Microfluidic approach for parallel capture and isolation of single cells

Standard molecular-analysis techniques provide averaged results from ensemble measurements based on large numbers of cells. With increasing evidence of variations among cells within the same population,^1,2 it is imperative to develop technologies for analysis at the single-cell level. We can only identify unique cells by understanding the variation inherent to normal cells. Such technologies will have implications for drug development, clinical diagnosis, cancer development, and stem-cell differentiation.

The first step toward developing high-throughput, single-cell analysis methods requires mechanisms to capture and fully isolate large numbers of individual cells. Most approaches employ microfluidic techniques, because microfluidic-device dimensions are of the same order as those of cells, and they can integrate cell capture, destruction (lysis), and detection. A number of recent studies have achieved isolation and analysis of single cells,^3–6 but these methods assess only one or a few cells at a time. They have demonstrated the importance of single-cell studies, but do not enable collecting the large amounts of data necessary to generate a proper statistical distribution. Others have demonstrated capturing large numbers of single cells,^7–10 but without the capacity to isolate individual cells.

Here, we describe a microfluidic device that is capable of parallel capture and isolation of single cells and in which downstream molecular analyses can be performed in situ. Our device incorporates a series of microfabricated metal electrodes to capture and lyse single cells using electric fields, combined with a network of fluidic channels and valves in polydimethyl siloxane (PDMS) layers to compartmentalize each cell (see Figure 1). Dielectrophoresis (DEP), i.e., manipulation of particles and cells in a nonuniform electric field,¹¹ can be used to capture and trap individual cells with minimal perturbations to the cell.^6,8,9 In addition, an electric field can also be used to lyse the cells to release their intracellular components.^6,12

PDMS is a commonly used silicone rubber in microfluidic applications.^13,14 PDMS channels and valve patterns are easily customizable, fabricated, and integrated with our fabricated electrodes. We employed a mechanism of PDMS valving that was derived from work by Unger and colleagues.¹⁵ In brief, valve lines are patterned into a PDMS layer above the fluidic lines, which are patterned in a second PDMS layer. When actuated, the valve lines expand down to close off the channels and form compartments. With proper alignment, each electrode capture region is centered within a given compartment. These compartments provide a small volume (~1nl) in which each cell can be lysed and its intracellular components analyzed in situ.

Figure 1. Microfluidic device for capture and isolation of single cells, showing the polydimethyl siloxane (PDMS) valve and channel layers on top of the microfabricated electrode substrate.

We have shown that we can capture, compartmentalize, and lyse individual Jurkat cells. We captured cells by DEP at 10MHz and a peak-to-peak voltage of 10V_pp using an AC sine function (see Figure 2). We formed compartments by actuating the valves using a pressure of 10–15psi (pounds per square inch) and subsequently lysed the cells within the compartments by increasing the voltage to 20V_pp and reducing the frequency to 10kHz.

Figure 2. Capture and lysis of a single Jurkat cell. (1) Bright-field image of a single cell captured by an AC field at 10MHz and peak-to-peak voltage 10V_pp. (2) Bright-field image of a cell after lysis with an AC field at 1kHz and 20V_pp. A bubble is seen that is caused by hydrolysis. (3) Fluorescent image of the same cell as in (1). (4) Fluorescent image of the cell undergoing lysis at 10kHz and 20V_pp. (5) Fluorescent image of the same cell as in (2).

In summary, we have developed a device capable of trapping, isolating, and lysing single cells in parallel. The cells are captured by DEP, compartmentalized by a series of PDMS valves, and lysed by applying an electric field. In our future work, we will further improve cell-capture and lysis efficiency and decrease the compartment size. We are also interested in incorporating techniques for on-chip molecular analysis of intracellular components, for example, protein analysis using antibody arrays prefabricated on-chip.