Unidirectional-flow assay strip fabricated via an integrated process for multiplex detection of inflammatory...

Lateral flow assay (LFA) is a low-cost and rapid immunoassay method for detecting inflammatory biomarkers and assessing severity of infection. Conventional LFA strips are composed of laminated porous components, such as conjugate pads and nitrocellulose (NC) membranes. However, micropores within these components impose resistance to the flow of sample solution, thereby reducing flow velocity and prolonging detection time. Additionally, LFA strips are usually housed in cassettes. When the cassettes are removed to reduce cost, the strip components may loosen under external disturbances. Loosening of components would disrupt the continuity of the flow path and cause the residue of the solution, thereby decreasing detection robustness and sensitivity. To overcome these limitations, we introduced an integrated process that fabricated all components on a barbed-arrow structure (BAS) membrane to develop a unidirectional-flow assay strip (UFA strip). The integrated components (e.g., conjugate ridge and test ridges) adhered tightly to the membrane, thereby providing a straight flow path and maintaining flow continuity. Moreover, the UFA strip achieveed a solution utilization of 35.7%, supporting detection with small-volume samples. Using the UFA strip, we developed a rapid, sensitive immunoassay method for multiplex detection of serum amyloid A (SAA) and C-reactive protein (CRP) within 7 min, with detection limits of 0.254 μg/mL and 0.285 μg/mL, respectively. In the detection of 26 clinical samples, this method showed a sensitivity of 93.8% (15/16) and a specificity of 90.0% (9/10). Therefore, the UFA strip demonstrated excellent performance for rapid and low-cost immunoassays, offering a promising platform for detection of inflammatory biomarkers.