Development of a lateral flow immunoassay with HRP enhancement for spiked SARS-CoV-2 protein N detection in human saliva

The Coronavirus (SARS-CoV-2) is an enveloped RNA virus that caused a dangerous COVID-19 pandemic following an outbreak in Wuhan, China in 2019. In response to the pandemic, the development of lateral flow assays (LFAs) has been crucial for the detection of viruses, commonly targeting the spike (S) or nucleocapsid (N) protein in nasopharyngeal swab (NS) specimens. COVID-19, caused by the SARS-CoV-2 virus, predominantly manifests as a respiratory tract infection-like illness, characterized by symptoms such as fever, dry cough, upper airway congestion, runny nose, sore throat, myalgia, headache, and exhaustion. This study presents the development of an LFA targeting the N protein to detect the coronavirus in saliva specimens, using a sandwich format. The use of 80 nm gold nanoparticles (AuNPs) has been investigated, and the application of horseradish peroxidase (HRP) and TMB substrate has been employed to enhance the limit of detection (LOD). The results demonstrate a significant 200-fold improvement in LOD, from 10 to 50 pg/ml, for N protein spiked in saliva samples after the application of HRP-TMB. This finding highlights an important advancement towards the utilization of saliva samples in diagnostic applications.