title: 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.
authors.affiliation: Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA.
authors.forename: K
authors.initials: K
authors.lastname: Du
authors.affiliation: School of Engineering, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA.
authors.forename: H
authors.initials: H
authors.lastname: Cai
authors.affiliation: Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA.
authors.forename: M
authors.initials: M
authors.lastname: Park
authors.affiliation: ECEn Department, Brigham Young University, 459 Clyde Building, Provo, UT 84602, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Wall
authors.affiliation: ECEn Department, Brigham Young University, 459 Clyde Building, Provo, UT 84602, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Stott
authors.affiliation: Department of Virology and Immunology, Texas Biomedical Research Institute, 7620 NW Loop 410, San Antonio, TX 78227, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Alfson
authors.affiliation: Department of Virology and Immunology, Texas Biomedical Research Institute, 7620 NW Loop 410, San Antonio, TX 78227, USA.
authors.forename: A
authors.initials: A
authors.lastname: Griffiths
authors.affiliation: Department of Virology and Immunology, Texas Biomedical Research Institute, 7620 NW Loop 410, San Antonio, TX 78227, USA.
authors.forename: R
authors.initials: R
authors.lastname: Carrion
authors.affiliation: Department of Virology and Immunology, Texas Biomedical Research Institute, 7620 NW Loop 410, San Antonio, TX 78227, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Patterson
authors.affiliation: ECEn Department, Brigham Young University, 459 Clyde Building, Provo, UT 84602, USA.
authors.forename: A R
authors.initials: AR
authors.lastname: Hawkins
authors.affiliation: School of Engineering, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA. Electronic address: hschmidt@soe.ucsc.edu.
authors.forename: H
authors.initials: H
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry, University of California at Berkeley, Berkeley, CA 94720, USA. Electronic address: ramathies@berkeley.edu.
authors.forename: R A
authors.initials: RA
authors.lastname: Mathies
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D029043:Ebolavirus / Q000302:isolation & purification*; D004867:Equipment Design; D019142:Hemorrhagic Fever, Ebola / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D057230:Limit of Detection; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009693:Nucleic Acid Hybridization; D019095:Point-of-Care Systems; D012367:RNA, Viral / Q000032:analysis*; D052616:Solid Phase Extraction / Q000295:instrumentation; D019809:Streptavidin / Q000737:chemistry
chemical_list: D012367:RNA, Viral; D019809:Streptavidin
keywords: Air-bubble mixing; Microfluidics; Point-of-care; Single molecule RNA detection