chemosensors
Article
Microwave Interferometric Cytometry for Signal Analysis of
Single Yeast Cells
Meng Zhang 1,† , Guangxin Huo 2,† , Juncheng Bao 1 , Tomislav Markovic 1,3, * , Patrick Van Dijck 2
and Bart Nauwelaers 1
Citation: Zhang, M.; Huo, G.; Bao, J.;
Markovic, T.; Van Dijck, P.;
Nauwelaers, B. Microwave
Interferometric Cytometry for Signal
Analysis of Single Yeast Cells.
Chemosensors 2022, 10, 318.
https://doi.org/10.3390/
chemosensors10080318
Academic Editors: Chunxiong Luo,
Chunyang Xiong and Wei Yang
Received: 30 June 2022
Accepted: 4 August 2022
Published: 8 August 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Chemosensors 2022, 10, 318. https://doi.org/10.3390/chemosensors10080318
1 WaveCore Division, Department of Electrical Engineering (ESAT), KU Leuven, 3001 Leuven, Belgium
2 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven,
3001 Leuven, Belgium
3 Department of Electronics, Microelectronics, Computer and Intelligent Systems,
Faculty of Electrical Engineering and Computing, University of Zagreb, Unska 3, 10000 Zagreb, Croatia
* Correspondence: tomislav.markovic@fer.hr
† These authors contributed equally to this work.
Abstract: Microwave dielectric sensing offers a rapid, label-free, and non-invasive way of characteri-
zation and sensing of biological materials at the microfluidic scale. In this work, a dielectric sensing
is achieved with a microwave interferometric setup that is applied to cytometric applications. A fast
way to analyze and design an interferometric system at microwave frequencies in software tools is
proposed together with a novel manufacturing and assembly process, which enables a short recovery
time and avoids extensive microwave-microfluidic chip fabrication. The simulation and measure-
ment results of the interferometric setup are in agreement with an excellent match at levels below
S21 = −60 dB. The sensitive microwave setup is evaluated on measurements of 3 µm polystyrene
spheres and finally applied for characterization of a widely used laboratory Saccharomyces cerevisiae
strain, the S288C, in a frequency range from 4 to 18 GHz.
Keywords: single cell; interferometer; permittivity; yeast; microfluidics
1. Introduction
Microwave dielectric characterization and heating techniques have drawn great in-
terest in microfluidics scale for rapid heating [1,2], polymerase chain reaction [3], and
detection of biogenic liquids and analysis of biological cells. The dielectric properties of
the cell, determined by the constitutions of the cell including its size and morphology,
the membrane formation, the cytoplasm, and the organelles, are representations of the
cell’s physiological status. Because electromagnetic signals at microwave frequencies go
beyond the cell membrane, microwave dielectric characterization has been studied in
many different scenarios and on numerous biological cells. For example, the interference
technique and a lock-in amplifier were used as a detection system on a resonant struc-
ture for measuring the dielectrophoretic response of the CHO cells [4]. The interference
technique was also used to distinguish the viability of single yeast cells [5]. The bulk
impedances were measured with electrodes to acquire membrane capacitance and cyto-
plasm conductivity for characterization and classification of human non-small cell lung
carcinoma cells [6] and so on. In other words, the dielectric properties of biological cells
and the corresponding sensing is being promoted as ideal for quantitative analysis of single
cell in microfluidics [7–11].
Interferometric dielectric spectroscopy technique is a highly sensitive, broadband,
and label-free method for lab-on-chip platforms [12–16]. It is flexible in terms of sensor
selection [17] and straightforward in permittivity extraction [14,18,19]. The interferomet-
ric technique utilizes the capability of power splitters and combiners, attenuators, and
phase shifters to create two signals from one source having almost identical amplitude
https://www.mdpi.com/journal/chemosensors
Chemosensors 2022, 10, 318 2 of 12

and opposite phase, through which a negative interference null at a certain frequency is
achieved. The two signals cancel out each other, forming a based line signal close to zero,
which is normally smaller than −60 dB from the source signal value. Thus, a small effective
permittivity change of the MUT caused by a single cell in a suspension medium can be
detected. However, the signal of the single cell and the extracted permittivity are sensitive
to the contrast of the dielectric properties between the cell and the medium, the size of the
cell, the topology of the sensing electrodes, and the flow height of the cell [12].
The state of art low-frequency impedance characterization techniques [4,20,21] mea-
sure the effects of ionic current flow, electrode polarization, and membrane potential of the
cell. However, the measurements using microwave frequency can detect the membrane
dielectric changes and cytoplasm ionic concentration changes [4]. In the meanwhile, the
broadband measurements specify the entire spectral response of the cell.
To tackle the challenge of analyzing the signal profile of a single cell from the system
response, we introduce in this paper a novel system modeling and signal profile simula-
tion technique for interferometric cytometry on single cell measurement. The Scattering-
parameter (S-parameter) measurements of every component are loaded in the Keysight
Advanced Design System (ADS) software tool for system modeling. Electromagnetic simu-
lations of a short length sensing electrode with a particle, cube with 3 um side length, on
top at different locations above the sensing electrodes are generated by means of Computer
Simulation Technology (CST) software tool. This is needed to model the interferometric sys-
tem as the signal varies once the particle is passing by the electrodes in flow. Furthermore,
we propose a new design and fabrication of the microfluidic channel for easy disassembly
and cleaning to solve the clogging issue and reuse of the electrodes. Finally, the 3 µm
polystyrene spheres (PSSs) were detected to validate the system setup. Afterwards, the
setup was applied to measure the yeast strain of S288C with a reported average diameter
of 4 µm to extract the permittivity change of material under test (MUT) caused by yeast
cells flowing over the sensor electrodes in the microfluidic channel.

2. Materials and Methods

2.1. System Setup and Sensor Design
2.1.1. System Setup and Measurement Principle
The proposed system setup of microwave interferometric cytometry is shown in Figure 1a,
which consists of four parts: (1) the proposed interferometric cytometry together with the
designed microfluidic sensor; (2) the vector network analyzer (VNA, Keysight M9375A,
Keysight, Santa Rosa, CA, USA) that generates high-frequency signals and provides the
readout of the two-port S-parameter measurements; (3) an inverted microscope (Olympus
IX73, Olympus Corporation, Tokyo, Japan, and Hamamatsu ORCA Flash 4 LT+ camera,
Hamamatsu, Hamamatsu, Japan) for recording images of the yeast cells passing through
the sensor to validate and correlate the microwave detection with the optical images; and
(4) a computer for saving the data of electrical detection and the images of the optical system.
Figure 1b shows the system setup during measurements.
During the single cell measurements, the high-frequency signals (4–18 GHz) generated
from VNA are separated at the port 1 by a 90-degree coupler (SigaTek, SQ16506, 4–18 GHz,
SigaTek LLC, Wayne, IN, USA) into two branches, the material-under-test (MUT) branch,
and the reference (REF) branch, with 90 degrees phase difference. The MUT branch
consists of the CPW sensor with a microfluidic channel and one attenuator (Narda 4799,
0–18 GHz, Narda-MITEQ, Hauppauge, NY, USA). While the REF branch contains only a
phase-shifter (Arra 9428a, 0–18 GHz, ARRA INC, Bay Shore, NY, USA). The readout at port
2 of another 90-degree coupler, combining the two signals from both branches, has a base
phase difference of 180 degrees. By respectively tuning the attenuator and phase-shifter to
match the losses of the two branches and make their phases reversed, the interference null
at a certain frequency can be achieved. This means that the base signal from the tuned setup
is small compared to the input signal (the transmission signal is attenuated by generally
−60 to −80 dB). Thus, small signal changes caused by single cells passing over the sensor
Chemosensors 2022, 10, 318 3 of 12

inside the microfluidic channel can be detected with the proposed setup. The S-parameters
of the system are measured and shown in Figure 2a, which is tuned at f 0 = 133 GHz
Chemosensors 2022, 10, x FOR PEER REVIEW of 12
to
−67 dB with a quality factor (Q = f 0 /∆ f 3dB ) equals to 3.8 × 104 . Then, the single cell
measurements can be carried out at f 0 with high sensitivity.

Figure 1. The schematic of the measurement system of the proposed microwave interferometric cy-
Figure 1. The schematic of the measurement system of the proposed microwave interferometric
tometry and the designed microfluidic sensor. (a) the schematic and (b) the picture of the measure-
cytometry and the
ment system designed
setup, includingmicrofluidic sensor.
the proposed (a) the schematic
microwave and (b)cytometry,
interferometric the pictureVNA,
of themicro-
measure-
ment system setup, including the proposed microwave interferometric cytometry, VNA,
scope, and computer; (c) The designed co-planar waveguide (CPW) sensor with tapering structure microscope,
and
andcomputer; (c) on
microfluidics Thetopdesigned co-planar
(a straight waveguide
channel with one inlet(CPW)
and onesensor
outlet);with tapering
(d) The structure
microscope imageand
of two yeast cells (marked inside the green circles) approaching the CPW sensor
microfluidics on top (a straight channel with one inlet and one outlet); (d) The microscope (the bright CPW
image
structure consisting of 3 parallel electrodes) in the microfluidic channel. The orange
of two yeast cells (marked inside the green circles) approaching the CPW sensor (the bright CPW rectangular
shows the region of interest (ROI) for microscope image intensity calculation.
Chemosensors 2022, 10, x FOR PEER REVIEW 4 of 12
structure consisting of 3 parallel electrodes) in the microfluidic channel. The orange rectangular
shows the region of interest (ROI) for microscope image intensity calculation.
During the single cell measurements, the high-frequency signals (4–18 GHz) gener-
ated from VNA are separated at the port 1 by a 90-degree coupler (SigaTek, SQ16506, 4–
18 GHz, SigaTek LLC, Wayne, IN, USA) into two branches, the material-under-test (MUT)
branch, and the reference (REF) branch, with 90 degrees phase difference. The MUT
branch consists of the CPW sensor with a microfluidic channel and one attenuator (Narda
4799, 0–18 GHz, Narda-MITEQ, Hauppauge, NY, USA). While the REF branch contains
only a phase-shifter (Arra 9428a, 0–18 GHz, ARRA INC, Bay Shore, NY, USA). The
readout at port 2 of another 90-degree coupler, combining the two signals from both
branches, has a base phase difference of 180 degrees. By respectively tuning the attenuator
and phase-shifter to match the losses of the two branches and make their phases reversed,
the interference null at a certain frequency can be achieved. This means that the base signal
from the tuned setup is small compared to the input signal (the transmission signal is
attenuated by generally −60 to −80 dB). Thus, small signal changes caused by single cells
passing over the sensor inside the microfluidic channel can be detected with the proposed
setup. The S-parameters of the system are measured and shown in Figure 2a, which is
tuned at 𝑓0 = 13 GHz to −67 dB with a quality factor (Q = 𝑓0 ⁄∆𝑓3𝑑𝐵 ) equals to 3.8 × 104 .
Then,2.
Figure
Figure 2.the
Thesingle
The cell measurements
S-parameters
S-parameters ofof(a)
(a)the can be carried
themeasurement
measurement system
system the𝑓the
outofat
of with high
proposed
0proposed sensitivity.
microwave
microwave interfero-
interferometric
metric cytometry and (b) the designed microfluidic sensor.
cytometry and (b) the designed microfluidic sensor.

2.1.2. Sensor Design

Design and
and Fabrication
Fabrication
For single cell
cell measurements,
measurements,aatapered
taperedco-planar
co-planarwaveguide
waveguide(CPW)
(CPW)sensor
sensorisischosen
chosen
structure which
for its planar structure which is
is compatible
compatible with
with the
the integration
integrationwith
withaamicrofluidic
microfluidicchannel
chan-
nel and
and connections
connections to thetointerferometric
the interferometric
system.system. The dimensions
The dimensions of the electrodes
of the electrodes should be
should be comparable
comparable to the dimensions
to the dimensions of the cellsofunder
the cells
test.under test. The
The width width
of the of thesignal
sensing sensing
line
signal
and line
gap is and
bothgap
6 µmis both 6 µm
and the and the
ground ground
is 20 is 20 µ m wide.
µm wide.
The tapered CPW structure is fabricated on a 1 mm Quartz wafer using photoresist
patterning, sputter coating, and lift-off techniques to form a 400 nm TiO2/Au/TiW conduc-
tor layer [22].
The microfluidic channel (Figure 3) is made by pasting the laser-cut super thin dou-
ble-coated adhesive tape (Nitto, No.5601, 10 µ m, Nitto Denko, Osaka, Japan) on top of the
sensor, then applying a 4 mm thick polydimethylsiloxane (PDMS) slab for interconnecting
the tubing with the microfluidic channel. The entire sensor and microfluidics structure is
 2.1.2. Sensor Design and Fabrication
For single cell measurements, a tapered co-planar waveguide (CPW) sensor is chosen
for its planar structure which is compatible with the integration with a microfluidic chan-
Chemosensors 2022, 10, 318
nel and connections to the interferometric system. The dimensions of the electrodes 4 of 12
should be comparable to the dimensions of the cells under test. The width of the sensing
signal line and gap is both 6 µ m and the ground is 20 µ m wide.
The tapered CPW structure is fabricated on a 1 mm Quartz wafer using photoresist
The tapered CPW structure is fabricated on a 1 mm Quartz wafer using photoresist pat-
patterning, sputter coating, and lift-off techniques to form a 400 nm TiO2/Au/TiW conduc-
terning, sputter coating, and lift-off techniques to form a 400 nm TiO2 /Au/TiW conductor
tor layer [22].
layer [22].
The microfluidic channel (Figure 3) is made by pasting the laser-cut super thin dou-
The microfluidic channel (Figure 3) is made by pasting the laser-cut super thin double-
ble-coated adhesive tape (Nitto, No.5601, 10 µ m, Nitto Denko, Osaka, Japan) on top of the
coated adhesive tape (Nitto, No.5601, 10 µm, Nitto Denko, Osaka, Japan) on top of the
sensor, then applying a 4 mm thick polydimethylsiloxane (PDMS) slab for interconnecting
sensor, then applying a 4 mm thick polydimethylsiloxane (PDMS) slab for interconnecting
the tubing with the microfluidic channel. The entire sensor and microfluidics structure is
the tubing with the microfluidic channel. The entire sensor and microfluidics structure
clamped by a polymethacrylate (PMMA) fixture on top and bottom with screws. As well,
is clamped by a polymethacrylate (PMMA) fixture on top and bottom with screws. As
this new microfluidic structure can be easily disassembled, cleaned, and replaced with a
well, this new microfluidic structure can be easily disassembled, cleaned, and replaced
new double-coated adhesive tape if the channel is clogged by the cells, making the elec-
with a new double-coated adhesive tape if the channel is clogged by the cells, making the
trode chipchip
electrode reusable. The The
reusable. S-parameter of theofsensor
S-parameter structure
the sensor with water
structure filled filled
with water liquidliquid
chan-
nel on top is shown in Figure 2b.
channel on top is shown in Figure 2b.

Figure 3.
Figure 3. The
Thetopology
topologyofofthe
thetapered
taperedCPW
CPW sensor
sensor structure
structure with
with microfluidic
microfluidic channel
channel on top
on top and and
the
the zoomed-in
zoomed-in sensing
sensing zone. Topology
zone. Topology of the
of the gold gold
coated CPWcoated CPW
sensor sensorchip.
on quartz on The
quartz chip. The
parameters of
the sensing CPW lines are S(ignal) = 6 um, G(ap) = 6 um, and Gnd = 20 um, and dimensions of the
PDMS liquid channel are L = 7 mm, W = 0.1 mm, and H = 10 um.

2.2. Modeling of the System

The microwave interferometric cytometry system is modeled in ADS (Figure 4). For
system modeling, the different components of the interferometric cytometry are presented
as independent units in ADS (Figure 4a), which are represented by the measured S-
parameters of the corresponding components. The loaded S-parameters are measured
with VNA, which is calibrated up to the ports of each component. The comparison of the
modeled (blue dots) and measured (orange line) system response S-parameters is shown
in Figure 5a, in which the system was tuned to 11 GHz to have the S21 be around −60 dB.
The modeled system in ADS is in good match with the measured system response. The
discrepancy in the S11 and S22 is mainly due to the reflections between different components
caused by the mismatches. The S12 and S21 differences in the interference nulls are mostly
coming from the inadequate sampling of the system model (frequency step is 5 MHz),
in other words, the frequency step needs to be smaller to acquire the deep interference
nulls. Thus, in Figure 5b, the system was modeled with a frequency step of 50 kHz in
the frequency range of 12–14 GHz. The measured interference null at 13 GHz is −65 dB,
while the modeled interference null is −62 dB, proving that the system model is in good
comparison with the real laboratory-grade measurements.
 crepancy
caused in the
by the S11 and S22The
mismatches. is mainly
S12 anddue to the reflections
S21 differences in the between different
interference nulls components
are mostly
caused by the mismatches. The S 12 and S21 differences in the interference nulls are mostly
coming from the inadequate sampling of the system model (frequency step is 5 MHz), in
coming
other from
words, thethe inadequate
frequency stepsampling
needs to ofbethe system
smaller model (frequency
to acquire step is 5 MHz),
the deep interference nulls.in
Thus, in Figure 5b, the system was modeled with a frequency step of 50 kHz in the nulls.
other words, the frequency step needs to be smaller to acquire the deep interference fre-
Thus, in
quency Figure
range 5b, the
of 12–14 system
GHz. Thewas modeled
measured with a frequency
interference step
null at 13 of 50
GHz kHzdB,
is −65 in the fre-
while
Chemosensors 2022, 10, 318 quency range of 12–14 GHz. The measured interference null at 13 GHz is −65
the modeled interference null is −62 dB, proving that the system model is in good com-5 of 12dB, while
the modeled
parison with the interference null is −62 dB,
real laboratory-grade proving that the system model is in good com-
measurements.
parison with the real laboratory-grade measurements.

Figure
Figure4.4.The
Themodeling
modelingofofthetheinterferometric
interferometriccytometry
cytometrysystem
system inin
ADS
ADS(a)(a)
without
without sensor
sensorsimula-
simulation
Figure
tion in 4. The
CST; and modeling
(b) with of the simulation
sensor interferometric
in cytometry
CST. QH system
stands for in ADS (a) hybrid,
quadrature withoutthe sensor simula-
90-degree
in CST; and (b) with sensor simulation in CST. QH stands for quadrature hybrid, the 90-degree
tion in TL
coupler; CST; and (b)
stands for with sensor simulation
transmission line, whichinisCST. QH stands
the tapered CPW forsensor;
quadrature
A1 is hybrid,
the 6 dBthe 90-degree
attenuator;
coupler;
coupler; TL
TL stands
stands for
for transmission
transmission line,which
line, which isisthe
thetapered
taperedCPWCPW sensor;
sensor; A1A1
is is the
the 6 dB6 attenuator;
dB attenuator;
A2 and PS are the tunable attenuator and phase shifter respectively.
A2
A2and
andPSPSare
arethe
thetunable
tunable attenuator andphase
attenuator and phaseshifter
shifterrespectively.
respectively.

Figure 5. The S−parameter comparisons between the modeled system in ADS with measurement
results for (a) the whole frequency range of 4−18 GHz, tuned at 11 GHz, in the frequency step of
5 MHz; and (b) the frequency range of 12−14 GHz, tuned at 13 GHz, in the frequency step of 50 kHz.

For the sensor simulation, a small sensing CPW section of the length T equals 10 µm
has been modeled in CST (Figure 6). To obtain fast and accurate simulation results of the
particle passing through the sensing CPW, the cubic particle with a side length R = 3 µm is
used in the model. The simulations are carried out for the particle at different positions
relative to the cross-section of the sensing CPW at a center height of 2.5 µm above the
substrate. The simulation results depicted in Figure 7 are the magnitude and the phase of
transmission signal S21 . The blue solid line and the orange ribbons around it are simulation
results and their error range, respectively. While the yellow solid line and blue ribbon
around it are respectively the mirrored simulation results and their error range of the left
half to demonstrate their symmetry with the right half of the simulation results. The particle
introduces signal variations when it passes through the region with high electromagnetic
(EM) fields. As well, in correspondence with the EM field intensity, which is highest at the
two gaps and relatively low at the center of the signal line, the variation signal caused by
the particle shows a profile of “M” shape for both the magnitude and phase of S21 .
 unit in the REF branch is loaded with simulation results of the particle above the ground
signal, while the one in the MUT branch is loaded with simulation results of the particle
at different positions relative to the sensing CPW. The overall simulation results are pro-
vided in Figure 8 for a 3 µ m cubic particle passing through the CPW sensor with signal
and gap widths equal to 6 µ m. The interferometric cytometry system tuned to −62 dB at
Chemosensors 2022, 10, 318 6 of 12
13 GHz and a magnitude change of ~0.1 dB and a phase change of ~3 degrees can be de-
tected.

Figure 6. The CST simulation model of the sensing CPW structure (Au) on quartz substrate with
Figure 6. The CST simulation model of the sensing CPW structure (Au) on quartz substrate with
microfluidic channel
microfluidic
Chemosensors 2022, 10, x FOR PEER REVIEW channel (DI
(DI water)
water) onon top
topand
andcovered
coveredwith withPDMS
PDMS slab. The
slab. Thecubic particle
cubic (polystyrene)
particle (polysty-
7 of 12
with a side length R of 3 µm is placed at a different position relative to the sensing
rene) with a side length R of 3 µ m is placed at a different position relative to the sensing CPW, CPW, with
withits
center
its height
center of of
height 2.52.5
µm. The
µ m. liquid
The channel
liquid channelHcstHis
cst 5 5 µ here
isµm in the
m here model
in the modelandand
the the
length T isT10isµm.
length 10
µ m.

Figure 7. The CST simulation results in the particle at different positions relative to the cross-section
of the
of the sensing
sensing CPW
CPW structure
structure to
to mimic
mimic the
the process
process of
of particle
particle passing
passing through
through the
the sensor.
sensor. (a)
(a) the
the
magnitude and (b) the phase of transmission signal S 21. The blue solid line and the orange ribbon
magnitude and (b) the phase of transmission signal S21 . The blue solid line and the orange ribbon
around it are simulation results and their error range respectively. While the yellow solid line and
around it are simulation results and their error range respectively. While the yellow solid line and
blue ribbon around it are respectively the mirrored simulation results and their error range of the
blue ribbon around it are respectively the mirrored simulation results and their error range of the left
left half to demonstrate their symmetry with the right half of the simulation results.
half to demonstrate their symmetry with the right half of the simulation results.

To have a particle signal profile of the modeled system response, the simulation results
of the sensor in CST are combined with the system modeling in ADS, as given in Figure 4b.
The TL_CST unit is introduced in both the two branches for a better match of the signals and
highlights the signal variation caused by the particle. The TL_CST_GND unit in the REF
branch is loaded with simulation results of the particle above the ground signal, while the
one in the MUT branch is loaded with simulation results of the particle at different positions
relative to the sensing CPW. The overall simulation results are provided in Figure 8 for a
3 µm cubic particle passing through the CPW sensor with signal and gap widths equal to
6 µm. The interferometric cytometry system tuned to −62 dB at 13 GHz and a magnitude
change of ~0.1 dB and a phase change of ~3 degrees can be detected.

Figure 8. The overall simulation results for the particle signal profiling of the system responses, at
different positions relative to the cross-section of the sensing CPW structure, to mimic the process
of particle passing through the sensor. (a) the magnitude and (b) the phase of transmission signal
S21.

2.3. Selection and Cultivation of the Yeast Strains

 Figure 7. The CST simulation results in the particle at different positions relative to the cross-section
of the sensing CPW structure to mimic the process of particle passing through the sensor. (a) the
magnitude and (b) the phase of transmission signal S21. The blue solid line and the orange ribbon
around it are simulation results and their error range respectively. While the yellow solid line and
Chemosensors 2022, 10, 318 7 of 12
blue ribbon around it are respectively the mirrored simulation results and their error range of the
left half to demonstrate their symmetry with the right half of the simulation results.

Figure
Figure 8.
8. The
Theoverall
overallsimulation
simulation results forfor
results thethe
particle signal
particle profiling
signal of the
profiling of system responses,
the system at at
responses,
different positions relative to the cross-section of the sensing CPW structure, to mimic the process
different positions relative to the cross-section of the sensing CPW structure, to mimic the process of
of particle passing through the sensor. (a) the magnitude and (b) the phase of transmission signal
particle passing through the sensor. (a) the magnitude and (b) the phase of transmission signal S21 .
S21.
2.3. Selection and Cultivation of the Yeast Strains
2.3. Selection and Cultivation of the Yeast Strains
As a widely used laboratory Saccharomyces cerevisiae strain, the S288C is selected for the
systemAs validation
a widely used laboratoryThe
experiments. Saccharomyces cerevisiaeby
S288C is designed strain, theMortimer
Robert S288C is selected for
for biochemical
the system validation experiments. The S288C is designed by Robert Mortimer for bio-
studies, and it is non-flocculent with a minimal set of nutritional requirements. Also, since
chemical studies, and it is non-flocculent with a minimal set of nutritional requirements.
it is a haploid strain, the diameter of it is around 4 µm, which has a closer volume to the
Also, since it is a haploid strain, the diameter of it is around 4 µ m, which has a closer
cubic particle in our simulation compared to other diploid strains like MDS130.
volume to the cubic particle in our simulation compared to other diploid strains like
During the preparation process of the experiments, the yeast cells were inoculated in
MDS130.
glass tubes containing YPDE medium (10 g/L yeast extract, 20 g/L bacteriological peptone,
During the preparation process of the experiments, the yeast cells were inoculated in
20 g/L glucose and 5 g/L ethanol) at 30 ◦ C with 200 rpm in a rotary shaker for 24 h. After
glass tubes containing YPDE medium (10 g/L yeast extract, 20 g/L bacteriological peptone,
which the medium was diluted to reach OD600 = 2 for measuring the signals of stationary
20 g/L glucose and 5 g/L ethanol) at 30 °C with 200 rpm in a rotary shaker for 24 h. After
phase cells.
which the medium was diluted to reach OD600 = 2 for measuring the signals of stationary
phase cells.
2.4. Experiment Workflow and Image Acquisition
After the yeast
2.4. Experiment cellsand
Workflow were cultivated,
Image the diluted medium with yeast cell samples were
Acquisition
extracted into syringes and kept in room temperature (~20 ◦ C) for 20 min. During which,
the interferometric cytometry system was tuned normally to a S21 of below −60 dB at the
desired frequency when the microfluidic channel was filled with deionized (DI) water. As
well, since the permittivity extraction algorithm requires two calibration measurements,
the system was measured when the channel is filled with DI water as calibration 1 (Cal1)
and when the channel is empty (or filled with air) as calibration 2 (Cal2). Then, the yeast
cell samples were pushed through microfluidic channel with the syringe, recording the
data readout from VNA and capturing the images from inverted microscope with software
(HCImageLive) on the computer.

3. Results
3.1. MUT Permittivity Extraction Algorithm
Similar to the permittivity extraction algorithm in [23], the measured S-parameters of
MUT, Cal1, and Cal2, namely S11(MUT/Cal1/Cal2) and S21(MUT/Cal1/Cal2) are presented here

S11( MUT ) − S11(Cal1) S11(CPW, MUT ) − S11(CPW, Cal1)

= (1)
S11(Cal2) − S11(Cal1) S11(CPW, Cal2) − S11(CPW, Cal1)

S21( MUT ) − S21(Cal1) S21(CPW, MUT ) − S21(CPW, Cal1)

= (2)
S21(Cal2) − S21(Cal1) S21(CPW, Cal2) − S21(CPW, Cal1)
Chemosensors 2022, 10, 318 8 of 12

in which, S11(CPW, MUT/Cal1/Cal2) and S21(CPW, MUT/Cal1/Cal2) are conformal mapping results
of the CPW in the sensing zone, taking the reflection effects caused by mismatch into
consideration.
 
Z(2CPW ) − Z02 sinhγl
S11(CPW,MUT/Cal1/Cal2) =   (3)
2Z(CPW ) Z0 cosh γl + Z(2CPW ) + Z02 sinhγl

2Z(CPW ) Z0
S21(CPW,MUT/Cal1/Cal2) =   (4)
2Z(CPW ) Z0 cosh γl + Z(2CPW ) + Z02 sinhγl

And Z0 is 50 Ω, l is the length of sensing CPW, while the characteristic impedance of

the sensing CPW √
εe f f (MUT/Cal1/Cal2)
Z (CPW, MUT/Cal1/Cal2) = (5)
Ctot c
where Ctot is the superposition of all four partial capacitance from the conformal mapping,
and εe f f (MUT/Cal1/Cal2) is the effective permittivity of the sensing CPW with MUT, Cal1 or
Cal2 filling the microfluidic channel.
Together with the propagation constant γ( MUT/Cal1/Cal2) = (αc + αd ) + jβ, which
consists of the conductive (αc ) and dielectric (αd ) loss, and phase constant (β) of the sensing
CPW, the complex permittivity of unknown MUT can be extracted based on their relation
with the effective permittivity and the propagation constant for better interpretation of the
detected single cell signals.

3.2. System Validation and Its Application of Yeast Strain—S288C Measurements

The validation of the microwave interferometric cytometry system is firstly carried out
by detecting 3 µm polystyrene particles (PSS), verifying the system modeling and single-
cell signal profiling. Then, the system is used to quantitatively measure the broadband
permittivity changes of MUT caused by single yeast cells of S288C flowing through the
CPW sensor in the microfluidic channel.
For system validation, the detection of the 3 µm PSSs, diluted in the DI water, was
performed by pumping the PSSs solution through the microfluidic channel with a syringe
when the system was tuned to around −69 dB at 11 GHz. The raw data of the system S21
in Figure 9a shows two particle detection signals with zoomed in views in the inset figures.
The maximum S21 changes of the two detection signal peaks are around 2 and 1.5 dB in
magnitudes, which is mainly due to the different heights of the PSSs above the sensor. As
well, the detection signals have the profile of the “W” shape, which is the inversion of the
“M” shape simulated in Section 2.1. Whether the decreasing of MUT effective permittivity
caused by PSSs will result in the “M” shape or the “W” shape depends on the different
tuning status of the system. It is the reason why the extraction of MUT permittivity is
necessary for analyzing single-cell signals.
To further demonstrate the proposed system, a quantitative analysis of MUT permit-
tivity is carried out with S288C yeast strain measurements. As described in Section 2.4, the
system was firstly tuned to around −67 dB at 4 GHz with DI water filling the microfluidic
channel, the system S21 was recorded as Cal1 at room temperature of 20 ◦ C. Secondly, the
diluted S288C solution with OD600 = 2 was pumped into the channel with a syringe, the
detected signals were saved as MUT (Figure 9b). The concentration of the yeast cell solution
is prepared as ~8 × 107 cells/mL, which makes most of the detected signals represent a
single cell passing through the sensor. As well, the flow rate of the cells is 1.5 mm/s on
average. At last, the channel was emptied by pumping air into it, and the system S21 was
noted as Cal2.
 ures. The maximum S21 changes of the two detection signal peaks are around 2 and 1.5 dB
in magnitudes, which is mainly due to the different heights of the PSSs above the sensor.
As well, the detection signals have the profile of the “W” shape, which is the inversion of
the “M” shape simulated in Section 2.1. Whether the decreasing of MUT effective permit-
tivity caused by PSSs will result in the “M” shape or the “W” shape depends on the dif-
Chemosensors 2022, 10, 318 9 of 12
ferent tuning status of the system. It is the reason why the extraction of MUT permittivity
is necessary for analyzing single-cell signals.

Figure
Figure9.
9.The
Theraw
rawdata
dataof
ofsystem
system SS2121when
when(a)(a)the
thesolution
solutionof
of33um
umPSSs
PSSsand
and(b)
(b)the
thesingle
singleyeast
yeastcell
cell
of
ofS288C
S288Cpassing
passingthrough
through the
the sensor
sensor in
in the
the microfluidic
microfluidic channel.
channel.

The
To detected
further signal of the
demonstrate a single
proposed yeastsystem,
cell is compared to that
a quantitative of 3 µm
analysis of MUT PSS permit-
and the
simulated cubic particle in Figure 10a. Despite the inverted detection
tivity is carried out with S288C yeast strain measurements. As described in Section 2.4, signals from the
simulated results, the signal of S288C has a larger variation
the system was firstly tuned to around −67 dB at 4 GHz with DI water filling the micro-at the center signal line of
the CPW sensor compared with the signal of 3 µm PSS. Because
fluidic channel, the system S21 was recorded as Cal1 at room temperature of 20 °C. Sec- S288C in general has
an average
ondly, diameter
the diluted of 4solution
S288C µm, which with is OD600
larger in = 2volume than the
was pumped PSSthe
into of channel
3 µm diameter.
with a
The volume
syringe, of S288C signals
the detected is large were
enough savedto influence
as MUT the electromagnetic
(Figure fields on topof
9b). The concentration ofthe
the
signal line of the CPW sensor. Figure 10b illustrates the correspondence
yeast cell solution is prepared as ~8 × 10 cells/mL, which makes most of the detected sig-
7 between the single
cell detection
nals represent of microwave
a single interferometric
cell passing through cytometry
the sensor.and As the optical
well, the flowmicroscope.
rate of the Within
cells
the three seconds of detection, there are three cells passing through
is 1.5 mm/s on average. At last, the channel was emptied by pumping air into it, and the the sensor. They are
captured by the microscope
system S21 was noted as Cal2. images as changes of overall intensity and recorded by the
proposed system, signal
The detected which of verifies
a singletheyeast
successful
cell isdetection.
comparedThe magnitude
to that of 3 µ mchanges
PSS and ofthe
the
first two cells
simulated cubicare larger in
particle compared
Figure 10a. to the third the
Despite one,inverted
meaningdetection
that the third
signals cell is most
from the
probably smaller than the first two. It is validated from the captured
simulated results, the signal of S288C has a larger variation at the center signal line of the images during the
experiment, since the third cell has a smaller intensity change
CPW sensor compared with the signal of 3 µ m PSS. Because S288C in general has an av- as a result of fewer pixels on
the
Chemosensors 2022, 10, x FOR PEER REVIEW images, meaning that it has a smaller size. In one measurement
erage diameter of 4 µ m, which is larger in volume than the PSS of 3 µ m diameter. set of 14 s, there
10 of 12 Theare
28 signal
volume ofprofiles
S288C isdetected by thetoproposed
large enough influencesystem, while 31 peaks
the electromagnetic were
fields on counted
top of the insig-
the
image intensity figure, resulting in a successful detection rate of around 90%.
nal line of the CPW sensor. Figure 10b illustrates the correspondence between the single
cell detection of microwave interferometric cytometry and the optical microscope. Within
the three seconds of detection, there are three cells passing through the sensor. They are
captured by the microscope images as changes of overall intensity and recorded by the
proposed system, which verifies the successful detection. The magnitude changes of the
first two cells are larger compared to the third one, meaning that the third cell is most
probably smaller than the first two. It is validated from the captured images during the
experiment, since the third cell has a smaller intensity change as a result of fewer pixels
on the images, meaning that it has a smaller size. In one measurement set of 14 s, there are
28 signal profiles detected by the proposed system, while 31 peaks were counted in the
image intensity figure, resulting in a successful detection rate of around 90%.

Figure
Figure10.
10.(a)(a)
The comparison
The comparisonof system S21 signals
of system between
S21 signals the single
between theyeast cellyeast
single of S288C,
cell the solu- the
of S288C,
tion of 3 um PSSs, and the simulated cubic particle in CST. (b) the optical correspondence with
solution of 3 um PSSs, and the simulated cubic particle in CST. (b) the optical correspondence with
electrical detection using the intensity changes of the microscope images.
electrical detection using the intensity changes of the microscope images.
The measurements of single yeast cells were conducted at different frequencies over
The measurements of single yeast cells were conducted at different frequencies over
the entire frequency range of the system. As well, their complex permittivities of MUT
the entire frequency range of the system. As well, their complex permittivities of MUT
with the single yeast cells present at the gap, which causes the signal peaks, are extracted
with the single yeast cells present at the gap, which causes the signal peaks, are extracted
with the algorithm mentioned in Section 3.1. The examples of extracted data points at 4,
with the algorithm mentioned in Section 3.1. The examples of extracted data points at 4, 8,
8, and 12 GHz are drawn in Figure 11a in comparison to the complex permittivity of DI
water at 20 °C. The differences in the extracted complex permittivity at a certain frequency
are mainly caused by the size variation of the S288C and the height difference when the
cells pass through the sensor.
 Figure 10. (a) The comparison of system S21 signals between the single yeast cell of S288C, the solu-
tion of 3 um PSSs, and the simulated cubic particle in CST. (b) the optical correspondence with
electrical detection using the intensity changes of the microscope images.

The measurements of single yeast cells were conducted at different frequencies over
Chemosensors 2022, 10, 318 the entire frequency range of the system. As well, their complex permittivities of MUT 10 of 12
with the single yeast cells present at the gap, which causes the signal peaks, are extracted
with the algorithm mentioned in Section 3.1. The examples of extracted data points at 4,
8, and
and 12 GHz
12 GHz are are drawn
drawn in Figure
in Figure 11a 11a in comparison
in comparison to the
to the complex
complex permittivity
permittivity of DI
of DI water
water◦ at 20 °C. The differences in the extracted complex permittivity at a certain frequency
at 20 C. The differences in the extracted complex permittivity at a certain frequency are
are mainly
mainly causedcaused by size
by the the size variation
variation of the
of the S288C
S288C andand
thethe height
height difference
difference when
when the
the cells
cells pass through the
pass through the sensor. sensor.

Figure11.
Figure 11.(a)
(a)The
Theextracted
extracted data
data points
points atat 4,
4, 8,
8, and
and1212GHz
GHzinincomparison
comparisontotothethecomplex
complexpermittiv-
permittivity
ity of DI water at◦ 20 °C [24]. (b) real0 (ɛ′) and (c) imaginary00(ɛ″) permittivity of the single S288C yeast
of DI water at 20 C [24]. (b) real (ε ) and (c) imaginary (ε ) permittivity of the single S288C yeast cells
cells in the frequency range of 4–18 GHz at the room temperature of◦20 °C. The blue error bars with
inmean
the frequency
values show range
the of 4–18 GHz
variations of at
thethe room temperature
detection signals. The of 20 C.
orange Theare
lines blue
theerror bars with
third-order mean
poly-
values
nomialshow theofvariations
fitting of the detection
the mean values. The blacksignals. Theare
solid lines orange linespermittivity
complex are the third-order polynomial
of DI water at 20
°C. of the mean values. The black solid lines are complex permittivity of DI water at 20 ◦ C.
fitting

The
Theextracted
extractedbroadband
broadbandcomplex
complexpermittivities
permittivities from
from the
the measured
measured signals,
signals, inin the
the fre-
quency
frequencyrange of 4–18
range GHzGHz
of 4–18 andand
at the room
at the temperature
room temperature of ◦20
of 20 C,°C,
areare
plotted in Figure
plotted in Figure11b,c.
The blue
11b,c. Theerror
bluebars
errorwith
barsmean values
with mean showshow
values the variations
the variations of the detection
of the detection signals.
signals.The
The orange
orange lines lines arethird-order
are the the third-order polynomial
polynomial fitting
fitting of the
of the mean mean values,
values, andandthethe black
black solid
solidare
lines lines
theare the complex
complex permittivity
permittivity of DIofwater at 20at◦ C.
DI water 20The
°C. The overall
overall looklook of the
of the ex-
extracted
tracted complex
complex permittivity
permittivity shows
shows that thethat the effective
effective permittivity
permittivity of MUT of reduced
MUT reducedwhenwhen the cells
the cells
pass throughpass the
through thesince
sensor, sensor,
thesince the heavily
heavily diluteddiluted
medium medium
of S288Cof S288C has similar
has similar permit-
permittivity values compared to DI water. As well, the permittivity
tivity values compared to DI water. As well, the permittivity change in absolute value ischange in absolute
valuein
larger is larger
lower in lower frequency
frequency for the
for the real realhowever,
part, part, however, it is larger
it is larger in higher
in higher frequency
frequency for the
for the imaginary
imaginary part. part.

4. Discussion
The detection of 3 µm PSSs has validated the functionality of system setup and appli-
cability of the signal profiling technique based on system modeling and sensor simulation.
The measurement and permittivity extraction of S288C yeast cells has demonstrated the
system’s capability of quantitative analysis of single-cell signals. The extracted complex
permittivity of S288C cells illustrated in Figure 11 shows an average decrease of 0.8–2 for the
real part permittivity and 0.9–2.1 for the imaginary part permittivity, which are 3–4 times
the permittivity change measured in [12]. The main reason is that despite the heights of
the channel being the same, the width of the electrode in [12] is 10 µm, while it is 6 µm in
this work. The smaller the electrode, the larger the volume ratio of the cell to the effective
volume of the channel, causing larger permittivity changes. The comparison between the
S21 signals of S288C cells, 3 µm PSSs, and the simulated cubic particle in Figure 10a shows
the sensitivity of the detection signal to the size of the cells, which can possibly be analyzed
in the future for size and height determination. Also, the toxicity of the adhesive material
from the double-coated adhesive tape needs to be further tested. As well, more importantly,
the sensitivities of the signal profile to the contrast of the dielectric properties between the
cell and the medium, the size of the cell, the topology of the sensing electrodes, and the
flow height of the cell should be analyzed in the next step.
Chemosensors 2022, 10, 318 11 of 12

Author Contributions: Conceptualization: M.Z., G.H. and T.M.; methodology: M.Z., G.H., J.B., T.M.;
software: M.Z. and G.H.; validation: M.Z. and G.H.; formal analysis: M.Z. and T.M.; investigation:
M.Z. and T.M.; resources, P.V.D., T.M. and B.N.; data curation: M.Z. and G.H.; writing—original draft
preparation: M.Z., G.H. and T.M.; writing—review and editing: T.M.; visualization: M.Z. and G.H.;
supervision: T.M., P.V.D. and B.N.; project administration: T.M.; funding acquisition: T.M., P.V.D. and
B.N. All authors have read and agreed to the published version of the manuscript.
Funding: This work is sponsored by the Research Foundation Flanders (FWO) research project
(G0A1220N).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank Matko Martinic, Marie Mertens, and Maede
Chavoshi for their help on chip fabrication and discussion, Johan M. Thevelein from the Laboratory
of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven for providing the
measurement samples, the Nitto Belgium NV for providing the super thin double-coated adhesive
tape samples, Donald Raddoux from MICAS, KU Leuven for wafer dicing and the Fablab Leuven for
technical support.
Conflicts of Interest: The authors declare no conflict of interest.

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