Full-electric Microfluidic Chip for Single-cell Trapping, Spectral Analysis, and Sorting

Microorganisms, such as yeast or bacteria, form heterogeneous populations comprised of multiple subpopulations. Over the past decades, numerous single-cell analysis technologies have been developed to study this phenotypic heterogeneity in a population and to identify cells with properties of interest. Optical techniques have widely and successfully been adopted for this purpose, but they suffer from some disadvantages. They are expensive, require sizeable equipment, and need sample preparation for cell labeling (flow cytometry), or they operate slowly, making sampling an entire population time-consuming (Raman spectroscopy). Electrical technologies are gaining interest due to their ability for label-free and real-time analysis of electrical cell properties, which can complement the already existing optical techniques. Moreover, they have the potential to become low-cost and compact, which can open avenues for portable tools performing on-site single-cell analysis.

Impedance flow cytometry (IFC) systems allow electrical characterization of microorganisms in suspension through high-throughput and single-cell resolution impedance measurements. However, most systems only measure impedance at two frequencies, severely limiting the information extracted from cells. Impedance spectroscopy over a broad frequency range is desirable since electrical properties of subcellular features can be more accurately probed. At low frequencies, for example, the measurement contains information about cell size, while at higher frequencies cell membrane and cytoplasm properties are probed.

CMOS-MEA (Complementary Metal-Oxide-Semiconductor Microelectrode array) systems can perform broadband impedance measurements on thousands of electrodes in a highly parallel way with single-cell resolution. However, applications are limited to adherent cells such as tissue or biofilms since cells must be close to the electrode surface. To enable the use of CMOS-MEA’s for cells in suspension, there is a need for a full-electric technique that can capture, analyze, and sort single cells. The technique should allow automation and on-chip integration to achieve a high-throughput and compact system.

Therefore, the aim of this thesis is to pioneer the development of the first fully electric system that incorporates three essential functionalities: i) trapping, ii) spectral impedance analysis, and iii) selectively releasing single cells. The system should have the potential for automation, integration on CMOS chip, and parallelization. The second aim is to use the system for the electrical characterization of microorganisms to validate its biological relevance, going beyond the state-of-the-art to explore novel biological applications where electrical techniques can be useful for single-cell analysis.

In this thesis, a microfluidic device is presented which demonstrates, for the first time, a combined method for full-electric cell capturing, analyzing, and selectively releasing with single-cell resolution. All functionalities are experimentally demonstrated on Saccharomyces cerevisiae. The microfluidic platform consists of traps centered around a pair of individually accessible coplanar electrodes, positioned under a microfluidic channel. The device uses a novel method for trapping single cells by positive dielectrophoresis (pDEP ), which was coined the “Two-Voltage” method. Cells are attracted to the trap when a high voltage (VH) is applied. A low voltage (VL) holds the already trapped cell in place without attracting additional cells, enabling full control over the number of trapped cells. After trapping, the cells are analyzed by broadband electrochemical impedance spectroscopy (EIS). These measurements allow the detection of single cells and the extraction of cell parameters. Additionally, the measurements show a strong correlation between average phase change and cell size, enabling the use of our system for size measurements in biological applications. Finally, the device allows selectively releasing trapped cells by turning off the pDEP signal in their trap.

The frequency band where cell properties can be measured by impedance spectroscopy is limited by two major effects which mask the cell impedance: the double layer impedance due to the electrode-electrolyte interface at low frequencies and the parasitic capacitance due to capacitive coupling between the metal tracks connected to the electrodes at high frequencies. This thesis presents a novel, extremely fast (1 s), and simple on-chip nanostructuring method for gold microelectrodes which results in a 40-fold reduction in double layer impedance at 1 kHz. Nanostructured electrodes experimentally showed an improved impedance stability and a higher sensitivity to cell impedance. The approach only required the immersion of the electrodes in a 100x diluted PBS solution and a DC voltage source to apply the voltage, without requiring extra fabrication or electrodeposition steps. Additionally, a custom designed PCB is presented which utilizes guarding techniques to minimize parasitic capacitance during cell impedance measurements.

Finally, single-cell measurements using the microfluidic device presented in this thesis show for the first time that Chlorella vulgaris and Microcystis aeruginosa, two common species in fresh water algal blooms, can be trapped and discriminated on a single-cell level by EIS. Control experiments validated that vacuoles, small pockets of air, in the cytoplasm of M. aeruginosa cells are the main cause of the difference in impedance when compared to C. vulgaris cells who do not have vacuoles.

In conclusion, the microfluidic device presented in this thesis validates, for the first time, full-electric single-cell trapping, spectral analysis and sorting. Impedance spectroscopy measurements of yeast and algal cells with the device show the potential of broadband impedance measurements in the characterization of cell properties. Although additional challenges remain, the full-electric device has the potential for automation and integration which will open new avenues towards small-scale, high-throughput single-cell analysis and sorting devices.