Single Cell Array Impedance Analysis for Cell Detection

and Classification in a Microfluidic Device

Emre Altinagac

, Selen Taskin

and Huseyin Kizil

Department of Nanoscience & Nanotechnology, Istanbul Technical University, Istanbul, Turkey

Faculty of Chemical and Metallurgical Engineering, Istanbul Technical University, Istanbul, Turkey

Keywords: Microfluidics, Impedance Analysis, Lab-on-a-Chip, Single Cell Detection.

Abstract: Impedance analysis of single cells is presented in this paper. Following the separation of a target cell type

by dielectrophoresis in our previous work, this paper focuses on capturing the cells as a single array and

performing impedance analysis to point out the signature difference between each cell type. Lab-on-a-chip

devices having a titanium interdigitated electrode layer on a glass substrate and a PDMS microchannel are

fabricated to capture each cell in a single form and perform impedance analysis. MDA-MB-231 and HeLa

cells are used in our experiments.

1 INTRODUCTION

Microfluidic based cell separation and capturing

systems with high efficiency, fast response time,

multi-functionality, high accuracy and repeatability

rates are emerged as a new diagnosis tool for many

applications in biotechnology, drug discovery,

medicine, chemistry and environmental problems

(Mateo et al., 2014, Karabacak et al., 2014). They

require minimal sample size, low cost to fabricate,

portable and most importantly allow early detection

of circulating cancer cells and can be used as a

point-of-care product compare to macro-scale cell

separation and diagnosis systems (Alix-Panabieres et

al., 2014, Jin et al., 2014). In literature, active and

passive cell separation systems could be found on

the basis of differences in cell’s geometry, chemical

and electrical properties. Passive systems are based

on the flow characteristics of the fluid with particles

inside the microchannel and separation is

accomplished using the geometrical differences of

the particles, whereas active systems require outside

source, like magnetic field, electrical field, acoustics

and optics, to sort/capture the particle inside

microchannel. The outside source applied in the

active systems must accomplish the task without

damaging the viability of cells (Hajba et al., 2014).

Impedance spectroscopy (IS) is an important

measurement system in cell biology in the analysis

of cellular structure, cell physiology and cell to

disease interaction studies (Park et al., 2010).

Cellular resistivity measurement without the need

for any molecular marker can provide significant

information to researchers on the mechanisms of cell

functioning, especially in the formation and

progression of a disease. For example, membrane

specific capacitances of cancer tissue cells are

different than that of the normal cell membranes.

And it is well known that white blood cells have

different capacitance values among themselves, due

to the surface geometry of the cell membrane, and

cell membranes with specific apoptosis or necrosis

condition have different capacitance and

conductivity values compare to the ones at the

normal mode of operation. In a recent study (Anh-

Nguyen et al., 2016) long-term monitoring of MCF-

7 breast cancer cell attachment, adhesion, spreading

and the response of those cells to anticancer drug

Cisplatin is presented within the same platform.

These cellular activities and responses of cancer

cells to drug treatment are indicated by impedance

spectra of target cells. Another study (Dastider et al.,

2016) presents a biosensor for detection of low

concentration (39 CFU/mL) foodborne pathogen,

E.coli, on a microfluidic platform consist of two

dielectrophoretic focusing and impedance sensing

sequentially. Positive dielectrophoretic force applied

to concentrate the bacteria towards to the center of

the microchannel and anti-E.coli coated

interdigitated electrode arrays detects the flowing

bacteria throughout the microchannel.

Altinagac E., Taskin S. and Kizil H.

Single Cell Array Impedance Analysis for Cell Detection and Classiﬁcation in a Microﬂuidic Device.

DOI: 10.5220/0006166700490053

In Proceedings of the 10th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2017), pages 49-53

ISBN: 978-989-758-216-5

The aim of our research is to fabricate a

microfluidic based lab-on-a-chip (LOC) device that

will consist of two main parts: In the first part, the

cells will be separated by dielectrophoresis (DEP)

based on their dielectric properties. In the second

part, cells are captured as single cell array by

hydrodynamic forces and the cell impedance is

measured. The fabricated LOC system has a

potential to be used for counting blood cells

(hemogram), stem cell count, drug resistance

detection, cell phenotype etc. These measurements

are currently made with restricted use of the flow

cytometer, which requires large volume of samples

which are fluorescently labelled and then excited by

the laser.

The two parts of our design are studied

separately to maintain the simplicity of our research

and this paper focuses on the capturing of target

cells by hydrodynamic forces and carrying out

impedance analysis for cell detection, classification

and characterization.

2 DESIGN

The idea is inspired from Tan’s study (Tan et al.,

2007) where target cells are trapped as a single cell

array throughout a two-dimensional microfluidic

channel. Interdigitated electrode couples are placed

under each trap site to detect captured cells via an

LCR meter. The width and the height of the

microchannel are close to the diameter of target cells

to maintain the stream of cells in a linear form.

There are two potential paths with different flow

resistances for cells to follow: Path 1 is the trap site

with a lower flow resistance when it is empty and

Path 2 is the fraction of the main channel. When a

cell is trapped in Path 1 consecutive cells are

directed to Path 2 due to increase in flow resistance.

Tan’s design consists of 5µm x 5µm narrow necks to

create a trap site in Path 1 and due to the fabrication

challenges of such structures we present a 3D

polydimethylsiloxane (PDMS) microchannel which

is shown in Figure 1 with a minimum feature size of

10µm. A narrow neck in Path 1 is created in vertical

direction by limiting the channel height to 5µm in

this region and the rest of the channel has a

thickness of 15µm.

Figure 1: There are two potential paths for cells to follow.

Path 1 has a lower flow resistance until a cell is trapped. A

triangular trap site has a lower flow resistance than a

circular one.

Different geometries for trap site are studied to

prevent multiple cell capturing in the same trap site

and clogging of main channel. The length and the

width of the Path 1 and Path 2 play a key role to

determine the flow resistance. Path 1 is designed to

be wider and shorter to maintain lowest flow

resistance possible. Fluid velocity variation is shown

in Figure 2 and it is shown that higher trapping

efficiency is achieved by just increasing the channel

length due to increase in the flow resistance of Path

2. But multiple cells could be captured at once in the

trap area in such structures.

Figure 2: Fluid velocity distributions for different Path 2

lengths. Colour bar: Fluid velocity (m/s).

We present a novel design to maintain capturing

single cells only in a graded triangular trap site as

seen in Figure 3. When a cell (1) is trapped in the

acute angled area the subsequent cells (2) are cleared

out of the trap site. This process repeats itself until

all the trap sites are filled with single cells.

BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices

Figure 3: Schematic view of the graded triangular trap

design. A) α=30°, β=60° B) When a cell is trapped in

narrow region subsequent cells are cleared out of the trap

site.

Interdigitated titanium electrode couples are

placed under each trap site. The width of the

microelectrode fingers is 15µm and the gap between

the fingers is 8µm. Schematic view of the whole

system is given in Figure 4. There are 40 triangular

trap sites in total divided in four parallel lines of 10

trap sites.

The signal is recorded for 2 of the 4

parallel lines at the same time by interdigitated

electrode array couples connected to an LCR meter.

Figure 4: Schematic view of the whole trapping area.

There are 40 triangular trap sites in total. Green lines:

microfluidic channel, orange lines: interdigitated

microelectrodes.

3 MATERIALS AND METHODS

Conventional optical lithography processes are used

to create 3D microchannel structures by aligning two

optical mask to create a master mold for PDMS

casting. SU-8 3010 negative photoresist is used for

both layers. First layer has a thickness of 5µm and

the second layer is 10µm. Path 1 has a continuous

opening (transparent) in the first mask only to create

a vertical narrow neck in trap sites.

Titanium microelectrodes with a thickness of

200nm are fabricated on glass substrate coated with

a 400nm AZ 1505 positive photoresist by DC

magnetron sputtering and following lift-off process.

PDMS microchannel and microelectrodes coated

glass slide are aligned under an optical microscope

and plasma activated bonding process applied. A

droplet of methanol is used to create a sliding layer

between the PDMS microchannel and glass slide

during the alignment. Aligned substrates are placed

in a vacuum oven at 50˚C for 15 minutes to

evaporate the methanol and a stable bonding is

achieved.

MDA-MB-231 (human breast cancer cell line)

and HeLa cells (cervical cancer cell line) with

different medium conductivities are used in our

experiments. Conductivity of medium is adjusted by

the concentration of PBS (phosphate-buffered

saline) in 200mM sucrose solution (1X PBS=15,84

mS/cm, 0.5X PBS=7,71mS/cm).

4 EXPERIMENTS & DISCUSSION

Fluid flow is controlled by a syringe pump to

achieve precise flow rates and experiment is

observed under an optical microscope. Agilent

E4980A Precision LCR Meter (Keysight

Technologies, USA) is used for the impedance

measurements. Impedance values are recorded for a

frequency range of 1-500kHz with an applied

potential of 1V

MDA-MB-231 and HeLa cells are efficiently

trapped in triangular sites and the signals are

recorded before and after the cells are trapped. Due

to the elastic nature of biological cells, some of them

are deformed through the bottom neck and slip

away. While the hydrodynamic trap design itself is

independent from the flow rate, it has been seen that

the design is most effective for flow rates below

2µl/min and cells with a minimum diameter of

10µm.

Single Cell Array Impedance Analysis for Cell Detection and Classiﬁcation in a Microﬂuidic Device

MDA-MB-231 cells are trapped as seen in

Figure 5. Yellow circles represent empty traps and

green ones for filled traps with a single cell. The

impedance shift is recorded with the cells in green

circles.

Figure 5: MDA-MB-231 cells in PBS. Graded triangular

traps are used. The cells with no contact with

microelectrodes or empty traps are shown in yellow circles

where the cells are in contact with the microelectrodes are

shown in green. Each trap site has a single MDA-MB-231

cell.

A detailed view of filled and empty trap sites is

given in Figure 6.

Figure 6: Single cell trap sites: A) filled traps with a single

cell B) empty traps.

The impedance readings are recorded

continuously before and after releasing the cells into

the microchannel. When a cell is trapped the

impedance is shifted depending on the cell

properties. It can be seen that the impedance shift

(∆Z ) varies for each cell line which can be used for

further analysis or diagnosis applications. ∆Z values

for MDA-MB-231 and HeLa cells in 1XPBS are

given in Figure 7, showing larger shifts for MDA-

MB-231 cells compare to HeLa cells. This result

further proved that the impedance measurements are

sensitive to cell types.

Figure 7: Impedance shift of MDA-MB-231 and HeLa cell

lines in 1XPBS medium.

∆Z values for MDA-MB-231 cells in 1XPBS and

0.5XPBS mediums are given in Figure 8. It is found

that the magnitude of ∆Z increases with decreasing

conductivity of medium. This result shows that

medium conductivity can be adjusted to achieve

higher sensitivity for a target cell line.

Figure 8: Impedance shift of MDA-MB-231 cell lines in

0.5XPBS and 1XPBS mediums.

1 10 100 1000

∆Z(kΩ)

freq (kHz) (log)

HeLa MDA-MB-231

1 10 100 1000

∆Z(kΩ)

freq (kHz) (log)

MDA-MB-231 in 1XPBS

MDA-MB-231 in 0.5XPBS

BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices

The signal is collected from the array of 20

single cells in our current design but it is proven to

maintain these target cells in these trap sites

throughout the impedance analysis. As a future work

of our research, individual signals will be collected

from individual interdigitated electrode couples

placed under each trap sites to gain the long-term

data of impedance analysis of individual cells.

There is a recent study also inspired by Tan’s

design (Zhou et al., 2016) that presents

hydrodynamic trapping and impedance spectroscopy

of single cells. It has been shown that with a similar

design of our own, it is possible to monitor dynamic

changes in electrical properties of individual cells

over long periods of time to investigate the external

effects on cells.

5 CONCLUSIONS

Our experimental results show that diagnosing of

different cell lines in mediums with an optimum

conductivity is achievable using current single cell

trap array. The impedance shift is sensitive to cell

type and it can be used for the estimation of the total

number of captured target cells. The cell would be

stimulated by different chemicals or drugs injected

to microsystem to see the effects on cell viability or

its electrical properties. Further studies will focus on

introducing the optimum medium conductivity and a

frequency value for a target cell line to record the

impedance shift with a minimum error. This

technique will be used for estimating the

physical/electrical properties of cell structures and

the separation efficiency by DEP will be increased

with gained knowledge of target cell lines.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the financial

support provided by the Scientific and

Technological Research Council of Turkey

(TUBITAK) under Grant No. 114M802.

REFERENCES

Mateo, J., Gerlinger, M., Rodrigues, D., de Bono, J. S.,

2014. The promise of circulating tumor cell analysis in

cancer management. Genome Biol, 15(8), 448.

Karabacak, N. M., Spuhler, P. S., Fachin, F., Lim, E. J.,

Pai, V., Ozkumur, E., Toner, M., 2014. Microfluidic,

marker-free isolation of circulating tumor cells from

blood samples. Nature protocols, 9(3), 694-710.

Alix-Panabières, C., Pantel, K., 2014. Technologies for

detection of circulating tumor cells: facts and vision.

Lab on a Chip, 14(1), 57-62

Jin, C., McFaul, S. M., Duffy, S. P., Deng, X., Tavassoli,

P., Black, P. C., Ma, H., 2014. Technologies for label-

free separation of circulating tumor cells: from

historical foundations to recent developments. Lab on

a Chip, 14(1), 32-44.

Hajba, L., Guttman, A., 2014. Circulating tumor-cell

detection and capture using microfluidic devices.

TrAC Trends in Analytical Chemistry, 59, 9-16.

Park, H., Kim, D., Yun, K.S., 2010. Single-cell

manipulation on microfluidic chip by dielectrophoretic

actuation and impedance detection. Sensors and

Actuators B: Chemical, 150(1), 167-173.

Anh-Nguyen, T., Tiberius, B., Pliquett, U., & Urban, G.

A., 2016. An impedance biosensor for monitoring

cancer cell attachment, spreading and drug-induced

apoptosis. Sensors and Actuators A: Physical, 241,

231-237.

Dastider, S. G., Barizuddin, S., Yuksek, N., Dweik, M., &

Almasri, M., 2016. Impedance biosensor for rapid

detection of low concentration of E. coli 0157: H7.

In 2016 IEEE 29th International Conference on Micro

Electro Mechanical Systems (MEMS) (pp. 302-306).

IEEE.

Tan, W.H., Takeuchi, S., 2007. A trap-and-release

integrated microfluidic system for dynamic microarray

applications. Proceedings of the National Academy of

Sciences, 104(4), 1146-1151.

Zhou, Y., Basu, S., Laue, E., & Seshia, A. A., 2016.

Single cell studies of mouse embryonic stem cell

(mESC) differentiation by electrical impedance

measurements in a microfluidic device. Biosensors

and Bioelectronics, 81, 249-258.

Single Cell Array Impedance Analysis for Cell Detection and Classiﬁcation in a Microﬂuidic Device