title: An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.
abstract: A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.
authors.affiliation: Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Dafeng
authors.initials: D
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Mauk
authors.forename: Xianbo
authors.initials: X
authors.lastname: Qiu
authors.forename: Changchun
authors.initials: C
authors.lastname: Liu
authors.forename: Jitae
authors.initials: J
authors.lastname: Kim
authors.forename: Sudhir
authors.initials: S
authors.lastname: Ramprasad
authors.forename: Serge
authors.initials: S
authors.lastname: Ongagna
authors.forename: William R
authors.initials: WR
authors.lastname: Abrams
authors.forename: Daniel
authors.initials: D
authors.lastname: Malamud
authors.forename: Paul L A M
authors.initials: PL
authors.lastname: Corstjens
authors.forename: Haim H
authors.initials: HH
authors.lastname: Bau
mesh_terms: D001409:Bacillus cereus / Q000302:isolation & purification; D002021:Buffers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D006678:HIV / Q000302:isolation & purification; D007202:Indicators and Reagents / Q000737:chemistry; D046210:Microfluidic Analytical Techniques; D009696:Nucleic Acids / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D052616:Solid Phase Extraction; D018511:Systems Integration; D013696:Temperature
chemical_list: D002021:Buffers; D004269:DNA, Bacterial; D007202:Indicators and Reagents; D009696:Nucleic Acids; D012367:RNA, Viral
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