NATIONAL CHUNG CHENG UNIVERSITY

COLLEGE OF ENGINEERING

Optimizing System Efficiency of an Open-Cathode Fuel Cell System

For unnamed UAV

By
Mr. Phuong-Long Le

A thesis submitted in partial fulfilment of the requirement

of National Chung Cheng University for degree of
Doctor of Science

Fuel cell laborary, Mechanical engineering, National Chung

Cheng University - Taiwan

September 2021
 Abstract

The open cathode PEM fuel cell OCPEMFCs is widely used like external DC
power source in industry energy with long-operation, low energy consumption and
high net power efficiency. Nertherless, A PEM fuel cell is not only have issue in
efficiency but the cost and duarability is criticle factor need a better understanding
application power demand. The main contribution of this study is to employ
experimental on Fuel cell stack H-100 to investigate the effect of hydrogen/oxygen
flow enhance good behavior of output performance to improve power propulsion for
annamed aircraft. To do this, the research study conducted two stage consists of faults
diagnosis and the system efficiency are proposed. In which the faults diagnosis low air
humidity, cathode flooding, low cell voltage and insufficiency hydrogen are
discussed, the system efficiency based on the cell voltage performance at stable
operation conditions with high efficiency and low auxiliary power consumption and
hydrogen ultilization. Under practicl working, the fuel cell operation at different fuel
cell load with the effect of. The impact of hydrogen/air, oxygen flow responds to
system efficiency was discusion:

1. The low air supply is caused fuel cell operation at high temperature with dry
membrane with low humidity and increased the membrane resistance because
insufficient water.

2. More air supply is caused water flooding blocked at the gas channel leading
to cell performance decreased due to fan’s duty cycle.

3. The optimization air flow rate under fan’s duty cycle was carried out to
discuss.

4. Too wet or too dry membrane is cause fuel cell degradation as cell voltage
performance reduce.

5. The insufficiency hydrogen sypply is caused current demand is drop due to

cell voltage decrease.

i
 6. Low hydrogen pressure is caused insufficient supplied hydrogen the sudden
current is effect to voltage respond.

7. High pressure is caused more hydrogen supply to the system leading to over
current and low cell voltage.

Keywords: OCPEMFCs, Flow rate hydrogen/oxygen, system efficiency, net

power efficiency, UAV.

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 Acknowledgements

First and foremost, I would like to express my sincerest gratitude to my

professor Yong-Song Chen who supported guidance in my research. Offering funding
for recomendation, full tuitiation scholarship and all research materials. Second, I am
also deeply thankful to D.r. Hien Lam-Thanh for their support to me, as well as, their
great motivation to undertake this research.

Over the course evaluating qualify examination, I would like to thank severals
professors: Professor Kua-Chun Pao (Inmediate thermodynamics and Engineering
analysis II), Professor Shyh-Leh Chen (Linear system) for their comment regarded to
my research. Special thanks are also due to all Professor related course in Mechanical
Department.

In addition, I would also like to thank the research group members from Fuel
Cell Laboratory for their their cooperation and technical support during experiment
test. Of course, I am grateful to the National Chung Cheng Universityand the Mold
Research Center, Chia-Yi, Taiwan, and all other administrative and academic staff
who supported completion of my study.

Finally, I want to express the deepest gratitude to my greatly family, my parents,

my brother, my young sister, and all my friends for their encouragements during my
study. Specialy, certainly to my wife and for her patience and take care my son during
the years of my study.

Sincerely,
Phuong-Long Le

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 Declaration

I would like to confirm that the research described in this thesis is my own work
an appropriate reference has not already been submitted for any other degree.

For copyright of this research. I certify that any quotation, reproduction of this
sources is not permit without accepted of authors and National Chung Cheng
University.

Taiwan, June 1st, 2019

Phuong-Long Le

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 Table of Contents

Abstract......................................................................................................................... i
Acknowledgements......................................................................................................ii
List of figures.............................................................................................................vii
List of tables..............................................................................................................viii
List of symbols and abbreviations...............................................................................ix
CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW..........................1
1.1. Background and literature review....................................................................1
1.2. Structure of a PEM Fuel cell...........................................................................1
1.2.1. Types and Applications of Fuel Cell............................................................3
1.3. Motivation of the research...............................................................................4
1.4. Objectives and aim of this thesis.....................................................................5
1.5. Scope-Format..................................................................................................6
1.6. Summary.........................................................................................................7

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 List of figures

Figure 1.1. Illustration of the metastasis development via CTCs. CTCs are detached
from a primary tumor and can enter into the bloodstream [33].....................................3

vi
 List of tables

Table 1.1: Dimensions and electric properties of red blood cell (RBC) and lung
carcinoma cell (A549) in the 8.62 wt% sucrose medium with dielectric permittivity
and conductivity be 78 and 1.76×10−3 S/m, respectively [40], [42]..............................8

vii
List of symbols and abbreviations

viii
 CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW

1.1. Background and literature review

In 1800, William Nicholson and Anthony Carlisle has been described the
process of using electricity to break water into hydrogen and oxygen. The fuel cell
system is a divice which converts energy in the hydrogen fuel into direct current.
Originals of fuel cell has been demonstrated by William Grove in 1839. Until 1889,
the prototype fuel cell has been performed various fuel cell experiments. In 1893, F.
Ostwald describes roles of fuel cell components. Willam Jacques constructed a carbon
battery in 1896. Early 1900, Emil Baur and studentsconducted experiments on high
temperature devices. 1939 Francis Bcon built the Alkaline Feul Cell. In 1940
O.Davtyan conducted electrolyte experiments (Molten carbonate and Solid oxide fuel
cells. In 1950, J.Broers and J.Ketelaar conducted MCFC research and K.Kordesch
created AFC at union Crbide. In 1959, AFC demonstration with Alilis-Chambers farm
tractor. In 1960. T.Grubb and L Niedrach invented PEMFC technology at general
electric. Texas instruments developed prototype MCFC technology. NASA uses
AFCs on Apollo spacecraft. In 1990 current worldwide extensive fuel cell research on
all fuel cell types.

The lack of advanced manufacturing technologies and high cost of catalyst

material such as platinum, avaible fossil fuel, have limited the interest in fuel cells as
auxiliary source of power. Consequently, commercial uptake in the technology has
been low. But, with recent developments in manufacturing technologies of electrolyte
membranes, electrode materials, effective use of catalyst materials, and with advances
in solid state power electronics and control systems, these factors have rejuvenated the
interest in the use of fuel cells as a prime source of power [2].

The increasing demand for electrical energy has resulted in increased production
which in turn has increased harmful emissions. Natural reserves of fossil fuels are
being depleted at an accelerated rate. Awareness of global warming, and the need for
alternative sources of energy that are reliable, safe and environmentally clean with
low cost has led to accelerated research [3, 4].

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 The efficiency of energy conversion-production for a fuel cell is comparatively
higher than combustion engines, both internal and gas turbines, because there is no
intermediate thermal conversion process that converts heat energy to mechanical
motion, which is ultimately used to drive electrical generators [5].

The pollutants such as carbon dioxide, nitrous oxides, and sulphur dioxide which
are produced from combusting hydrocarbon fuels are eliminated when using fuel
cells. Also, the absence of the intermediate mechanical conversion process makes a
fuel cell a quiet device [3].

Compared to conventional storage elements such as lead acid, and some other
forms of batteries, fuel cells produce higher energy density. In practical applications,
batteries need to be charged, and this process is time consuming. In contrast,
recharging of the fuel cell reactants, namely oxygen and hydrogen, is a quick process
[2]. Fuel cells are deployed in many industries such as transportation, aviation, navy,
microelectronics, and combined heat and power (CHP) applications, and their
applicability in diverse application areas is on the increase [6]. However, fuel cells are
characterised as highly energy dense devises, but with low power density, therefore a
heavy and large size of stack is required to fulfil an application with a high power
demand [7]. Furthermore, fuel cells respond slowly to sudden changes in power
demands which limit their dynamic performance.

The first use of fuel cells as an auxiliary power source device was in the 1960s.
The device was used in the Gemini space flights programme directed by NASA. In
particular, PEM fuel cells were developed and deployed for this programme [16].
Early fuel cells proposed to produce electricity as an alternative technology for the
electric aircraft were suggested in 1974, where the configuration employed fuel cells
and batteries for driving the motors of propellers [21].

The first fuel cell powered aircraft was built and tested by AeroVironment, Inc.
in 2003, using PEM fuel cells run by hydrogen extracted from sodium borohydride. It
had a very short flying endurance of less than 15 minutes. Then, in 2005,
AeroVironment, Inc. demonstrated a second version of UAV using PEM fuel cells
operated by liquefied hydrogen; this had an endurance of about 2.4 hours. A solid

2
oxide fuel cell powered UAV using propane fuel was constructed in 2006 by
Advanced Materials, Inc. with a flying endurance of about 4 hours. However, since
2003, a significant increase has been observed in the research and investigations into
the use of fuel cells in aviation [22, 24]. In the literature, the acronym UAV is an
abbreviation for unmanned aircraft/aerospace/aerial vehicles. However, the aviation
industry has now adopted UAS rather than UAV as a preferred acronym for
unmanned aerial/aircraft systems, “as UAS encompasses all aspects of deploying
these aircraft and not just the platform itself” [25].

1.2. Structure of a PEM Fuel cell

A PEM fuel cell is an electrochemical device where the hydrogen is fed to the
anode and, air/oxygen is fed to the cathode. in the most element sate, the fuel cell
consists mainly of one electrolyte and two electrodes. But practically a fuel cell may
consist of more than these components in order to increasesits efficiency and output
power, which makes the fuel cells commercially viable. A schematic structure of
single PEM fuel cell is shown as Fig x. It consists of a membrane electrode assembly
(MEA) sandwiched by two parallel flow field plates called bipolar plates, which serve
as current collectors and to connect cell in series for greater stack voltage. The MEA
consists of three players, (1) polymer electrolyte membrane layer (i.e. proton
exchange membrane), (2) catalyst layer and, (3) gas diffusion layer (GDL). The
electrolyte is a proton conduction polymer, bounded by a catalyzed porous electrode
on each side. The anode, electrolyte, and cathode are assembled as one very thin
piece. Usually, these components are manufactured individually and then pressed
together at high temperature and pressure.

Figure 1.1. Schematic diagram of a single PEM fuel cell

The electrolyte plays an important role in a PEM fuel cell, as it conducts protons from
the anodic electrode catalyst layer to the cathode one while blocking electrons. The
electrical ohmic resistance propertices and the protonic conductivity of the membrane
has a great impact on the voltage and perfoamce of a PEM fuel cell [].

3
As a result of continuos development, in the late 1960s, a new polymer membrane
Nafion became a standard electrolyte membrane for a PEM fuel cell []. Development
of catalysts, membrane electrolyte assembly (MEA) components, and bipolar plates
are vital for overcoming the concerns of cost and durability. Materials with high
degradation and corrosion resistantce and low platinum loading are essential to
achieve lifetime nd cost target. Improment an optimization of the materials used in the
gas diffusion layer (GDL) and gas flow channel (GFC) interface are also important, so
as to provide efficient water removal and flow of gases and to avoid flow
misdistribution, hence achieving and maintaining high fuel cell performance []. Fig
1.2 shows different sizes of H-series 100W-5kW PEM fuel cell stacks produced by
Horizon Fuel Cell Technologies.

Figure 1.2. Different sizes of H-series 100W-5KW PEM fuel cell stacks produced by Horizon
Fuel Cell Technologies

1.1.1. Types and Applications of Fuel Cell

According to the type of the electrolyte that has been deloyed, the reactants, and
temperature has been separated six types of fuel cell. PEMFC, DMFC, SOFC, AFC,
MCFC, PAFC.

The proton exchange membrane (PEM) fuel cell, also know as a polymer
electrolyte membrane fuel cell requires a high purity of hydrogen fuel and operates at
low temperature range between 30 – 100 degree. Hydrogen fuel is processed at the
anode where electrons are separated from protons on the surface of the platinum-
based catalyst. The protons pass through the membrane to the cathode side of the cell
while electrons travel in the external circuit, generating the electrical output of the
cell. On the cathode side, onother precious metal electrode combines the protons and
electrons with oxygen to produce water, which is expelled as the only waste product;
oxygen can be provided in a purified form, or extracted at the electrode directly from
the air.

4
 A variant of the PEMFC which operates at elevated temperatures is known as
the high temperature PEMFC (HT PEMFC). By changing the electrolyte from being
water-based to a mineral acid-based system, HT PEMFCs can operate up to 200
degrees Celsius. This overcomes some of the current limitations with regard to fuel
purity with HT PEMFCs able to process reformate containing small quantities of
Carbon Monoxide (CO). The balance of plant can also be simplified through
elimination of the humidifier

One of the alternative solutions to replacing the use of pure hydrogen in the
PEM fuel cell is to supply the fuel cell directly with liquid menthol, such a fuel cell is
called a direct menthol uel cell (DMFC), where the hydrogen is extracted from
menthol. It is similar to the PEM cell in that it uses a polymer membrane as an
electrolyte. However, the platinum-ruthenium catalyst on the DMFC anode is able to
draw the hydrogen from liquid methanol, eliminating the need for a fuel reformer.
Therefore, pure methanol can be used as fuel, hence the name.

The problem of a slow reaction rate is avoided by using an alkaline fuel cell
(AFC) which has the ability to operate at high pressures and temperatures of about
200 degree, AFC requires pure oxygen and hydrogen [].

The phosphoric acid fuel cell (PAFC) operates at a fairly high temperature 220
degree, which results in an increased reaction rate. The issue of hydrogen fueling is
solved by reorming natural gas (mostly methane) to hydrogen and carbon dioxide, but
this process adds more cost, size and complexity to the fuel cell. PAFC can operate
for long periods with very minor maintance and high reliability, with an opportunity
to be integrated to the combined heat and power (CHP) system [].

Molten carbonate fuel cell (MCFC) operate at temperatures of around 650 degree and
require carbon dioxide in the air in order to operate. While solid oxide fuel cells
(SOFC) operate at temperature levels of 500-1000 °C and can be supplied with low
purity hydrogen (i.e. hydrogen containing carbon monoxide). These high temperatures
offer high reaction rates without the need to use an expensive catalyst. Hydrocarbon
fuel such as propane, kerosene, methane, and coal gas can be used after a reforming
process. Reforming and filtering processes work to break the bonds between the

5
molecules of hydrocarbon fuel in order to separate hydrogen molecules from carbon
monoxide and carbon dioxide molecules in order to produce the desired purity level of
hydrogen. However, this technique introduces extra costs, complexity, weight and
volume to the system. Moreover, as they operate at very high temperatures, the costs
of heat tolerant materials and the associated cooling system costs make the system
very expensive and complex [1, 10]. PEM fuel cells and SOFCs are currently
attracting the greatest interest and development. SOFCs offer higher overall system
efficiency but lower power density and lower load dynamic responses in comparison
with PEM fuel cells. SOFCs take a longer time to reach high operational
temperatures, which make these more convenient for stationary power generation
applications [10]. When compared with other types of fuel cells, PEM fuel cells have
several features, such as high power density, high conversion efficiency, long
operational life hours, low corrosion, low weight and compact size, low operational
temperature, and faster transient response, which makes PEM fuel cells suitable for
transport and can also be used in stationary applications. Therefore, research and
development are on-going in academia and industry in order to produce commercially
viable PEM fuel cell systems for different applications [11-14].

Despite increased efforts to develop PEM fuel cells technology, durability and cost

remain the major barriers against PEM fuel cell commercialisation and exploitation.
The target of the US Department of Energy toward fabrication cost and operational
lifetime of a PEM fuel cell for transportation applications is $30/kW and 5000 hours
by 2015, in order to compete effectively with the conventional technology of internal
combustion engines [15].

Fuel cell classification.

Characteristic Polymer Phosphoric Molen
Alkaline Solid oxide
s electrolyte acid carbonate
FC operating
40-80 65-220 205 650 600-1000
temperature
Hydrated Immobilize
Mobilized or Immobilize
polymeric d liquid
immobilizedpotassi d liquid Perovskites
Electrolyte ion molten
um hydroxide in phosphoric (ceramics)
exchange carbonate in
asbestos matrix acid in SIC
membrane LiAIO2
Perovskite and
Nickel and
Electrodes Carbon Platinum Carbon Perovskite/metal
nickel oxide
cermet
6
 Electrode Electrode
Catalyst Platinum Platinum Platinum
material material
Stainless
Carmon or Nikel, Ceramic
Interconnect Metal Graphite steel or
metal or steel
nickel
Charge carrier H+ OH- H+ CO3+ O-

Fuel cell reactions

Fuel cell kind Reaction at the anode Reaction at cathode
Polymer electrolyte and 
H 2  2 H  2e 
O2  4 H   4e   2 H 2 0
phosphoric acid
Alkaline H 2  2OH   2 H 2O  2e  O2  2 H 2O  4e   4OH 
Mollten carbonate H 2  CO32  H 2O  CO2  2e  O2  CO2  4e  2CO32
CO  CO32  2CO2  2e 
Solid oxide H 2  O 2  H 2O  2e  O2  4e   2O 2
CO  O 2  CO2  2e 
CH 4  4O 2  2 H 2O  CO2  8e 

1.3. Motivation of the research

The investigation based on system efficiency and fault diagnosis to enhance fuel
cell protection during current demand for system stable. In order to reduce auxiliary
power consumption for the fuel cell system consists of MCU controller and fan power
consumption. The MCU controller has made by fuel cell lab which applied to
implement serial experiment to test the system efficiency and fuel cell performance.
The output perormace has been compaired with buil-in controller to determine
auxiliary power consumption and amount of hydrogen consumption. In order to
supply sufficient hydrogen and oxygen to PEM fuel cell when operating at different
fuel cell load under practical working. The fault diagnosis was determined to improve
the system. the variation environment such as too hot or to dry air, air saturation, dư
thừa air, were directly effected to membrane leading to reduce membrane conductivity
as increased membrane resistant as increased voltage losses ohmic as the cell
performance drop. The supplied sufficient hydrogen is not only generating enough
current demand but also keep fuel cell operation stable. The insufficient hydrogen is
caused reduce Enert and increased voltage losses activation leading to cell voltage
performance drop. Both of theses are the main contribution of research project and the

7
application can be used for UAV project with high current when the take off and low
current consumption for landing. With purpose reducing the weight of the system
device and increasing the fuel cell performance the control strategy based faults
diagnosis and the system efficiency can be reached high improment in UAV project.

1.4. Objectives and aim of this thesis

The airm of this study is to develop strategy control based MCU controller lab
made and fauls diagnosis to improve power demand for UAV project. Reducing size
and weight of fuel cell system is critical factor to improve th supplied
hydrogen/oxygen, and propulsion system. the objectives can be summarized as
follows:

1. To carried out a critical literature review of the relevant published literature

review relating to using fuel cell as a power source for drone and UAV applications.
The strategy control output performance of fuel cell stack operation under variations
fuel cell loads.

2. To develop programing employed on MCU controller to conduct experiment

by MATLAb/Simulink.

3. The validate value is carried out by 100W PEM fuel cell stack (H-100)
developed by Horizon Fuel Cell Technologies. The experimental will be applied to
examine all of the operational and performance variables under various operational
conditions, and fuel cell losses are considered as well.

4. To investigate both of cathode and anode effect to fault diagnosis on the

operation and fuel cell performance.

5. To propose and examine a controller that can efficiently optimize and supply a
sufficient flow of hydrogen and air/oxygen to the PEM fuel cell.

6. To develop early faults detection to reduce weight, space, and combined

power demand to proposed control strategy with hydrogen and oxygen to improve
propulsion system of UAV project.

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1.5. Scope-Format

The optimal auxiliary power consumption and faults diagnosis to develop of PEM fuel
cell for AUV project will presented in Chapter Two of this thesis. In the chapter three,
the thesis will look the effect of air supply on cell voltage performance. Chapter four
will examine fault diagnosis, while chapter five will validate the combination between
system efficienct and faults diagnosis of the system. chapter six will discus oxygen
supply and hydrogen supply. Chapter seven presents the conclusion and discusses
furture work.

Matlab and Simulink are registered trademarks of The MathWorks, Inc. Matlab is a
high level language and interactive environment used by scientists and engineers for
technical computing integrates computation, programming, and visualisation where
problems and solutions are expressed in a familiar mathematical form. “Simulink is a
block diagram environment for multi-domain simulation and model-based design. It
supports system-level design, simulation, automatic code generation, and continuous
test and verification of embedded systems. It provides a graphical editor, customizable
block libraries, and solvers for modelling and simulating dynamic systems. Simulink
is integrated with Matlab, enabling to incorporate Matlab algorithms into models and
export simulation results to Matlab for further analysis” [30].

LabVIEW program is a registered trademarks of National Instruments and is

described as a virtual instruments software because their operation and appearance
and imitate sort of physical instruments, such as multi-meters and oscilloscopes.
LabVIEW consist of a set of comprehensive tools for acquiring, displaying, storing,
and analysing data, as well as tools to help the user to troubleshoot the codes.

1.6. Summary

This chapter presented historical overview relating to the use and development
of fuel cells. A summary of the major six types of fuel cells and their applications in
different fields was demonstrated. The structure of the PEM fuel cell and background
regarding the use and development of electrical aircraft was presented. Fuel cell
powered aircraft are generally characterized by a low power to weight ratio (W/kg).

9
the propulsion system of of an unmanned aircraft needs a large range of power and
fast response to fulfil the requirements of different flight phases and to balance the
variations in the load demand. PEM fuel cells suffer from limited power density and
slow dynamic responses when sudden change in power demand is presented.

A stable and robust controller and fuel supply system that can provide fast and
sufficient flow of hydrogen and air/oxygen to the reaction of the fuel cell is one of the
critical objectives.

In the next chapter, the literature relating to many developed models of PEM
fuel cells, different techniques of managing and controlling the output power of a fuel
cell stack, and managing the operational variables of PEM fuel cells under steady and
transient states of load variations will be reviewed and presented. Also, the
deployment of fuel cells as primary or auxiliary power sources for UAV applications
will be presented and critically discussed.

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 Chapter 2. DISTINGUISHING CYTOLOGICAL STAGES OF
ESOPHAGEAL CARCINOMA CELLS BY DIELECTROPHORETIC-
BASED IMPEDANCE MEASUREMENT METHOD IN THE
MICROFLUIDIC CHAMBER

2.1. Introduction

In recent years, esophageal cancer is one of the six highest causes of cancer
death worldwide, and it is the eighth most common cancer [119]. Esophageal
squamous cell carcinoma (ESCC) and adenocarcinoma (ADC) are two main
histological types in high incidence areas in the world. In which, ESCC is a typical
histological type of cancer lesion in Asia, Africa, and America [120]. A combination
of surgical biopsy, radiology, and pathological assessment of tissue samples can be
utilized to diagnose esophageal cancer. However, the mortality rate of ESCC still
remains high due to the lack of reliable diagnosis methods in primary cancer
metastasis [121]. Thus, early detection of a cancerous cell is natural desirability to
reduce mortality among patients with ESCC.

Several works have been explored to identify the different carcinoma cell lines,
or between normal cells and cancer cells [122], [123]. Numerous current typical
methods required high costs, surface biomarkers labeling complexity, and time-
consuming. Non-invasive and label-free approaches have recently been attracted as an
alternative technique. Among them, electrical impedance monitoring microfluidic
devices have been developed for distinguishing poorly and highly stages of metastatic
cancer cells, or between them with the normal cells. Using an electrical cell-substrate
impedance measurement system, the stages of squamous cell carcinoma cells could be
discriminated by the difference in the impedance-based cell index, and its change rate
[123]. However, most of these devices still demand the culture conditions for cell
spreading, the rather long time. In this chapter, the design of a rapid, label-free, and
sensitive microchip using the DEPIM technique within the interdigitated
microelectrodes array is presented. Two ESCC lines are investigated, including
CE81T and CE81T-4. Therefore, these two cell strains are distinguished by simply
differentiating the conductance or admittance properties of the sensing electrodes, as

11
well as, by the difference of the slopes of the curves between the admittance variation
and the number of cells. The obtained results demonstrated the potential of our chip
design as a useful analytical method for cancer research.

2.2. Microchip design and fabrication

1.1.2. Chip design and operation

Figure 2.1. Schematic diagram of the proposed microfluidic device for DEP-based cell
concentration and impedance-based cell identification: (a) Illustration of the chip design
with the material layers, including a glass substrate, an ITO interdigitated microelectrode
array, and a PDMS chamber; (b) The top view of the electrode structure and its dimensions.

An illustration of the microfluidic device and its dimensions are shown in

Fig.2.1. The structure has three main parts: an interdigitated electrodes (IDE) array, a
glass substrate, and a cell sample chamber. The design of microelectrodes in circle-
on-line shape is employed [73], [116], [122]. The IDE array consists of 20
microelectrodes with the length of each electrode finger be 6 mm. The detail
dimensions are shown in Table.2.1. The chip operation includes two main steps, the
concentration of cells into the electrodes by DEP manipulation, and recognition of the
cell line by measuring the electrical impedance of the chip. Under a sinusoidal
excitation signal at a high frequency, pDEP force is generated on each cell particle in
the suspension medium. Under the influence of DEP response, the cells can be pulled
to higher gradient areas of the electric field. As a result, the cells are collected around
the tips of the microelectrodes. After concentrating cell, the impedance-based
measurements within the same device are performed to identify the stage of the cell
sample in the chamber.

Table 2.1: Geometry parameters of the DEPIM microfluidic chip

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1.1.3. Fabrication of the microchip

The microchip was fabricated via the conventional photolithography and lift-off
process, etching, casting, and molding techniques (see in Fig.2.2 a-c). The electrodes
were displayed by wet etching an indium tin oxide (ITO) glass substrate using a
hydrochloric acid solution. First, the ITO glass side was cleaned with acetone,
methanol, and deionized water. Subsequently, the photomask was aligned on the
substrate surface coated by a layer of S1813/ S1818 positive photoresist. Following a
process of exposure photography, developing, baking, and etching, the proposed
electrode configuration was generated on the glass substrate. A biocompatible
material, polydimethylsiloxane, was adapted to fabricate the open-top microlitter
chamber. The volume of the chamber was about 50 µL. After the PDMS prepolymer
mixture (by mixing PDMS monomers and a curing agent in a 10:1 ratio) was cured
and peeled off, the chamber was created using a puncher. The chamber was
subsequently bonded to the ITO-glass substrate after the treatment in an oxygen
plasma cleaner. Fig.2.2(d) shows the image of a fabricated chip after the
microfabrication process.

(a)

(b)

(c)

(d)
Figure 2.2. The standard photolithography processes for chip fabrication: (a) ITO
microelectrodes patterned onto a glass substrate; (b) SU-8 mold; (c) PDMS chamber

13
generation; (d) A fabricated chip after bonding PDMS chamber onto the ITO-glass
substrate.

2.3. Cell sample preparation

Two esophageal cancerous cell lines (CE81T, and CE81T-4 cells) were cultured
to carry-out the experiments of the microchip. The cells were grown in Dulbecco’s
modified Eagle’s medium, supplemented with NaHCO 3 of 45 mM, 10% fetal bovine
serum and 1% penicillin/streptomycin. The cell culture dishes were incubated in an
incubator with a humidified atmosphere containing 5% carbon dioxide at a
temperature of 37°C; the medium was replaced every 1 to 3 days. Cells grown to sub-
confluence were washed with phosphate-buffered saline and harvested following a 5-
min treatment of 0.02% ethylenediaminetetraacetic acid and 0.25% trypsin. The cells
were then prepared in an 8.62 wt% sucrose solution. The conductivity of the medium
solution is measured about 1.76×10-2 S/m. For the DEP-based cell operation, the
sucrose medium was utilized to go up the osmolarity to normal physiological levels.
Fig.2.3 shows the images examined under a microscope of two esophageal cancer
cells types.

Figure 2.3. Microscopic images of two cell samples corresponding to different stages of
human esophageal cancer cells.

2.4. Apparatus

The experimental system for the microchip is schematically described in Fig.2.4.

The electrodes were driven by a signal generator (Agilent 33220A) that provide
sinusoidal excitations in a large frequency and amplitude range. A voltage signal of 5
Vpp with a frequency of 1 MHz between the electrodes was applied for DEP-based cell
manipulation in the microchamber. Therefore, the pDEP response appeared on each
cell suspended in the medium. A specific cell number was dropped into the chamber
of the chip for each investigation time. The cell concentration was monitored and
captured using a microscope with a mounted CCD camera that was connected to a
computer running Olympus DP Controller image software. After capturing the cells
by the DEP force, the impedance of the chip was then measured and recorded. A
voltage signal from the waveform generator with an amplitude of 1 Vpp and a
14
frequency of 4 kHz [124], [125] was used to conduct the impedance measurement.
The output signal of the device was collected through a data acquisition NI USB card.
The data were transferred to the computer and were then processed using LabVIEW
software to determine the alteration in the impedance or the admittance of the cell
samples in the working chamber.

Figure 2.4. Schematic diagram of the experimental system for the dielectrophoretic
impedance measurement device.

2.5. Results and discussion

In experiments, esophageal cell samples were evaluated with various cell

numbers in a range from 1400 to 11200 cells, separately. At each time, the DEP
application was examined for ten minutes. The cells appeared in the medium chamber
were driven by the pDEP force, and then magnetized into the tips of the
microelectrodes. Thus, the DEP implement could increase the sensitivity of the
sensing part because of the significant signal enhancement of the electrode-solution
interface impedance [13]. When the DEP manipulation was completed, the
microscopic images of cells on the electrodes were captured at different cell numbers
(see in Fig.2.5). The next, the capacitive and conductive components of the chip were
measured through the imaginary and the real parts of the measured impedance. The
admittance of the chip was then calculated from the basic formula. However, the
capacitive element was ignored because the imaginary value was very smaller too
much than the real value at the same cell number. As a result, the amplitude of the
admittance was approximately equal to the conductance value of the chip.

(a)

(b)

(c)

15
Figure 2.5. Distribution of the esophageal cancerous cells after applying DEP manipulation
at different numbers of cell: (a) CE81T cell samples; (b) CE81T-4 cell samples; and (c)
CE81T-4 cells at before and after DEP application for 10 minutes with the cell number of
8400 cells.

The initial chip that no cell and only the sucrose medium was injected in the
chamber, was assumed as the reference sample. In this case, the admittance was
recorded about 50±2 μS. The next, the admittance was recorded at each test
corresponding to a defined number of the cell. The admittance change was derived
from the variation of the admittance of the chip with the cell sample in comparison
with the reference sample. Fig.2.6 presents the graph of the admittance change with an
increase in cell concentration. The experimental results showed that the admittance is
higher with increasing the number of cells. Moreover, the variation of this parameter
increased linearly with cell number with the regression coefficient up to 0.99 in both
two esophageal cell lines. It can be seen a significant difference in the admittance
could be observed between the two cell lines. The slopes of the respective curves were
0.0129 and 0.0321 for the CE81T and CE81T-4 cells, respectively. This difference is
the result of cellular morphological changes resulting from alterations in the
cytoplasm conductivity, the cytoskeleton structure, and, specifically, the membrane
topography of cells [126]. The increase in cell dimension also results in a decrease in
cytoplasmic conductivity [127]. Differences in the membrane permeability, the
intracellular volume fraction, the concentration of ion species in the cytoplasm, and
the exit or entry levels of the ion into the cytoplasm are the reasons that lead to
differences in cytoplasmic conductivity. Furthermore, CE81T-4 cells (more invasive)
took a higher admittance than CE81T cells (less invasive) at the same cell number.
From Fig.2.5, CE81T-4 cells collected to the clusters more strongly than CE81T cells.
Therefore, these high linear characteristic lines could be used to differentiate the
ESCC cells at different stages herein. In this study, the microdevice indicated the
capability of discriminating the cytological stages based on the impedance-based
measurement, which requires a shorter time while the sensitivity still reached high
efficiency. Finally, the proposed design demonstrated that the DEPIM method could
provide a potential approach to recognize the stage of different cancerous cells.

16
Figure 2.6. Linear relationships between the calculated admittance change and the cell
number of the two ESCC cell lines. The curves indicated that the microdevice is possible to
distinguish different cytological stages of cells.

2.6. Conclussions

A microdevice that combines both cell concentration and identification of cells

was designed and fabricated, based on the DEPIM approach. The pDEP cells in
sucrose medium could be focused onto the gaps between the microelectrodes by the
applied signal of 5 Vpp and 1 MHz. Human esophageal cancerous cell lines at two
different cytological stages were successfully concentrated by DEP manipulation.
Subsequently, the electrical impedance measurements were performed in various cell
numbers of ESCC cells with the sinusoidal signal of 1 Vpp and 4 kHz. Using the
admittance change lines to recognize and distinguish CE81T and CE81T-4 esophageal
cell lines was also demonstrated. The microchip has potential as a label-free, rapid,
highly sensitive device for the detection of cancer. The proposed device can be
integrated in biological assays, clinical diagnostics, and biomedicine fields.

17
 CHAPTER 3. A BUILT-IN ELECTRONICS MODULE IN THE
DEPIM DEVICE FOR HUMAN LUNG CELL DETECTION AT
DIFFERENT STAGES

3.1. Introduction

Impedance-based measurement fluidic devices have been developed in some

researches for characterization, identification, and detection of biological cells [14].
Several studies have been conducted to recognize the different stages of cancerous
cells, like as breast [128], bladder [129], oral [116], [122], [123], ovarian [126],
prostate [130], urothelial [131], and lung cancer cells [22]. In which, lung cancer is
estimated with about 1.8 million new cases a year, accounting for over 10% of total
cancer diagnoses [119]. This work reports the simple and rapid DEPIM chip given in
the previous chapter for human lung cell line identification. The microdevice was
revealed to be easy in fabrication, simple in cell concentration, and quick in
conducting time. Cell suspended in the sucrose solution is first dropped into the
chamber with a microliter volume. The electrodes are then excited by the potential at
high frequency for pDEP application to collect the cells to the clusters around the tips
of the microelectrodes. The electrical impedance parameters of the microchip are
successively sensed. The DEP effect leads to faster recruitment of cells in the electric
field, which increases the conductivity of the sensing materials between the electrodes
during the measurement.

Signal generator, impedance analyzer and conventional measurement instrument

are really large, cumbersome, inexpensive, and thus limited in practical applicability
[4], [130], [132], [133]. Development of the smaller experimental equipments are
interested in our study [115], [134]. In this present work, a hand-held electronics
module that based on a high sensitivity lock-in amplification technique via analog
integrated devices, is designed and built for the DEPIM chip. Two human lung cell
lines, A549 (lung carcinoma cells), and MRC-5 (normal cells) are explored in the
experiments. A comparison between the data from the conventional equipment system
and the data from the PCB module are performed. The results indicate that the
developed module is possible and reliable to response for both DEP manipulating and

18
impedance measuring of the microchip. In addition, the lung cancer cells can be
distinguished with the lung normal cells due to the slope difference of the impedance-
based characteristics.

3.2. Microchip design and equivalent electrical circuit model

The microchip design and its primary parameters are described in the previous
chapter. Fig.3.1 shows the main steps in the fabrication and experiment process of the
device.

Figure 3.1. Block diagram of the experimental process for the proposed DEPIM chip.

A simplified equivalent electrical circuit model (see in Fig.3.2) for the impedance
sensing electrode design is used in this study [135]–[137]. The model is composed of the
medium resistance Rs and capacitance Cs, the electrode-cell solution interface impedance
(Zsc), and the parasitic resistance Rp. Zsc consist of the cell sample solution resistance Rsc, and
the double layer capacitances Cint. The parasitic resistance is often related to the series
resistors of the linking wires, while the other electrical elements depend on the shape and
dimension of the electrode structure, the permittivity and conductivity coefficients of the cell
type, and the medium solution in the chamber. The total impedance Z of the chip can be
given as:

1
Z  j   2 R p 
 1 1  (3.1)
 jCs   
 Rs Z sc 

where

2
Z sc   Rsc (3.2)
jCint

with ω is the frequency, and j is the imaginary unit ( j  1 ).

Figure 3.2. Electrical equivalent circuit model of the sensing electrodes of the microchip.

Changes in the cell number and the solution medium lead to alterations in Rs, Cs,
Rsc, and Cint of the equivalent model. As we have known the impedance magnitude
decreases with increasing frequency in a low frequency range due to the improvement

19
of the interface impedance parameters [13]. The total impedance Z mainly depends on

the Zsc, and it can be thus approximately as Z  Z sc [135]. The admittance that is
derived as the inverse of the impedance:

1 jCint  2Cint2 Rsc  j 2Cint

Y    G  jX (3.3)
Z 2  jCint Rsc 4   2Cint2
Rsc2

in which, conductance G and reactance X are the real part and the imaginary part of
the admittance, respectively. The conductance, reactance, admittance and impedance
properties of the chip can be measured to confirm the appearance of cells.

3.3. Experimental system and cell sample preparation

A549 lung carcinoma cells were cultured in Dulbecco’s modified Eagle’s

medium (DMEM), supplemented with 3.7 g of NaHCO 3 per liter of medium, 1%
penicillin/streptomycin, and 10% fetal bovine serum. MRC-5 lung epithelial cells
were cultured in minimum essential medium (MEM), supplemented with 2.2 g of
NaHCO3 per liter of medium, 1% penicillin/streptomycin, and 10% fetal bovine
serum. Culture dishes were incubated in the standard incubator, and the medium was
changed every 1 to 3 days. Before conducting the experiments, the cells were washed
and mixed in the 8.62 wt% sucrose solution. The cell concentration was counted using
a hemocytometer with two counting grids under a microscope. Fig.3.3 shows the
microscopic images of the two different lung cell lines.

Figure 3.3. Microscopic images of two different human lung cell types, including A549 lung
carcinoma cells (cancer cells) and MRC-5 lung epithelial cells (normal cells).

A high precision experimental system is shown in Fig.3.4. DEP-based cell

concentration was carried out by a waveform generator (Agilent 33220A). An
impedance analyzer (Wayne Kerr 6420) connected the electrodes of the microchip via
BNC cables was used for electrical impedance spectroscopy measurements. A cell
sample could be captured using a microscope (CKX41 or BX43) with a mounted
CCD camera (DP71) connected to a computer running Olympus DP Controller image
software. The measured signals of the impedance analyzer were collected and
transferred to a computer through a data acquisition card (GPIB-USB-HS). The data

20
were then analyzed by LabVIEW tool to determine the changes in the impedance or
the admittance of the cell samples. The impedance spectroscopy measurements on the
microchip were first carried out using the impedance analyzer. Subsequently, the
corresponding experiments were repeated using the built-in electronics module
instead of the types of cumbersome equipment.

Figure 3.4. Block diagram of the high precision conventional experiment system.

3.4. Electronic circuit board design

A printed circuit board (PCB) module was designed and manufactured using
small and cheap electronics components (see in Table 3.1). The module with the
dimensions of 8 cm × 12 cm was built. A DC power supply of ±12 V was used for all
elements on the board. Block diagram and layout of the PCB module are illustrated in
Fig.3.5(a-b). A fabricated PCB module is shown in Fig.3.5(c). The circuit comprises
two main parts: a sinusoidal signal generator and an impedance detector. A signal
generator chip (ICL8038) and a pre-amplifier (AD817) were employed to create
excitation sources for both DEP driving and IM sensing. The IC ICL8038 chip allows
for the creation of the voltage signals in a wide frequency range.

Table 3.2: The main electronic components of the PCB module

21
 The current PCB module was designed to form possibly the sinusoidal waves within
the frequency range from 1 to 200 kHz. As we have known that the Clausius-Mossotti factors
for biological cells are the same as the frequencies from 50 kHz to 50 MHz [115]. Thus, a
signal wave of 5 Vpp and 100 kHz could be utilized for DEP manipulation. The efficiency of
the cell concentration process was similar to the results of the waveform generator machine.
For the impedance measurement, cell membrane breakdown may appear at a high voltage
[4], [138], [139], so the IC AD817 chip was used to decrease the operating potential. Two
electrodes of the microfluidic chip were connected to the V1 and V2 pins of the readout
board. The signal current developed across the microchip flows into the V1 pin and created a
voltage signal at the V2 pin via an appropriate shunt resistor R. The impedance detector part,
which consists of a high-accuracy instrumentation amplifier (IC AD620) and a lock-in
amplifier combined with a low-pass filter (ICs AD630 and TL082), was then sensed. A
switch could be used to choose the modes (the imaginary or real part of the complex
impedance). The output voltage signal from the PCB model was recorded to calculate the
admittance or the impedance of the cell sample. The data were displayed on an LCD screen
through the UNO Arduino board, or transferred to a computer via the NI-USB card and the
jt
LabVIEW toolkit. It assumed that an input signal Vi e was set from the wave generator part
of the module. The output signal of the impedance measurement obtained through the filter

and amplifiers was V0e 

j t  
. In which, V0 and φ depend on the selected switching mode.

The measured impedance of the chip could be calculated by:

 Vi e j 
Z  RG  1 (3.4)
 V0 

22
where G is the total gain factor of the amplifiers. The present module was tested by
the sample capacitors and resistors with the impedance magnitudes from 500 Ω to 1
MΩ. It indicated that the module was suitable for the DEPIM proposed chip.

(a)

(b)

(c)
Figure 3.5. Circuit board for the DEPIM chip. (a) Block diagram of basic parts. (b) Layout
the components on the PCB. (c) The fabricated module for DEPIM chip.

3.5. Results and discussion

1.1.1. Cell concentration by DEP

Figure 3.6. Distribution of MRC-5 and A549 cell samples before and after DEP application.

In this study, Lung A549 carcinoma cells and MRC-5 epithelial cells at different
cell numbers ranging from 1,400 to 11,200 cells were used. Each cell sample at a
specific cell concentration was prepared in sucrose solution and dropped into the
microchamber of the chip. For DEP manipulation herein, the IDE array was driven by
a sinusoidal potential 5 Vpp and 1 MHz for about 10 minutes. The pDEP forces have
occurred on cells in the suspension medium. The cells were moved into the electrodes,
they were connected together by electric field lines between adjacent microelectrodes.
Fig.3.6 shows the microscopy images of cell samples before and after the application
of DEP-based cell concentration.

1.1.2. Impedance spectroscopy measurement of the biochip

(a)

(b)

23
 (c)
Figure 3.7. Electrical impedance spectroscopy response of various lung A549 cell samples
in the DEPIM microchip using the impedance analyzer. (a) Impedance amplitude, (b) Phase
angle, (c) Bode plots of impedance spectra from the measured and simulated data in a
frequency range of 1-100 kHz for the two cases without and with A549 cells.

For the impedance measurement of the microfluidic chip, a small sinusoidal

excitation from the impedance analyzer with the voltage amplitude of 100 mV and the
frequencies from 1 to 100 kHz was first applied. We have seen that the measured
impedance reached a stable amplitude after the DEP manipulation period as for being
10 minutes, in all the cases at different cell numbers. Because almost cells were
settled to the bottom of the chamber, and then attached around the electrodes.
Impedance magnitude and phase angle were measured and recorded. Resistance and
reactance components of the impedance were also displayed. Fig.3.7 (a and b) show
the obtained results from the impedance analyzer as a function of frequency in various
A549 cell numbers. It is transparent that the total impedance magnitude decreased as
the frequency increased in the investigation frequency range. In addition, the phase
angle increased in the lower frequencies, then decreased in the other higher range.
Fig.3.7(c) shows the measured impedance and the simulated data for two cases, which
are no cell and with 11,200 A549 cells captured on the IDE array. Besides, the data
were in good agreement with the theoretical model. The values of components fitted
the above model are given in Table 3.2. When there were no cells in the chamber, the
obtained relative standard deviations of Rs, Cs, Rsc, and Cint were 2.182%, 0.257%,
5.185%, and 10.88%, respectively. Since the highest error of Cint, it can derive that the
electrode-solution interface capacitance changes were dominant in the impedance of
the chip. Therefore, the experimental results were proved to be reliable.

Table 3.3: Simulated values of the electric components in the equivalent impedance
circuit model for A549 lung cell line.

24
1.1.3. Detection the cytological stages of cell
In here, the impedance of the chip was continuously observed at a reliable
frequency of 4 kHz [124], [125], [140]. Both the impedance analyzer and the
electronics PCB module were employed for impedance measurement. The admittance
change between the electrode was calculated in each repeated experiment. Each
measured point is the average value of at least three separate measurements, and the
error bar expresses the standard error of the mean. The admittance alteration curves of
the chip with respect to the cell number is shown in Fig.3.8. A highly linear
relationships between the admittance variation and the number of cell were found,
with the regression of the lines being up to 99% for the both two lung cell lines (A549
and MRC-5). A significant difference in the curves of the two lung cell lines were
observed; the slopes were about 0.003 and 0.004 for the MRC-5 and A549 cells,
respectively. The divergence between the curves increased with increasing cell
number. At a given cell concentration, MRC-5 cells had a lower admittance than that
of A549 cells. It demonstrated that the DEPIM chip can discriminate lung cancerous
cells from lung normal cells. Moreover, the obtained data from the PCB module were
compared with the data from the conventional system. The results indicated a close
agreement between the two measurement systems. It appears that cancerous and
normal cells can be distinguished directly and quickly through the proposed
electronics module.

Figure 3.8. Admittance variation at the frequency of 4 kHz vesus the number of cell for two
human lung cell lines (A549 and MRC-5 cells), using the impedance analyzer and the
electronics module for the microchip, respectively.

3.6. Conclusions

The DEPIM microchip using the IDE array that combines cell collection and
detection within a chamber was effectively proven for identification of A549, and
MRC-5 human lung cells. An impedance analyzer was added in the experimental
system. Electrical impedance spectra of the chip with various cell numbers of each
25
cell line were observed in the frequency range from 1 to 100 kHz. A hand-held PCB
module was also designed and built for both driving DEP manipulation and sensing
electrical impedance. The measured impedance values from the PCB module were
consistent with the data from the impedance analyzer at the frequency of 4 kHz. The
electronics module is possible completely improved for applications with the lower
cell concentrations, as well as detection of rare tumor cells. Finally, the DEPIM
device is a potential tool for characterizing cancerous cell lines.

26
 Chapter 4. DEVELOPMENT OF A DIELECTROPHORESIS
ENRICHMENT MICROFLUIDIC PLATFORM WITH IMPEDANCE
DETECTION OF LUNG CARCINOMA CELLS IN THE CHANNEL

4.1. Introduction

Enrichment, separation, isolation, and purification of specific cancerous cells,

like as rare stem cells, and circulating tumor cells (CTCs) are essential for subsequent
detection in microfluidic devices [8], [29], [30]. Collection and enrichment
manipulation can raise CTCs concentration, thereby enhancing detection efficacy and
accuracy, as well as, providing a reliable diagnosis for cancer treatment [141]. CTCs
could be isolated from normal blood cells due to one or several cell properties, such as
shape, size, morphology, deformability, electrical mechanical or magnetic properties,
surface biomarkers, etc [7]. Dielectrophoresis (DEP) has become a potential
manipulation technique that has been expanded in microfluidic platforms for many
types of biomolecules, such as DNA, protein, virus, bacteria, and cell [10], [12],
[142]–[145]. A DEP-based device was often high throughput, inexpensive, simple to
fabricate, and good compatibility with the miniaturized systems [10], [142]. Several
DEP-based microchips have been developed and achieved high capture rates for
cancer cell separation and isolation. The microchips that based on the DEP
manipulation to move the target cells to the desired electrodes have been designed and
tested in many our previous works [39]–[41], [113]–[116], [134]. The numerical
simulation could be also performed to evaluate the performance of the design. The
pDEP-based cell concentration was applied by the excitation signals at a high
frequency within a range from 1 to 10 MHz, and a low potential within a range from 5
to 16 Vpp. Most of the cancerous cells were focused on the center of the working area.

After separation and enrichment process, several techniques can be employed to

identify CTCs in fluidic devices, including electroosmosis, mass spectrometry,
fluorescence, electromagnetic spectroscopy, or electrochemical spectroscopy [13],
[14], [61], [146]. Among them, the impedance-based detection method has emerged
as a non-invasive, and label-free approach. Electrical impedance spectroscopy (EIS) is
a powerful technique for living cell analysis. Impedance-based measurement has not

27
only monitored the cell numbers in medium, but also enabled to determine the electro-
mechanical and chemical properties of the cell line, that can provide information on
cytoplasm permittivity and conductivity, and membrane resistance and capacitance
[14], [12]. The combination of DEP operation and impedance-based measurement
(DEPIM) is an effective method for the improvement of cell capture efficiency and
detective sensitivity of the microchip [20], [21]. Cell pearl chains by the DEP
manipulation often lead to the enhancement of permittivity or conductivity parameters
of the media in contact with the electrodes. The conductance or admittance of the chip
has increased with increasing numbers of cells [116]. Therefore, different electrode
configurations using the DEPIM method with a chip have been proposed for
cancerous cell characterization and identification in our studies.

In this work, a rapid and simple microfluidic platform based on the DEPIM
method is built for A549 human lung CTC enrichment and detection. A549 line is a
typical cancerous cell type at the primary cancer stages [100]. Thus, early detection of
A549 CTC cells plays an important role for treatment of lung cancer. The device uses
the circular electrodes configuration to trap and focus target cells from the outside
electrodes to the inside sensing electrodes by the pDEP response. The electrical
impedance signal between the sensing electrodes is consequently improved. We also
design a differential electrodes structure to enhance detective sensitivity. The double
pairs of symmetrical sensing electrodes in the working region can take off the
common-mode signals due to the surrounding environment parameters. In our
measurements, the single type of A549 CTCs sample was employed to reliably
evaluate the sensing efficiency and the detection limit of target cells. Different cell
numbers at low concentration are investigated in a large frequency range by the
device. The differential analysis technique-integrated DEPIM method within a
microchip is revealed as a potential method for detection of rare cancerous cells.

4.2. Microchip design and operation

The configuration and operation diagram of the microfluidic platform is

illustrated in Fig.4.1. Target cell concentration and detection based on the DEPIM
method within a microchip is applied. The chip design consists of eighteen gold-on-
chromium circular electrodes, a SU8 protection layer, a PDMS channel, and a glass

28
substrate (see in Fig.4.1(a)). Its geometrical dimensions are shown in Table 4.1. The
outside blue electrodes in termed focusing electrodes are utilized for DEP
manipulation. Two pairs of symmetrical red electrodes at the central area in termed
sensing electrodes are applied for attracting and sensing cells. An insulation layer is
covered on the focusing electrodes to avoid cell adhesion and consequently enhance
the performance of DEP-based cell concentration. A simple straight PDMS
microchannel is placed on the working region of the electrodes. The electrodes can be
functionalized by selectively, and affinity probes such as an antibody for effective
capture of the desired cells [147].

Table 4.4: Geometry parameters of the DEPIM microfluidic platform

The working principle of the microfluidic device is represented in Fig.4.1(b-c).

The cell collection and detection of target CTC cells can be carried out within the
electrodes region of the device. First, a mixture sample including target and non-target
cells is injected into the microfluidic channel with a low-speed flow. A549 lung target
cells are illustrated by the yellow particles while non-target cells (like as RBCs) are
described by the green particles. All cells are expected to be captured into the two
outmost circular electrodes by a combination of pDEP and hydrodynamic drag forces.
An inward stepping electric field is subsequently generated due to switching the
electric field between the adjacent electrode pairs from the outermost to innermost in
the working region [115]. In the suspension medium, the cells exhibit the pDEP
effects at a stimulus signal of high frequency [39]. However, the corresponding
magnitude of the DEP force for CTC cells is much greater than that on RBC cells
under the same electric field distribution [134]. As a result, the CTCs are separated
from the RBCs, and moved toward the singsing electrodes in the central area.
Thereafter, the EIS of the two sensing electrodes are measured and recorded. The
29
measured impedance data are processed with the differential analysis techniques to
recognize the presence of CTCs.

Figure 4.1. Design and operation of the microfluidic device for the concentration and
detection of A549 lung CTC cells (a) Schematic of the microdevice. (b) When injecting cell
sample solution into the channel, almost cells are trapped at the outermost circular
electrodes by pDEP and drag forces. (c) Target cells are then isolated into one of the two
pairs sensing electrodes by DEP effect and the size-based difference in stepping electric
fields. Subsequently, EIS measurement is performed to detect the appearance of the CTCs.

4.3. Microdevice fabrication

The fabrication process of the microfluidic device is shown in Fig.4.2(a). First,

chromium (50 nm) and gold (100 nm) metal layers were sequentially deposited onto a
glass surface. Next, the substrate side was cleaned by immersing in piranha solution
(96% H2SO4: 30% H2O2 with a ratio of 3:1) for 30 min, rinsed with DI water, and
dried with nitrogen. The positive photoresist (S1813/ S1818, MicroChem) was spun
on the slide (first spread at 500 rpm for 10 s, then spun at 2000 rpm for 20 s, and soft-
baked at 90 °C for 4 min). The mask design was drawn in AutoCAD. Subsequently,
the substrate was exposed under UV light (90 mJ/cm 2) by using an exposure machine,
then underwent development for 10 s in developer mixture (1:5 MicroChem’s MP351
and DI water, respectively), and hard-baked at 120 °C for 90 s to define the etching
mask. Wet etching was then applied using Gold Etchant and Chromium Etchant
(Sigma-Aldrich) to form the electrode structure on the slide. The substrate was then
cleaned with isopropyl alcohol, acetone, methanol, and DI water. After nitrogen
purging and drying, a 2 µm-thick SU-8 2002 negative photoresist layer (first spread at
500 rpm for 20 s, then spun at 2000 rpm for 50 s, and soft-baked at 90 °C for 4 min)
was continuously deposited onto the substrate. Following a similar process of
alignment, exposuring, developing, and hard baking, the protective layer is generated
to cover the actuating electrodes.

The PDMS channel was fabricated by casting, and molding techniques. A silicon
wafer was cleaned with the piranha solution followed by deionized water rinsed and
dried prior to applying negative photoresist (SU-8 2050, MicroChem). The clean

30
wafer was coated with a layer of ∼25-40 μm-thick photoresist on the surface (first
spread at 500 rpm for 20 s, spun at 5000 rpm for 100 s, then baked at 65 °C for 15
min, and at 95 °C for 30 min). A SU-8 master mold with channel pattern was finally
obtained using the soft-lithography process (exposed with an exposure energy of 200
mJ/cm2, post-exposure baked at 65 °C for 5 min, then at 95 °C for 15 min, developed
for 5 min with MicroChem’s SU-8 developer, rinsed in isopropyl alcohol for 10 s,
dried with pressurized nitrogen, at the end hard-baked at 120°C for 30 min. The
PDMS prepolymer mixture was made by mixing PDMS monomers and a curing agent
in a 10:1 ratio. The mixture was poured and cured on the mold master for 2 hours at
75 °C to replicate the patterned structures. After the PDMS replica had been peeled
off, inlet and outlet ports of the channel were created by a puncher, and the PDMS
replica was bonded with glass substrate via treatment (P[O 2] = 500 mTorr, for 60 s) in
the oxygen plasma cleaner (model PDC-32G, Harrick Plasma). Fig.4.2(b) shows the
image of an fabricated microchip.

(a)

(b)
Figure 4.2. (a) Process of the microchip fabrication. (b) Image of a fabricated microchip.

4.4. Cell sample preparation

For the experiments, A549 human lung circulating tumor cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 3.7 g of NaHCO 3
per liter of medium, 1% penicillin/streptomycin, and 10% fetal bovine serum. The cell
culture dishes were incubated in an incubator with a humidified atmosphere
containing 5% carbon dioxide at a temperature of 37°C; the medium was replaced
every 1 to 3 days. Cells grown to sub-confluence were washed with PBS and
harvested following a 5-min treatment of 0.02% ethylenediaminetetraacetic acid and
0.25% trypsin. Before conducting the experiments, the cells were washed, and
suspended in an 8.62 wt% sucrose buffer with a measured conductivity of 1.76×10 -2
S/m. Prior to the experiment, cell concentration was determined using a
hemocytometer with two counting grids under a microscope. The viability of the cells
31
was assessed prior to use by trypan blue dye exclusion. In some experiments, A549
cells were stained using a standard fluorescence assay with calcein AM (Molecular
Probes, Eugene, OR, USA) before pumping into the working channel. Calcein AM is
a cell tracker fluorescent dye which is able to penetrate the cell membrane into the
cytosol and transform into a fluorescent form when it is hydrolyzed by esterase's
located inside cells. Because viable tumor cells were brightly fluorescent, the viability
of tumor cells was verified, and cell ratios, as well as concentration efficiencies, were
also counted. The fluorescence intensity was quantified using NIH ImageJ software.
The efficiencies of cellular concentration were evaluated by calculating the
fluorescence intensity of the whole electrodes region at the beginning and at the
sensing electrodes after finishing the DEP concentration. The previous reports
demonstrated that all the cells still recovered their viability after DEP concentrating in
our microchip. Because the applied electric field strength (approximate 5×10 5 V/m, 1
MHz) was lower than the threshold strength of cell lysis to maintain the viability of
cells.

4.5. Experimental setup

Figure 4.3. Experimental system for the DEPIM microfluidic platform.

A high precision experimental system used in investigations is shown in Fig.4.3.

The buffer solution with a fixed volume of the cell sample was injected into the
channel using a syringe pump (Model KDS 101). The flow rate was able to be
controlled from 0.1 to 100 µL/min. DEP-based cell concentration was carried out by
the suitable excitation signals from a waveform generator (Agilent 33220A) or the
electronics module in the previous chapter. The focusing electrodes were connected to
the output signal of the generator through the switches to control the stepping electric
fields. An impedance analyzer (Wayne Kerr 6420) connected the electrodes of the
microchip via BNC cables was used for electrical impedance spectroscopy
measurements. Cell sample and distribution were captured using a fluorescence
microscope (CKX41) with a mounted CCD camera (DP71) connected to a computer
running Olympus DP Controller image software. The measured signals of the
impedance analyzer were collected and transferred to a computer through a data

32
acquisition card (GPIB-USB-HS). The data were then analyzed by LabVIEW tool to
determine the changes in the impedance value of the sensing electrodes.

4.6. Theoretical model analysis

In the microfluidic channel, the cells follow the streamlines of fluid flow until
they approach the walls or trapping structures. Cell motion is generally determined by
the DEP force (FDEP), and the hydrodynamic drag force (Fdrag) [148]. Considering
Newton’s law, the differential equation describing cell trajectory is frequently
expressed as:

dv p (4.1)
FDEP  Fdrag  m p ,
dt
where mp and vp are the mass and velocity of the cell, respectively.

DEP is a type of electrokinetic effect that causes the movement of polarizable

particles placed in a non-uniform electric field [36]. When the electric field is
computed in the frequency domain, the dielectrophoretic force acting on a spherical
particle of radius rp, suspended in a liquid medium is given as [149]:

FDEP  2 rp2 m Re al  fCM   Erms , (4.2)

2

where Erms is the root-mean-square of the external electric field, p and m are the
represented characters of the particle and the medium, respectively, and fCM is called
the Clausius–Mossotti factor, which relates to the effective polarizability of a particle.
This factor depends on the complex dielectric properties of the particle and the
surrounding medium and on the frequency of the applied field.

The Fdrag can be defined by Stokes’ law:

Fdrag  6 rp  vm  v p  , (4.3)

where η is the dynamic viscosity of medium and vm is the fluid velocity.

In coplanar electrodes configuration, FDEP is locally constant, whereas the

influence of inertia is neglected. Cell-sensing yield depends on the ratio of FDEP/Fdrag
[148], [149]. Cells are trapped at the surface of electrodes calculated as follows:

FDEP / Fdrag  1 , (4.4)

33
 In impedance measurements applied for biological particles, a small alternating
source is connected to the electrodes of the microchip at a particular frequency f [14].
The complex impedance (Z) of the system is calculated as the quotient of the voltage-
time function and the resulting current-time function:

V0 sin(2 ft ) (4.5)
Z f    Z ' jZ ",
I 0 sin(2 ft   )
where V0 and I0 are the voltage and current signal amplitudes, t is the time. The
real (Z’) and imaginary (Z”) parts are the resistance and reactance of the impedance,
respectively. The amplitude (|Z|) and phase angle (θ) are determined using:

(4.6)
 Z '   Z " ,
2 2
Z 

 Z" (4.7)
  arctan   .
 Z'
For most cases, target cells are suspended in a medium or adhered to a substrate.
Resistance and capacitance are the typical components in the models of these
biosensors. The changes in resistive and capacitive parameters, which depend on the
electrode geometry and the properties of materials inside the electrical field between
the electrodes, lead to impedance alteration. Cell EIS is a common theoretical model
on impedance measurement that could be considered as a steady-state analysis
technique. Numerical simulations of the electric field were conducted using the
commercial software package CFD-ACE+ and COMSOL Multiphysics reported in our
previous literature [39], [147], [150].

4.7. Results and discussion

1.1.4. DEP-based cell concentration

Separation of CTCs from blood samples could be done by the circular electrodes
structure in the previous studies. An applied frequency and potential were set at 1
MHz and 16 Vpp respectively. The time interval for DEP manipulation on each pair of
electrodes was 20 seconds. The RBCs to CTCs ratio was 13 when the cell
concentrations of CTCs and RBCs were 2.5 × 105 cells.mL-1, and 3.25 × 106 cells.mL-
1
, respectively [39]. CTCs were selected into the center of the microchamber with a
recovery rate of around 80% [115]. At a CTC cell concentration of 104 cells.mL-1, a

34
higher ratio of RBCs to CTCs was achieved 325. CTCs could be isolated from the
RBCs even with only a single CTC cell in the working region [39].

Figure 4.4. The process of DEP-based cell concentration for A549 CTC cells. (a) The initial
chip with no cell. (b) Microfluidic stream direction was from left to right, with the flow speed
of 1.2 µL/min, and pDEP effect at the voltage of 10 Vpp and the frequency of 1 MHz, to trap
all cells at the outermost electrodes. (c) CTC cells were concentrated by DEP response in the
stepping electric fields from outermost to innermost electrodes. (d) Trapped cells were
collected at the left sensing electrodes for EIS measurement.

In this investigation, A549 CTCs samples at the same cell concentration of 5×104
cells.mL-1 were used to indicate the main working principles of the microchip. Just the
single type of target cell sample was used in order to enhance the cell enrichment
factor, as well as conveniently calculate the limit of cell detection. For DEP-based cell
manipulation, an excitation signal was applied with a frequency of 1 MHz and a
voltage amplitude of 10 Vpp. A549 cells were consequently pulled into the gap and the
edge of the circular electrodes due to pDEP responses in the sucrose solution [39],
[115], [116], [134]. Fig.4.4 shows the microscopy images of a cell sample on the
electrodes at the main steps of the DEP manipulation process. First, the microfluidic
channel was cleaned with the sucrose solution for 30 minutes to block hydrophobic
interactions between the PDMS surface and cell sample solution in the experiments.
The next, the cell sample was injected into the channel of the chip at different
volumes from left to right. The fluid flow rate was set 1.2 µL/min by the syringe
pump. Using the electric field at the peripheral, almost cells were attached in the gap
between the two highest electrodes. Subsequently, CTCs were collected from the
outermost electrodes toward the left sensing electrodes by the DEP application with
the sucrose buffer flow at a low speed of 0.2 µL/min in the channel. Finally, CTCs
were located on the left sensing electrodes group. The cell capture efficiency was
evaluated by estimating the fluorescence intensity of the whole electrodes region at
the beginning and at the sensing electrodes after finishing the process of applying the
stepping electric field from the outermost to central electrodes. As a result, the cell
enrichment coefficient by the microdevice was accounted for over 90%. The time

35
period for DEP driving on the outermost electrodes was about 5 minutes. The stepping
electric field was held for 20 seconds for each pair of the electrodes before switching
to the next adjacent electrodes. Following EIS measurement was conducted less 2
minutes. Therefore, the total duration of the whole process was approximately 10
minutes. Furthermore, there were few cells that still kept to the outermost electrodes.
This finding revealed that the cells would be firmly captured to the outer electrode
circles without insulation layer.

1.1.5. Electrical impedance spectroscopy measurement

Following the process of DEP-based cell concentration, A549 cells were attached
to the left sensing electrodes. The cell samples at the cell concentration of 5×104
cells.mL-1 were injected into the channel with specific volumes in a range from 0 to 10
µL with a step by step of 2 µL. The pumped cell number could be estimated ranging
from 0 to approximately 50 cells. Two pairs of symmetric sensing electrodes were in
the central working region of the structure. This design aimed to trap target cells into
the left sensing electrodes pair called the cell-sensing electrodes. The other pair did
not capture any cell called the un-capturing electrodes. The experimental results
showed that A549 CTCs were trapped in the cell-sensing electrodes, whereas the un-
capturing electrodes were all kept empty (see in Fig.4.5).

Figure 4.5. Microscopic images of trapped cells into the left sensing electrodes at different
cell numbers ranging from 0 to 50 cells, respectively. A549 CTC cells were trapped in the
left sensing electrodes (cell-sensing electrodes), while the right sensing electrodes (un-
capturing electrodes) were empty.

For EIS analysis, fluid flow was stopped during the measurement. Each pair of
the sensing electrodes was connected to the probes of the impedance analyzer,
separately. An AC sinusoidal signal with a voltage of 500 mV pp within a frequency
range from 1 kHz to 1 MHz was applied for all experiments. Fig.4.6. shows the EIS
responses for the central sensing electrodes at 60 points per decade. Data are
represented in the form of amplitude Z (Fig.4.6a), phase angle θ (Fig.4.6b), and
Nyquist plots (Fig.4.6c) at different numbers of the A549 cells. Both impedance
amplitude and phase angle decreased when the frequency increased at each level of

36
the cell number. It can be seen, the variation was minimal in the un-capturing
electrodes. By contrast, significant differences could be observed in the cell-sensing
electrodes. In addition, there were three specific zones in the impedance amplitude
spectra of the cell-sensing electrodes. With a higher number of captured cells, the
impedance magnitude increased rapidly in the low-frequency range (1-10 kHz), while
it decreased in the intermediate frequencies (10-100 kHz), and altered not much in the
high-frequency range (100 kHz - 1 MHz). For the un-capturing electrodes, the
impedance difference appeared less at the low-frequency range.

(a)

(b)

(c)
Figure 4.6. EIS measurements for A549 CTCs detection by the microdevice using the
impedance analyzer in sucrose medium, with a frequency range from 1 kHz to 1 MHz, at an
applied potential of 250 mV. (a) Amplitude Z, (b) Phase angle θ, and (c) Nyquist diagram of
the electrical impedance in comparison between two pairs of sensing electrodes; at different
trapped cell numbers: 0, 10, 20,) 30, 40, and 50 cells, respectively.

The phase angle describes the relative contributions of the real Z’ and imaginary
Z” components to the impedance. A phase angle of -90° results from a purely
capacitive element, while a phase angle of 0° is the product of a structure that is pure
resistance. In these measurements, the phase angle value was close to -10° at the low-
frequency range of the EIS spectra. Whereas, the phase angle approached -45° to -90°
in the high frequencies. Therefore, the capacitive element was dominant at the high-
frequency range, whereas the resistive element of the impedance dominated the low
frequencies where high magnitude change could be seen. Impedance alteration was up
to hundreds of kilo-ohms in the frequency ranging from 1 to 100 kHz, at which the
angle phase was ranged from -10° to -30°.

In this work, the electrodes were in contact with the electrolyte solution medium.
Thus, the Randles equivalent circuit model was used to characterize the experimental
37
results of EIS [151]. The model was comprised of the surface modification
capacitance Cs, the solution resistance Rs, the charge transfer resistance Rct, and the
Warburg impedance ZW. The values for Rs, Rct, and Cs could be easily defined by the
Nyquist plots in Fig.4.6(c). Rs was the minimum real element value of the impedance.
Rct corresponded to the semicircle diameter of the Nyquist plot. Cs could be calculated
from the frequency at the maximum of the semicircle (2πf=1/RctCs). The Warburg
impedance resulted from the impedance of the current due to diffusion from the bulk
solution to the interface. In this work, the Rs, Rct, and Cs values were respectively
determined approximate 0, 500 kΩ, and 6 pF, when the channel was filled full in
sucrose medium without cells. For the cell-sensing electrodes, the Nyquist responses
changed gradually with the increasing number of trapped CTC cells from 0 to 50
cells. Meanwhile, the EIS graphs of the un-capturing electrodes expressed negligible
differences. These variations in the impedance properties demonstrated that the A549
cells were captured at the left cell-sensing electrodes.

1.1.6. Differential impedance spectra analytical methods

The impedance and phase angle were then converted to the parameters termed the
differences in all the explored frequency range. Fig.4.7. shows the differential
impedance spectrum in two analytical methods at the various volume of the injected
CTC cell sample. T and U are expressed for the pairs of cell-sensing electrodes and
un-capturing electrodes, respectively. The impedance change within the pair of cell-

sensing electrodes, in terms of the amplitude (∆ZT) and phase angle ( ) shown in

Fig. 4.7(a) are defined by:

Z T  Z 0T  f   Z CN
T
 f, (4.8)
 T   0T  f    CN
T
 f, (4.9)

where the indicators and are the representative of cases without and with cell

numbers, respectively, at each individual frequency (f).

38
 Similarly, the impedance and phase angle difference spectrum between the two
pairs of cell-sensing and un-capturing electrodes called the differential impedance
(∆ZD), and the differential phase angle (∆𝜃D) given in Fig. 4.7(b) are defined using:
Z D  Z CN
U
 f   Z CN
T
 f, (4.10)
 D   CN
U
 f    CN
T
 f, (4.11)
In this second analysis method, the cell-sensing electrodes pair was used as the
working group, while the un-capturing electrodes pair was considered as the reference
group [13]. Ideally, no cell was captured (all the central electrodes were empty, or
CN=0), and only the sucrose medium was sensed. The impedance between the cell-
sensing electrodes was thus the same as that across the un-capturing electrodes. The
magnitude and phase angle differences were almost zero, resulting in straight
differential spectrums in the whole frequency range. A trend of impedance alterations
was observed upon increasing the volume of the cell sample. When the cells captured
to the sensing electrodes, the impedance difference was non-zero. The impedance
spectra lines were similar to each other at different low cell numbers. The spectra
amplitudes were expanded as cell number increases in both analytical methods. The
tendency of impedance responses is positive in the frequency range of 10 kHz to 100
kHz, in which the impedance difference increased for additional trapped cells.

(a)

(b)
Figure 4.7. Differential impedance spectrum with different numbers of the trapped cells. (a)
Impedance variation (∆ZT and ∆𝜃T) for the cell-sensing electrodes, (b) Differential
impedance (∆ZD and ∆𝜃D) between the cell-sensing electrodes and un-capturing electrodes.

1.1.7. Limit of cell detection

Fig.4.8. shows the impedance alteration versus the number of the A549 cells
corresponding to the volume of the injected cell sample. The linear regression
equations are expressed in the graphs. At each cell number, the impedance magnitude
at high frequencies changed more than that at low frequencies. The divergences in

39
impedance amplitude (both ∆ZT and ∆ZD) among the five frequencies increased with
increasing cell concentration. This finding revealed the linear relationships at some
frequencies, in which the phase angle difference was approximately zero (Fig. 4.7).

Figure 4.8. High linear relationships between impedance variation and number of A549
CTCs: (a) Impedance change (∆ZT) of the cell-sensing electrodes at the frequency range
from 8.9 to 14.1 kHz, (b) Differential impedance (∆ZD) between the cell-sensing electrodes
and un-capturing electrodes at the frequency range from 39.8 to 63.1 kHz. The coefficient of
linear regression R2 is over 0.99 at several frequencies in both cases.

Measurements were repeated three times for each cell number, and the error bar
was shown to depict the standard error of the mean. The results show that highly
linear relationships could be achieved with the correlation coefficient (R 2) up to over
99% for both methods of calculation. In addition, the sufficient frequency ranges were
proposed for the detection of A549 cells based on EIS data. The range from 10 kHz to
12.5 kHz is proposed for the analysis method using the impedance variation of the
cell-sensing electrodes, whereas 45 kHz to 55 kHz is proposed for the differential
impedance analysis method between the cell-sensing and un-capturing electrodes. It
also shows clearly that the sensitivity of the differential analysis method is higher than
that of the first method. With the same change in the number of cells, both the
impedance variation and the appropriate frequency range of the second method are
larger than those of the first one. In this way, the detective sensitivity of the device
could be enhanced using a four-electrode design instead of the commonly used two-
electrode structure [152]. Limit of detection (LOD) was calculated from the formula
3σ/slope, where σ is the standard deviation and slope is found from the linear response
range. Herein, a LOD of approximately 3 cells was achieved frequencies near 50 kHz.
The obtained results indicated the feasibility of using DEP-based enrichment and
impedance-based measurements to detect A549 human lung CTC cells.

4.8. Conclusions

Herein, a DEPIM microdevice was successfully developed for the rapid, label-
free, and highly sensitive detection of A549 human lung CTC cells by using a simple
microfluidic channel with the circular electrodes structure. DEP-based cell

40
manipulation was conducted to concentrate CTC cells in the central region with the
cell capture coefficient accounted for over 90 %. Such cells were captured by cell-
sensing electrodes and then identified by EIS measurements. EIS data of the sensing
electrodes were displayed in the frequency range from 1 kHz to 1 MHz. Two
differential analysis methods for the detection of target cells were applied. Linear
relationships were found between impedance alteration and cell number with the
correlation coefficient up to 99%. Higher detection accuracy of the sensing part was
observed at the frequency of 50 kHz using differential analysis between the cell-
sensing electrodes and the un-capturing electrodes. These results also indicated that
the sensing design was capable of detecting CTC cells at low cell concentration. The
proposed DEPIM device could be a potential approach to identify cancer cell types.

41
 CHAPTER 5. SPECIFIC APTAMER-BASED IMPEDANCE
BIOSENSOR FOR IDENTIFICATION OF LUNG CARCINOMA CELLS
IN THE MICROFLUIDIC DEVICE

5.1. Introduction

Micro/nanofluidic devices combined with biological probes have been attracted

great attention in cancer diagnosis and treatment. An aptamer that is a single-stranded
deoxyribonucleic acid or RNA oligonucleotide is currently known as a typical type of
the membrane surface-specific molecules for the cancer subtypes recognition, with
high specificity, selectivity and affinity [23], [65], [66]. Aptamer molecules are
synthesized by a selection protocol called the systematic evolution of ligands via
exponential enrichment (SELEX). To date, aptamer-based technique has emerged as
an attractive approach for the identification of many types of objects, such as amino
acids [95], enzymes [105], [109], peptides [99], [153], [154], proteins [68], [82],
[155]–[157], antibiotics [27], [108], viruses [91], [158], [159], bacteria [108], [160],
whole or part of cells [33], [71], [112], [161]–[165]. Aptamer has obtained several
special advantages because of its unique three-dimensional structure. Aptamer probe
often has high structural flexibility, is nonimmunogenic, nontoxic, stable in harsh
biological environments, and smaller in size compared with an antibody [23], [63],
[64], [166]. From these benefits, aptamer types have been applied to many fields, such
as therapeutics, diagnostics, microfluidics, biosensors, and analytical fields [167].

During the past few years, electrical impedance-based detection method has been
preferably developed for recognizing the appearance of biological cells in
microfluidic devices [13]–[15], [168], [169]. In which, microfluidic EIS sensor is an
inexpensive approach due to its simple fabrication, miniaturization capability, rapid
response, and high sensitivity [16]–[18], [170]–[173]. The changes in medium
properties inside the electric field area could be estimated by measuring interfacial
resistance and capacitance of the impedance model. Cancerous and normal cell lines
could be distinguished using these impedance sensors [22], [73]. However, the
experiments in there have just employed a single type of cell samples. Several
aptamer-modified substrates were demonstrated to be capable of separating human

42
lung target cells from the normal cells [97]. A biosensor based on MCU1 aptamer was
built for the sensitive and selective detection of A549 lung cancer cells [89]. The
device achieved a high selectivity for A549 cells in comparison with the other human
non-target cells, including HepG2 liver cancer cells, PC3 prostate cancer cells, and
MRC-5 lung normal cells. An aptamer-coated slide for trapping A549 CTC cells was
indicated with the capture coefficients over 90% for target cells, while less than 10%
for the non-target cells [112]. Aptasensor that combines aptamer and impedance-based
measurement method within a microchip has also been well accomplished for the
classification of specific cells in many studies [33], [91], [97], [174].

The aptasensor field has achieved remarkable advances by the conjugation of the
aptamers with nanomaterials, such as nanostructures, nanowires, nanotubes, and
nanoparticles [24], [26], [77], [104]. This combination allows improving the signal
generation and amplification, the surface-to-volume ratio, and the sensitivity of the
sensor [175]. Gold nanoparticle (AuNP) is one of the most common nanomaterial
types for the development of the aptasensors due to its electrical conductivity, and
reactivity properties [26]. AuNPs could be used as a label for signal amplification, or
be attached on the working surface to extend the sensing area [176]. This approach
might increase the number of capture probes, thus enhancing electrochemical signal
intensity. The simple surface self-assembly process of aptamers and AuNPs on the
sensing microelectrodes have been applied in the microfluidic devices with high
reproducibility [106]. Several aptasensors have been built for the detection of human
rare cancerous cells, such as AGS gastric cancer cells [104], MCF-7 breast cancer
cells [77], and CT 26 colorectal cancer cells [75].

In this study, a microfluidic platform is developed for the capture of the A549
human lung cancer cells using the specific DNA aptamers and the EIS measurements.
The immobilization processes of self-assembled monolayers (SAMs) onto the gold
electrode surface are represented. The efficiency of the cell capture process is
estimated the microscopic images and the fluorescence intensities of the cell
distribution on the working region. EIS analysis is conducted within a large frequency
range to indicate the main binding events. The impedance responses can be explored
at various cell concentrations to evaluate the performance of the sensing device. The

43
obtained results may promise a simple and rapid method for the identification of CTC
cell lines with high specificity and selectivity.

5.2. Chemicals and reagents

Most of the chemicals were purchased from Sigma-Aldrich, including DNA

aptamers with 5'-thiol (5’-C6SH) or 5’-amine (5’-NH2) modification, thiol PEG
carboxylic acid (HS-PEG-COOH), N-hydroxysuccinimide (NHS), bovine serum
albumin (BSA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), TE buffer,
MES buffer, and phosphate-buffered saline (PBS) solution. The aptamer that its
sequence is 5′-ACGCTCGGAT GCCACTACAG GGTTGCATGC CGTGGGGAGG
GGGGTGGGTT TTATAGCGTA CTCAGCTCAT GGACGTGCTG GTGAC-3’, has
given a selective binding for A549 human lung CTC cells [177]. The aptamer was
received in a dried form. The 100 µM aptamer stock solution was prepared using the
TE buffer solution (10 mM Tris, 1 mM EDTA, and pH 8.0) and kept at −20 °C. The
HS-PEG-COOH, EDC, and NHS solutions were diluted in 100 mM MES buffer,
respectively. A washing buffer solution was made by adding 4.5 g of glucose, 0.475 g
MgCl2 into 1 L of PBS 1× (pH 7.4), and could be stored at 4 °C for up to 3 months. A
binding buffer by adding 1 mg of tRNA and 10 mg of BSA into 10 mL of the washing
buffer, could be stored at 4 °C for 1 month. The aqueous solutions were diluted with
deionized water (18.2 MΩ.cm) from a Milli-Q machine.

5.3. Microfluidic chip design and fabrication

The microfluidic device is designed with three basic parts, including a glass
substrate, a simple PDMS channel, and a coplanar gold microelectrodes configuration.
The main parameters and dimensions of the microchip is shown in Table 5.1.

Table 5.5: Geometry parameters of the aptamer-based impedance microchip

44
 The microchip was fabricated using a similar soft lithography procedure reported
in the previous chapter (illustrated in Fig.5.1(a)). Following a process of cleaning,
S1813 photoresist spin coating, aligment and exposure, development, baking, etching,
and washing, the gold electrode structure patterned on glass substrate was finally
formed for the self-assembly and impedance measurement processes.

(a)

(b)
Figure 5.1. (a) The standard photolithography process of the microfluidic chip. (b) Picture
of a fabricated microdevice.

Using the similar casting and molding techniques, an SU-8 2050 master mold
with the channel pattern was created on a silicon wafer. A mixture of PDMS was
poured and cured on the prepared master mold. After curing, a PDMS block was then
released from the channel mold. Finally, the PDMS piece was punched with inlet and
oulet fluidic ports, and was bonded to the gold-glass substrate using an oxygen plasma
machine. The obtained channel length, width, and height were approximately 20 mm,
1 mm and 50 µm, respectively. Fig.5.1(b) shows the image of a fabricated device.

5.4. Self-assembly process of aptamers onto the gold electrodes surface

The layer by layer self-assembly process on the gold surface is sketched in

Fig.5.2. The aptamer probes are prepared by immobilization of the amine-terminated
aptamers onto the carboxylic acid-functionalized gold surface. The gold-sulfur
interaction formed between thiols and gold surface can generate the binding forces to
form SAMs for the aptamer conjugation. The substrate is firstly washed by DI water,
PBS buffer solution, and dried by nitrogen flow. The gold substrate is incubated by
100 µM HS-PEG-COOH solution and stored overnight at 4 °C for 18 h. The modified
substrate is then lightly washed by the buffer solution and is activated in a solution
containing 100 mM NHS and 400 mM EDC for 30 min to form stable amine-reactive
esters of carboxylate groups for linking with the NH2 aptamers. Subsequently, the
initial 100 µM NH2 aptamers is diluted to the required concentration by the binding

45
buffer. The mixture solution is heated at a temperature of 95 °C for 5 min. Then, the
tube is left on a bench at room temperature for 15 min. The gold substrate is washed
again with the washing buffer, followed by the incubation of refreshed aptamer
solution at room temperature for 1 h. A cell sample at a defined concentration is
injected and incubated on the modified gold substrate in the channel. Target cells are
then trapped onto the gold surface by the specific aptamers. Finally, the channel is
washed by the buffer solution to remove nonspecific molecules.

Figure 5.2. Illustration of aptamers immobilization procedure on gold surrface.

5.5. Modification procedure of aptamer-conjugated AuNPs on the sensing area

Figure 5.3. Outline the aptamer modification process onto the SAMs of AuNPs at the sensing
area in the channel for the capture of the target cells.

The assembly process using AuNPs on the glass–gold substrate is represented in

Fig.5.3. The self-assembly procedure using AuNPs has been reported in our previous
publications [178], [179]. First, the square area for the SAM of AuNPs is created at
the sensing position by an S1818 photoresist layer via standard photolithography
steps. The APTES salinization on the substrate surface is carried out for 1 min. The
substrate is then rinsed by DI water, dried by nitrogen, and reacted in a 179 ppm
AuNPs solution for 1 h. The next, the substrate is cleaned to remove the photoresist
layer using acetone, methanol, and DI water and is dried with nitrogen. After the
attachment of AuNPs in the sensing area, the PDMS channel replica is bonded on
there using the oxygen plasma machine.

Subsequently, the aptamer immobilization steps on the AuNP layers in the

channel are performed. The channel is rinsed with the washing buffer solution. The
initial 100 µM SH-aptamer solution is diluted to the required concentration using the
binding buffer. The refreshed aptamer solution is heated at temperature of 95 °C for 5
min and is left on a bench at room temperature for 15 min. Next, the channel is fully
filled with the aptamer solution and then incubated at room temperature for over 1 h.
Then, a cell sample at a defined concentration is injected and incubated in the channel.

46
Finally, the channel is washed by the buffer solution to remove nonspecific adsorbed
molecules and to retain the target cells at the sensing area.

5.6. Cell sample preparation and experimental system

1.1.8. Cell lines and cell culture

For the experimental demonstrations of the proposed microdevice, A549 human

lung adenocarcinoma cancer cells, Hela human cervical cancer cells, MKN45 human
gastric cancer cells, and Caco-2 human colorectal cancer cells were cultured in
DMEM or MEM, made with 3.7 g/L of NaHCO3 medium, 1% penicillin, or 1%
streptomycin, and 10% FBS. The cell dishes were stored in an incubator with the
humidified conditions containing 5% carbon dioxide at a temperature of 37°C; the
culture medium was exchanged every 1 to 3 days. Cells were washed with PBS and
harvested following a 5 min treatment of 0.02% ethylenediaminetetraacetic acid and
0.25% trypsin. Prior to the experiments, the cells were collected by the trypsinization
process. The cell samples were then rinsed three times with the washing buffer
solution by centrifugation method. Cell concentration of the sample was determined
using a hemocytometer under a microscope by a trypan blue dye exclusion protocol or
a standard fluorescence assay with calcein AM. The fluorescence intensity was
quantified using NIH ImageJ software.

1.1.9. Instrumentation

Figure 5.4. The main parts of the experimental system containing a syringe pump, a
microscope, a precision impedance analyzer, and a controller computer.

A high precision experimental system for the experiments is shown in Fig.5.4.

The solution could be pumped into the microfluidc channel of the chip by a syringe
pump (Model KDS 101). The flow speed in the microfluidic channel was set about 10
µL.min-1 in all the experiments. Cell sample and distribution were captured using a
fluorescence microscope (CKX41 or BX43) with a CCD camera (DP71) connected to
a computer running Olympus DP Controller image software. A precision impedance
analyzer (MICROTEST 6630) connected the two electrodes of the aptasensor via
coaxial cables was used for the EIS measurements within a wide frequency range. All
the impedance measurements were carried out at room temperature in the 10 mM PBS

47
buffer solution. The measured signals of the impedance analyzer were recorded,
selected and transferred to the computer through a data acquisition USB card. The
data were then analyzed by LabVIEW tool to determine the changes in the impedance
value of the sensing electrodes.

5.7. Results and discussion

1.1.10. Aptamer-functionalized gold surface with microscopic observation

(a)

(b)
Figure 5.5. Optical microscopic images of the cell distribution onto the aptamer-modified
gold-glass slides with various cell lines at the same concentration of 2×105 cells.mL-1, under
the same immobilization process: (a) A549 lung cancer cells, before and after the cell
incubation and capture process. (b) Control cell line samples after the cell incubation and
capture process, including Hela cervical cancer cells, Caco-2 colorectal cancer cells,
MKN45 gastric cancer cells, and Red Blood Cells.

To assess the selectivity of the aptamer probes, the investigations on the gold-
glass slides were performed with different human cell lines from various cancer
tissues, including A549 lung carcinoma cancer cells, Hela cervical cancer cells, Caco-
2 colorectal cancer cells, and MKN45 gastric cancer cells. The cell samples were
prepared at the same cell concentration of 2×105 cells.mL-1. A549 cell line was chosen
as the target cells, while Hela cells, MKN45 cells, Caco-2 cells, and red blood cells
(RBCs collected from volunteers) were chosen as the control cells. The cell
distribution on the aptamer-modified gold-glass surface was observed by using the
microscope. The microscopic images shown in Fig.5.5 were taken between before and
after the cell binding process, at the same exposure mode, objective scale, and
sensitivity. The results show that many A549 CTC cells were trapped stably, while the
other non-target cells were removed out the modified gold-glass substrate. The results
revealed a significantly high specificity of the aptamer probes toward A549 human
lung carcinoma cells, and a successful modification process of the SAMs onto the
gold surfaces.

48
 In practical surveys, various parameters related to the aptamer probes were
explored to build the optimal conditions of the immobilization procedure, including
medium pH and temperature, aptamer concentration and incubation times. In general,
the aptamer probes could be normally operated at room temperature in a medium pH
ranging from 7.0 to 7.4. In this work, the response of the aptamers is gradually
enhanced by increasing the aptamer concentration. The stable states could be reached
at the aptamer concentrations from 10 to 20 µM. The aptamer concentration of
approximately 10 µM was chosen in subsequent experiments. In addition, the
response of the probe gradually increased with increasing incubation times. However,
a too long time of the cell incubation could make more much cells being adhesives
onto the glass surface. Thus, a reasonable incubation time of the cell sample less than
3 min was chosen.

1.1.11. EIS observations

Fig.5.6 shows the structure of the capacitive sensing microdevice. The NH2
aptamer-attached gold microelectrodes were used to bind the A549 target cells, while
the control cells were moved out of the substrate. The EIS measurements were carried
out within the chip to identify the appearance of the cells. Herein, a coplanar two-
microelectrode structure was utilized. Its advantages were simple to fabricate, easy to
monitor the electrical changes inside the electrodes zone. In addition, the electrode
surface was designed large enough for the observation of trapped cells.

Figure 5.6. Schematic of the capacitive sensing microfluidic device based on aptamers for
specific detection of A549 human lung cancer cells.

To confirm the immobilization procedure of aptamers on the gold electrodes,

EIS investigations were performed within a frequency range from 0.1 kHz to 1 MHz,
at the voltage amplitude of 100 mV. EIS values were measured at 40 points per
decade. The flow of the PBS buffer solution in the microfluidic channel was stopped
during the measurements. Fig.5.7 shows the measured EIS responses in three cases,
consisting of the original chip (bare gold electrodes), after the modification of the
aptamer (aptamer-modified gold electrodes), and after the capture process of the A549
cells at the concentration of 2×10 5 cells.mL-1 (cells captured on the aptamer-modified

49
gold electrodes). Data were shown in the form of impedance magnitude Z (Fig.5.7a),
and phase angle θ (Fig.5.7b). Each measured point is the average value of at least
three separate measurements, and the error bar expresses the standard error of the
mean. The amplitude increased as the frequency decreased in the applied range. The
difference value at each frequency point increased up to tens of kΩ after trapping the
aptamers onto the gold surfaces. Especially, the variations were observed more clearly
after the cells were bound onto the modified electrodes at lower frequencies. The
phase angle approached -55° at the low-frequency range, while it was close to -5° in
the high-frequency range of the spectra. Therefore, the resistance was dominant of the
impedance at the high-frequency range, whereas the capacitive element dominated the
low-frequency range at which the high magnitude variations were observed.

(a) (b)

Figure 5.7. Measured EIS responses of the microchip in three cases, including bare gold
electrodes, aptamer-modified gold electrodes, and captured A549 target cells onto the
aptamer-modified electrodes within a frequency range from 0.1 kHz to 1 MHz: (a)
Impedance magnitude, (b) Impedance phase angle.

1.1.12. Equivalent circuit model

An electrical equivalent circuit model of the impedance measurement in the
microdevice is shown in Fig.5.8. The impedance consists of the interface resistance Rs
and capacitance Cs, the medium solution resistance Rm, and the parasitic capacitance
Cp and resistance Rp. The main components of the impedance depend on the
conductivity and permittivity constants of the binding cells, and the medium solution
inside the electric field between the two electrodes. The parasitic resistive and
capacitive elements are related to the original electrode design and the connecting
wires. By ignoring these parasitic elements, the total impedance Z of the chip is given:
Rs  Rs2Cs
Z     Rm   j, (5.1)
 2 Rs2Cs2  1  2 Rs2Cs2  1
in which, ω is the angle frequency, and j is the imaginary unit ( j 2  1 ). We can
easily realize that the impedance Z is approximate Rm at the high-frequency range.
Impedance alteration is mainly influenced by the changes of the electrode-solution

50
surfaces at the low frequencies. In which, the capacitance is the dominant component
of the impedance. It is clearly shown that the measured data are in good agreement
with the analytical model.

Figure 5.8. Electrical equivalent circuit model of the impedance-based measurement of the
microfluidic chip.

1.1.13. Capacitive sensing of the aptamer-based biosensor

The capacitance component of the biosensor was calculated by the imaginary

part of the impedance. The A549 cell samples at various cell concentrations were
investigated. Fig.5.9(a) shows the plots of the capacitance with different cell
concentrations ranging from 1×105 to 5×105 cells.mL-1. The results show that the
capacitance value increased as a lower cell concentration at the low frequencies. Then,
the capacitance variation was defined as the change of the capacitance value of each
test in comparison with the case of the modified electrodes without a cell. A linear
regression relationship of the capacitance change versus the cell concentration was
found at a frequency of approximately 5 kHz. The equation is expressed in Fig.5.9(b)
with the correlation coefficient (R2) up to 99%. Limit of cell detection was achieved
approximately about 1.5×104 cells.mL-1. To evaluate the stability of the probe storage,
aptamer-modified electrodes were stored in the refrigerator at 4 °C. After 15 days,
impedance signals still maintained over 90% in comparison with its initial response.

(a) (b)
Figure 5.9. (a) Capacitive responses for A549 cells captured on the aptamer-modified
electrodes in the microfluidic channel at various cell concentrations, (b) The capacitance
variation of the microchip at frequency about 5 kHz with different cell concentrations.

In this work, the main operating principles of the microfluidic device have been
indicated. The chip has used the aptamer immobilization procedure on the gold
surfaces, and the capacitance-based cell detection for A549 lung cancer cells. The
results indicated that the aptamers were attached well on the gold surface by the
proposed assay. Besides, the capacitive sensing method was proven to be a simple and
convenient approach to present each step of the modification process of the aptamer.
51
While the other existed methods have required expensive and complex equipment
systems, such as surface plasmon resonance (SPR) measurements [180], quartz crystal
microbalance (QCM) [78], and atomic force microscopy (AFM) [181]. This study
also allows us to develop a DEPIM microfluidic chip combined with a highly
sensitive capacitance method for detection of the circulating tumor cells.

1.1.14. Aptamer-conjugated SAMs of AuNPs in the impedance microchip

Fig.5.10 sketches the design of the microdevice. A simple gold microelectrode

structure was used to confirm the immobilization steps in the channel. An assembly
procedure was given to attach AuNPs and aptamers on the sensing zone. The thiol-
labeled aptamers were conjugated with the SAM region of AuNPs generated around
the working electrodes for the capture of the A549 cells. The binding performance of
the probes was monitored by the fluorescence microscopic images. The obtained
results indicated a simple and rapid assay for the identification of the cancerous cells.

Figure 5.10. Schematic of the microfluidic device, using a coplanar gold electrode structure,
a glass substrate, and a simple PDMS channel. Thiol- aptamers are conjugated onto the
SAM of AuNPs deposited around the electrodes to capture A549 cells in the channel.

(a) SAM of AuNPs

In this work, the microelectrodes configuration was used to mark the desired
SAM area of AuNPs in the channel and to reveal the stepwise modification process.
The structure consisted of a center electrode of 50 µm in diameter, an outer circular
electrode of 30 µm in width, with the gap of 30 µm. The AuNPs with a mean diameter
of 13 ± 1 nm, were used herein. The picture of the SAM area around the electrodes is
shown in Fig.5.11(a). Fig.5.11(b–d) shows the SAM of AuNPs on a substrate captured
by a scanning electron microscopy (SEM) system at magnifications of 20,000,
100,000, and 200,000 times, respectively. The results indicated that AuNPs could be
attached to both gold and glass surfaces with a high density. The experiments were
then carried out to confirm that A549 cells can be captured by the aptamer-coated
AuNPs layer in the microfluidic channel.

52
Figure 5.11. (a) SAM of AuNPs at the sensing area after the silanization steps; (b–d) SEM
images of the SAM of AuNPs on the gold-glass substrate at the magnifications of 20k, 100k,
and 200k times, respectively.

(b) Cell Specificity and selectivity

In the experiments, the A549 cancer cell samples were prepared at the same cell
concentration of 5×102 cells/µL. Following the immobilization of aptamers onto the
sensing electrode region, the A549 cell samples were injected into the channel. The
duration of 2 min was chosen as the stability incubation time of the cell samples.
Fig.5.12 shows the fluorescence microscopic images of the cell distribution at the
domain of the SAM area around the sensing electrodes between before and after the
final washing step. The efficiencies of cell capture were calculated via the
fluorescence intensities. These images demonstrated that the A549 cells were
removed out the channel without the SAM of AuNPs on the electrode area, whereas
they were still bound on the SAM of AuNPs area modified by aptamers with a cell
binding coefficient over 90% with the cell concentration ranging from 105 to 106
cells.mL-1. It also indicated a successful self-assembly assay of the AuNPs and
aptamers on the gold–glass substrate.

(a)

(b)
Figure 5.12. Fluorescence microscopic images of A549 cell distribution on the sensing area
between before and after the capture process with the cell concentration of 5×102 cells/µL:
(a) Non-use SAM of AuNPs; (b) Use SAM of AuNPs around the sensing microelectrodes.

To explore the application capability of the microchip for the detection of A549
cells in complicated cell samples, A549 cells were mixed with Hela cells and 5%
whole blood sample (collected from healthy donors) in the buffer solution. Before the
mixture of cells, A549 and Hela cells were labeled by Calcein green and red-orange,
separately. To obtain the cell mixture, 500 µL of A549 cell solution at a concentration
of 106 cells.mL-1 was combined with 500 µL Hela cell solution at a concentration of
2×106 cells.mL-1. Thus, the concentrations of A549 cells and Hela cells were 5×10 5
cells.mL-1 and 106 cells.mL-1, respectively. Similarly, 5 µL of whole blood was diluted
in 995 µL of A549 cell solution at a concentration of 5×10 5 cells.mL-1. The cell
53
mixture samples were then assayed, as in the above process. As can be seen in
Fig.5.13, most of the A549 cells were trapped onto the SAM section, whereas the
other non-target cells were washed away. These results confirmed that the microchip
is a promising tool for the capture A549 lung CTC cells with high selectivity.

(a)

(b)
Figure 5.13. Fluorescence microscopic images of the cell mixtures around the modified
electrodes between before and after the final washing step with the SAM of AuNPs area: (a)
A549 cells (green) of 5×102 cells/µL, and Hela cells (red) of 10 3 cells/µL; (b) A549 cells of
5×102 cells/µL in 5% whole blood sample.

(c) EIS measurements in the SAM immobilization process

In this work, the EIS measurements were repeated within a frequency range from
1 kHz to 100 kHz, with a potential of 50 mV. The A549 cell samples at the same
concentrations of 5×102 cells/µL were used herein. EIS data were recorded at 125
points per decade. The obtained results that represent the average values of at least
three separate experiments are shown in the form of impedance amplitude Z
(Fig.5.14a), and impedance phase angle θ (Fig5.14b). A comparison of the responses
in two cases was assessed in terms of the use and non-use of SAM of AuNPs, with
four investigations, including the initial gold electrodes without the SAM generation,
the 10 µM aptamer-modified electrodes without SAM of AuNPs after the cell capture
process, the gold electrodes with SAM of AuNPs, and the 10 µM aptamer-
functionalized electrodes with the SAM of AuNPs after the cell capture process. The
impedance magnitude increased, whereas the phase angle decreased with the decrease
in applied frequency. Besides, a distinct difference can be realized in the EIS graph
trends in the two mentioned cases. For the case without SAM of AuNPs, the
impedance magnitude of the aptamer-modified electrodes increased insignificantly
compared with the original electrode at each frequency, whereas the phase angle was
almost constant at low frequencies then slightly decreased at higher frequencies. In
this case, aptamers only covered the surface of the gold microelectrodes without
catching on the glass surface or the gap between the two electrodes. In the other case,
the impedance magnitude decreased sharply during the modification process using

54
AuNPs. The impedance value at each frequency point decreased by tens of kilo Ohms
after the attachment of the AuNP layer and especially after aptamer incubation. The
changes were observed clearly at low frequencies ranging from 1 kHz to 10 kHz. At
such frequencies, the measured impedance values were high, whereas the phase angle
values were low, indicating that the capacitive elements of impedance were dominant
in a low frequency range. By contrast, the resistive components dominated at high
frequencies. The immobilization process resulted in an increase in the conductivity of
the electric field between the electrodes and a reduction in the measured impedance
magnitude at each frequency point because of the envelopment of the aptamers and
AuNPs on the gap and surface of the electrodes. The obtained results also indicated
that the aptamers were successfully covered on the AuNP layer using the proposed
SAM functionalization method.

The performance of the SAM method was investigated on the basis of the EIS
differential ratios in the investigated frequency range. The factors were defined in

terms of amplitude difference factor Z  1  Z A  f  / Z I  f  and phase angle

difference factor   1   A  f  /  I  f  separately, where the indicators I and A are

representative of the initial electrodes and the aptamer-modified electrodes at each
individual frequency (f) after the cell capture process, respectively. Fig.5.14(c, and d)
shows the impedance variation in cases non-use and use SAM of AuNPs, respectively.
In general, the coefficient lines with the use of the AuNPs were higher than those
without the AuNPs in the impedance magnitude and phase angle analysis, especially
in the low-frequency range. Results indicated that the EIS signal with the use of
AuNPs achieved a higher sensitivity compared to without SAM of AuNPs.

(a) (b)

(c) (d)
Figure 5.14. EIS graphs of the sensing electrodes in the microfluidic channel with a
frequency range from 1 to 100 kHz in 4 cases, respectively: the original gold electrodes, the
aptamer-modified gold electrodes without SAM of AuNPs after the cell capture process, the
gold electrodes with SAM of AuNPs, and the modified electrodes with SAM of AuNPs after
the cell capture process: (a) Impedance magnitude; (b) Impedance phase angle; (c,d)

55
Impedance and phase angle difference factors of the cell-trapped electrodes with respect to
the initial electrodes in two cases, non-use and use SAM of AuNPs, respectively.

In this study, we focused on the capture ability of the microdevice for A549 lung
carcinoma cells using the aptamer-conjugated SAM of AuNPs. The EIS signal
improvement of the sensing electrodes has been explored in the two cases of with
SAM of AuNPs, and without SAM of AuNPs. The above experimental results showed
that A549 target cells were trapped with high affinity and selectivity onto the aptamer-
conjugated AuNP SAM in the microfluidic channel. However, the design still has
some drawbacks in the application of the impedance measurement for cell detection.
For instance, the CTC abundance in the real cell sample is extremely low. As a result,
the extremely low numbers of the target cell could be captured into the electric field
between the microelectrodes. Thus, the chip must be continuously improved in future
works, including the optimization of the sensing electrode structure and the
microfluidic channel design. In addition, the extension of the SAM layer area and
application of the DEP-based cell manipulation will be proposed to manipulate the
target cells onto the sensing electrodes conveniently [39], [182]. Although its
detection capacity has been still drawbacks, the microfluidic device exhibited some
attractive features, such as biocompatibility, cost-effectiveness, simplicity, rapidity,
high affinity, and selectivity for the of lung cancer diagnosis.

5.8. Conclusions

A simple microfluidic device using the cell-specific aptamer-modified gold

electrodes was presented for the detection of A549 human lung carcinoma cells. Two
protocols of the aptamer immobilization were reported. First, the amine-labeled
aptamer probes were created on the gold surface without of AuNPs. Second, the thiol-
terminated aptamer probes were conjugated onto the SAM zone of AuNPs around the
sensing electrodes in the channel. The responses of the microchip were examined by
optical microscopic images and EIS measurements. From the microscopic
observations, the chip achieved a highly selective capture performance against A549
cells compared with several control cells. The measured impedance data indicated that
the successful binding of the molecules was achieved by the proposed modification
assays. The equivalent circuit model was used to evalute the impedance-based

56
detection of the chips. The detection ability of the sensing part for target cells could be
estimated through the capacitance variation at the appropriate frequency. In addition,
the microchip using SAM of AuNPs was also applied for the isolation of A549 cells
in the whole blood samples. In addition, the microdevice still displayed several
attractive features, as follows: biocompatible, selectivity, specificity, simplicity,
sensitivity, rapidity, and low-cost in the development of aptasensors for cancer
diagnosis. The electrode and channel design would be continuously developed for the
cell quantification in the proceeding steps.

57
 Chapter 6. CONCLUSIONS AND PROSPECTS

Electrical impedance-based detection technique has attractive advantages, such

as cheap price, rapid response, high sensitivity, and suitable to the microsensors.
Combining impedance-based measurement (IM) and dielectrophoresis (DEP) is an
effective strategy to reduce the processing time and enhance the sensitivity of the
microfluidic device. The DEPIM method is also proposed as a useful approach in
biosensors to identify cancerous cells. In addition, the specific aptamers are currently
the prominent candidates for isolation target cells from the non-target cells. The
aptamer probes can be immobilized on the sensing electrodes surface to bind the
target cells. In this thesis, we have built up the capability the microfluidic devices
exhibited several attractive features, as follows: biocompatible, simplicity, low-cost,
rapidity, sensitivity and selectivity toward the detection of lung cancer cells. The main
conclusions are described as follows:

A microchip with the interdigitated microelectrodes array based on the DEPIM

method was designed for both manipulation and detection of cell types. The DEP-
based cell collection and impedance-based measurement are performed within a
fluidic chamber. Positive DEP response driven by the excitation wave at the
frequency of 1 MHz and the amplitude of 5 V pp, pulled the cells in the suspension
medium onto the tips of the microelectrodes. Subsequently, electrical impedance
properties of the chip were measured with different cell densities, using the sinusoidal
waveform signal at the frequency of 4 kHz. The experimental results demonstrated
that the DEPIM chips were able to identify and distinguish human esophageal and
lung cells at several cytological stages. A hand-held electronic module was also built
and integrated with the microchip. The measured data of the module were agreement
with the results from the high-reliability system. As a result, CE81T and CE81T-4,
MRC-5 and A549 cell lines could be identified by the admittance characteristics from
the measurements.

The DEPIM method was also successfully developed in the microfluidic device
based on the straight channel with circular electrodes for the enrichment and detection
of A549 lung cancer cells. The target cells were concentrated into the sensing zone by

58
the DEP application using the potential signal at the frequency of 1 MHz and
amplitude of 10 Vpp. The cell enrichment coefficient was achieved over 90% after the
DEP process. Before and after the cell capture at the desired area, EIS measurements
in the frequency range from 1 kHz to 1 MHz were performed for the two sensing
electrode groups. The A549 cell samples were investigated at different injected cell
volumes corresponds to a small number of captured cells at the cell-sensing
electrodes. Highly linear relationships of the impedance alteration versus the captured
cell number were found between via the differential analysis methods. From the
differential analysis between the cell-sensing and the un-capturing electrodes, a high
detective sensitivity of the chip was achieved at the frequency of approximately 50
kHz. These results demonstrated that the proposed device was capable of both
isolating, concentrating and detecting CTC cells.

Finally, a simple microfluidic device using the cell-specific aptamer-modified

gold electrodes was presented for the detection of A549 lung cancer cells. The
protocols of the aptamer immobilization on the sensing area were reported. The
aptamer probes were created on the gold surface without of AuNPs. Or, the aptamer
probes were conjugated onto the SAM zone of AuNPs around the sensing electrodes
in the channel. The responses of the microchip were examined by microscopic
observations and EIS measurements in the frequency range from 1 to 100 kHz. The
microchips achieved a highly selective capture efficiency against A549 cells
compared with several control cells. In addition, the use of SAM of AuNPs enabled
the sensor to obtain higher sensitivity, and be applied for the isolation of A549 cells in
the whole blood samples.

Designing the novel microfluidic channel and electrode structures, verifying the
cell isolation performance, and developing the electronics circuit modules would be
continuously optimized for the sensitive detection and quantification of cancerous
cells. In addition, the conjugation of aptamer probes with AuNPs or magnetic beads
could be considered in the electrochemical signal intensity enhancement, as well as,
the excellent improvement in the sensitivity of the aptasensor in the future works.

59
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 Glossary and List of Abbreviations

Symbol Description Units

 Water activity in the membrane Olympus
A Flowing gas area m2
A Rotational area of the propeller m2
Afc Membrane active area of the fuel cell cm2

F Faraday’s constant (F = 96485) coulomb / mol

CO2 Concentration of oxygen at the catalyst interface mol / cm3

Cp Specific heat of the fluid kJ / kg .K

CW Molar concentration of water in the membrane kmol / m3

DW Diffusion coefficient of water molecules in the membrane -
E Weld joint efficiency code of the metal -
h Altitude m, km
h Specific enthalpy kJ / kg
hshell Overall height of the cylindrical vessel Cm, m
h0 Stagnation enthalpy kJ / kg
I Fuel cell current Ampere (A)
i Current density of fuel cell mA / cm2
im Maximum current density of fuel cell mA / cm2
I st Current drawn from fuel cell stack Ampere (A)
k Constant of specific heat ratio
K sm,out ,an Nozzle constant of supply manifold outlet of anode kg.s 1 / kPa

K sm,out ,ca Nozzle constant of supply manifold outlet of cathode kg.s 1 / kPa

L Thickness of membrane cm
M air Molar mass of air kg / kmol

man Mass of hydrogen inside the anode kg

mca Mass of gas inside the cathode kg

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M H2 Molar mass of hydrogen kg / kmol

mH 2 , rct Mass of reacted hydrogen in the anode kg

mO2 Mass of oxygen inside the cathode kg
M O2 Molar mass of oxygen kg / kmol

mO2 ,react Mass of reacted oxygen in the cathode kg

mN 2 Mass of nitrogen inside the cathode kg
M N2 Molar mass of nitrogen kg / kmol

M shell Mass of the cylindrical vessel kg

mW Mass of water vapour inside the cathode kg
MW Molar mass of water or water vapour kg / kmol

mW , gen Mass of produced water in the cathode kg

mW ,mbr Mass of water vapour across electrolyte membrane kg
mW ,out Mass of water vapour exit the cathode kg
n Number of fuel cells in the stack -
nd Electro-osmotic drag coefficient of water molecule -
N Number of moles in a volume of gas -
P Internal pressure of gas inside the vessel psi
Pn Pressure of denoted gas kPa
Pan,in Pressure of hydrogen enter the anode kPa
Pan,out Pressure of hydrogen exit the anode kPa
Pca,in Pressure of air enter the cathode kPa
Pca,out Pressure of air exit the cathode kPa
Pd Power delivered by the motor to the propeller Watts (W)
PH 2 Pressure of hydrogen in the anode kPa
Pi propeller induced power Watts (W)
PO 2 Partial pressure of oxygen in the cathode kPa
Pr Necessary power required by the propeller Watts (W)
Ps Electrical DC power supplied to the motor Watts (W)

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Psat Saturation pressure of water vapour kPa
Psf Fan static pressure kPa
Psm ,in ,an Pressure of supply H2 to the supply manifold of anode kPa
Psm ,in ,ca Pressure of supply air to the supply manifold of cathode kPa
Psm ,out,an Pressure of H2 exit the supply manifold of anode kPa
Psm ,out,ca Pressure of air exit the supply manifold of cathode kPa
Pt Total pressure of fan air stream kPa
Po Pressure of ambient air at sea level kPa
Po Stagnation pressure of gas kPa
r Radius of the cylindrical shell cm
R Universal gas constant (R = 8.31441) kJ/kmol.K

kPa.m3 / kmol.K
RD Relative density of air at certain altitude -
Relectrons Electronic resistivity of fuel cell Ohm()

ri Inner radius of the cylindrical shell cm

Rions Ionic resistivity of fuel cell Ohm()

Rm Membrane specific resistivity ohm.cm

ro Outer radius of the cylindrical shell cm

Rs Specific constant of the fluid kJ / kg .K
kPa.m3 / kg.K

S Allowable code stress of the metal Psi

Sy Yield stress of the metal Psi
t Thickness of the shell cm
T Temperature of ambient air Kelvin (K)
T Fuel cell temperature Kelvin (K)
T Required thrust produced by the propeller Newton (N)
Ts Static thrust produced by the propeller Newton (N)

Tsm Maximum static thrust produced by the propeller Newton (N)

Tst Stack temperature Kelvin (K)

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To Temperature of ambient air at sea level Kelvin (K)

To Stagnation temperature of gas Kelvin (K)

v Velocity of the flowing gas in a section m/s
v Velocity of the stream air through the propeller m/s
V Volume of gas m3
Vact Activation voltage losses of fuel cell Volt (V)

Van Volume of fuel cell anode m3

Vca Volume of fuel cell cathode m3
Vcon Concentration voltage losses of fuel cell Volt (V)

VOC Open circuit voltage of fuel cell Volt (V)

Vohm Ohmic voltage losses of fuel cell Volt (V)

V fc Fuel cell output voltage Volt (V)

Vshell Volume of the cylindrical vessel m3

Vo Velocity of free-stream air far ahead from propeller m/s
w Mass flow rate of gas kg/s
Wan ,out Mass flow rate of hydrogen exit the anode kg/s

Wca ,in Mass flow rate of air enter the cathode kg/s

Wf Volume flow rate of air through the fan of the stack m3 / min

WH 2, rct Mass flow rate of reacted hydrogen in the anode kg/s

WO 2,in Mass flow rate of oxygen enter the cathode kg/s

WO 2,out Mass flow rate of oxygen exit the cathode kg/s

WO 2,rct Mass flow rate of reacted oxygen in the cathode kg/s

WN 2,in Mass flow rate of nitrogen enter the cathode kg/s

WN 2,out Mass flow rate of nitrogen exit the cathode kg/s

Wsm ,in ,an Mass flow rate of H2 enter the supply manifold of anode kg/s

Wsm ,in ,ca Mass flow rate of air enter the supply manifold of cathode kg/s

Wsm ,out,an Mass flow rate of H2 exit the supply manifold of anode kg/s

Wsm ,out,ca Mass flow rate of air exit the supply manifold of cathode kg/s

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Ww, gen Mass flow rate of produced water in the cathode kg/s

Ww,mbr Mass flow rate of water vapour across the membrane kg/s

Ww,out Mass flow rate of water vapour exit the cathode kg/s

n Empirical parametric coefficient of fuel cell -

 Water content in the membrane -
 Density of gas kg / m3
m Density of the material g / cm3
o Density of air at sea level kg / m3
Pf Rise in the pressure of air generated by the fan kPa

V Change in fuel cell voltage Volt (V)

 an Relative humidity in the anode

 ca Relative humidity in the cathode

m Efficiency of the motor

L Stress applied in the longitudinal direction psi

t Stress applied in the tangential direction psi

84