Taking Your Microfluidics EIS Measurement to the Ultra-High Frequency Domain

Introduction

The HF2IS Impedance Spectroscope from Zurich Instruments is a versatile tool suitable for label-free electrical impedance spectroscopy (EIS) of single cells in the domain of cell counting and discrimination. The high frequency range, from DC to 50 MHz, and the multi-frequency capability of the instrument enable microfluidics researchers not only to count, but also to discriminate between different types of cells flowing through a microfluidic channel. This is accomplished by measuring the dynamic impedance variations when cells pass by electrodes embedded in the channel walls.

More recently, one team of microfluidics researchers has found ways to utilize the 600 MHz UHFLI lock-in amplifier from Zurich Instruments in a microfluidic impedance cytometer. With more than ten times the frequency range of the HF2IS, the UHFLI represents a breath of fresh air in the domain of microfluidic EIS. It is now possible to implement an impedance cytometer that can measure at frequencies up to hundreds of mega-hertz.  Where and why would one need such high frequencies for  microfluidic EIS? This blog aims to quickly answer both questions with examples from published references.

Vacuole-based yeast-strain discrimination

Figure 1 below, taken from a review of microfluidic impedance cytometry by Sun and Morgan [1], illustrates the complex impedance as a function of frequency of a cell as measured by an impedance spectroscope.

Figure 1: The impedance of a cell as a function of frequency.

Figure 1: The impedance of a cell as a function of frequency

(Source: T. Sun and H. Morgan, Microfluidics and Nanofluidics, 2010)

As it can be seen, the impedance reveals information about different aspects of cells depending on the frequency:

  • size at low frequency
  • membrane capacitance at medium frequencies
  • cytoplasm and subcellular components at high frequencies.

With the HF2IS Impedance Spectroscope as part of an impedance cytometer, it is possible to discriminate cells based on differences in conductivity of the cytoplasm up to tens of megahertz. However, the 50 MHz limitation of the instrument also means that the subcellular features, like vacuoles, will likely not be ‘visible’. Asami et al. [2] have done static impedance characterizations at frequencies up to 200 MHz, which have demonstrated a difference in dielectric properties of wild-type yeast cells compared to vacuole-deficient mutant-strain. Thanks to the UHFLI, the same type of experiment can now be performed with a dynamic microfluidic impedance cytometer setup, as demonstrated by a research group in Switzerland [3]. The technique of using the UHFLI in an ultra-high frequency impedance cytometer for discriminating different yeast strains will be introduced below. Then, an experimental microfluidic EIS setup using the integrated phase-locked loop technique from the same group will be described as well.

UHFLI-based Microfluidic Impedance Cytometer

Researchers from the Department of Biosystems Science and Engineering (ETH Zurich, Basel) have presented an implementation of an UHFLI-based microfluidic impedance cytometer. An illustration of the setup is given in Figure 2 below. The figure also shows the characteristic double-peaked waveform of the measured signal as a particle or a cell passes between the measurement electrodes. The complex difference between the two peaks (i.e. peak-to-peak voltage) is detected and extracted for each passing cell or particle. This peak-to-peak voltage is directly related to the dielectric properties of the object passing the electrodes.

Figure 2: UHFLI-based microfluidic impedance cytometer.

Figure 2: UHFLI-based microfluidic impedance cytometer

(Source: Haandbaek et al., Lab on a Chip, 2013)

The UHFLI generates a multi-frequency signal to electrically excite the electrodes in the microfluidic device. These signals are buffered by a power-amplifier (PA). The PA is necessary since the impedance mismatch at high frequencies between the signal generator output impedance (i.e. 50 Ω) and the microfluidic channel impedance will create significant reflections. The close proximity of the PA to the microfluidic device will minimize the effect of reflections. At the output electrodes of the microfluidic device, a high frequency current-to-voltage converter (C2V) is also required to provide not only the trans-impedance gain, but also the high frequency 50 Ω termination.

The measurement results using the setup described above is given in Figure 3 below. One can see that it is possible to distinguish the wild-type yeast from a vacuole-deficient mutant strain by considering the opacity at frequencies above 50 MHz. The opacity, which is also a complex value, provides a volume-independent measure of the dielectric properties of the detected objects. It is defined as the peak-to-peak voltage at a given frequency normalized to the peak-to-peak voltage at 0.5 MHz. Incidentally, the multi-frequency (UHF-MF) capability of the UHFLI is exactly the right tool for the opacity measurement, because simultaneous measurements at multiple frequencies are required.

Figure 3: Magnitude (A) and phase (B) of the opacity of 4-µm beads, wild-type and vacuole-deficient yeast cells

Figure 3: Magnitude (A) and phase (B) of the opacity of 4-µm beads, wild-type and vacuole-deficient yeast cells

UHFLI Phase-Locked Loop

In this implementation, also from the ETH Zurich group in Basel [4], the microfluidic device is configured such that it actually forms an LC resonance circuit with an off-chip inductor. This is shown in Figure 4. As a result, a change in the dynamic impedance creates an immediate shift in the resonance frequency. Instead of directly measuring the impedance, like in the previous impedance cytometer setup, the impedance change can now be detected by tracking the resonance frequency shift using a phase lock loop (UHF-PLL). The major advantage is the potentially improved measurement sensitivity. This is due to the fact that the presence of the resonance results in an increased signal and, therefore, an improved signal-to-noise ratio, compared to a pure amplitude-measurement.

Figure 4: Microfluidic impedance cytometer using resonance frequency modulation for detection of cells and particles.

Figure 4: Microfluidic impedance cytometer using resonance frequency modulation for detection of cells and particles

(Source: Haandbaek et al., 17th Intl. Conf. on Miniaturized Systems for Chemistry and Life Sciences, 2013)

The measurement data in Figure 5 below shows the different resonance frequency shifts according to whether cells or particles pass the electrodes. These results validate the principle of using a phase-locked loop in a microfluidic EIS setup. A further improvement in sensitivity can be achieved by optimizing the quality factor of the LC resonator.

Figure 5: (A) Frequency shift as cells and particles pass the measurement electrodes. (B) Histogram of the frequency shifts of a complete measurement

Figure 5: (A) Frequency shift as cells and particles pass the measurement electrodes. (B) Histogram of the frequency shifts of a complete measurement

Conclusion

The ultra-high frequency capability of the UHFLI lock-in amplifier enables researchers to obtain new information such as the detection of subcellular features. The fact that the UHFLI lock-in amplifier can be configured either as an impedance spectroscope or a phase-locked loop offers EIS microfluidics researchers the possibility to experiment with different setups or microfluidic devices in a very flexible manner.

Acknowledgements

I would like to thank Niels Haandbaek for his scientific insights and contributions, as well as Professor Hierlemann and the Department of Biosystems Science and Engineering from ETH Zurich, Basel for agreeing to share the results from the UHFLI lock-in amplifier measurement.

References

[1]    T. Sun and H. Morgan, “Single-cell microfluidic impedance cytometry: a review,” Microfluid. Nanofluidics, vol. 8, no. 4, pp. 423–443, Mar. 2010.

[2]    K. Asami and T. Yonezawa, “Dielectric behavior of wild-type yeast and vacuole-deficient mutant over a frequency range of 10 kHz to 10 GHz.,” Biophys. J., vol. 71, no. 4, pp. 2192–200, Oct. 1996.

[3]    N. Haandbæk, S. C. Bürgel, F. Heer, and A. Hierlemann, “Characterization of subcellular morphology of single yeast cells using high frequency microfluidic impedance cytometer.,” Lab Chip, vol. 14, no. 2, pp. 369–77, Jan. 2014.

[4]    N. Haandbæk, O. With, S.C. Bürgel, F. Heer, and A. Hierlemann, Microfluidic sensor using resonance frequency modulation for characterization of single cellsProceedings of the 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS), 2013, Freiburg, Germany, pp. 1680-1682, ISBN 978-0-9798064-6-9.