetylcholinesterase and choline oxidase on gold nanoparticles: stoichiometry, activity, and reaction efficiency.
abstract: Hybrid structures constructed from biomolecules and nanomaterials have been used in catalysis and bioanalytical applications. In the design of many chemically selective biosensors, enzymes conjugated to nanoparticles or carbon nanotubes have been used in functionalization of the sensor surface for enhancement of the biosensor functionality and sensitivity. The conditions for the enzyme:nanomaterial conjugation should be optimized to retain maximal enzyme activity, and biosensor effectiveness. This is important as the tertiary structure of the enzyme is often altered when immobilized and can significantly alter the enzyme catalytic activity. Here we show that characterization of a two-enzyme:gold nanoparticle (AuNP) conjugate stoichiometry and activity can be used to gauge the effectiveness of acetylcholine detection by acetylcholine esterase (AChE) and choline oxidase (ChO). This was done by using an analytical approach to quantify the number of enzymes bound per AuNP and monitor the retained enzyme activity after the enzyme:AuNP synthesis. We found that the amount of immobilized enzymes differs from what would be expected from bulk solution chemistry. This analysis was further used to determine the optimal ratio of AChE:ChO added at synthesis to achieve optimum sequential enzyme activity for the enzyme:AuNP conjugates, and reaction efficiencies of greater than 70%. We here show that the knowledge of the conjugate stoichiometry and retained enzyme activity can lead to more efficient detection of acetylcholine by controlling the AChE:ChO ratio bound to the gold nanoparticle material. This approach of optimizing enzyme gold nanoparticle conjugates should be of great importance in the architecture of enzyme nanoparticle based biosensors to retain optimal sensor sensitivity.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology , Gothenburg 41319, Sweden.
authors.forename: Jacqueline D
authors.initials: JD
authors.lastname: Keighron
authors.forename: Sebastian
authors.initials: S
authors.lastname: Åkesson
authors.forename: Ann-Sofie
authors.initials: AS
authors.lastname: Cans
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D015374:Biosensing Techniques; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: D004800:Enzymes, Immobilized; D006046:Gold; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D000110:Acetylcholinesterase