Synergistic HRP/Biotin encapsulation in DNA nanoflowers via RCA: A path to stabilized, streamlined ELISA platform

Enzyme-linked immunosorbent assay (ELISA) requires the simultaneous maintenance of enzyme activity and the efficient labelling of the enzymes on the antibodies. Challenges in ELISA include the enzyme decomposition under harsh conditions and the requirement for secondary antibodies for binding. Preserving enzyme activity and simplifying antibody binding is a crucial prerequisite for expanding the applications of ELISA. Most encapsulation cases focus on enzyme immobilization in MOFs/COFs, while the synergetic encapsulation of enzyme-biotin in DNA nanoflowers (DNFs) and their labeling of capture antibodies to construct stable streamlined ELISA platforms was few. It is clear that a novel protocol is needed that is facile, capable of preserving both high enzymatic activity and efficient antibody binding for the stable ELISA.

We proposed a novel horseradish peroxidase (HRP)/biotin co-encapsulated DNA nanoflowers (HRP/biotin@DNFs) for the synergistic implementation of the robust enzyme immobilization using rolling circle amplification and secondary antibody-free antibody binding using biotin-streptavidin ligation in ELISA. Enzyme immobilization, abundant antibody binding sites, and superior protection of enzymes were achieved in synchrony. High enzyme activity, excellent stability and chemical tolerance of HRP/biotin@DNFs were obtained. Especially, HRP/biotin@DNFs provides 95 %-85 % and 91 %-81 % of its original activity after the exposure at 25 °C and 37 °C in 5 days, respectively, whereas that of free HRP was drastically reduced to only 40 % and 20 % after the exposure at 25 °C and 37 °C for 5 days, respectively. Using tetrabromobisphenol A bis-(2-hydroxyethyl) ether (viz. TBBPA-BHEE) as the target model, high performance for the assay was achieved, with high enzymatic activity, good tolerance and sensitive limit of detection of 0.66 ng mL^-1 that was 5-fold more sensitive than that of common ELISA. Stable and accurate ELISA for TBBPA-BHEE was realized with recovery yields of 91.4 %-112.8 % with RSD at 1.6-3.4 %.

This study pioneers a simple yet powerful strategy that simultaneously achieves both affordable, biologically encapsulated enzymes with high activity and secondary antibody-free antibody conjugation. It expands the toolbox of enzyme immobilization and facilitates enzyme binding on antibody without the need of secondary antibody, enabling cost-effective and robust ELISA for environmental assays.