ViPER: A visual bipolar electrochemical biosensor based on isothermal addition of a universal tag for detection of SARS-CoV-2.

Emergence of recent pandemics/endemics e.g. COVID-19 and Dengue fever, demonstrated the necessity of development of strategies for swift adaptation of present biosensor for detection of the new emerging pathogens. However, development of a biosensor for a new target is time- and labor-consuming. In this study, we aimed to integrate the primer exchange reaction (PER), an isothermal technique that extends an initiator DNA with a user-defined single-stranded DNA tail, with bipolar electrochemistry. This integration led to the development of a universal biosensor, termed ViPER. We demonstrated the utility of the developed system to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic RNA as a model. The genomic RNA was reverse transcribed to a short cDNA and was tailed with a universal tag, consequently, the tagged cDNA was applied to an electrochemiluminescence integrated bipolar electrochemical biosensor (BPE-ECL). ECL signals were recorded using a digital camera and analyzed by ImageJ. The platform demonstrated a linear response over a wide dynamic range of 10^-7-10^-17 M for the target nucleic acid with a detection limit of 2.31 × 10^-17 M for synthetic targets. The biosensor could also successfully discriminate between biological RNA samples from infected and non-infected individuals. This study introduces the potential of DNA-based visual biosensors for detecting single-stranded RNAs in low-equipped environments, and it holds promises for further development of an ultrasensitive method for various human RNA-based viral pathogens. Moreover, we can design a platform with a predetermined DNA probe sequence for a vast variety of different targets, simply by changing the PER input.