Abstract
ABSTRACT
Lateral flow assays (LFAs) are widely used for point‐of‐care diagnostics, but their sensitivity is often limited by inefficient antibody orientation and poor signal transduction. Herein, we report a universal strategy integrating orientation‐controlled antibody conjugation at the immunoprobe with a biotin‐streptavidin‐modified test line to enhance LFA sensitivity, specificity, and analytical reliability. Using an orientation‐controlled conjugation approach (P‐6, Scheme 1), antigen accessibility was maximized while nonspecific adsorption and false‐positive signals were suppressed. Compared with conventional randomly conjugated probes (P‐3, Scheme 1) on unmodified test lines, the combined P‐6/biotin‐streptavidin configuration achieved limits of detection of 0.0352 ng/mL for cytokeratin 19 fragment (CYFRA21‐1) and 0.0808 ng/mL for carcinoembryonic antigen (CEA), corresponding to sensitivity enhancements of approximately 14–19 fold. However, probe rankings get inverted across capture chemistries, indicating the importance of dual‐site orientation. The universality of this strategy was further validated using alpha‐fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 15‐3 (CA15‐3), and prostate‐specific antigen (PSA). Furthermore, multiplexed LFAs using fluorescent polymer dots enabled simultaneous detection of CYFRA21‐1 and CEA with minimal cross‐reactivity and were validated in 28 clinical serum samples against the hospital reference method.