Gold nanoflowers (AuNFs) as enhancers for improved LAMP performance

AuNFs (gold nanoflowers) are widely applied in the field of biosensing; their unique morphological characteristics and high specific surface area enable them to provide a large number of active sites, thereby enhancing the efficiency of catalytic reactions. These features make AuNFs a promising material for improving the performance of loop-mediated isothermal amplification (LAMP). Firstly, the reduction of 4-NP catalyzed by nano-gold (NG) with different morphologies showed that AuNFs made the yellow solution almost transparent in 4 min, similar to AuNPs (gold nanoparticles), with higher catalytic efficiency compared to AuNRs (gold nanorods) (> 8 min). The incorporation of AuNFs increased the activity of Bst DNA polymerase, and the kinetic parameters showed a V_max/K_m of 0.021, which was higher than that of AuNPs (0.015), AuNRs (0.013), and the LAMP system without NGs (0.013). Moreover, at ambient temperature, the UV-vis intensity of the characteristic peak indicates that the loading capacity of ssDNA is approximately 1.6 × 10⁵ per AuNF, and when the temperature rises to 65 °C, ssDNA is almost completely released from the AuNFs. This mechanism may be beneficial for suppressing the generation of non-specific amplification. Dynamic light scattering (DLS) and circular dichronism (CD) results indicate that AuNFs alter the α-helix structure upon binding with Bst, potentially thereby enhancing enzyme activity. LAMP amplification was performed at different temperatures, and the results illustrate that the Tt values of AuNFs@LAMP were lower than those of AuNRs@LAMP (P < 0.0001), AuNPs@LAMP (P = 0.0082), and classical LAMP (P < 0.0001) at 40, 45, 50, 55, and 60 ℃. Thus, we proposed the AuNFs@LAMP (AuNF-mediated LAMP) method and applied it to the detection of AHSV (African horse sickness virus) standard plasmid sample and spiked dust sample with a sensitivity as low as 10 copies/μL. Intra-day, inter-day, and intra-batch precision showed RSD < 5%, with RSD ranging from 2.51 to 8.75% across low, medium, and high plasmid template concentrations, respectively. Moreover, the specific AuNFs@LAMP system inhibits non-specific amplification in negative samples and in samples containing other equine virus plasmids (WNV, JEV, VSV, NIV, EEEV, WEEV).