Catalytic and Gating Nanoreactors: Cu(I)/Cu(II)-MOF@HMS for Size-Selective DNA-Templated Click Ligation Chain Reaction and Detection of Nucleic Acids

Distinguishing short nucleic acid targets from their highly homologous precursors remains a formidable challenge in precision diagnostics, primarily due to the inherent sequence embedding. Herein, we report a spatially confined nanoreactor strategy that synergizes a rigorous size-selective kinetic filter with DNA-templated click ligation chain reaction (DT-CLCR) to achieve highly specific exponential amplification. We engineered a core–shell architecture comprising a mixed-valence Cu(I)/Cu(II)-MOF core encapsulated within a hollow mesoporous silica (HMS) shell. By leveraging the HMS shell as a tunable molecular sieve and the confined MOF core as a robust catalyst for Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) involved in DT-CLCR, the nanoreactor exhibits superior catalytic efficiency in CuAAC with turnover frequencies reaching 264.4 h^–1 and excellent structural durability in aqueous media. Crucially, the rigid mesopores function as a stringent size-selective kinetic filter. For instance, the reaction rate constant for DNA-templated click ligation in 4 nm pores exhibits a sharp kinetic cutoff, dropping distinctively from 0.031 s^–1 for 20 nt templates to 0.002 s^–1 for 50 nt analogues. Consequently, the 4 nm nanoreactors enable exponential signal amplification for 20 nt templates, achieving an amplification efficiency of 69% and a limit of detection of 57.5 aM, while effectively suppressing the signal from 30 to 50 nt analogues. To validate the specificity for biological targets, this mechanism affords over 20-fold discrimination of miRNA-21 against its longer pre-miRNA-21 precursor, confirming that the hydrodynamic diameter mismatch effectively resolves the fundamental challenge of nested sequence interference.