Programmable PNA-nanoparticle hybrids as nanoscale recognition architectures for amplification-free nucleic acid recognition.

Rapid, field-deployable nucleic acid diagnostics require robust recognition elements and simple signal transduction. We present a programmable peptide nucleic acid-gold nanoparticle (PNA-AuNP) platform for amplification-free detection of Schistosoma spp. DNA. Thiolated PNAs were conjugated to citrate-stabilized AuNPs, forming a nanointerface that translates sequence-specific hybridization into a plasmonic colorimetric signal. Structural and thermodynamic validation confirmed duplex integrity: X-ray photoelectron spectroscopy revealed N 1s (+0.4 eV) and O 1s carbonyl (-0.2 eV) shifts, while FTIR showed amide I/II changes and new sugar-phosphate bands (921-1045 cm-1) with a phosphate fingerprint at 1237 cm-1. Zeta potential shifted from -35 mV (DNA) to -15 mV (PNA-DNA complex), indicating charge neutralization. Isothermal titration calorimetry demonstrated strong binding (Kd ≈ 20.9 nM, ΔH ≈ 252.4 kcal/mol, ΔS ≈ 880.4 cal/mol·K), nearly twice that of ASO-DNA. Guided by these insights, a tri-probe plasmonic lateral flow assay achieved a detection limit of 0.01 ng. mL-1 within 20 min tenfold more sensitive than ASO-based LFAs and comparable to RT-PCR while maintaining high specificity, reproducibility (CV <10 %), and six-week stability. This work establishes PNAs as high-affinity, enzymatically stable probes, offering a versatile framework for rapid, amplification-free diagnostics in resource-limited settings.