Bidirectionally Favorable Platform: A Dual-Targeting Probe-Encoded Maple Leaf-Type Fluorescent Lateral Flow Immunoassay for Multiple Biomarker Detection

In the traditional multiplexed lateral flow immunoassay (LFIA), different detection probes against different targets are necessary. However, the relative complexity and high cost of probe preparation, as well as the insufficient user-friendliness, limit the application of the multiplexed LFIA in disease diagnosis. Here, we reported a bidirectionally favorable LFIA (BDF-LFIA) platform to maximize convenience for both manufacturers and users. Red-emitting time-resolved fluorescent nanoparticles were coated with different antibodies to recognize multiple targets simultaneously, which greatly simplified probe preparation by the manufacturers. Ultrabright green-emitting gold nanoclusters were pre-embedded on the test line as a reference signal to achieve a target concentration-dependent maple leaf-type hue readout from green to yellow to red, which was quite user-friendly. Taking cancer biomarkers alpha-fetoprotein and carcinoembryonic antigen as examples, this assay achieved a visual detection limit of 2 ng/mL. Compared with the conventional fluorescent LFIA, the BDF-LFIA could generate a more discernible signal around the threshold concentration of the targets. Moreover, the assay successfully diagnosed 54 clinical samples. Overall, the BDF-LFIA showed bidirectional benefits for both manufacturers and users and provided a new concept for the LFIA in multiplexed detection.