An assay for a target single strand 20-base sequence of DNA coding for the anthrax lethal factor, based on conjugated polymer fluorescence superquenching, is reported. The assay employs a platform in which the receptor (a biotinylated complementary sequence "capture strand") and polymer (two components: an anionic poly(phenylene ethynylene) (PPE) and a biotinylated -PPE) are co-located on streptavidinderivatized polystyrene microspheres. A conjugate of the target strand with the energy transfer quencher QSY-7 (DNA-QTL) is used to construct competition assays for the target. A direct competition assay between the target-DNA and DNA-QTL for the microsphere-bound capture is only marginally successful due evidently to greater kinetic affinity of the polymer-capture ensemble for the conjugate. However a sequential addition of target, followed by DNA-QTL affords a quantitative assay for the target by attenuation of PPE fluorescence quenching by the DNA-QTL. Likewise a direct competition in solution between the target and DNA-QTL for the biotinylated capture strand followed by addition of microspheres provides a sensitive and quantitative assay for the target single strand DNA.

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 3′-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.

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