During this reporting period, substantial progress was made in the development and characterization of DNA aptamers for the four malaria biomarkers central to MalDiProT. The work began with the fabrication and preliminary testing of a microfluidic SELEX chip; however, due to reproducibility issues, including clogging, incomplete fabrication of the amplification chamber, and contamination risks, the workflow was transitioned to a more reliable magnetic bead–based SELEX strategy. Using this optimized protocol, eight rounds of selection were performed independently for Ang-1 and Ang-2, incorporating extensive counter-selection steps (His-tag peptides, bare beads, and 10% serum) to eliminate non-specific binders. Next-generation sequencing identified highly enriched candidates, and biolayer interferometry confirmed strong, specific binding for several sequences. Two aptamers were ultimately selected as lead candidates: Ang-1.1 with picomolar affinity (KD = 11.7 pM) and no cross-reactivity, and Ang-2.13 with nanomolar affinity (KD = 3.76 nM) and similarly excellent specificity. These represent a major achievement, as no previously reported pairs have been shown to discriminate well between Ang-1 and Ang-2 without cross-reactivity.
In parallel, SELEX campaigns for PfHRP2 and PfLDH were completed using the same selection protocol, generating abundant candidate sequences. However, none exhibited detectable affinity for their targets. After repeating SELEX without improvement and considering project timelines, the contingency plan was implemented: previously published high-affinity aptamers for both biomarkers were retrieved and characterized. For PfHRP2, aptamer HRP2-702 produced a clear competitive LFA response spanning clinically relevant concentrations, with analytical parameters (LoD, LoQ, EC50, dynamic range) suitable for differentiating mild from severe malaria. For PfLDH, literature-reported aptamers P11-30 and P11-35 were validated by biolayer interferometry, confirming affinities of 18 and 39 nM, respectively. These aptamers are now ready for implementation in the electrokinetically driven LFA system.
Throughout the SELEX and validation stages, multiple technical challenges were addressed, including the optimization of PCR amplification steps, the adaptation of biolayer interferometry methods for screening large aptamer libraries, and the refinement of counter-selection strategies to ensure clinical specificity. In addition, I provided significant technical leadership within the host environment, training group members in AuNP conjugation, LFA strip dispenser operation, and aptamer-based assay fabrication. Overall, the project achieved its principal scientific goals for the outgoing phase: establishing a reproducible SELEX workflow, identifying two high-performance aptamers for Ang-1 and Ang-2, and preparing validated bioreceptors for PfHRP2 and PfLDH to be integrated into the next stages of assay development.
