Bisintercalating DNA redox reporters for real-time electrochemical qLAMP

The electrochemical detection methods have emerged as a potential alternative to the bench-top optical systems in monitoring nucleic acid amplification. DNA intercalating redox reporters play a crucial role in such monitoring schemes. Here, a series of bisintercalating redox probes have been tailor-made to meet specific requirements of electrochemical quantitative loop-mediated isothermal amplification (qLAMP). The probes composed of two naphthoquinone-imidazole (NQIM) derivatives as signal motifs that are covalently bridged by different linkers (R). They are bis-NQIM-R; R = Alkane (Ak), ethylene glycol (EG) and phenyl (Ph). The linkers allow the probes to be fine-tuned for securing ideal redox reporter. DNA binding studies via electrochemical and fluorescence techniques demonstrate that the bis-NQIM-R probes possess better ds-DNA bisintercalating ability compared to their mono-analogs. The bis-NQIM-Ph was implemented in a real-time electrochemical qLAMP, for which a prototype custom-made device that can perform fully automated multiplexed analyses is devised. A single copy of Salmonella DNA was quantified in just 10 min and the performance is comparable to the benchtop fluorescence method. Thus, the bisintercalating redox reporters incorporated electrochemical detection schemes hold great promise in qLAMP.