Lab-on-a-chip for botulinum neurotoxin a (BoNT-A) activity analysis

Abstract

A Lab-on-a-chip (LOC) was designed, fabricated and tested for the in vitro detection of botulinum neurotoxin serotype A (BoNT-A) activity using an assay that measures cleavage of a fluorophore-tagged peptide substrate specific for BoNT-A (SNAP-25) by the toxin light chain (LcA). LcA cleavage was detected by Förster Resonance Energy Transfer (FRET) fluorescence. FRET fluorescence was measured by a newly developed portable charge-coupled device (CCD) fluorescent detector equipped with multi-wavelength light-emitting diodes (LED) illumination. An eight V-junction microchannel device for BoNTs activity assays was constructed using Laminated Object Manufacturing (LOM) technology. The six-layer device was fabricated with a Poly(methyl methacrylate (PMMA) core and five polycarbonate (PC) layers micromachined by CO2 laser. The LOC is operated by syringe and is equipped with reagents, sample wells, reaction wells, diffusion traps (to avoid cross contamination among channels) and waste reservoirs. The system was detected LcA at concentrations as low as 0.5 nM, which is the reported sensitivity of the SNAP-25 in vitro cleavage assay. Combined with our CCD detector, the simple point of care system enables the detection of BoNTs activity and may be useful for the performance of other complex medical assays without a laboratory. This approach may realize the potential to enhance the quality of health care delivery for underserved populations.