New insights into the photophysical properties and interaction mechanisms of Janus green blue dye with polyanions and its applications in colorimetric visualization of loop-mediated isothermal amplification and polymerase chain reaction

Abstract

In this investigation, the photophysical properties and interaction mechanisms of Janus green blue (JGB) dye with polyanions were systematically studied using spectroscopic techniques. The absorption spectral analysis revealed that JGB binds cooperatively to sodium alginate, leading to dye stacking along the polymer chain. The interaction of JGB dye with DNA was characterized by the emergence of a metachromatic peak at 564 nm, indicating the formation of dye aggregates. The analysis of absorption data reveals that JGB dye interacts with DNA at multiple binding sites, including at least one high-affinity site. The AutoDock Vina based blind docking approach was used to analyze the most probable binding location of JGB dye in DNA. By making use of the DNA-induced metachromasia, a colorimetric approach was developed for the visualization of loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The LAMP-colorimetric assay, targeting the Streptococcus pneumoniae gene, demonstrated a noticeable colour change with a detection limit of 1 pg μL⁻¹. The practical applicability was validated by detecting S. pneumoniae in artificial urine. In addition to LAMP, we tested the JGB dye based colorimetric assay for applicability in PCR reactions. The colorimetric PCR assay using the metal-responsive transcription factor (MTF-1) gene achieved a detection limit as low as 0.1 pg μL⁻¹. The study highlights the potential of DNA binding metachromic dye to significantly enhance colorimetric assays, offering a robust and sensitive tool for molecular diagnostics.