Automated processing integrated with a microflow cytometer for pathogen detection in clinical matrices

Abstract

A spinning magnetic trap (MagTrap) for automated sample processing was integrated with a microflow cytometer capable of simultaneously detecting multiple targets to provide an automated sample-to-answer diagnosis in 40 min. After target capture on fluorescently coded magnetic microspheres, the magnetic trap automatically concentrated the fluorescently coded microspheres, separated the captured target from the sample matrix, and exposed the bound target sequentially to biotinylated tracer molecules and streptavidin-labeled phycoerythrin. The concentrated microspheres were then hydrodynamically focused in a microflow cytometer capable of 4-color analysis (two wavelengths for microsphere identification, one for light scatter to discriminate single microspheres and one for phycoerythrin bound to the target). A three-fold decrease in sample preparation time and an improved detection limit, independent of target preconcentration, was demonstrated for detection of Escherichia coli 0157:H7 using the MagTrap as compared to manual processing. Simultaneous analysis of positive and negative controls, along with the assay reagents specific for the target, was used to obtain dose-response curves, demonstrating the potential for quantification of pathogen load in buffer and serum.

Fig. 1

Spinning magnetic trap concentrates immunomagnetic microspheres from a sample stream and moves them continually. (a) Photo of MagTrap wheel showing strip magnets. (b) Pulling the microspheres upstream against the flow while simultaneously moving them from one side of the channel to the other concentrates the microspheres and enhances interaction with reagents (trap). (c) When exposure to the reagents is complete, the rotation of the magnets is reversed, and the microspheres are released downstream into an interrogation device (release).

Fig. 2

System overview diagram depicting the fluidic and optical connections for the MagTrap-microflow cytometer assembly.

Fig. 3

ID plot for nonmagnetic microspheres (a) and dose–response for E. coli (b) in PBSTB buffer (square) and 10% serum (triangle). Cluster 56 microspheres were specific for the E. coli assay. Assays for chicken and BSA on microsphere sets 54 and 75, respectively, were positive and negative controls. The E. coli signal shown in (b) was normalized to the chicken signal, above the BSA threshold. Error bars are SEM. Threshold value was calculated to be 3 standard deviations above the blank signal (gray line).

Fig. 4

ID plot for magnetic microspheres (a) and dose–response curve for E. coli (b) in PBSTB buffer (square) and 10% serum (triangle) using the MagTrap and three sets of magnetic Luminex microspheres for automated sample preparation. Cluster 100 microspheres were prepared for the detection of E coli, while cluster 93 was for detection of chicken (positive control). Cluster 56 microspheres were coated with BSA and served as the negative control. E. coli response was normalized to the chicken signal, above the BSA threshold. Error bars are SEM. Threshold value was calculated to be 3 standard deviations above the blank signal (gray line).