Base-Stacking-Driven Catalytic Hairpin Assembly: A Nucleic Acid Amplification Reaction Using Electrode Interface as a Booster for SARS-CoV-2 Point-of-Care Testing

Electrochemical DNA (E-DNA) biosensors based on interface-mediated hybridization reactions are promising for point-of-care testing (POCT). However, the low efficiency of target recycle amplification and the steric hindrance at the electrode interface limit their sensing performance. Herein, we propose a base-stacking-driven catalytic hairpin assembly (BDCHA), a nucleic acid amplification reaction strategy, for POCT. The introduction of the base-stacking effect in this strategy increases the thermodynamic stability of the product, thereby effectively improving the recycling efficiency. Also, it enables the interface-mediated hybridization to maintain stability with even fewer bases in the reaction-binding domain, hence minimizing DNA secondary structure formation or intertwining at the electrode surface and ameliorating the steric hindrance limitation. The introduced base-stacking effect makes the electrode serve as a booster by integrating the advantages of homogeneous and heterogeneous reactions, giving BDCHA an increased reaction rate of about 20-fold, compared to the conventional catalytic hairpin assembly. As a proof of concept, our BDCHA was applied in constructing a portable E-DNA biosensor for the detection of a SARS-CoV-2 N gene sequence fragment. A simple 30 min one-pot incubation is required, and the results can be readily read on a smartphone, making it portable and user-friendly for POCT.

Automated summary

Electrochemical DNA (E-DNA) biosensors based on interface-mediated hybridization reactions are promising for point-of-care testing (POCT). This study proposes a base-stacking-driven catalytic hairpin assembly (BDCHA) nucleic acid amplification strategy for POCT. The strategy improves recycling efficiency by increasing the thermodynamic stability of the product and addresses steric hindrance limitation by minimizing DNA secondary structure formation or intertwining at the electrode surface. The introduced base-stacking effect makes the electrode serve as a booster, resulting in an increased reaction rate compared to conventional catalytic hairpin assembly.