Multiplexed efficient on-chip sample preparation and sensitive amplification-free detection of Ebola virus

Abstract

An automated microfluidic sample preparation multiplexer (SPM) has been developed and evaluated for Ebola virus detection. Metered air bubbles controlled by microvalves are used to improve bead-solution mixing thereby enhancing the hybridization of the target Ebola virus RNA with capture probes bound to the beads. The method uses thermally stable 4-formyl benzamide functionalized (4FB) magnetic beads rather than streptavidin coated beads with a high density of capture probes to improve the target capture efficiency. Exploiting an on-chip concentration protocol in the SPM and the single molecule detection capability of the antiresonant reflecting optical waveguide (ARROW) biosensor chip, a detection limit of 0.021pfu/mL for clinical samples is achieved without target amplification. This RNA target capture efficiency is two orders of magnitude higher than previous results using streptavidin beads and the limit of detection (LOD) improves 10×. The wide dynamic range of this technique covers the whole clinically applicable concentration range. In addition, the current sample preparation time is ~1h which is eight times faster than previous work. This multiplexed, miniaturized sample preparation microdevice establishes a key technology that intended to develop next generation point-of-care (POC) detection system.

Keywords: Air-bubble mixing; Microfluidics; Point-of-care; Single molecule RNA detection.

Figure 1

(a) Design of the sample preparation multiplexer (SPM) with six incubation reservoirs for target preparation. Reagents and metered air bubbles are introduced into the incubation reservoirs by microvalve pumps. (b) Photograph of the SPM. (c) Schematic of the solid-phase extraction process and assay.

Figure 2

(a) Schematic of how air bubbles are generated and used to effect mixing in the SPM. (b) Photographs of magnetic bead solution before (0 second) and after (30 seconds) air bubble mixing. A video of mixing is available in the supplement information. (c) Capture efficiency of synthetic nucleic acid target vs. mixing time. Air mixing was applied every 10 minutes during the overall 40 minute incubation time.

Figure 3

Optimization of incubation conditions for 100-mer synthetic DNA nucleic acid target capture as a function of incubation time (10 to 60 minutes) and release time (2 to 10 minutes). The release temperature is 80 °C. Thirty seconds of air bubble mixing was applied every 10 minutes during incubation for all samples.

Figure 4

Capture efficiency results for 1 nM synthetic DNA nucleic acid (1X) as a function of incubation time compared with similar experiments performed with the 3X target concentration procedure. (1X: input 20 μL, release 20 μL; 3X: input 45 μL, release 15 μL)

Figure 5

Detection of Ebola virus with silicon-based optofluidic ARROW chip. Concentration-dependent fluorescence counts for Streptavidin beads (off-chip and on-chip) and 4FB beads (off-chip and on-chip) target capture. No peaks were detected above dye background for negative control samples. (Dark blue and green lines are from reference 12). (Inset) Segments of digitized fluorescence signal above SYBR^® Gold dye background indicating single RNA detection events.