A new sequential dual flow lab-on-a-chip with a lyophilized one-component chemiluminescence substrate for high-sensitive microchannel lateral flow assay (mLFA)

Recently, there has been a growing demand for the development of lab-on-a-chip (LOC) platforms with new assays and detection protocols for point-of-care-test (POCT) applications. So far, chemiluminescence (CL) detection-based immunoassays have shown promising performance for the high-sensitive POCT, but they require automated machines or multiple manual steps to perform the CL-based assay. In this work, a fully automated CL-based immunoassay was developed using a new sequential dual flow LOC with on-chip lyophilized CL substrate, and then a highly specific and sensitive immunoassay using a pair of single chain variable fragment (scFv) capture and detection antibodies was successfully performed. The concept of sequential and automatic control of dual flows, which was strongly desired for ensuring that the reconstituted detection antibody conjugated with horseradish peroxidase flowed first through the reaction zones and then the reconstituted CL substrate flowed, was newly developed and implemented on the LOC. In addition, a new one-component CL substrate in liquid format was introduced and lyophilized for the on-chip lyophilized substrate, developing a new lyophilization process. To evaluate the assay performance on the developed new LOC platform, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was chosen as a demonstration vehicle. The nucleocapsid (N) protein of SARS-CoV-2 was analyzed using the custom-developed scFv antibody pair from a phage display library system, which showed a better limit of detection (LoD) over the commercially available rapid diagnostic test (RDT) kits for detecting SARS-CoV-2. Finally, a portable reader for reading the CL signal from the CL-based microchannel lateral flow assay (CL-mLFA) was developed and used for evaluating the performance of the SARS-CoV-2 assay on the developed LOC platform. An LoD of approximately 1.6 ng mL⁻¹ was achieved, which was acceptable for the early diagnosis of SARS-CoV-2 infection. The new CL-mLFA platform developed in this work, adopting the sequential dual flow LOC and the lyophilized one-component CL substrate, can be applied to other high-sensitive immunoassays in POCT for diagnosing various chronic or infectious diseases.