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ABSTRACT: A laboratory-on-a-chip system for pathogen detection is presented that integrates cell preconcentration, purification, polymerase chain reaction (PCR), and capillary electrophoretic (CE) analysis. The microdevice is composed of micropumps and valves, a cell capture structure, a 100 nL PCR reactor, and a 5 cm long CE column for amplicon separation. Sample volumes ranging from 10 to 100 microL are introduced and driven through a fluidized bed of magnetically constrained immunomagnetic beads where the target cells are captured. After cell capture, beads are transferred using the on-chip pumps to the PCR reactor for DNA amplification. The resulting PCR products are electrophoretically injected onto the CE column for separation and detection of Escherichia coli K12 and E. coli O157 targets. A detection limit of 0.2 cfu/microL is achieved using the E. coli O157 target and an input volume of 50 microL. Finally, the sensitive detection of E. coli O157 in the presence of K12 at a ratio of 1:1000 illustrates the capability of our system to identify target cells in a high commensal background. This cell capture-PCR-CE microsystem is a significant advance in the development of rapid, sensitive, and specific laboratory-on-a-chip devices for pathogen detection.
Analytical Chemistry 05/2009; 81(9):3523-8. · 5.86 Impact Factor
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ABSTRACT: A cell concentration microdevice for immunomagnetic pathogen isolation from a dilute sample is presented. Cells are driven by integrated on-chip pumps through a fluidized bed of immobilized immunomagnetic beads. Off-chip polymerase chain reaction and capillary electrophoretic analysis are used to determine capture efficiencies of E. coli and to optimize the system. Beads are immobilized after each split in a bifurcated channel system to ensure a balanced distribution of beads in all the capture channels. The addition of a pumping flutter step to repeatedly drive sample through the bead bed was found to enhance capture. Capture efficiencies of 70% and a limit of detection of 2 cfu/microL were achieved; specific capture of E. coli at a concentration of 100 cfu/microL in a 100-fold background of S. aureus is shown. This capture/concentration system is an important step in overcoming the macro-to-micro interface challenge in the development of microdevices for pathogen detection.
Biomedical Microdevices 09/2008; 10(6):909-17. · 3.03 Impact Factor
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ABSTRACT: A portable forensic genetic analysis system consisting of a microfluidic device for amplification and separation of short tandem repeat (STR) fragments as well as an instrument for chip operation and four-color fluorescence detection has been developed. The microdevice performs polymerase chain reaction (PCR) in a 160-nL chamber and capillary electrophoresis (CE) in a 7-cm-long separation channel. The instrumental design integrates PCR thermal cycling, electrophoretic separation, pneumatic valve fluidic control, and four-color laser excited fluorescence detection. A quadruplex Y-chromosome STR typing system consisting of amelogenin and three Y STR loci (DYS390, DYS393, DYS439) was developed and used for validation studies. The multiplex amplification of these 4 loci with 35 PCR cycles followed by CE separation and 4-color fluorescence detection was completed in 1.5 h. All the amplicons can be detected with a limit of detection of 20 copies of male standard DNA in the reactor. Real-world forensic analyses of oral swab and human bone extracts from case evidence were also successfully performed. Mixture analysis demonstrated that a balanced profile can be obtained even at a male-to-female template ratio of 1:10. The successful development and operation of this portable PCR-CE system establishes the feasibility of rapid point-of-analysis DNA typing of forensic casework, of mass disaster samples or of individuals at a security checkpoint.
Analytical Chemistry 04/2007; 79(5):1881-9. · 5.86 Impact Factor
