An electrochemiluminescence biosensor based on tetrahedral DNA framework anchoring covalent organic frameworks/gold nanoclusters with intra-cluster hydrogen bonding for anti-interference and ultrasensitive detection of mecA genes.

The mecA genes pose a serious threat to human health. Their widespread detection in actual water samples is greatly hindered by their low concentration and complex matrix interference. Therefore, an electrochemiluminescence (ECL) biosensor based on a tetrahedral DNA framework (TDF) anchoring high-performance ECL luminophores is developed for ultrasensitive and anti-interference detection of mecA genes. The introduced TDF can immobilize probes that specifically recognize mecA genes and enhance the stability and durability of the biosensor. Importantly, the high-performance ECL luminophores combine ultrabright gold nanoclusters under the protection of Cys-Arg-Arg-Arg peptides and 6-aza-2-thiothymine (CR4/ATT/AuNCs, ultrahigh quantum yield), resulting in a ∼266-fold improvement in ECL intensity (compared with ATT/AuNCs). The conformational rigidity of CR4 and ATT is attributed to intra-cluster hydrogen bonds between them. This promotes ordered π-π stacking and markedly enhances the ECL quantum efficiency of CR4/ATT/AuNCs (∼3.26-fold). Benefiting from the unique nanoconfinement effect, uniformly sized covalent organic frameworks (COFs), as supporting substrates to inhibit the aggregation of CR4/ATT/AuNCs, contributes to the accumulation of reactive radicals, while its outstanding adsorption capacity efficiently increases the local concentrations of co-reactants and CR4/ATT/AuNCs to promote the decomposition of co-reactants to generate more reactive radicals. The biosensor with a low limit of detection (LOD, S/N = 3) of 0.72 fM has excellent repeatability, stability, and selectivity after further amplifying the reaction signal using catalytic hairpin assembly (CHA), achieving direct detection of mecA genes in wastewater samples.