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

• Published in: Biosensors & bioelectronics Vol. 91; pp. 489 - 496
• Main Authors: Du, K., Cai, H., Park, M., Wall, T.A., Stott, M.A., Alfson, K.J., Griffiths, A., Carrion, R., Patterson, J.L., Hawkins, A.R., Schmidt, H., Mathies, R.A.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15-05-2017
• Subjects: Air-bubble mixing; Biosensing Techniques - instrumentation; Ebolavirus - isolation & purification; Equipment Design; Hemorrhagic Fever, Ebola - diagnosis; Hemorrhagic Fever, Ebola - virology; Humans; Limit of Detection; Microfluidic Analytical Techniques - instrumentation; Microfluidics; Nucleic Acid Hybridization; Point-of-care; Point-of-Care Systems; RNA, Viral - analysis; Single molecule RNA detection; Solid Phase Extraction - instrumentation; Streptavidin - chemistry; Air-bubble mixing; Single molecule RNA detection; Point-of-care; Microfluidics
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2016.12.071

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.
•An automated microfluidic sample preparation multiplexer (SPM) has been introduced for Ebola virus detection.•Metered air bubbles injected through pneumatically actuated microvalves are used to enhance mixing.•A significantly improved sensitivity for clinical samples (0.021 pfu/mL) is reported without target amplification.