Engineering High-Performance Aptamers via Optimized SELEX and Structure-Guided Dimer Assembly for Clinical Malaria Diagnostics.

Malaria's high transmissibility and significant mortality demand reliable point-of-care diagnostics, yet aptamer-based detection has been hindered by the moderate affinity and poor nuclease stability of existing aptamers. To overcome these limitations, we developed a two-step strategy that integrates an optimized SELEX workflow with structure-guided dimer engineering to generate high-performance aptamers targeting Plasmodium falciparum lactate dehydrogenase (PfLDH). Initial enrichment was performed using bead-based SELEX, followed by gel-based isolation to preserve the native structure of PfLDH and reduce bead-induced selection biases. This hybrid approach yielded monomeric aptamers with substantially enhanced affinities unattainable through conventional bead-only SELEX, and these were subsequently engineered into a symmetric inverted dimeric aptamer (IDA) featuring a 3'-3' linkage. The IDA architecture is precisely matched to the symmetric scaffold of the PfLDH homotetramer, enabling picomolar affinity (>800-fold higher than existing aptamers), while the 3'-3' linkage provides greatly enhanced nuclease resistance. When incorporated into a cascading colorimetric amplification system, the IDA enabled highly sensitive PfLDH detection in human serum with a limit of detection of 3.3 ng/mL. Importantly, clinical evaluation using 52 human blood samples demonstrated 81.8% sensitivity and 100% specificity, which compares favorably with current commercial rapid diagnostic tests, thereby underscoring the strong translational potential of IDA for point-of-care malaria diagnostics.