Elucidating the mechanism of fulvic acid-mediated PCR inhibition and its mitigation through nanoparticle-assisted...

Fulvic acid is a common humic contaminant found in soil-matrix forensic samples and is a highly effective PCR inhibitor. This work uses computational analysis to investigate its molecular interaction with Taq DNA polymerase and evaluate nanoparticle-based facilitation strategies. Molecular docking and molecular dynamics simulations proposed selective binding of fulvic acid with catalytic residues (ARG587, ASP610, PHE667, TYR671, ASP785, GLU786), affecting DNA binding, active-site geometry, and Mg²⁺ coordination. Tryptophan quenching assays confirmed a low-affinity (∼251.6 ± 2.2 µM), reversible interaction. Functionally, fulvic acid reduced the peak height by > 80 % within the DNA profile and affected critical loci-D18S51, Penta D, D22S1045, FGA, CSF1PO, and D21S11. Nano-based facilitators-bare and BSA-coated AuNPs-were compared with the commonly employed BSA; the latter showed a statistically significant (p < 0.0001) 25 % TPH improvement compared to the inhibited level but did not affect allelic balance. These findings establish the inhibitory mode for fulvic acid and confirm the effectiveness of BSA-coated AuNPs as a scalable solution to restore PCR efficiency to environmentally compromised forensic DNA samples over highly concentrated application of BSA as a facilitator.