Controlled Assembly for Well-Defined 3D Bioarchitecture Using Two Active Enzymes

Abstract

This paper reports that a bioarchitecture with two different active enzymes can be fabricated conveniently on a prepatterned surface by highly selective surface-templated layer-by-layer (LBL) assembly by coupling a bilayer of avidin/biotin-lactate oxidase (biotin-LOD) with a bilayer of avidin/biotin-horseradish peroxidase (biotin-HRP). The two different active enzymes can be utilized as excellent building blocks for the fabrication of well-defined 3D nanostructures with precise control of the position and height on the surface. In addition, the LBL assembled bienzyme structures are highly functional, and bioarchitectures based on LOD and HRP-mediated coupling reaction can be employed in a number of viable biosensing applications.

Constructing LBL-assembled functional bio-architecture using gold nanorods for lactate detection

“…Avidin acts as a biocompatible linker between the biotin-HRP and the biotin-LOx due to a strong biotin-avidin interaction (K d $ 10 À15 M) [20]. This bienzyme system can be utilized as a mediatorless electrochemical lactate sensing using a coupled reaction of LOx and HRP [21]. L-lactate is oxidized into pyruvate via dehydrogenation reaction of LOx (Scheme 1B).…”

Bienzymatic nanoreactors composed of chloroperoxidase–glucose oxidase on Au@Fe3O4 nanoparticles: Dependence of catalytic performance on the bioarchitecture

“…The gold surface was first functionalized by streptavidin (SA), and then used as a template for LBL assembly of the biotinylated enzymes. Biotinchloroperoxidase (B-CPO), each conjugated with an average of 2.6 biotins, and biotin-glucose oxidase (B-GOx), each conjugated with an average of 8.8 biotins [16,31], were used to fabricate a well-defined nanostructure of the bienzymatic reactor. SA acts as a biocompatible linker between the B-CPO and B-GOx owing to the strong biotin-avidin specific interaction (K d ≈ 10 −15 M).…”

“…As mentioned above, each GOx was conjugated with an average of 8.8 biotins, whereas each CPO molecule was conjugated with only an average of 2.6 biotins. As a result, a cage-like configuration can form at the GOx layer, in which a unit of 6 streptavidin (SA) molecules presumably form a nonregular stable hexagon [34,16], and one GOx molecule occupies the center of this hexagon (Scheme 2a). Conversely, a similar cage-like structure is difficult to form at the CPO layer (Scheme 2b).…”

“…Specific interaction between the host and guest molecules is a facile driving force for the binding of an enzyme on a carrier. The specific interaction between avidin and biotinylated biomolecules has been used as an ideal force owing to the specific and strong interaction (K d ≈ 10 −15 M) [16] of avidin-biotin. It is also possible to position two different enzymes next to each other on the carrier through the specific interaction of avidinbiotin.…”

“…It combines two functions: the generation of H 2 O 2 in situ by GOx for the CPO catalytic reaction and easy separation of the enzyme from the reaction solution by an applied magnetic field. Moreover, the use of the specific and strong interaction of avidin-biotin to construct the double enzyme reactor has been reported [24,16], but the relationship between the structure and enzyme activity of the bienzymatic nanoreactorhas not been studied in detail.…”