Integrated capture, concentration, polymerase chain reaction, and capillary electrophoretic analysis of pathogens on a chip

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.

Figure 1

Cell capture-PCR-CE microdevice that integrates a capture structure with PCR and capillary electrophoresis. Immunomagnetic beads are immobilized by an external magnet in the capture channels as sample solution is driven through the bead bed. After capture and washing, the bead-cell duplexes are pumped into the 100 nL PCR reactor, where an external heater drives the reaction. Finally, the PCR amplicons are injected onto the CE column, separated, and detected near the anode using LIF. Channels in blue are enclosed using a PDMS-glass sandwich. Channels in red are enclosed by thermally bonding the etched glass wafer to the RTD wafer. Green features delineate the Ti/Pt resistance temperature detector features.

Figure 2

Detection limit of the cell capture-PCR-CE microdevice using E. coli K12 as a target. Immunomagnetic beads with polycloncal antibodies specific to E. coli were used to capture the cells from the sample, and primers designed against island KI#128 on the E. coli K12 genome were used in the PCR. The presence of the 259 bp amplicon demonstrates a detection sensitivity of 10 cfu/μL or 100 cfu in the 10 μL input sample.

Figure 3

Detection limit of the cell capture-PCR-CE microdevice using E. coli O157 as a target. Commercial immunomagnetic beads specific to E. coli O157 (Invitrogen) were used to capture the cells from the sample, and primers designed against island OI#43 in the E. coli O157 genome were used in the PCR. The presence of the 191 bp amplicon demonstrates a detection sensitivity of 10 cfu/μL or 100 cfu in the 10 μL input sample.

Figure 4

Dilution study using the cell capture-PCR-CE microdevice. Using a fixed number of input E. coli O157 cells (10 cfu), the cell capture-PCR-CE system was tested with input sample volumes of 10, 20, 50 and 100 μL. Positive detection is demonstrated in the 20 μL and 50 μL traces, corresponding to a lower detection limit of 0.2 cfu/μL. No product peak is present in the most dilute 100 μL trace.

Figure 5

Detection of E. coli O157 in a background of E. coli K12. The ratio of O157 to K12 used in this test is 1:1000. The 10 μL sample input contains 10 cfu/μL O157 and 10⁴ cfu/μL K12.