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Herein, we report a universal strategy integrating orientation\u2010controlled antibody conjugation at the immunoprobe with a biotin\u2010streptavidin\u2010modified test line to enhance LFA sensitivity, specificity, and analytical reliability. Using an orientation\u2010controlled conjugation approach (P\u20106, Scheme\u00a01), antigen accessibility was maximized while nonspecific adsorption and false\u2010positive signals were suppressed. Compared with conventional randomly conjugated probes (P\u20103, Scheme\u00a01) on unmodified test lines, the combined P\u20106\/biotin\u2010streptavidin configuration achieved limits of detection of 0.0352\u00a0ng\/mL for cytokeratin 19 fragment (CYFRA21\u20101) and 0.0808\u00a0ng\/mL for carcinoembryonic antigen (CEA), corresponding to sensitivity enhancements of approximately 14\u201319 fold. However, probe rankings get inverted across capture chemistries, indicating the importance of dual\u2010site orientation. The universality of this strategy was further validated using alpha\u2010fetoprotein (AFP), carbohydrate antigen 125 (CA125), carbohydrate antigen 15\u20103 (CA15\u20103), and prostate\u2010specific antigen (PSA). Furthermore, multiplexed LFAs using fluorescent polymer dots enabled simultaneous detection of CYFRA21\u20101 and CEA with minimal cross\u2010reactivity and were validated in 28 clinical serum samples against the hospital reference method.<\/jats:p>","DOI":"10.1002\/adhm.71815","type":"journal-article","created":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T07:46:42Z","timestamp":1791532002000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rational Control of Antibody Orientation for Enhancing Sensitivity and Specificity in Lateral Flow Immunoassays With Clinical Validation"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-6458-8390","authenticated-orcid":false,"given":"Pin\u2010Hao","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Applied Chemistry National Yang Ming Chiao Tung University  Hsinchu Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-0232-1012","authenticated-orcid":false,"given":"Shunmuga Nathan","family":"Shunmuga Nainar","sequence":"additional","affiliation":[{"name":"Department of Materials Science and Engineering National Yang Ming Chiao Tung University  Hsinchu Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bing\u2010Han","family":"Zhuang","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry National Yang Ming Chiao Tung University  Hsinchu Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shun\u2010Mao","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Surgery National Taiwan University Hospital  Hsinchu Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong\u2010Cheu","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Materials Science and Engineering National Yang Ming Chiao Tung University  Hsinchu Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6496-2362","authenticated-orcid":false,"given":"Yang\u2010Hsiang","family":"Chan","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry National Yang Ming Chiao Tung University  Hsinchu Taiwan"},{"name":"Center For Emergent Functional Matter Science National Yang Ming Chiao Tung University  Hsinchu Taiwan"},{"name":"Department of Medicinal and Applied Chemistry Kaohsiung Medical University  Kaohsiung Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,9]]},"reference":[{"key":"e_1_2_10_2_1","unstructured":"J.Horgan Justinian's Plague (541\u2010542 CE)In 2014; Updated December 26."},{"key":"e_1_2_10_3_1","unstructured":"WHO Coronavirus (COVID\u201019) Data2023 https:\/\/data.who.int\/dashboards\/covid19\/more\u2010resources(accessed 2026 14 January)."},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac0302859"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acssensors.7b00299"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/adhm.201900244"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/adhm.202401677"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2024.117798"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.4c06564"},{"key":"e_1_2_10_10_1","volume-title":"Discontinue the Use of Antigen Testing in Skilled Nursing Facilities until Further Notice","author":"Azzam I. 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