Enzyme-loaded Fe³⁺-doped ZIF-90 particles as catalytic bioreactor hybrids for operating catalytic cascades

Fe³⁺-doped ZIF-90 (Fe³⁺-ZIF-90), a metal-organic framework (MOF), was synthesized and characterized. The MOF particles reveal peroxidase-like activity reflected by catalyzing the H₂O₂ oxidation of 3,3',5,5'-tetramethylbenzidine, TMB, to TMB˙⁺. Integration of the two enzymes, β-galactosidase, β-Gal, and glucose oxidase, GOx, in the Fe³⁺-ZIF-90 provides an organized framework allowing the operation of a three-catalyst cascade, where the β-Gal-catalyzed oxidation of lactose yields glucose and galactose, and the resulting glucose is aerobically oxidized by GOx to gluconic acid and H₂O₂, followed by the Fe³⁺-ZIF-90-catalyzed H₂O₂ oxidation of TMB to TMB˙⁺. The coupled bienzyme/nanozyme cascade in the MOFs is ca. 5-fold enhanced, as compared to a homogeneous mixture of the catalytic constituents. The enhanced catalytic activity of the enzyme cascades in the MOFs is attributed to the confined reaction framework, allowing product channeling across the multienzyme constituents and overcoming diffusion barriers. Moreover, the enzymes, acetylcholine esterase, AChE, and choline oxidase, ChOx, are encapsulated in the confined porous Fe³⁺-ZIF-90 particles. The catalytic cascade where the neurotransmitter acetylcholine is hydrolyzed by AChE followed by the stepwise ChOx-catalyzed oxidation of choline to betaine and H₂O₂, and the Fe³⁺-ZIF-90-catalyzed oxidation of TMB to colored TMB˙⁺ by H₂O₂ is demonstrated. The three-catalyst cascade is ca. 5-fold enhanced as compared to the mixture of separated catalysts. The integrated three-catalyst AChE/ChOx/Fe³⁺-ZIF-90 particles are applied as colorimetric sensors detecting the neurotransmitter acetylcholine and probing AChE inhibitors. The novelty of the systems is reflected by the assembly of multienzyme catalytic Fe³⁺-ZIF-90 hybrids in confined environments as bioreactor frameworks driving effective biocatalytic cascades.