A universal fluorescent aptasensor based on AccuBlue dye for the detection of pathogenic bacteria

We report a universal fluorescent aptasensor based on the AccuBlue dye, which is impermeant to cell membranes, for the detection of pathogenic bacteria. The sensor consists of AccuBlue, an aptamer strand, and its complementary strand (cDNA) that partially hybridizes to the aptamer strand. We have fabricated two models by changing the sequence of the reaction between the elements in the system. One is the "signal on" model in which the aptamer is first bound to the target, followed by the addition of cDNA and AccuBlue, at which time the cDNA hybridizes with the free unreacted aptamer and forms a double-stranded DNA (dsDNA) duplex. Such hybridization causes AccuBlue to insert into the dsDNA and exhibit significantly increased fluorescence intensity because of the specific intercalation of the AccuBlue into dsDNA rather than single-stranded DNA (ssDNA). The other model, "signal off," involves hybridization of the aptamer with cDNA first, resulting in high fluorescence intensity on the addition of AccuBlue. When the target is added, the aptamer binds the target, causing the cDNA to detach from the dsDNA duplex and resulting in low fluorescence as a result of the liberation of AccuBlue. Because this design is based purely on DNA hybridization, and AccuBlue is impermeant to cell membranes, it could potentially be adapted to a wide variety of analytes.