Nucleic acid-templated toluidine blue O assemblies as colorimetric probes for point-of-care nucleic acid tests

This study systematically investigated the metachromatic properties of toluidine blue O (TBO) in the presence of polyanions using spectroscopic techniques. Spectroscopic analyses demonstrated the formation of DNA-templated H-type aggregates of TBO. Fourier-transform infrared (FT-IR) and optical microscopy analyses were carried out to study the binding of TBO to DNA and subsequent dye aggregation. Auto-docking studies were performed to identify the binding sites of TBO in DNA. DNA-templated metachromasia of TBO was used to develop a colorimetric method for monitoring polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). The TBO colorimetric assay showed a significant color shift from blue to purple for PCR and LAMP reactions targeting the metal-responsive transcription factor 1 (MTF-1), a biomarker for osteoarthritis, and the Streptococcus pneumoniae gene, respectively, with a limit of detection as low as 1 pg/μL (for both MTF-1 and S. pneumoniae). The aggregation of TBO was highly specific to the DNA amplicons and was not influenced by the PCR or LAMP reagents. The practical utility of this colorimetric method for point-of-care testing in pediatric diagnosis was confirmed by its successful application in the detection of S. pneumoniae in artificial urine samples.