Monitoring and tracking infection is required in order to reduce the spread of the coronavirus disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, the development and deployment of quick, accurate, and sensitive diagnostic methods are necessary. The determination of the SARS-CoV-2 virus is performed by biosensing devices, which vary according to detection methods and the biomarkers which are inducing/providing an analytical signal. RNA hybridisation, antigen-antibody affinity interaction, and a variety of other biological reactions are commonly used to generate analytical signals that can be precisely detected using electrochemical, electrochemiluminescence, optical, and other methodologies and transducers. Electrochemical biosensors, in particular, correspond to the current trend of bioanalytical process acceleration and simplification. Immunosensors are based on the determination of antigen-antibody interaction, which on some occasions can be determined in a label-free mode with sufficient sensitivity.

Lateral flow immunoassays (LFIAs) have wide application in point-of-care testing, particularly in resource-poor settings. To achieve signal amplification in a gold nanoparticle-based lateral flow assay without an additional procedure or the need for complex fabrication, a new and simple method was developed for using a “stacking pad” configuration that adds an additional membrane between the conjugation pad and test pad to the conventional AuNP-based LFIA format. This design helps to accumulate the antibody and antigen on the stacking pad, hence extending the antigen/antibody binding interactions to enhance the test’s detection sensitivity. With the enhanced lateral flow assay, as low as 1 ng/mL of Protein A and 15.5 ng/mL of C-reactive protein can be visualized with the naked eye. We also successfully applied the stacking pad system in the analysis of C-reactive protein in human serum and synovial fluid samples. These results suggest that this stacking pad LFIA can provide sensitive and on-site prognosis for detection in synovial fluid and serum samples in resource-limited settings.