High-throughput and label-free multi-outlet cell counting using a single pair of impedance electrodes

There are increasing number of cell separation applications where cells needs to be separated into multiple outlets. Quantification of sorted or separated cells and particles in microfluidic systems flowing through multiple outlet channels are typically conducted off-line through microscopic image analysis, or by first collecting cells from each outlet and counting them afterwards. However, these methods do not provide real-time analysis, are time consuming, and can lead to significant error in analysis when handling and collecting a small number of cells (such as rare cells). Here, we present a low-cost, label-free, and real-time on-chip cell counting and quantifying method for sorted/separated cells flowing through multiple microfluidic outlets using only a single pair of microelectrodes. The single staircase-shaped electrode design positioned perpendicular to the outlets subjects cells flowing through different outlets to different electric field strength, thus resulting in different impedance signals depending on which outlets the cell passes through. This design was enhanced by studying and comparing the results of both simulations and experiments. To analyze whether cells passing through each of the five outlets can be correctly classified based on their impedance peak height and width, three different classification methods were tested and compared. The developed design was successfully utilized to distinguish cells flowing through 5 different outlets using only a single pair of impedance electrodes, showing classification error rate of only 1.91%. This single-pair staircase-shaped electrode design can be applied to any cell separation system, regardless of the separation methods utilized, and thus have extremely broad application space in the field of microfluidic cell separation.