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. Author manuscript; available in PMC: 2011 Apr 15.
Published in final edited form as: Anal Chem. 2010 Apr 15;82(8):3183–3190. doi: 10.1021/ac902683t

Figure 4.

Figure 4

High-throughput digital multiplex detection of E. coli O157 in a background of E. coli K12 with various input ratios and average cell concentrations (Cavg) using MEGAs. (A, B) With Cavg≤ 0.2 cpd, flow cytometry profiles of beads show four distinct populations: negative (blue), FAM positive beads specific for K12 (green), Cy5 positive beads specific for O157 (red), and double positive beads for both cells (orange). Small populations are expanded along the event axis for better visualization, as indicated. The gray regions mark the population gating. The measured O157 ratios (O157 positive beads/total positive beads) are (A) 0.48 (expected: 0.5) and (B) 0.11 (expected: 0.1). (C, D) When Cavg≥ 10 cpd while keeping O157 cells at 0.01 cpd, double positive beads quantify O157 since a single O157 cell is co-compartmentalized into a droplet with multiple K12 cells. In this case the ratios of double positive beads divided by Cavg give the measured O157 ratios: (C) 0.92/103 (expected: 1/103) and (D) 0.98/104 (expected: 1/104). Up to 3000 beads can be analyzed using a 4-channel device for ~25 min. run time. Increasing Cavg to 100 cpd reduces the number of beads required, improving the detection sensitivity to 1/104 without excessively extending run time. (E, F) O157 detection using a 96-channel MEGA shows the measured O157 ratios consistent with the inputs: (E) 0.94/103 vs 1/103 and (F) 0.85/104 vs 1/104. Up to 104 events were processed within 5 min. run time. The capability offered by 96-channel MEGA (up to 3.4 × 106 droplets per hour) greatly increased analysis throughput and decreased processing time necessary for detecting a statistically significant population of a low-frequency sample. Bead concentration was 0.1 bpd for all cases.