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Optofluidic Analysis System for Amplification-Free, Direct Detection of Ebola Infection


Optofluidic Analysis System for Amplification-Free, Direct Detection of Ebola Infection

H Cai et al. Sci Rep.


The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.


Figure 1
Figure 1
(a) Modular approach to hybrid optofluidic integration with individual chips dedicated to sample preparation and single nucleic acid detection; intersecting solid-core (SC-WG) and liquid-core (LC-WG) waveguides are highlighted in the optofluidic layer along with the planar optical beam geometry for single nucleic acid detection; (b) photographs of silicon-based optofluidic ARROW chip (bottom) and PDMS-based microfluidic automaton (top); pneumatic/sample channels are filled with red/blue dye for visualization of channel layers and layout; chips connect with flexible tubing; (c) Solid-phase extraction assay used for target isolation and detection. The dashed box indicates the steps that were implemented on the automaton chip.
Figure 2
Figure 2. On-chip target preconcentration.
(a) Particles to be concentrated: target oligomers are bound to pull-down recognition sequence on magnetic microbeads; molecular beacons specifically bind to target and cause beads to fluoresce; (b) particle fluorescence counts detected on ARROW chip before preconcentration step and (c) after preconcentration on automaton. A 335x concentration increase is observed.
Figure 3
Figure 3. Amplification-free detection of Ebola virus on optofluidic chip.
(a) Segments of digitized fluorescence counts above background showing concentration-dependent numbers of single RNAs; (b) concentration-dependent particle counts for off-chip (open squares) and using the automaton (solid circles) sample preparation. Negative controls (SUDV, MARV) did not create any counts (note the broken vertical scale). Dashed line: Predicted particle count determined from initial concentration and experimental parameters (tested sample volume, excitation and detection mode areas, liquid-core channel cross section). The lowest two concentrations for on-chip sample prep were reached by 50x and 460x preconcentration steps, respectively.

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