Facile construction of a reusable multi-enzyme cascade bioreactor for effective fluorescence discrimination and quantitation of amino acid enantiomers

Effective discrimination and quantitation of Amino acid (AA) enantiomers is enormously pivotal in diverse fields ranging from pharmaceutics to biological systems. We herein proposed a one-step strategy to construct a novel cascade bioreactor for chiral sensing of multiple AA enantiomers by coupling L-amino acid oxidase (LAAO) and Horseradish peroxidase (HRP) into zeolitic imidazolate framework-8 (ZIF-8) (LAAO/HRP@ZIF-8). LAAO specifically catalyzes L-AAs to alpha-keto acid, ammonium and H2O2, and then the yielded H2O2 oxidizes o-phenylenediamine (OPD) to strong yellow fluorescence signal of 2,3-diaminophenazine (DAP) by wrapped HRP. ZIF-8 not only guaranteed free enzymes stability owing to the steric confinement effect of the framework, but also reinforced multi-enzyme cascade catalytic activity due to a preconcentration effect of the analyte inside the cavities. In view of this, the fabricated chiral sensor allowed highly sensitive enantioselectivity discrimination a range of chiral AA enantiomers, especially tryptophan (Trp), valine (Val), methionine (Met), phenylalanine (Phe), isoleucine (Ile), leucine (Leu) and alanine (Ala). According to the linear correlation between the content of L-AAs and fluorescence intensity, this sensor achieved the quantitative determination of Enantiomeric excess (ee) value with high accuracy, and the results were well matched with those obtained by High performance liquid chromatography (HPLC). Additionally, the prominent stability and satisfactory reusability of this sensor were favorably elucidated by using Trp as the model molecule. Our work provides an unprecedented protocol for enantioselective fluorescence discrimination and quantitative determination of ee value for AA enantiomers, and immensely advances the promising application field of multi-enzyme@metal-organic framework biocomposites.

Automated summary

This study presents a one-step strategy to construct a novel cascade bioreactor for chiral sensing of multiple amino acid enantiomers by coupling LAAO and HRP. The chiral sensor allows highly sensitive discrimination of various amino acid enantiomers and quantitative determination of ee value. The stability and reusability of the sensor are well demonstrated, making it a promising tool for enantioselective fluorescence discrimination and quantitative determination of ee value for amino acid enantiomers.