A New Chemiluminescence-Based Rapid Diagnostic Testing Platform with Sequential Dual-Flow Strips for Cardiac Troponin I (cTnI)

Although the most commonly used method for enhancing a limit of detection (LoD) in immunoassay is adopting chemiluminescence (CL), the liquid form of CL substrates has hindered its use for rapid diagnostic testing (RDT). In order to use the CL-based immunoassay in RDT with minimal user intervention, the liquid CL substrate should be converted to a dry form. In addition, a new RDT platform that is able to perform two sequential flows needs to be developed for the sequential flow control of the CL substrate. In this work, we have successfully developed a new dry form of CL substrate on the strip using a lyophilization process, as well as new lateral flow strips using an additional membrane pad for a time delay to achieve the desired sequential dual flows. Thus, on the dual-flow RDT strips, first the detection antibody conjugated with an enzyme flows over the test and control lines, and then the reconstituted CL substrate flows later. A hydrophilic PVDF membrane was selected as a pad material for the time delay to achieve the sequential dual flows through two flow paths, and flow introduction timing was functionally controlled to secure the time delay of approximately 5 minutes desired between the two flows. A CL-based cardiac troponin I (cTnI) assay was successfully performed on the new dual-flow RDT platform with a sample volume of 120 μL, achieving a LoD of 100 pg/mL. The achieved LoD is better than those possible with most of the currently available RDTs on the market. The new CL-based RDT platform with the capability of dual flows developed in this work can be used for numerous other immunodiagnostic platforms which need further high-sensitivity detection, envisaging a new RDT platform for point-of-care testing with further quantitative analysis.