Multi-wavelength Spatial LED illumination based detector for in vitro detection of Botulinum Neurotoxin A Activity

A portable and rapid detection system for the activity analysis of Botulinum Neurotoxins (BoNT) is needed for food safety and bio-security applications. To improve BoNT activity detection, a previously designed portable charge-coupled device (CCD) based detector was modified and equipped with a higher intensity more versatile multi-wavelength spatial light-emitting diode (LED) illumination, a faster CCD detector and the capability to simultaneously detect 30 samples. A FITC/DABCYL Förster Resonance Energy Transfer (FRET)-labeled peptide substrate (SNAP-25), with BoNT-A target cleavage site sequence was used to measure BoNT-A light chain (LcA) activity through the FITC fluorescence increase that occurs upon peptide substrate cleavage. For fluorescence excitation, a multi-wavelength spatial LED illuminator was used and compared to our previous electroluminescent (EL) strips. The LED illuminator was equipped with blue, green, red and white LEDs, covering a spectrum of 450-680 nm (red 610-650 nm, green 492-550 nm, blue 450-495 nm, and white LED 440-680 nm). In terms of light intensity, the blue LED was found to be ~80 fold higher than the previously used blue EL strips. When measuring the activity of LcA the CCD detector limit of detection (LOD) was found to be 0.08 nM LcA for both the blue LED (2 s exposure) and the blue EL (which require ≥60 s exposure) while the limits of quantitation (LOQ) is about 1 nM. The LOD for white LED was higher at 1.4 nM while the white EL was not used for the assay due to a high variable background. Unlike the weaker intensity EL illumination the high intensity LED illumination enabled shorter exposure times and allowed multi-wavelength illumination without the need to physically change the excitation strip, thus making spectrum excitation of multiple fluorophores possible increasing the versatility of the detector platform for a variety of optical detection assays.