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<p>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) (&gt; 8 min). The incorporation of AuNFs increased the activity of Bst DNA polymerase, and the kinetic parameters showed a <i>V</i><sub>max</sub>/<i>K</i><sub><i>m</i></sub> 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 × 10<sup>5</sup> per AuNF, and when the temperature rises to 65&#xa0;°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 (<i>P</i> &lt; 0.0001), AuNPs@LAMP (<i>P</i> = 0.0082), and classical LAMP (<i>P</i> &lt; 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 &lt; 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).</p> Graphical Abstract <p></p>

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Gold nanoflowers (AuNFs) as enhancers for improved LAMP performance

  • 期刊论文

  • 发表日期：

    2025年11月24日

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  • Yirui Li,
  • Xiaodong Sun,
  • Jiale Gao,
  • Yan Wang,
  • Zhenghui Li,
  • Bing Niu,
  • Qin Chen

摘要

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 Vmax/Km 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).

Graphical Abstract