An improved assay for the detection of a 20-mer DNA sequence coding for the Anthrax Lethal Factor sequence that utilizes fluorescence superquenching and peptide nucleic acids (PNAs) is reported. The basis for this assay is a microsphere sensor that is coated with both Neutravidin (a biotin-binding protein) and biotinylated poly(phenylene)ethynylene (a fluorescent conjugated polymer). A 15-mer PNA tethered through its N-terminus to a biotin serves as a capture ligand for DNA oligonucleotides. When mixed, the PNAs and microspheres form a strong complex through the biotin-avidin interaction, creating a sensor for DNA detection. The 20-mer DNA target strand and a 17-mer DNA-QTL (QTL = quencher tether ligand, where the quencher is a QSY-7 label at the X-terminus of the DNA strand) of a similar sequence to the target strand are then used to develop an assay for DNA detection. A sequential addition of target, followed by DNA-QTL, yields a sensitive and selective assay for DNA detection. This study compares PNA to DNA in the ability to perform as a capture ligand, evaluates the importance of assay temperature, and illustrates resolution of single base-pair mismatches using this novel detection platform.