Let Us Divide Twice: A New Design of Duplex Quantitative Lateral Flow Immunoassay

Simultaneous determination of salivary cortisol and testosterone levels could provide complementary information on the physiological status of the human body. However multiplex immunoassays are challenging because the two analytes occur at different concentration levels and the corresponding immunochemical reagents can interfere with one another. Here, we developed a duplex lateral flow immunoassay (LFIA) for simultaneous colorimetric determination of cortisol and testosterone. The assay was constructed on a single nitrocellulose membrane containing two spatially separated competitive immunoreaction zones. A distinctive feature of the immunoassay design was a selective interception zone that was located between the two test lines and functioned as an immunochemical sink. The optimized assay provided working ranges of 0.38–5.41 ng/mL for cortisol and 27–845 pg/mL for testosterone, with calculated detection limits of 0.18 ng/mL and 10.1 pg/mL, respectively. The analysis took 20 min and enabled the detection of both signals using a single test strip and colorimetric signal processing. Analysis of spiked artificial saliva samples yielded recoveries of 96.4–105.7%. The results demonstrate that spatial organization of immunochemical reactions may represent an alternative strategy for multiplexing LFIAs in cases where spectrally distinct labels are undesirable or when the analytical requirements of individual assays differ significantly.