Thermodynamic Determinants in Antibody-Free Nucleic Acid Lateral Flow Assays (AF-NALFA): Lessons from Molecular Detection of Listeria monocytogenes, Mycobacterium leprae and Leishmania amazonensis

Antibody-free nucleic acid lateral flow assays (AF-NALFA) are an established approach for rapid detection of amplified pathogens DNA but can yield inconsistent signals across targets. Since AF-NALFA depends on dual hybridization of probes to single-stranded amplicons (ssDNA), site-specific thermodynamic (Gibbs free energy-ΔG) at probe-binding regions may be crucial for performance. This study investigated how <i>site-specific</i>-ΔG and sequence complementarity at probe-binding regions determine Test-line signal generation, comparing native and synthetic amplicons and assessing the effects of local secondary structures and mismatches. Asymmetric PCR-generated ssDNA amplicons of <i>Listeria monocytogenes</i>, <i>Mycobacterium leprae</i>, and <i>Leishmania amazonensis</i> were analyzed in silico and tested in AF-NALFA prototypes with gold-labeled thiol probes and biotinylated capture probes. T-line signals were photographed, quantified (ImageJ version 1.4k), and statistically correlated with <i>site-specific</i>-ΔG. While native ssDNA from <i>M. leprae</i> and <i>L. amazonensis</i> failed to produce AF-NALFA T-line signals, <i>L. monocytogenes</i> yielded strong detection. <i>Site-specific</i>-ΔG below -10 kcal/mol correlated with reduced hybridization. Synthetic oligos preserved signals despite structural constraints, whereas ~3-4 mismatches, especially at capture probe regions, markedly impaired T-line intensity. The performance of AF-NALFA depends on the synergism between thermodynamic accessibility, <i>site-specific</i>-ΔG-induced site constraints, and sequence complementarity. Because genomic context affects hybridization, target-specific thermodynamic in silico evaluation is necessary for reliable pathogen DNA detection.