Crystalline COFs Assembled in an Enzyme-Compatible Milieu for Cascade Biosensing

The assembly of covalent organic frameworks (COFs) in enzyme-compatible media is highly desirable yet challenging. Herein, we proposed an electron donor–acceptor (EDA)-complex-assisted strategy for the aqueous ambient construction of β-ketoenamine COFs under additive-free condition simultaneously with the in situ encapsulation of enzymes. EDA complexes, formed by an electron-rich 2,2′-bipyridine-5,5′-diamine (Bpy) with electron-deficient 1,3,5-triformylphloroglucinol (Tp), prearranged monomers in specific orientation and significantly lowered the reaction activation energy, thus enhancing the error correction and yielding highly crystalline TpBpy COFs. Beyond affording a protective scaffold, the Bpy units within COFs also served as anchoring sites for the chelation of various metallic ions to establish biomimetic enzymatic centers. On this basis, a spatially coupled nanoreactor was designed by encapsulating the natural creatinine deiminase (CD) followed by biomimetic laccase (Bpy-Cu) installation within COFs. The product generated by the encapsulated CD activated the neighboring Bpy-Cu within the adjacent pores, creating a proximity-induced cascade amplification and enabling the entire cascade to respond specifically to one of the key chronic kidney disease (CKD) biomarkers of creatinine. The constructed detection platform exhibits a wide linear range, with a detection limit as low as approximately 7.7 μM. This platform can provide valuable supporting information for the assessment of kidney function and holds promise for applications in the early screening and disease course monitoring of CKD. By simply altering the encapsulated natural enzymes and biomimetic active sites, this modular and generalizable platform is expected to offer a promising tool for early diagnosis of various diseases.