Visual detection of single-stranded target DNA using pyrroloquinoline-quinone-loaded liposomes as a tracer

The preparation of DNA-tagged liposomes containing an encapsulated prosthetic group tracer, pyrroloquinoline quinone (PQQ), and their application to the development of a sandwich-type hybridization assay for the visual detection of single-stranded DNA are described. Capture DNA is conjugated to the surface of microtiter plate wells through a biotin-streptavidin interaction. Target DNA is incubated with the plate in high salt concentrations. The reporter DNA-tagged liposomes encapsulating PQQ, the prosthetic group of the apo-enzyme glucose dehydrogenase (GDH), are used as the label to probe for bound target DNA. After washing away unbound liposomes and subsequent lysis of the bound fraction by surfactant, PQQ is released and available to activate the apo-enzyme. In the presence of glucose and a redox dye, 2,6-dichlorophenol indophenol (DCPIP), the dye is reduced to yield an optical color change from blue to colorless. This transition is observed visually or spectrophotometrically. The degree of optical change is proportional to the amount of PQQ present, which directly relates to the number of liposomes and, thus, the total amount of target DNA. An arbitrary target DNA sequence is used as a model system, and a limit of detection of 62 fmol is achieved.