Detection of botulinum toxins: micromechanical and fluorescence-based sensors

Botulinum neurotoxins (BoNTs) are the most lethal of known human toxins, exerting their actions by cleaving the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) required for neurotransmitter release. Early detection of these toxins is important for appropriate medical treatment. To detect BoNT activity, traditional assays monitor the effects of the toxins on a mammalian organism (observing signs of botulism in mice), or identify cleaved substrate molecules (electrophoresis and immunoblot). Similarly, enzyme-linked assays were used for screening potential toxin inhibitors in vitro in attempt to select antitoxins that could be used for therapeutic purposes. Here we review two recently developed sensor systems for detection of toxin activity in vitro and in living cells. In vitro detection was carried out using a micromechanosensor that relies on the attachment of a bead to the micromachined cantilever through the interactions between SNARE proteins, with synaptobrevin 2 deposited onto beads and syntaxin 1A deposited onto cantilevers. The presence of toxin is indicated by the detachment of the bead, resulting from cleavage of synaptobrevin 2. Additional in vitro detection is possible using fluorescent sensors constructed by inserting linkers, containing fragments of SNARE proteins acting as toxin substrates, between cyan and yellow fluorescent proteins (CFP and YFP). Toxins cause the cleavage of these linkers and thereby abolish fluorescence resonance energy transfer (FRET) between CFP and YFP. This approach, combined with an additional sensor based on subcellular redistribution of YFP fluorescence in cells, was used for cell-based screening of toxin activity.