The value of electrochemical ratiometry in immunosensing: A systematic study.

Reluctant reproducibility and accuracy make electrochemical immunosensors suffering from high possibility of false negative/positive results, and it is the main obstacle that hinders them into an eligible alternative technology to the gold-standard method. It has been demonstrated sporadically previously that ratiometry helps deal with this issue but to what extent this could be beneficial and why it could fulfill is yet to be explored. In this study, to the best of our knowledge, for the first time, we have attempted to answer these questions through comprehensive experiments. For this purpose, labeled and label-free electrochemical immunosensors for SARS-CoV-2 pseudovirus quantification are constructed as a model electrochemical immunosensor. Conventional and ratiometric immunosensors are prepared by using electrochemically synthesized graphene modified electrodes coupled with various electrochemical probe pairs. It was found that the electrocatalyst modification at the electrode interface makes the predominant contribution to immunosensor sensitivity, while appropriate ratiometry provided electrochemical immunosensors with significantly enhanced reproducibility, accuracy, as well as sensing stability. Further, the experiments confirmed that the improvement in sensor performance achieved by ratiometry is primarily through overcoming the inherent errors and dynamic variations of the base electrode. It is also demonstrated electrochemical immunosensors made thereof could easily rival the performances of the gold-standard PCR method, in the view of immunoassay diagnosis. Therefore, it is of great promise to evolve electrochemical immunosensors into an eligible substitute technique towards the prevalent nucleic acid detection method in point-of-care testing (POCT), with the aid of electrochemical ratiometry.