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

Abstract

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.

Figure 1. LED-CCD multi-wavelength detector

(A) A schematic configuration of the multi-wavelength LED detector. (B) A digital camera image of the actual detector platform. The detector system components highlighted in the schematic (A) are [1] an SXVF-M7 CCD camera mounted in a homemade acrylic shelf box [2], which was designed to hold the filters and the sample chips. The camera is equipped with a Tamron manual zoom CCTV 4-12 mm, f1.2 C-mount lens [3] with a green band pass emission filter [4] mounted on the end of the lens. The black acrylic 30-well sample chip [5] (also panel E) is placed on a shelf in the camera box above the blue band pass excitation filter [6]. The camera shelf box is placed on the top of the multi-wavelength LED illuminator [7] (also panel C) with light switches to operate the red, blue, green and white LEDs [8]. (C) Digital photograph of the multi-wavelength LED illuminator prior to attachment of the shelf box. (D) Spectra of the white (W), blue (B), green (G) and red (R) LEDs that comprise the multi-wavelength LED illuminator. (E) The black acrylic 30-well chips designed to hold aqueous samples for imaging.

Figure 2. Light characteristics of the EL and LED illumination sources

The spectra (measured by a spectrometer) and the light intensity of the LED were measured for the (A) white LED and the (B) blue LED illuminators (exposure time 100 ms) compared with the (C) white EL and (D) blue EL strips (exposure time 5 s). In panels A-D, the upper plots are measurements of the whole spectra (I) and the lower plots (II marked with arrows) are spectra measured with the blue filter, used for excitation of the FITC dye, in place. To evaluate the actual signal from the assay, the fluorescence signal of 5 nM unquenched SNAP-25 peptide (labeled with FITC) was measured with various exposure times (ranging from 10 ms to 10 s) using a cooled CCD camera equipped with a blue emission filter and a green excitation filter. (E) The signal intensity measured at the CCD plotted as a function of exposure time for the different excitation sources white LED (white circles), blue LED (black circles), and the blue EL panel (black triangles). Note that both the exposure time axis and the relative intensity axis are plotted on a log scale.

Figure 3. CCD Images and 3D analysis of EL-CCD and LED-CCD detection of unquenched FITC-SNAP peptide

The CCD images and the corresponding 3D imageJ analysis of the CCD emission images of nine concentrations of a 50% dilution series of the unquenched SNAP-25 peptide ranging from 0.019 nM to 5 nM and a control are shown for: (I) blue EL excitation exposure of 60 seconds, (II) blue EL excitation exposure of 300 s, (III) blue LED excitation exposure of 2 s and (IV) white LED excitation exposure of 3 s.

Figure 4. LED-CCD fluoresce analysis of unquenched SNAP-25 peptide

A 50% dilution series of the unquenched SNAP-25 (labeled with FITC) and a control (no SNAP-25) was prepared, giving 10 samples with a final concentration range of 0 nM-5 nM. The dilution series was loaded in triplicate into each 30-well chip and the fluorescence excited using either the LED or EL illuminators. The CCD camera was used for detection. The signal-to-noise (S/N) ratio for CCD measured intensity as a function of the SNAP concentration was plotted for each of the EL and LED excitation method used. (A) Blue LED illumination (1 s-diamond, 2 s-triangle and 3 s-rectangle) and blue EL illumination (5 min-circle). (B) Expansion of the lower concentration range, 0 nM-0.625 nM, of SNAP-25 dose response curve from (A). (C) White LED illumination (1 s-X, 2 s-vertical line and 3 s-diamond/dashed line) and the shorter exposure times used for the blue EL (15 s- triangle, 30 s,- circle and 60 s- rectangle). (D) Expansion of the lower concentration range for the SNAP-25 dose response plotted in (C). (E) White EL illumination (15 s- circle, 30 s-diamond and 60 s- triangle). (F) Expansion of the lower concentration range for the SNAP-25 dose response plotted in (E). Standard deviations were determined from three separate readings of each chip.

Figure 5. In vitro activity analysis of BoNT-A light chain (LcA) cleavage of FITC/DABCYL-SNAP-25 peptide detected by FRET

Various concentrations of LcA (0 nM-20 nM) were used to cleave FITC/DABCYL-SNAP-25 (5 μM). The cleavage products were excited by LED or EL and fluorescence intensity measured by CCD. (A) Correlation between the S/N ratio of the cleavage assay measurements in chip B and chip C. LcA dose response curves plotted as a function of the excitation source for (B) chip B and for (C) chip C. For both plots; blue LED exposure of 2 s (black circle), blue LED exposure of 3 s (white circle), white LED exposure of 3 s (black triangles), white LED exposure of 2 s (white rectangle), blue EL exposure of 30 s (black rectangle), blue EL exposure of 60 s (white rectangle), blue EL exposure of 300 s (black diamond). Note that the LcA concentration axis is plotted on the log scale, so the zero (blank) concentration is not present. Standard deviations were determined from three separate readings of each chip.