Multi format compatible visual and fluorometric detection of SEB toxin in nanogram range by carbon dot-DNA and acriflavine nano-assembly

A simple, sensitive biosensing platform for visual and fluorometric detection of Staphylococcal enterotoxin B (SEB) was developed using SEB-binding aptamer as recognition element and Carbon Dot (CD) and Acriflavine (Acf) as fluorescent probes. The assay is based on FRET between CD and Acf, the latter being intercalated in DNA duplex. SEB aptamer and partially complementary ssDNA constitute the duplex that denature in presence of SEB due to selective binding of the aptamer with SEB. SEB binding to aptamer lead to release of Acf in solution and disrupt FRET between CD and Acf. Subsequently, fluorescence emission due to Acf disappeared with emergence of CD fluorescence upon UV irradiation. A linear spectrophotometric response in the range of 0.5 ng/mL to 10 ng/mL of SEB concentration was obtained within 15 min of incubation. Interestingly, FRET inhibition and recovery of blue emission of CD is visually detectable in multiple format like test tube, 96 well-plate and microfluidic channels when irradiated with a hand held UV lamp only and without any machine intervention at 5 ng/mL sensitivity level. UV irradiated microfluidic channel is able to display visible changes from green to blue emission with as low as 0.01 ng of the toxin in 20 μL sample volume.