Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry

Abstract

A microfabricated fluidic device was developed for the automated real-time analysis of individual cells using capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS). The microfluidic structure incorporates a means for rapid lysis of single cells within a free solution electrophoresis channel, where cellular constituents were separated, and an integrated electrospray emitter for ionization of separated components. The eluent was characterized using mass spectrometry. Human erythrocytes were used as a model system for this study. In this monolithically integrated device, cell lysis occurs at a channel intersection using a combination of rapid buffer exchange and an increase in electric field strength. An electroosmotic pump is incorporated at the end of the electrophoretic separation channel to direct eluent to the integrated electrospray emitter. The dissociated heme group and the alpha and beta subunits of hemoglobin from individual erythrocytes were detected as cells continuously flowed through the device. The average analysis throughput was approximately 12 cells per minute, demonstrating the potential of this method for high-throughput single cell analysis.

Figure 1

Schematic diagram showing the design of the cell lysis CE-MS microchip. Reservoirs are labeled C (cells), B (buffer), and SC (EO-pump side channel). The separation channel is 4.7-cm long, measured from the cell lysis intersection to the electrospray corner. A nanojunction (~100-nm deep) connects the EO pump side channel to the separation channel.

Figure 2

Schematic diagram showing the basic operation of the cell lysis CE-MS microchip. Reservoirs are labeled as described in figure 1. The arrows indicate the direction and relative magnitude of electroosmotic flow. The cells migrate toward the lysis intersection, at which point the increased electric field and the rapid dilution of the cell buffer causes the cells to quickly lyse. An electrophoretic separation then occurs as the cell contents migrate toward the electrospray orifice where electrospray ionization occurs.

Figure 3

1 minute summation of the background MS signal acquired with no cells present in the cell buffer. The major peaks correspond to protonated and sodiated sucrose clusters. The top trace shows the full m/z range measured (300–2000), and the bottom trace shows the m/z range used for the cell lysis experiment (600–1000). In the bottom trace the y-axis range was expanded to better show the less intense ions.

Figure 4

Frame capture from a CCD video taken on a fluorescence microscope showing Oregon Green stained erythrocytes moving (from left to right) from the cell reservoir through the lysis intersection and into the separation channel. The cell on the right of the injection intersection has already lysed. The cell buffer contained Oregon Green dye that was not fully washed from the cell suspension, so the flow out of the different channels can be seen. The arrows indicate the direction of EOF in the channels.

Figure 5

Raw (top) and background subtracted (bottom) total ion count measured over 10 minutes of continuous erythrocyte lysis/CE-MS. The peaks correspond to the α and β subunits of hemoglobin.

Figure 6

The ion count at 616 m/z corresponding to the dissociated heme groups (top) and the background subtracted total ion count (bottom) for a 2 minute segment of the continuous lysis/CE-MS experiment. The dissociated heme groups migrated faster through the separation channel, so each lysis event generated a 616 m/z peak approximately 10 seconds before the major peak (visible in the TIC) corresponding to the α and β hemoglobin subunits. The peaks corresponding to the first 4 cell lysis events observed in this detection window are labeled in both traces to help illustrate the trend which continues throughout the entire run. The gray rectangles correspond to the data that was used to generate the spectra labeled with the same letters in figure 7.

Figure 7

Mass spectra generated by summing the data in each of the 3 second wide windows illustrated by gray rectangles in figure 6. The top trace (A), corresponds to the baseline, the middle trace (B) corresponds to a single cell lysis event, and the bottom trace (C) either corresponds to the lysis of a cell with greater than average hemoglobin content, or the simultaneous lysis of multiple cells.