Abstract
To decrease the burden of laborious and reagent-intensive screening of modified aptamers, their binding function requires assessment in assay formats compatible with the end diagnostic application. Here, we report on the use of an alternative and cost-effective approach: a rapid lateral flow assay (LFA) utilising streptavidin-conjugated gold nanoparticles (AuNP) as reporter molecules to screen novel ssDNA aptamers for their ability to detect CD4. Crossover-SELEX was employed to identify CD4-targeting aptamers from a ssDNA library enriched against a recombinant human CD4 protein (hCD4) conjugated to magnetic-beads and to endogenous CD4 expressed by U937 cells. Counter-selection with IgG-conjugated beads and CD4-negative Ramos RA-1 cells was employed. Following SELEX, four sequences (U4, U14, U20 and U26) were selected for candidate screening. Fluorescence confocal microscopy showed comparable localization of the Cy5-labeled aptamer U26, compared to antibodies binding CD4's cytoplasmic domain. Aptamer-hCD4 binding kinetics were evaluated by a qPCR-based magnetic-bead binding assay to unmodified aptamers. U26 exhibited the highest binding affinity (Kd = 2.93 ± 1.03 nM) to hCD4-conjugated beads. Citrate-stabilized gold nanoparticles (mean particle diameter, 10.59 ± 1.81 nm) were functionalized with streptavidin to allow immobilization of biotin-labeled aptamers. Except for U4, the aptamer-gold nanoparticle conjugates (Apt-AuNP) remained stable under physiological conditions with their size (approx. 15 nm) appropriate for use in the LFAs. Lateral-flow based screening was used to evaluate the suitability of the Apt-AuNPs as CD4-detecting reporter molecules by immobilizing hCD4 and flowing the nanoparticle conjugates across the LFA. Using this approach, two novel sequences were identified as being suitable for the detection of hCD4: visual detection at 9 min was obtained using U20 or U26. After 20 min, equivalent colorimetric hCD4 responses were observed between anti-CD4 monoclonal antibody (ΔI = 94.19 ± 3.71), an existing CD4 aptamer F1-62 (ΔI = 90.31 ± 19.31) and U26 (ΔI = 100.14 ± 14.61) LFA's, each demonstrating high specificity to hCD4 compared to IgG. From the above, Crossover-SELEX allowed for the successful identification of ssDNA aptamers able to detect hCD4. Streptavidin-conjugated AuNPs, when bound to candidate aptamers, show potential application here as screening tools for the rapid evaluation of aptamer performance in low-cost lateral flow diagnostics.