Chunxia Chen, Zhan Yang, Yangguang Ren, Menghan Xue, Jinbo Wu, Jianing Ji, Yan Zhao, Ruyan Meng, Yao Ma, LI Xiao-quan, Yonghong Wang, Xiaohui Chen, Z Y Li, Xiaogang Lin, Zhizeng Wang, Yang Luo
Conventional colloidal gold lateral flow immunoassays (AuNPs-LFIA) frequently experience limitations in sensitivity and accuracy as a result of ineffective, random immobilization of antibodies on gold nanoparticles (AuNPs), which obstructs antigen-binding sites. An effective strategy is presented that exploits the distinct isoelectric points (pI) of antibody Fab and Fc domains. By accurately adjusting the pH close to the pI of the Fc region, antibodies are oriented in a specific direction: the Fc region effectively binds to AuNPs, while the Fab region remains optimally exposed for antigen binding. The pH-controlled Fab-up orientation enhances antibody coating efficiency and antigen-binding accessibility, leading to substantial improvements in analytical sensitivity, stability, and quantitative accuracy, facilitated by an integrated smartphone artificial intelligence (AI) system. Validated for acute myocardial infarction biomarker heart-type fatty acid-binding protein detection across 781 diverse clinical samples, the optimized assay achieved exceptional diagnostic performance (94.2% sensitivity and 100.0% specificity), demonstrating strong correlation ( R 2 = 0.982 5) with laboratory chemiluminescence assays. This innovative orientation strategy paves the way for next-generation ultrasensitive point-of-care biosensors.