Signal enhancement methods for naked-eye lateral flow immunoassays by designing colored nanoparticles

Abstract

Lateral flow immunoassays (LFA) detected by naked-eye readout are widely used as point-of-care testing (POCT) platforms due to their simplicity and low cost. However, their primary limitation is poor sensitivity, which hinders their use in high-performance diagnostics. This study introduces signal enhancement strategies to overcome this issue, focusing on two distinct approaches. First. I explored the application of Biotin-assisted dual gold clusters(Biotin-DGC) and BSA-assisted dual gold clusters(BSA-DGC) to enhance the optical signal for Troponin I (TnI) detection. Characterization revealed that BSA-DGC possessed a larger size and a broader absorption range, resulting in enhanced LFA signal intensity compared to single 80 nm AuNPs. Specifically, the naked-eye detection limit was 10 pg/mL for BSA-DGC, whereas it was 50 pg/mL for 80 nm AuNPs. However, Biotin-DGC did not exhibit significantly improved LFA signals, despite showing a higher spectral peak compared to single 80 nm AuNPs and less redshift from 80 nm single AuNPs than that of BSA-DGC. Second, I utilized Platinum Nanoparticles (PtNPs) as a nanozyme to catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine(TMB), significantly enhancing the colorimetric signals. By optimizing the reaction conditions for superior PtNP activity and color stability, this LFA utilizing PtNPs-TMB reaction achieved an improved performance for Influenza B and SARS-CoV-2 detection. It demonstrated an 8-fold signal enhancement and lowered the naked-eye limit of detection (LOD) to 102 PFU/mL, making the results clearly distinguishable by naked-eye. I also confirmed that Na2O2 could be effectively used as a dried substrate for TMB, which is critical for integrating the enhancement mechanism into a stable, automatic TMB reaction-utilizing LFA format. The signal enhancement effect of the automatic TMB reaction-utilizing LFA was also confirmed. It produced a clearly enhanced visual signal at 103 TCID50 mL-1and above for SARS-CoV-2 detection.