Vajpayee, KamayaniKamayaniVajpayeeSrivastava, ShriyanshShriyanshSrivastavaSharma, ShivkantShivkantSharmaGupta, SwadhaSwadhaGuptaSrivastava, AshutoshAshutoshSrivastavaPaida, VidhiVidhiPaidaDash, Hirak RanjanHirak RanjanDashPappachan, AnjuAnjuPappachanShukla, Ritesh K.Ritesh K.ShuklaSengupta, SouvikSouvikSengupta2026-04-092026-04-092026-03-010937-982710.1007/s00414-026-03782-5https://repository.iitgn.ac.in/handle/IITG2025/34984Indigo, a naturally occurring dye that is currently synthesized in large quantities for modern textile uses, has been found to be a strong inhibitor of the polymerase chain reaction (PCR).The present study aims to understand the molecular basis of inhibition through a multifaceted approach that integrates in silico, spectroscopic, and amplification experiments. Molecular docking as well as dynamics simulations proved the preferential binding of Indigo inside the catalytic cleft of Taq DNA polymerase (3KTQΔDNA with a ΔG binding of -8.916 kcal/mol), which generated stable Mg2⁺-facilitated contacts as well as salt bridges along with significant residues (Asp610, His784, Lys663, K831). The contacts led to DNA site occlusion, which was inferred as steric interference with catalytic enzyme catalysis. Tryptophan fluorescence quenching (Kd = 616 ± 3.7 µM) confirmed moderate affinity within the site of action of the polymerase in its immediate vicinity. Amplification of STR markers exhibited substantial inhibition at loci TH01, D3S1358, D16S539, D13S317, D18S51, vWA, TPOX, D8S1179, and FGA, with up to 60% peak height reduction ratios as well as global signal losses (PHR < 0.30). Mechanistically, Indigo induced a tri-modal inhibitory effect through Mg2⁺ sequestration, DNA intercalation, and fluorescence quenching through π–π conjugation, collectively impairing polymerase activity and detection sensitivity. The application of bovine serum albumin-coated gold nanoparticles presented significantly enhanced recovery of amplification yield and allelic balance compared to free BSA. In contrast, free BSA-a universal facilitator-required high concentrations to achieve partial recovery of inhibition and exhibited batch-dependent variability, whereas BSA-coated AuNPs ensured predictable performance at sub-micromolar levels due to their enhanced surface reactivity, catalytic stability, and efficient inhibitor sequestration. The present study identifies Indigo as a multi-pathway inhibitor of PCR and presents a field-applicable nanoPCR-based protocol for generating profiles from dye-contaminated DNA samples.en-USPCR InhibitorIndigo DyeSTR profileGold nanoparticlesBSABSA-coated AuNPsArticle1437-1596