Lab-on-a-chip for botulinum neurotoxin a (BoNT-A) activity analysis

Abstract

A Lab-on-a-chip (LOC) was designed, fabricated and tested for the in vitro detection of botulinum neurotoxin serotype A (BoNT-A) activity using an assay that measures cleavage of a fluorophore-tagged peptide substrate specific for BoNT-A (SNAP-25) by the toxin light chain (LcA). LcA cleavage was detected by Förster Resonance Energy Transfer (FRET) fluorescence. FRET fluorescence was measured by a newly developed portable charge-coupled device (CCD) fluorescent detector equipped with multi-wavelength light-emitting diodes (LED) illumination. An eight V-junction microchannel device for BoNTs activity assays was constructed using Laminated Object Manufacturing (LOM) technology. The six-layer device was fabricated with a Poly(methyl methacrylate (PMMA) core and five polycarbonate (PC) layers micromachined by CO2 laser. The LOC is operated by syringe and is equipped with reagents, sample wells, reaction wells, diffusion traps (to avoid cross contamination among channels) and waste reservoirs. The system was detected LcA at concentrations as low as 0.5 nM, which is the reported sensitivity of the SNAP-25 in vitro cleavage assay. Combined with our CCD detector, the simple point of care system enables the detection of BoNTs activity and may be useful for the performance of other complex medical assays without a laboratory. This approach may realize the potential to enhance the quality of health care delivery for underserved populations.

Fig. 1

LED-CCD multi-wavelength detector. A schematic configuration of the multi-wavelength LED detector (A), photograph of the detector (B). The elements in the schematic and the photograph of the actual detection platform are: SXVF-M7 CCD camera (1) 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 pass band emission filter (4) mounted on the end of the lens. The 8-channel LOC (5) 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) with light switches to operate the red, blue, green and white LEDs (8).

Fig. 2

Functional elements of multi-channel LOC system. An expanded single channel (A) where the sample reservoir (1) and the reagent reservoir (2) are joined via V junction (3) to the joining channel (4). The cleavage reaction is monitored in the detection wells (5) designed with air traps to minimize air bubbles, followed by a mixing trap (6). The full schematic of eight-channel LOC chip (B), including the eight channel negative pressure distribution splitter (7) connected to a waste chamber (8) to the outlet for a pump or syringe (9). The alignment holes (10) were designed to simplify fabrication. A clear PMMA LOC device (C) photographed with blue illumination to increase contrast.

Fig. 3

Outline of layers for the six layers LOC. Outline of individual layers for the six layer LOC (A), side view of the layers showing fluid flow (B). Layers are numbered in order from the top layer down (I to VI). All the layers are 250 μm PC except the core layer III which is 3.2 mm PMMA. The main elements shown are: sample reservoirs (1), reagent reservoirs (2) connected to the V junction (3) via connecting slits (1A and 2A), through layer pass (3A) to the joining channel (4), detection wells (5) with air bubble traps (5A), mixing traps (6) negative pressure distribution splitter (7) a waste chamber (8) to the outlet for a pump or syringe (9).

Fig. 4

Optical and fluidics cross-talk between channels. A clear LOC with fluidics trap (A), a black LOC with fluidics trap (B) and a black LOC without fluidics trap (C) were loaded with fluorescein (channels 1, 3, 5 and 7) and water (channels 2, 4, 6 and 8). The LOCs were incubated for 120 minutes at room temperature and the signal from the detection wells marked with an arrow was measured every 15 minutes. The plot of diffusion cross talk for the water channels (D) was calculated as a signal/noise ratio where noise is signal at T0 (before diffusion started) without trap (a), with trap (b).

Fig. 5

LOC in vitro activity detection of BoNT-A light chain (LcA using the SNAP-25 cleavage assay. The SNAP-25 peptide substrate for BoTN-A is labeled with the FITC donor/DABCYL acceptor FRET pair. Interaction of the substrate with the toxin light chain LcA results in cleavage of the peptide sequence, disrupting the FRET and resulting in increased FITC donor emission as measured by CCD with 500 ms exposure. FRET activity assay was performed in a nine well plate (A) and the 8-channel LOC (B) measured with different concentrations of 2 fold LcA serial dilution in the range 0 to 16 nM nm (with 0 nM used as control) using four replicas. The assay was carried out for two hours in room temperature. The Signal to Noise of the FRET activity assay was calculated as the ratio between the value of the measurement to the value of the control (no LcA) and was plotted (C) for the nine well plate (a) and the LOC (b).