Key significance of DNA-target size in lateral flow assay coupled with recombinase polymerase amplification

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.