Molecular arrangements of Thionine within the DNA scaffolds enable machine learning-assisted colorimetric LAMP...

In the present work, we describe the distinctive molecular arrangement behavior of the phenothiazine dye - thionine (Th) - in the presence of LAMP amplicons and demonstrate its potential use as a visual indicator for LAMP-based detection. To better understand the mechanism responsible for DNA-templated stacking of Th molecules, detailed photophysical investigations were carried out in the presence of DNA and poly(acrylic acid) (PAA). Molecular docking analysis using AutoDock suggested that the planar phenothiazine aromatic core of Th is positioned along the groove region with the -NH₂ groups oriented toward the outer region of the groove, remaining partially exposed to the solvent. Taking advantage of this interaction behavior, Th was further utilized to develop a colorimetric assay for nucleic acid amplification using LAMP. The dye exhibited a distinct color transition from violet to blue in the presence of LAMP amplicons generated by targeting the pneumolysin (ply) gene of Streptococcus pneumoniae. This visual change was accompanied by a bathochromic shift in the absorption spectrum, indicating DNA-induced molecular arrangements of Th molecules. The developed Th-based LAMP assay showed a limit of detection of 0.1 pg/μL. In addition, a machine learning-assisted image analysis approach was explored to quantitatively evaluate the colorimetric responses under standardized imaging conditions.