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Nanotechnol. Precis. Eng. 6, 023003 (2023); https://doi.org/10.1063/10.0017649 6, 023003
© 2023 Author(s).
Tunable microfluidic chip for single-cell
deformation study
Cite as: Nanotechnol. Precis. Eng. 6, 023003 (2023); https://doi.org/10.1063/10.0017649
Submitted: 15 December 2022 • Accepted: 01 March 2023 • Published Online: 03 April 2023
Ruiyun Zhang, Xuexin Duan, Shuaihua Zhang, et al.
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Tunable microfluidic chip for single-cell
deformation study
Cite as: Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649
Submitted: 15 December 2022 •Accepted: 1 March 2023 •
Published Online: 3 April 2023
Ruiyun Zhang,1,2Xuexin Duan,1,2Shuaihua Zhang,1,2Wenlan Guo,1,2Chen Sun,1,2and Ziyu Han1,2,a)
AFFILIATIONS
1College of Precision Instrument and Opto-electronics Engineering, Tianjin University, Tianjin 300072, China
2State Key Laboratory of Precision Measuring Technology and Instruments, Tianjin University, Tianjin 300072, China
a)Author to whom correspondence should be addressed: ziyu_han@tju.edu.cn
ABSTRACT
Microfluidic phenotyping methods have been of vital importance for cellular characterization, especially for evaluating single cells. In order to
study the deformability of a single cell, we devised and tested a tunable microfluidic chip-based method. A pneumatic polymer polydimethyl-
siloxane (PDMS) membrane was designed and fabricated abutting a single-cell trapping structure, so the cell could be squeezed controllably
in a lateral direction. Cell contour changes under increasing pressure were recorded, enabling the deformation degree of different types of
single cell to be analyzed and compared using computer vision. This provides a new perspective for studying mechanical properties of cells at
the single cell level.
©2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/10.0017649
KEYWORDS
Microfluidics, Cell deformation, Single cell, Computer vision
I. INTRODUCTION
The single-cell study is of vital importance for understand-
ing the mechanism of cell behaviors, such as invasion, migration,
and diffusion of malignant tumor cells,1,2 in which cell mechan-
ical properties3have emerged as a label-free characteristic of cell
phenotyping. Cell mechanical properties, including deformability,
recoverability, and cell stiffness, have been investigated for cell
recognition,4,5 disease diagnosis,6cell biophysical study, and so on.
Conventional methods, such as atomic force microscopy (AFM),7–11
micropipetting,12–14 and optical stretching15–19 have been widely
used for measuring cell deformation when subjected to external
forces, giving precise characterization of the mechanical properties
of cells. However, the need for skilled operators and the high costs
of these techniques still limit their widespread use.
Recently, microfluidic technology has developed rapidly.
This has led to the adoption of two techniques as mainstream
microfluidic strategies for cell mechanical phenotyping: contactless
hydro-stretching deformability cytometry, and contact constriction
deformability cytometry. Compared to conventional methods, the
constriction of microfluidic channels20–26 allows gentle pressures
to squeeze the cells by physical contact with the inner wall of the
microchannel, avoiding cell damage.
By measuring the passage time and deformation degree of
cells under the external forces in the constricted microchannel, the
mechanical properties of cells can be further evaluated and com-
pared. Abkarian et al.27 designed a microfluidic system composed
of 64 narrow channels to measure the passage time of white blood
cells, and used a high-speed imaging system to study the deforma-
bility of different types of these cells. Urbanska et al.28 studied the
mechanical properties of different cells by measuring cell area, con-
tour, roundness, and shape variables when cells flowed through a
constricted channel. However, cell deformation is usually fixed to a
certain degree or range when the constriction channel is fabricated,
which limits the study of the cell mechanics.
In order to achieve various degrees of deformation within
a single chip, a constriction microchannel with tunable channel
dimension is one potential solution. Previous reports discussed tun-
able polymer polydimethylsiloxane (PDMS) microfluidic chips for
microbead and cell screening29,30 and tunable microfluidic channels
Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649 6, 023003-1
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for cell capture.31 In this paper, we propose a tunable microfluidic
chip with a pneumatic PDMS membrane, fabricated to abut a single-
cell trapping structure. The tunable chip enables controllable lateral
compression on the single cell by adjusting the air pressure.
First, the lateral displacements of the PDMS membrane were
verified and optimized by finite element method (FEM) simulation.
The single-cell trapping structure was then integrated with the tun-
able PDMS membrane to perform single-cell squeezing. The whole
device was in a single layer and could be photographed in real time
using a high-speed camera, making it straightforward to measure
cell deformation. The U-net neural network automatically measured
and analyzed the cell contour changes under successive increases in
applied pressure. The deformation of different types of cell was com-
pared, showing the potential of the proposed system to automatically
and efficiently study single-cell mechanical properties.
II. EXPERIMENTAL METHODS
A. Experimental setup
A schematic of the microfluidic chip and system setup is shown
in Fig. 1. Compressed dry air was introduced as a pressure source,
which was adjusted precisely by a pressure-regulating controller
(MFCS-EZ, Fluigent, France). The cell suspension was injected into
the chip through the inlet of the liquid channel, and the compressed
air source was connected to the inlet of the dead-end dry chan-
nel to deform the PDMS membrane. In each experiment, a PC and
the pressure controller adjusted the working pressure on different
channels. The pressure controller contained a pressure sensor for
measurements in real time. The microfluidic chip was mounted
on an inverted optical microscope (IX53, Olympus, Japan) with a
40×phase contrast objective lens, which allowed a sufficient field
of view for observing the deformation of single cells. A high-speed
camera (Mini UX50, FASTCAM, Japan) captured images from the
microscope.
B. Device design and fabrication
The microfluidic chip consisted of a liquid and an air
microchannel, with widths of 60 μm and 200 μm, respectively. The
height of the microchannels was 20 μm. The microfluidic chip was
fabricated by the PDMS soft lithography process.32 The PDMS mold
was fabricated on the silicon substrate with SU-8 2015. The PDMS
FIG. 1. Schematic of the microfluidic system.
base and curing agent were mixed at volume ratios of 25:1,33 20:1
and 10:1 to make the PDMS layer sufficiently easy to deform; the
different ratios of base and curing agent resulted in different elas-
tic moduli. The uncured PDMS mixture was then poured on the
molded wafer, degassed in a vacuum chamber, and baked in an oven
at 90○C for 1 h to further cure the PDMS. The cross-linked PDMS
was cut into individual chips and punched with inlet and outlet
holes. The PDMS channel layer was air plasma treated, then irre-
versibly bonded to a glass slide and baked at 80 ○C for 1 h to reinforce
the bonding strength.
C. Cell culture
The EJ human bladder carcinoma cell line and the hCMEC/D3
human endothelial cell line were used in the cell squeezing experi-
ments. The EJ cells were cultured in 1640 medium at a concentration
of approximately 1 ×105cells/mL in the cell incubator at 37○C
with 5% CO2. After cell adhesion and growth for 48 h, the adherent
cells were suspended using 1 mL trypsin-EDTA solution (Biosharp).
Then, EJ cells were collected using centrifugation and resuspended
in 1640 medium for the experiments. The hCMEC/D3 cells were cul-
tured in ECM medium at a cell concentration of 1 ×105cells/mL in
a cell culture incubator at 37○C and 5% CO2. After 72 hours of cell
adhesion and growth, the adherent cells were suspended with 1 mL
of trypsin-EDTA solution (Biosharp). Cells were then collected and
resuspended in ECM medium for the experiments.
III. RESULTS AND DISCUSSION
A. Numerical simulation study of the tunable channel
The behavior of the tunable PDMS membrane was simu-
lated using a simplified microfluidic structure. Figure 2(a) shows
a schematic of the microfluidic chip with a tunable PDMS mem-
brane. Two dead-end T-shaped microchannels (air channels) were
placed symmetrically either side of a straight microchannel (liquid
channel), forming a “sandwich” of channels (air-liquid-air) so that
the pressure in the air channels could squeeze the liquid channel.
The air and liquid channels were separated by a PDMS membrane.
When pressure was applied to the air, the dead-end air channels were
forced to expand and deform, reducing the diameter of the liquid
channel. To predict the degree of deformation of the PDMS mem-
brane under various air pressures, finite element method (FEM) sim-
ulation (COMSOL Multiphysics 5.6) was introduced.23 Figure 2(b)
shows the simulation results for stress and strain added to a 10 μm
PDMS membrane under 2 bar air pressure.
The maximum displacement of membranes of 10 μm to 20 μm
was investigated. Figures 2(c) and 2(d) show the position displace-
ment curves of PDMS membranes of 20 μm and 10 μm thickness
respectively under increasing pressure. Figure 2(e) shows the degree
of deformation for different membrane thicknesses under constant
pressure. As expected, a larger displacement was obtained with a
thinner membrane.
The deformation of PDMS membranes with different elastic
moduli was investigated. The moduli were varied by adjusting the
ratio of PDMS base to curing agent. Ratios of 25:1 and 10:1 were
used, resulting in elastic moduli of 0.20 MPa and 0.75 MPa respec-
tively. Figure 2(f) presents the results. A 10 μm thick membrane
Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649 6, 023003-2
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FIG. 2. Numerical simulations of PDMS membranes. (a) Schematic of tunable PDMS membrane. (b) FEM simulation of the stress and deformation of a PDMS membrane,
10 μm thick. (c) The displacement of a 20 μm-thick PDMS membrane under a series of pressures. (d) The displacement of a 10 μm-thick PDMS membrane under a series
of pressures. (e) The maximum displacement of PDMS membranes with different thicknesses under varying pressure. (f) The maximum displacement of 10 μm thick PDMS
membranes with different PDMS base:curing agent mix ratios.
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made with a mix ratio of 25:1 (smaller elastic modulus) showed a
larger maximum displacement under the same pressure.
B. Experimental verification of PDMS membrane
lateral deformation
A tunable “sandwich” microfluidic chip was fabricated. Exter-
nal air pressures between 0 bar and 1.5 bar were introduced into
the air channels to deform the PDMS membrane so that it squeezed
the liquid channel. To better characterize the contour of the liquid
channel under squeezing, red ink was injected into the liquid chan-
nel, and microscopic images were taken by a high-speed camera.
Figure 3(a) shows the images of the deformable microchannel under
air pressure of 0 bar to 1.4 bar. As the added pressure increased, the
liquid channel shrank until it was fully closed at the middle of the
channel. The Java image processing program ImageJ was employed
to quantify the maximum deformation of the membrane by extract-
ing the boundary of the deformable liquid channel. The same was
done for different PDMS mix ratios (25:1 and 20:1) and membrane
thicknesses (10 μm, 15 μm, and 20 μm), as shown in Figs. 3(b) and
3(c). It is clear that the 10 μm-thick membrane with a 25:1 PDMS
mix ratio exhibited the maximum deformation, which corresponded
well to the results of the FEM simulation. However, it is also worth
noting that there was a non-linear deformation range when the
applied pressure was below ∼0.6 bar, which was due to the compress-
ibility of air in the sample reservoir that may have counteracted the
applied pressures. The maximum deformation shows a linear trend
when the pressure is greater than 0.6 bar, which corresponds to the
simulation results.
Therefore, in order to realize controllable deformation within
the microfluidic chip, for subsequent experiments a PDMS mem-
brane was selected at 10 μm thick and a PDMS mix ratio
of 25:1.
C. Design of single-cell squeezing chip
According to the simulation and experimental results for
the deformable PDMS membrane, the lateral deformation of the
microchannel can be well-tuned by adjusting the added air pressure.
The tunable PDMS membrane was then integrated with a single-
cell trapping structure to investigate single-cell deformation under a
series of squeezing conditions. The layout of the microfluidic chip is
FIG. 3. (a) Microscopic images of the deformable microchannel under increasing air pressure. The white scale bar at bottom right is 100 μm. (b) Comparison of maximum
deformation of different membrane thicknesses with a PDMS mix ratio of 25:1 and (c) 20:1.
Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649 6, 023003-4
© Author(s) 2023
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FIG. 4. (a) Layout of the microfluidic
chip for single-cell deformation study
with a hydrodynamic trapping structure.
(b) Schematic of single-cell trapping and
squeezing. (c) Microscopic image of a
single cell trapped in the structure and
(d) deformed as pressure was applied to
the structure.
shown in Fig. 4(a). Single-cell trapping was realized by the hydrody-
namic principle.34 A bypass channel with a relatively high initial flow
resistance was designed in parallel with the semicircular single-cell
trapping structure so that a single cell was more likely to flow straight
into the trapping structure. Once the trapping structure was blocked
by a single cell, subsequent cells flowed into the bypass channel. A
dead-end air channel was fabricated at the side of the single-cell
trapping structure to squeeze the single cell in the trapping struc-
ture. As shown in Fig. 4(b), after cell trapping, the air channel was
pressurized in order to deform the PDMS membrane. Images of the
squeezed cells were captured in real time by the high-speed cam-
era and analyzed using computer vision. Figures 4(c) and 4(d) show
microscopic images of a single cell in the microfluidic chip before
and after squeezing, respectively.
D. Computer vision assisted single-cell
deformation study
The contours of the squeezed cell were studied by sequen-
tial analysis. Automatic cell contour extraction is usually achieved
by image processing to improve accuracy and efficiency. However,
the cell and channel wall had overlapping contours. The Attention
U-Net neural network was applied to segment the cells from the
channel background, as shown in Fig. 5(a). The cells were manu-
ally segmented using LabelMe software to generate data labels. The
single-cell images were normalized and inputted into the Attention
U-net for data training. The Attention U-Net neural network was
trained on the TensorFlow-based Keras framework.35,36 A stochas-
tic gradient descent (SGD) optimizer was employed during training,
with the initial learning rate set to 1 ×10-3, and the network trained
for 200 epochs. Training was done using 90% of the dataset, and
the remaining 10% was used for validating all the input data. As
shown in Fig. 5(b), the segmented image has an intersection over
union (IOU) of 0.85, so the trained neural network can be applied for
accurate extraction of single-cell contour in the microfluidic chan-
nel. The segmented cell contour was then applied for analysis of
cell deformability by extracting the cell’s short axis length and long
axis length using the fitEllipse algorithm in OpenCV. Furthermore,
to verify the feasibility of the proposed method for cell deforma-
tion study, two types of cell, a cancer cell line (EJ) and a human
endothelial cell line (hCMEC/D3) were measured and their cell con-
tours analyzed under increasing pressure. As shown in Fig. 5(c), 30
groups of single-cell deformation experiments were carried out on
EJ and hCMEC/D3 cells. The extracted deformation degree (long
axis length divided by short axis length) of EJ cells and hCMEC/D3
cells were plotted against applied pressure. As the pressure increased,
the EJ cells deformed more, and increasingly so, compared with the
hCMEC/D3 cells, due to cancer cells being less rigid than normal
cells.37,38
Thus, by analyzing the deformation degree of single cells under
increasing pressure, the mechanical properties of different types of
cells could be evaluated and compared using a single-chip design.
Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649 6, 023003-5
© Author(s) 2023
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FIG. 5. (a) Diagram of the Attention U-Net segmentation model. (b) Original images of single-cell deformation and segmented images with extracted contour. The scale bar
is 15 μm. (c) Deformation degree of single EJ cells and hCMEC/D3 cells as pressure increases (N =30).
IV. CONCLUSIONS
In this paper, a tunable microfluidic chip with a deformable
PDMS membrane was proposed to squeeze single cells laterally
and study their deformation degree. The feasibility of the proposed
method was first verified by FEM simulation. A device was fabri-
cated and experimental tests conducted. By integrating a deformable
PDMS membrane at the side of the single-cell trapping structure, a
series of tunable, lateral squeezes were applied to a single cell. The
images of a single cell under squeezing were analyzed by extract-
ing the cell contour using computer vision, and a characteristic
curve of single-cell deformation degree was efficiently obtained. The
deformation degree curves for two different types of cell (EJ cells
and hCMEC/D3 cells) were obtained, indicating the feasibility of
the proposed method for cell deformation studies on different cells
with a single chip design. The proposed method for controllable
single-cell squeezing and efficient acquisition of single-cell defor-
mation degree can potentially be applied for studying mechanical
properties of various types of cell, as well as studies on dynamic cell
morphology at single-cell level.
ACKNOWLEDGMENTS
The authors gratefully acknowledge financial support from
National Key R& D Program of China (2018YFE0118700), the
National Natural Science Foundation of China (NSFC No.
62174119), the 111 Project (B07014), and the Foundation for Tal-
ent Scientists of Nanchang Institute for Micro-technology of Tianjin
University. Quanning Li, Xuejiao Chen, Bohua Liu, and Chongling
Sun are thanked for the help with device fabrication.
AUTHOR DECLARATIONS
Conflict of Interest
The authors have no conflicts of interest to declare.
DATA AVAILABILITY
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649 6, 023003-6
© Author(s) 2023
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Precision Engineering ARTICLE scitation.org/journal/npe
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Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649 6, 023003-7
© Author(s) 2023
Nanotechnology and
Precision Engineering ARTICLE scitation.org/journal/npe
Ruiyun Zhang received her B.E. degree in
engineering from China Agriculture Uni-
versity, Beijing, China, in 2020. She is cur-
rently pursuing the master’s degree at State
Key Laboratory of Precision Measuring
Technology and Instruments, Tianjin Uni-
versity, Tianjin, China. Her current research
interests include tunable microfluid chips.
Xuexin Duan received a Ph.D. degree from
the University of Twente, Netherlands in
2010. After postdoctoral studies at Yale Uni-
versity, he moved to Tianjin University,
Tianjin, China. Currently, he is a full profes-
sor at the State Key Laboratory of Precision
Measuring Technology and Instruments,
Department of Precision Instrument Engi-
neering of Tianjin University. His research
concerns MEMS/NEMS devices, microsys-
tems, and microfluidics, and their inter-
faces with chemistry, biology, medicine, and
environmental science.
Shuaihua Zhang received the B.S. degree
from Northeastern University, Qinhuang-
dao, China, in 2019. He is currently work-
ing toward a Ph.D. degree at Tianjin Uni-
versity, Tianjin, China. His research inter-
ests include biosensors, microfluidics flu-
orescence and impedance flow cytometry,
droplet technology, and acoustic actuators.
Wenlan Guo received her M.S. in food
science from Tianjin University in 2013.
She is currently an engineer at the School
of Precision Instruments and Opto-
Electronics Engineering, Tianjin Uni-
versity. Her research interests include
Physical Vapor Deposition based various
materials, and Semiconductor package
processing.
Chen Sun received his B.S. degree in
optical information science and technol-
ogy from Nanjing University of Science
and Technology, in 2006, and his M.S.
degree in optics from Tianjin University
in 2015. He is currently an engineer at
the School of Precision Instruments and
Opto-Electronics Engineering, Tianjin
University. His research interests include
wet processing of silicon-based materi-
als and structures, photomechanics, and
digital image processing.
Ziyu Han received a Ph.D. degree
from Tianjin University, China in
2022. Currently, he is a postdoctoral
fellow at the College of Precision
Instrument and Opto-Electronics Engi-
neering, Tianjin University. His research
concerns microfluidic impedance cytom-
etry, nanofluidic devices, biosensing
microsystems and platforms.
Nano. Prec. Eng. 6, 023003 (2023); doi: 10.1063/10.0017649 6, 023003-8
© Author(s) 2023