Abstract
AbstractA portable fluorescence assay for direct endopeptidase activity detection has been developed with the use of a cyclophaneâbased conductive conjugated fluorescent polymer and peptide substrates. The substrates, carrying internal quenching amino acid, were designed to be cleaved in a sequenceâspecific manner by a protease of interest. Intact substrates were incapable of quenching the fluorescence of the polymer due to steric constraints. Upon specific cleavage, the quencher became exposed and could interact with the ring structure of the fluorescent polymer, disrupting the conjugation and quenching the fluorescence along the polymer chain. The approach was developed using a model GluâC endopeptidase from Staphylococcus aureus strain V8, detected in the picomolar to micromolar concentration range. The developed assay was tested for the detection of endopeptidase activity of botulinum toxin. The feasibility study showed that botulinum neurotoxin (BoNT)âA could be detected down to picomolar concentration, with limit of detection of 5 pg, or 33 amol in 5 ”L of sample, and a total assay time under 2 h. The assay exhibited high specificity and no crossâreactivity with BoNTâB was detected. Following this proofâofâconcept work, the assay can be further optimized and expanded to differentiate between various botulinum toxin serotypes in their active proteolytic form, or modified for detection of proteases with other specificities. © 2015 Society of Chemical Industry
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