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IMPEDANCE SPECTROSCOPY FOR CHARACTERIZATION AND COUNTING

Microfluidics allows for the precise monitoring and control of chemical or biological events at the microscale level. At this scale, comparable to the dimensions of the biological cell, microfluidic detection and analysis of single cells is of great interest, allowing lab-on-a-chip and point-of-care applications1. Cell detection is generally performed using optical methods such as FACS (Fluorescent Activated Cell Sorting). However, these methods require additional and usually time-consuming labelling steps.

Electrical impedance spectroscopy (EIS) is a label-free technique that enables real-time, high-throughput measurements, and eases the process of data extraction and processing2. Continuous flow microfluidic devices with embedded microelectrodes for electrical measurements can be employed for detecting and classifying single cells or particles (e.g. beads or droplets) in a high throughput manner1. Further, since the dielectric properties of a biological cell are defined by its cellular characteristics such as cell volume, composition and architecture1, impedance spectroscopy can be used to differentiate between cell types.

There are several key advantages of this technique in the microfluidic environment including:

  • Fast throughput: ~1000 particles/s
  • Multiple parameter analysis
  • Probe impedance of the analyte at multiple frequencies simultaneously
  • Integrates well with other analysis methods (e.g. optical detection)
  • Label free analysis method

We present in this application note our Electrical Impedance Spectroscopy Platform (or EISP) consisting of microfluidic flow controllers from Fluigent to maintain precise flow control, a chip from Micronit Microtechnologies B. V to localize impedance measurements, and a lock-in amplifier from Zurich Instruments to perform impedance measurements. We demonstrate the system efficiency by determining the size of micrometer beads and by measuring the generation rate water-in-oil droplets.

MATERIAL AND METHODS

An external pressure source is connected to two Fluigent Flow EZ flow controllers that are connected to the Fluigent EZ Drop microfluidic chip via tubing. The tubing passes through Flow Units to allow flow rate measurements. The droplets generated flow into the Micronit Electrical Impedance Spectroscopy (EIS) chip and pass through the sensing region. The Zurich Instruments HF2LI lock-in amplifier provides voltage excitation to the differential electrode pairs on the EIS chip, and measures the returning current via the HF2TA transimpedance current amplifier. Visualization of the EZ Drop chip channels is performed using an optical microscope.

Materials

Microfluidic flow controller

Particles alignement and localization

Impedance measurements

Droplets generation

Impedance measurements of microbeads and water-in-oil droplets

A scheme of the microfluidic setup is presented in figure above. An external pressure source is connected to the LineUp System consisting in two Flow EZ, which in turn are connected to two reservoirs to set the pressure drop between the input and output of the system. The system is thus pressurized allowing for enhanced flow control. One reservoir contains the microbead suspension to be injected (inlet solution) and the second reservoir is used to collect the solution coming out from the chip (pressurized waste). The inlet pressure is always higher than the outlet pressure to maintain a unidirectional flow from the input to the output. The reservoirs are connected to the EIS microfluidic chip using tubing. The microbeads pass the electrode pairs within the chip and impedance measurements are performed using the HF2LI lock-in amplifier coupled with the HF2TA current amplifier. The experiment is performed using beads of 3 µm and 5 µm diameter.

Here, an external pressure source is connected to the LineUp System consisting in two Flow EZ, which in turn are connected to two  reservoirs containing water and 3M™ Novec™ 7500 with dSurf. The reservoirs are connected to the two inlets of the EZ Drop microfluidic chip via tubings. The tubing passes through Flow Units to allow flow rate measurements. Pressure is applied on the two reservoirs: water is injected in the inner channel and the oil phase is injected in the surrounding channel of the microfluidic chip. Visualization of the chip channels is performed using an optical microscope.
Once generated, the droplets flow through the outlet tubing and are injected into the EIS microfluidic chip. The droplets pass the electrode pairs within the chip and impedance measurements are performed using the HF2LI lock-in amplifier coupled with the HF2TA current amplifier.

Results

Impedance measurements of microbeads

Using the same microfluidic system presented in last part of the above “Materials and Method” section,  microbead suspensions are injected into the EIS chip, where beads pass electrodes pairs surrounding the microfluidic channel allowing impedance measurements to be performed.

The figure above shows that the signals from the  beads (blue trace) display consistently larger amplitudes than the 3 ?m beads (red trace) in both X and Y. The 5 ?m beads show a peak-peak amplitude change between 75 and 120 mV while the 3 ?m beads between 20 and 30 mV. These results correspond nicely to the difference in volume between the two beads (a factor of 4.6). Thus, using this impedance spectroscopy signal can discriminate particles or cells according to their sizes. Using our microfluidic system, it is thus possible to differentiate between 3 µm and 5 µm beads.

Impedance measurements of water-in-oil droplets

To delve one step further, the experiment is repeated using the same microfluidic system presented in the last part of the “Materials and Method” section. Water-in-oil droplets are injected into the EIS chip, where the droplets pass electrodes pairs surrounding the microfluidic channel allowing impedance measurements to be performed.

The attached figure on the left shows the impedance measured at 10 MHz. Clear peaks can be seen in the current amplitude and in the phase as each droplet passes the electrode pair. The phase information indicates a clear change from resistive (fluid only) to capacitive behavior as the droplets pass the sensing region of the electrode pair. Additionally, the density of peaks in the time-domain chart gives useful information on the droplet generation rate and velocity. Hence this technique can be used to count even fast moving droplets, beads or cells. Simultaneous multi-frequency measurements can be seen in the lower figure of the attached figure. Here, the HF2LI equipped with the HF2-MF option measures the impedance signal (current) at six different frequencies simultaneously. The imaginary current signal varies with frequency, changing in both magnitude and phase.  This simultaneous multi-frequency measurement offers a fuller picture of the frequency-dependent dielectric properties of the passing droplets (beads, or cells), while saving the overall measurement time by a factor of 6.

Conclusion

We have demonstrated the efficiency of our cost-effective EISP by determining the size of micrometer beads and by measuring the generation rate and velocity of waterin-oil droplets. Combining the LineUp system with the EIS-chip and the HF2LI lockin amplifier enables fast detection and discrimination of individual cells or particles in flow at a speed unavailable to camera-based solutions. In addition, this label-free technique can distinguish particle sizes and cell types thanks to high sensitivity at different frequencies. Ultimately, the use of EISP on the microfluidic scale is diverse and includes applications such as: 

  • Quality control in the Food industry
  • Flow cytometry for counting and sorting of cells or droplets, marker-free detection and protein engineering
  • Blood analysis

References

  1. Panwar, J. & Roy, R. Integrated Field’s metal microelectrodes based microfluidic impedance cytometry for cell-in-droplet quantification. Microelectron. Eng. 215, 111010 (2019).
  2. Zhou, Y. et al. Characterizing Deformability and Electrical Impedance of Cancer Cells in a Microfluidic Device. Anal. Chem. 90, 912–919 (2018).
  3. Cahill, B. P. Optimization of an impedance sensor for droplet-based microfluidic systems. Smart Sensors, Actuators, MEMS V 8066, 80660F (2011).

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