A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity

Abstract

Current biodetection illumination technologies (laser, LED, tungsten lamp, etc.) are based on spot illumination with additional optics required when spatial excitation is required. Herein we describe a new approach of spatial illumination based on electroluminescence (EL) semiconductor strips available in several wavelengths, greatly simplifying the biosensor design by eliminating the need for additional optics. This work combines EL excitation with charge-coupled device (CCD) based detection (EL-CCD detector) of fluorescence for developing a simple portable detector for botulinum neurotoxin A (BoTN-A) activity analysis. A Förster Resonance Energy Transfer (FRET) activity assay for BoTN-A was used to both characterize and optimize the EL-CCD detector. The system consists of two modules: (1) the detection module which houses the CCD camera and emission filters, and (2) the excitation and sample module, containing the EL strip, the excitation filter and the 9-well sample chip. The FRET activity assay used in this study utilized a FITC/DABCYL-SNAP-25 peptide substrate in which cleavage of the substrate by BoTN-A, or its light chain derivative (LcA), produced an increase in fluorescence emission. EL-CCD detector measured limits of detection (LODs) were similar to those measured using a standard fluorescent plate reader with valves between 0.625 and 1.25 nM (31-62 ng/ml) for LcA and 0.313 nM (45 ng/ml) for the full toxin, BoTN-A. As far as the authors are aware this is the first demonstration of phosphor-based EL strips being used for the spatial illumination/excitation of a surface, coupled with CCD for point of care detection.

Deciphering Enzyme Function Using Peptide Arrays

“…This technique was evaluated using combinatorial peptide libraries and substitutional analyses of substrate peptides incubated with trypsin [170,171] and subsequently employed to determine the substrate specificity of the integral membrane protease OmpT of Escherichia coli [171]. A similar assay principle was used for the fluorescence-based detection of botulinum neurotoxin A activity on peptide arrays [172]. A sophisticated but rather tedious procedure involves peptides coupled to cellulose membranes by their C-terminus and having an antibody epitope tag with a biotinylated lysine residue at the N-terminus (see Fig.…”

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…For instance, FRET-based biosensors have led to a deeper understanding of a number of biological phenomena such as integrin adhesiveness and signaling dynamics [53], plasma membrane biophysical interactions [54], host-pathogen interactions [55], and protein folding geometries and conformational states [56]. Similar dye combinations are also useful for FRET-based biosensing, including glucose sensors [57] and biological agent detection [58].…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…This principle was used by different groups to detect the activity of different BoNT serotypes with sensitivities between 35 pg/mL and 150 ng/mL depending on serotype, FRET substrate, and assay time used (Anne et al 2001;Dong et al 2004;Rasooly and Do 2008;Pires-Alves et al 2009;Poras et al 2009;Gilmore et al 2011;Ruge et al 2011). The principle has also been implemented into portable devices with sensitivities in the ng/mL range (Sapsford et al 2008;Kostov et al 2009;Sun et al 2010;Balsam et al 2011) and is the basis for commercial substrates like SNAPtide Ò (Shine et al 2002).…”