A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky prospect 33, 119071, Moscow, Russia.
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, Leninsky prospect 33, 119071, Moscow, Russia.
Anal Chim Acta. 2020 Mar 15;1102:109-118. doi: 10.1016/j.aca.2019.12.048. Epub 2019 Dec 19.
The combination of isothermal nucleic acid amplification and lateral flow assay (LFA) provides highly sensitive non-laboratory ("point-of-care") detection. The aim of this study is to investigate the recognition on lateral flow membranes of DNA targets with different lengths as products of recombinase polymerase amplification (RPA). We produced double-stranded DNA with lengths of 50, 100, 150, 200, and 300 bp. Each DNA target was functionalized with biotin and fluorescein (FAM). Kinetic and equilibrium constants of the interaction of FAM at the 5'-end of DNA with anti-FAM antibodies did not depend on DNA length. Gold nanoparticles (GNPs) with diameters of 17.4 ± 1.0 nm were conjugated with anti-FAM antibodies and streptavidin. LFA was performed in two schemes: 1) anti-FAM antibodies immobilized in the test zone, GNP-streptavidin conjugates recognized as DNA; 2) streptavidin immobilized in the test zone, GNP‒anti-FAM antibodies conjugates recognized as DNA. Considering that the components of the RPA mixture caused the aggregation of the GNP-streptavidin conjugate in contradistinction to conjugate with anti-FAM antibodies, we found that 150 bp was the most promising length for the DNA target. For this length, a detection limit was achieved up to 70 pM that was approximately 10 times lower than for 50-bp DNA in the same scheme. Moreover, we showed that high concentrations of primers containing FAM or biotin competed with the DNA target on lateral flow membranes. These results demonstrated that a DNA length should be considered when designing RPA-LFA systems to detect DNA targets with high sensitivity.
等温核酸扩增与侧流分析（LFA）的结合提供了高度敏感的非实验室（“即时检测”）检测。本研究旨在研究不同长度的 DNA 靶标作为重组酶聚合扩增（RPA）产物在侧流膜上的识别。我们合成了长度为 50、100、150、200 和 300 bp 的双链 DNA。每个 DNA 靶标均用生物素和荧光素（FAM）功能化。FAM 在 DNA 5'末端与抗 FAM 抗体相互作用的动力学和平衡常数不依赖于 DNA 长度。直径为 17.4±1.0nm 的金纳米颗粒（GNPs）与抗 FAM 抗体和链霉亲和素偶联。LFA 以两种方案进行：1）抗 FAM 抗体固定在测试区，GNPs-链霉亲和素缀合物识别为 DNA；2）链霉亲和素固定在测试区，GNPs-抗 FAM 抗体缀合物识别为 DNA。考虑到 RPA 混合物的成分导致 GNP-链霉亲和素缀合物聚集，与抗 FAM 抗体缀合物不同，我们发现 150 bp 是最有前途的 DNA 靶标长度。对于这个长度，检测限达到 70 pM，大约比相同方案中 50 bp 的 DNA 低 10 倍。此外，我们表明含 FAM 或生物素的高浓度引物在侧流膜上与 DNA 靶标竞争。这些结果表明，在设计具有高灵敏度检测 DNA 靶标的 RPA-LFA 系统时，应考虑 DNA 长度。
