Selection of high-affinity optimer aptamers for A-type inclusion proteins and designing of optimer-enabled photonic crystal paper device for recognition of poxviruses.

We present an optimer-enabled photonic crystal paper device (OPCD) for rapid, multiplexed detection of orthopoxviruses, alongside a fundamental insight into aptamer structure-function relationships. By systematically truncating computationally designed 40-nucleotide DNA aptamers targeting A-type inclusion protein (A27L) proteins from cowpox, monkeypox, and vaccinia viruses, we identified a structural threshold at 32 nucleotides that defines the minimal functional binding core. Level 1 optimers (32 nt) retain 88-92% of the parent aptamer affinity while reducing synthesis costs by ∼20%, achieving dissociation constants (Kd) of 11.3-18.4 nM. However, further truncation to 26 nt results in a significant loss of activity. Multi-technique validation using isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and computational modeling reveals that this threshold arises from a bipartite architecture comprising an essential protein-binding core and flexible terminal regions with minimal functional contribution. Thermodynamic analysis indicates that truncation enhances binding efficiency by reducing entropic penalties via structural pre-organization. These optimized aptamers were integrated into an optimer-enabled photonic crystal paper device (OPCD) composed of optimer-functionalized poly(methyl methacrylate) microspheres assembled into photonic crystal arrays on nitrocellulose membranes. The device simultaneously detects three orthopoxviruses via visible colorimetric shifts, with an LOD of 10 ng/mL, intra-assay coefficient of variation (CV) of 4.3 ± 1.2%, inter-assay CV of 6.8 ± 1.5%, and high specificity (<5% cross-reactivity). The device maintains >98% functionality after one month of storage at room temperature. This work establishes a strategy for rational aptamer optimization. It demonstrates a scalable, low-cost platform for point-of-care diagnostics, highlighting the potential to remove non-functional sequence elements without compromising performance.