Preparation And Enzyme Immobilization Of Graphene-based Nanostructured Composites

Graphene,as two-dimensional nanomaterials with unique physicochemical properties,exhibits great potential in biological-related applications.Graphene oxide and reduced graphene oxide have been widely explored as carriers to immobilize enzymes,for the preparation of nanobiocatalysts.However,the nanoscale character of GO and rGO usually leads to extremely high difficulty in recycling for the immobilized enzymes.Incorporation of magnetic nanomaterials was proven as one of the most effective ways to prepare the graphene-based immobilized enzymes.Alternatively,three-dimensional?3D?graphene-based hydrogel with monolithic macroscale also offered a feasible way for the acquirement of easily recovered immobilized enzymes.3D graphene-based hydrogel could not only inherit the high surface area of 2D graphene,but also retain large percentage of water entrapped in the porous structure that implied an adequate environment for enzymes.Biominerals that acquired through biomolecule-induced nucleation and growth of inorganic phase at ambient pressure,temperature,and neutral pH commonly exhibited unique physicochemical properties.The bioinspired silica research has made the most progress in the variety of silica structures and morphologies including monodispersed nanoparticles and silica coating on surfaces for device fabrication.In this study,three kinds of graphene-based nanocomposites,such as rGO loaded with magnetic nanoparticles and 3D graphene-based hydrogel designed by synergy of 3D graphene-based hydrogel and biominerals,were prepared and then used for the construction of stable monolithic biocatalytic systems.The details in this study are summarized as follows:Firstly,the rGO/Fe₃O₄ nanocomposites are synthesized by the simultaneous reduction of graphene oxide and in situ deposition of Fe₃O₄ nanoparticles?ca.20 nm?enabled by Fe²⁺ ions.The as-prepared rGO/Fe₃O₄ nanocomposites integrate the magnetic property of Fe₃O₄ nanoparticles and the large specific surface area of rGO nanosheets.Catalase?CAT?,can be efficiently immobilized on the rGO/Fe₃O₄ nanocomposites through physical adsorption.The CAT loading capacity is as high as 312.5 mg?g of support?-1,while the activity recovery of CAT can be high up to nearly 98%,the immobilized CAT exhibits zero leaching,desirable stability and excellent reusability.Secondly,the rGO/FeOOH hydrogel is firstly prepared through metal ion-induced reduction/assembly of GO nanosheets,which is then utilized to adsorb cationic polyethyleneimine?PEI?.This cationic PEI,as the inducer,catalyzes the condensation of silicate to form silica?biomimetic silicification?on the rGO surface,where enzyme is simultaneously entrapped.Combined with monolithic macroscale of the rGO/FeOOH/silica hydrogel,the acquired biocatalytic systems display easy recyclability and elevated pH/thermal/recycling/storage stabilities during the catalytic production of 6-aminopenicillanic acid?6-APA?.Particularly,the activity can be retained up to 93.3% of its initial activity after 11 reaction cycles for the biocatalytic systems constructed by the rGO/FeOOH/silica hydrogel.Thirdly,we present a biomimetic silicification approach to synthesize rGO/PEI/silica aerogels under electrostatic interaction and hydrogen bonding between GO and PEI.In detail,PEI is utilized to reduce GO and acted as a cross-linker for forming the aerogel.On the other hand,PEI could induce the process of biomimetic silicification to form a layer of silica on the surface of the aerogel.The structure and mechanical strength of the hydrogel could be tailored through changing the concentration of GO as well as the mass ratio of GO to PEI.The rGO/PEI/silica hydrogel is utilized to immobilized penicillin G acylase?PGA?,the specific activity of which is 13.8 U enzyme-1.Specifically,the initial enzyme activity can be retained 89.2% after 16 cycles by using rGO/PEI/silica hydrogel.