Rapid and highly sensitive luciferase reporter assay for the automated detection of botulinum toxin in the centrifugal microfluidic LabDisk platform

Abstract

We developed a system for the detection of botulinum neurotoxin (BoNT) type A based on a highly sensitive luciferase reporter assay automated by the centrifugal microfluidic LabDisk platform. The assay is based on the detection of BoNT's proteolytic activity and generation of a bioluminescent signal due to the release of firefly luciferase, pre-bound to microbeads via a cleavable peptide linker, in response to BoNT. It detected purified BoNT, BoNT in complex with neurotoxin associated proteins, and the recombinant enzymatic BoNT light chain in buffer and whole milk in the concentration range of 8 pM to 6 nM with an analytical sensitivity and limit of detection of 10–39 pM and 6–14 pM, respectively. The intra-disk, intra-day and inter-day variability were in the range of 1–13%, 1–7% and 10–13%, respectively. The developed assay correlated well with the conventional microwell plate assay. It is superior to the conventional BoNT assays in terms of portability, cost-effectiveness, lesser sample requirement, lesser number of steps and detection of a broad spectrum of BoNT serotypes and subtypes. It takes only 30 minutes, which is ideal for point-of-need BoNT detection in the case of security threats and food monitoring.