Luận văn nghiên cứu phát triển hệ thống vi lỏng thao tác và cảm biến tế bào ung thư

1.1 Introduction of biological cell assay and current situation 1.1.1 Biological cell assays 12 Biological cell sensing systems hased on microfluidic 1.2.1 Overview of the cancer situati

VIETNAM NATIONAL UNIVERSITY VNU UNIVERSITY OF SCIENCE

Dé Quang Léc

DEVELOPMENT OF A CANCER CELL MANIPULATING

AND SENSING MICROFLUIDIC PLATFORM

(Nghiên cứu, phát triển hệ thống vi lỏng

thao tác và cảm biến tế bào ung thir)

PhD ‘THESIS IN PHYSICS

1lanoi - 2019

VIKTNAM NATIONAL UNLVERSITY VNU UNIVERSITY OF SCTENCE

Đỗ Quang Lộc

DEVELOPMENT OF A CANCER CELL MANIPULATING

AND SENSING MICROFLUIDIC PLATFORM

(Nghiên cứu, phát triển hệ thing vi lông thao tac và cảm biến tế bào ung thư)

Major : Radiophysies and Electronics

Pledge - Lời Cam đoan

Thereby declare that thie thesis is solely my own wark The data in this thesis are the resnlts of my personal research and have not been used in other publications

Acknowledgement

Toremost, T wonld like to express my sineere gratituđe io my advisor, Prof, Chủ Due Trính, for the cantinuons suppam of my Ph.D study and research, for his pac tience, motivation, enthusiasm, and immense Imowledge My research would have been impossible without his guidance and supports

Besides, 1 would also like to express any appreeiation Wo Dr Bui Thauh Tung whose office door was always open whenever I ran into a trouble spot or had a question about my research or writing He consistently allowed this paper to be my own work, but steered me in the right the dircction whenever he thought I necded it My sincere than alse gous Wo Dr Do Trung, Kiet He hus offered ine uuy good oppertuuitics in

my life and Ph.D study

Further, T must express my very profound graritude Prof Chim-Ping Jen and T would like to thank all of my friends at Department of Mechanical Engineering and Automution for Uheir support duriug wy intcruship program in National Chung Choug University

Tam forever thankfnl ta my colleagues at the Faculty af Physica for their friend- ship and support, and for creating a cordial working environment

Last but not the least, I would like to thank my family: my mom and my sisters Jor cneouruging; aud supporting ue spiritually throughout say life Most importantly,

I wisk to Ghauk my loving and supportive wife, Phau Thi Tuyel, aud aay beloved

daughter, Anh ‘I'hu who provide unending inspiration

1.1 Introduction of biological cell assay and current situation

1.1.1 Biological cell assays

12 Biological cell sensing systems hased on microfluidic

1.2.1 Overview of the cancer situation and the urgency of cancer cell

diagnosis „ 1.2.2 Current studies on the detection cancer cells 1.2.3 Material for microfluidic systems

1.3 Conclusion of the chapter and the direction of research

2 Biological ccll manipulation methods

2.1 Overview of biological cell manipulation 2.0.0.0

BLL Mechanicalimethod ©0002

2.1.2 Optical method 2.1.3 Magnetical method

2.1.4 Filectrieal merhod 3

2.2.1 Dielectrophoresis cell manipulation theory | : 43

Numerical Calculations and Simulations» 2.2 47 3.8 Devigu and Experimental preparation 2 Leese 8?

2.3.2 Microelectrade structure fabricarion prooess : 87 23.3 Microchannel fabrication process 6 ee 60

234 Cell vample prepation co cee 8

24 Resnits and Disenssions ` va : đã

Biological cell sensing, detection und measurement systems 70

BL Overview of biological cell detection aud euumeration techniques ZL

3.1.1 Flow Cytometry hased on fluorescence activation method 2

2.1.3 Biclogival cull impedance sncayurerments in stutic Muidic 80

1 Biological cell sensing theary ` Lee BR

3.3.1 Microfluidic flow measurement and detection 2 - 90 3.3.2 Biological cell concenlzution ønđ đetoeiion áo 95

34.1 Object detection in millimeter scale fluidic flow chaunel 97

Biological cell detection in microfluidic flow 104

4 Aptamer immobilization based on gold surface 118

4.2 - A549 call line deteetion on aptamer immobilized gold suriaee 122

42.1 Aptamer immobilization ơn gold surÏaoc proeess "-

Apbamor spooilieity with A549 ucll line experiment 128 4.2 Cell preparation results - : : 123 4.4 A5AQ cell line detection on gold surface based on aptamer immobilization

44.1 A549 coll linc immobilintion on gold substrate 187 4.4.2 Aptamer specification experiment with A549 eell line 128 AA3 Aptamer specification experiment with A549 cell Tine versus time 129

Molecular structure of Polydimethylsiloxane (PMS)

(A) Lmage scanning electron microscope system of micro gripper: (B) Bright field microscopy shows the operation of separating Meoli K-12 bactwria from blood volutivns contuiniug red bleed cells

Magnetic manipulation methods

Dielectrophoresis manipulation methods

Electrophoresis and dielectrophoresis: (a) ‘Lhe force exerted on charged particles und uucharged puclicles in the uniform eleetric field; (b) A neutral particle in a nonuniform elecwic field creates a total force acling

on the particle hecanse the magnitnde of the electric field at the two ends of the electric dipole is different

Siumlution program iuterluee finite elcuenits using, COMSOL

Sketch view of the simulation chamber

Proposal of biochip aensor based on finiđic chambar

Process of fabricating microelectronic ebructure and packing with micro- channel structure

The process of mvld fabrication with SU-§ muturial 2

2.10 Process of manufacturing PDMS chip from SU-8 mold

Images of fabricated clevice (a) Fabricated chip; (b) working chamber:

{c) fabricated clectrode pattem 22 eee

The syuare of the clustric [eld (B®) on micro-cleclrode pairs with inward

stepping electric field in a numerical simulation

The simnlation results of the manipulation of cancer cells from a hlaod

sample using inward (16 V of peak-to-peak voltage - 1 MHz of frequency)

A549 calls concentration experimental results utilizing step electric field

applied to: (a) 5* electrode pair; (bh) 4% electrode pair; (c) 3°? electrade

pair; (d) 2" electrode pair

Components and operating principles of flow cytometry in flow call

7; side scatter

counts (photomultiplier tubes - PM]; forward seatter - ÈÌ

- 88G) ¬

Biological cell impedance seusing in Muidie low 2.0.00 oe

Biological cell impedance sensing in static fluidic

Model of coplanar capacitor,

leetrieal model of (u) a single-cell in suspension with complete cleetrie

cirenit model; (b) single-cell in suspension with Foster and Schwan’s sim-

plified cirenit: model; (¢) Fquivalent electric cirenit, af the biological cell

detection structure in microfluidic channel; (d) quivalent electric cir

cuil of seusing sysicmn when the cell is located on one sensing, cleclrodes

pair; () The simplified circnir madel for comting system

Microfluidic platform for hialogieal cell detection in mierolfnidic flow (a)

Sample is mixed at mixing area (1), then separated at cell manipulation

area (2) before passing through the counting area (3); (b) ‘lie detailed

design of mieruchip proposed for cell impedanee eylumelry purpose

Block diagram of the measurement selup ee

Block diagram of proposed dietectropharesis micraflnidic enrichment

platform with bnilt-in capacitive sensor for cireulating tumor cell de-

tection

Mcasurement sctup: (a) measurement methods; (b} measurement setup

Block diagram design of the DC4D finiđie sensor,

fa)The DCAD ontput voltage respanse when a plastic particle crosses

electrodes watcr channel ond salt sulution chennel; {b} The DC4D out-

pul voltage amplitude versus particle voluine in various conveutration

of salt solution

fa) Capacitance change of sensor with three different materials; (b) Ob-

ject’s diameters are 25 ym and Maximum differential capacitance output

Experimental result showing the output signal when uly bubbles cross

the sensing area

Working principle of C4) sensor system

(a) Variation of resonance frequeney when air bubbles move through

channel filled with differeut NaCl concentrations solution; (b) Depen-

dence of resonance frequency change on the size of} air bubble moving

throngh DT water channet, air bubble moving throngh oil channel, and

water droplet moving through oil channel

Fabricated replaceable mmicrofluidie chíp

Simulation results: Admittance change when a cell with radius of 10 gan

moves across the sensing electrodes in: (a) Suerose 8.6 % sohnion; (b}

PBS LX solution

Simulated admittance change according to: (a) the cells vertical posi-

Output signal variaion when an A549 cell passing over sensing ele

trodes (Insets are the position of the cell corresponcling to the output

voltage at point A, B, € in the graph (scale: 100 zmm)

Measured voltage trace of 4549 cells in continons flow in 8nerose 8.6%

solution and PBS 1x solution,

Simulation results Hlectrie potential distribution of model: (a) top-down

and {b)front cross-section views; (c} Capacitance change versus number

{a) and (b): Bode plot of sensor; (co) The impedance spectrosenpy change

versna number of cells on sensing area; (d) Microscopic image of electrode

3-180 cull detection operation, A, B,C, und D correspond te inercasus

in cell number on sensing electrodes (error har shows the accuracy of

Measurement equipment)

(a) Equivalent circuit of contactless impedance sensor: (b) Block diagram

of circuit for differential impedance measurement © 2 «

A3AY cells immobilization on gold substrate utilizing NH® aptamer pro:

too 2 ee ¬

Microscopy nage of A549 cell line culture provess: (2) Before culture;

{b} After culture; (c) Before using Trypsin; (d) After using Trypsin

ells counting of the A549 cell line for the experiment using counting

ASG cells immobilization perforinsave ufter being washed: (w) 254M

NH2-Aptamer; (b} 10M NH2-Aptamer

Positive control experiment

Negative control experiments

Aptuiner spuuificution experiment with A549 cell fine versus tie: (a)

After cell injection; (b) After 13 minntes inenbatian and washing

Schematic diagram of dielectrephoresis inanipulavion voutzol circuit

Schematic diagram af signal processing circuit for cell detection in fluidic

AB Schematic diagram af signal processing circuit for cell detection in fluidie

hamber

Comparison between cammon cytometry methods | | 83

Material permittivity parameters used in simulation [162] 87 Qeometry parameters used ìn simulation - 8 Simulation material parameters (162, 179 co 89

Coulrol experiment for uplumer spceiliciiy đócrminalion 124 A549 call viability vereus tìme when euspended in PDS 1X aolution and

Sucrose 8.6% solution c we "¬ + + 128

Bovine Scrum Alburnir Capacitively Coupled Contactless Condnetivity Detection

Charge Coupled Device

Contactless Dielectrophoresis Capillary Electrophoresis Circulating, Tumor Cells Cirenlating Tumor DNA

Circulating Tumor Microemboli Circulating ‘lumor Materials

10

Epithelial Cell Aclhesion Molecule Epithclial Splicing Regulator 1

Inductor Capacitor

Magnetic-Activated Cell Sorting

Madin-Darby Canine Kidney Epithelial

MEMS /NEMS Micro/Nano Elcctro-Mcchanieal Systems

nDEF

PS

CAD

Negative Dielectrophoresis Phosphate-Buffered Saline

assive Capacitively Coupled Contactless Conductivity Detection

T1

Photomultiplier ‘lubes Red Blood Cells

Reverse Transcription Polymerase Chain Reaction Systematic Evolntion Of Ligands Ry Txponential Rnrichment Superparamagnotic Beads

Side Scatter White Toad Cells Wheat Germ Agglutinin Micro Electrical Impedance Spectroscopy Microfluidic Maguetic Activated Cell Sorting

Abstract

The urgency of the project:

Currently, cell assays are one of the indispensable proc

a for diagnosing diseases

or evaluating pathological conclitions of patients In Vietnam, 23 well a3 im many other

countrics around the world, the ecll assays for detecting, analyzing and evaluating

the number und types of cells presenting in clined samples are oflen performed ina medical center where fully equipped with facilities and services Ilowever, when taking traditional tests, patients often have to pay high costs and spend a lot of time waiting

at crowded medical centers In cell assays, in addition to classifying and labeling target celly, Uke process counting the numbers, ssaussing deusily, aud cell concentration in

patient’s samplea alsc plays an important role in influencing to subsequent tests Th medical centers and research laboratories, cell conmting, as well as the determination of

target cell concentrations in patient’s samples, are often performed using some common

aulheds such ux limiting dilution (limiting -dilutiou culture), ELISPOT test meted or Flnorescence-Activated Cell Sorting (FACS) method based on flew cytometry principle With the cell assays that require high throughput, such as CD4 — T lymphocyte eonnts

in JUV diagnosis and monitoring, flow cytometry is often preferred due to the ability

ta analyze cells quickly and capable of providing high throughput

However, the commercial machine and devieus ure often high cust, requiring tech- nivally qualified leclnivians, not suilable for using im limited resourves areas, such ax

in lower-level hospitals Vor that reason, many research groups around the world are

interested in ceducing costs by restricting the use of expensive optics and optical de-

viees using aulabeled detection (labe-free) approach, simplifying the strucbure, cle

Along with the development of science and technology, specifically, the emergence of

18

microelectromechanieal systems integrated in mierofluiđic simetnres, miniaturized oell assays hased on microfluidic have been develaped by many research groups to perform cell enumeration on micro-chip platform toward simpler, quicker and more reasonable, especially suitable for using in limited substance and facilities

‘The integration of vell detection aud counting ystems based ou inierolluidies not only brings advantages in terms of reducing time performing tests, sample volume,

as well as minimizing the chemical substance needed for the tests, these microfluidic devices also help paticnts shortening the testing time, minimizing the number of sam- ples needed and reducing tke cconomie burden Bevides, Uke usicrofluidie platform also allows the integration of components and sub-processes pretreatment such as cell lysis

or cell staining on microfluidic chips

On the other hand, the microtluidic chips may disposable, that can provide a disinfection environment, avoiding the risk of cross-contamination, the risk of danger in 1naipulutdng the samples The development of microfluidic biclogieul call devection cus carry this device to laboratories, enabling biological stndies in cancer detection, drug development, genetic research and possibly brings new opportunities for biomedical research ‘Lherefore, conducting research to develop living cell counting and detection systems for rapid cell tost upplicutions is u highly practical and scicutific issue

Based ou analyzing Une requirements as mentioned above, in this thesis, the au- thor has studied and proposed a system capable of testing living cella hased on mi-

crofluidic techniques to enrich, detect and quantify biological cell concentration ‘I'he

proposed systern includes of a suiero-munipulution structure und embedded impedanee sengor This is perfectly suitable for using for living cell fast detection and diagnostic Kits where the sensing chips are used only ance and are replaced after each nse

Scientific and practical significance

‘Yhis is an interdisciplinary topic, involving many ereas such as electronics, con- trol, microfluidic, physics, biology and uticrofabrication The senyer syste will detect the presence of a specific cell and evaluate the number of that cells in the sample solu- tion The successful implementation of this study will provide a tool that can quickly

detect the number of cells and cell population can be monitored without the use of

14

very expensive eammercial eqnipments With regard to medical devices, the prodnet

of the proposed device also provides a tool to detect and count the number of cells

Object and purpose of research

The object of the dissertation is the biological cell onrichment and detection sys- bom; ypecifivully in this case, A549 und Sarcoma 180 cell lines The proposed sLructure integrates with microfluidic systems that allow enrichment cell detection in fluid sam- ples with a very small volume and a short duration of the experiment The objective

of the research is to research, design and perform the experimental tost on proposed system using micro seusor ehipy based on the principle of impedances sensors, The sensor chip is able to detect the presence of cancer cells in patient's samples based on the change impedance between built-in sensor electrodes The output signal data of the sensor chip is recorded and processed to provide the number of ceils in the microfluidic channel and chamber of the sensor chip

Methods and scope of research

In arder to realize the specific objectives, the thesis implements the main research contents ineluding an overview of research, simulation, fabrication, and experimental measurement to evaluate simulation results Specifically, the study of the design of the unicrofluidie yuructure integrated with manipulating cleelrode structures und sensing electrodes Modeling aud simulating the response of objects in tuicrofluidic environ menta including microflnidic chamber and microfluidic channel In addition, this study implements circuit control design, manipulate cells in the microfluidic chamber and signal procowing, circuit bu deWwet Une prescaee of culls iu the sensor urcu of the systen Finally, the biological cell counting is experimental performed in standard solution nsing bie-chips and mannfactared cirenit, board

‘The thesis structure

"Lhe thesis consists of 4 main chapters

Clupler 1: Tu this chapter, firs), » brief introduetion to cell aysuys ud cell assays applications is presented Then, review of researches and applications of inierofluidic technology in cell assays in domestic and international research groups is provided

Finally, the results of developing fluidic flow detection systems in millimeter and mi

Tã

crometer channels are given

Chapter 2: This chapter delve into the stmeture of a microfluidic chip used in cell manipulation and enrichment ‘Chen, theory and application of dielectric electrophoresis foree to manipulate biological colts in fluidic chamber arc presented Basie theories used

iu simulation Luols by COMSOL Multiphysics utilizing Uhe finite clement wethod are also provided Next, information of experimental processes including the fabrication

of microfluidie chips based on microfabrication technology, biological call preparation process is introduced Finally, experimental ccll manipulation performance is reported und compared wilh simulation results

Chapter 3: Tn this chapter, two microfluidic system structures to derect: cells in the microfluidic chamber and in the microfluidic channel based on impedance sensing approach are presented Numerical calculation results based on impedance technique followed by experimental results are reported In this chapter, experimental investiga- tion revully on object wud fluidic flow detection based ou impedance upprouch ure also presented and discussed

Chapter 4: In this chapter, the issue of cell detection based on aptamer- biosensor

is presented, including the combination of impedance sensor methods with gold elec trode surface funetioualiaation process with aptamer specille to target cell Exper imental resully of the cell culluze process and exauiues biological cell viabilily after enltnre and preparation are given Then, in order to determine ability of aptamer im- mobilization on gold electrodes, several control experiments have been performed and reported

Finally, the author concludes the research study and proposes the next research

direction.

Chapter 1

Tntroduction

1.1 Introduction of biological cell assay and current

situation

1.1.1 Biological cell assays

is defined as the measurement and analyzation of biological cell's re-

Cell assay

sponse according to the external physical and chemical stimulations The cell's re- sponses are divided into the biochemical alterations of intracellular and extracellular media, cell morphology, motility, and growth properties [4] 'Lhhese responses of biologi cal culls cun assist Lo distinguish coll pheuoty pe and ure typically monitored ina culture dish or a multi-well plate, while nore revemly uticrefluidie devives lave been utilized

A cell assay performed in a microfluidic device is alternarively termed an on-chip assay

Biological cells are the primary elements of life and are often considered to repre sent busic models for complex biological systcis Some studies, such ay worphologival analysis and cell growth, cau only Le evaluated by cell assays Other studies, such ax cell biachemical analysia, can also be performed by simple tests snch as molecular assay

to analyze molecular interactions Molecular testing is faster and less complex but can lead to confusing conclusions because it is inpossible ta mimic the complex and special propertice of the intraccilular environment Therefore, ccll oxperiment analysis is more

suitable for the stndy of living aystems However, there are some limirations sch as

17

costly implementarion, time consnming and much more complex than other types of analysis

Gell tests are usually performed in culture plates based on multi-well plates (molds containing 96, 384 wells, ctc.) While culture plates require large volumes of solutions and chewivl substanee, mulli-wall plates require only uieroliter of sumple volume and allow simultaneous analysis of varions cell types Readers will read optical ine formation designed to analyze samples at the same time using technologies such as

fluorescence and polarization, luminescence or absorption Multi-well discs and disc

readers have become the staudard Wol for sucdium und high-throughpub ser

cell and biamolecular tests

Microfinidic technology is utilized in micrachip fabrication whereas it provides the micrometer fluidic channel, which allows integrating numerous processes in only one platform Hence, the experiment time and sample volume can be reduced when

experiments ure performed un the microfluidic platlorin Thus, inicrofluidies ullows ex- periments to be carried ont that cannot be performed simply by miniatnrizing and mechanizing conventional laboratory procedures using robotics and microplates Mi crofluidic technology creates new opportunities for high throughput cell screening de vicos [36]

Otherwise, :nicrofluidic devices provide various benefits in cell assays due to Lhe homologous between cell size and the microfinidie channel dimensions (the depth and width are normally 10 - 100 ym) At the micrometer-in-size channels, the fluidic flow is choraeturized by laminar flow, The combination belween laminar flow nud the diffusion provides the formation of highly resolved chemical gradients across small distances An- other advantage of micraflnidic devices is the mereased surfaoe-to-valume tatio which facilitates the heat and mass transfer, as well as scaling the electrical and magnetic fields that are often used in cell analysis, Hence, the cell assays performed in a mi- crofluidie devive require 6 saul volume (i.c., sub-microliler volues} of cell sample, the concentration of cell sample is kept at high conceutration, which facilitates the detection sensitivity

For

However, microfluidic system does have some disadvantages for cell assays

18

instances, the difficulty of controlling many reagents simultaneously; the high surface- to-volnme ratio of microchannel enhances the adsorption of molecules anto channel walls which directly reduces the effective concentration of reagents [4]

Typically, micro flow provides flow rates usuolly smollcr than 10 pil/min Hence,

it provides the ability of continuous und uniform perfusion of media as well us steady cnlturing conditions When designing the microfluidic channels for cell assays, the flow

is designed to have small shear stress impacts on the adherent cells while the cells in suspension can be assaycd while carried by bulk micro flow Manipulation techniques can be used for suspension cells sueb uy hydredy namic Wrapping or eell immobilization

transmitted light, making it easier Lo moniler the process in miicro-systems using opli-

cal methods For long-term teata, the sterilization of micro-channel i needed to avoid infection in the cell solution ‘ests involving adherent cells often require the eanal wall

tv be pre-treated by utlaching suitable protcins (e.g Fibroncetin, collagen, or laminin)

to enhance adhesion On the other hand, when experimenting with suspension cells, channel walls can he coated with bovine serum albumin (BSA) to prevent nonaperific adhesion [102, 187]

Wheeler et al focusect on the use of microfluidic technology for single cell anal ysis und deseribed in detuil thet cell manipulation is a problem thai can bè solved with inicrofluidie systems [187] Phowlithography Lechniques using masks and pho- toresist materials are used to create micro-channel systems based on l’M§ materials

Studer et al have developed a microfluidic device that can be used to organize and

19

manipulate biological cells |I60| They built a device based an PMS material nsing multi-layer soft etching The device consists of many active nite (mixing pumps) con- trolled by pressure through micro valves ‘he use of microfluidic techniques in cell experiments has become popular and is widely used duc to many outstanding advan- ingos of microfluidic techuology in iulugrating inte micro-systemy Especially, with Uae strong development of technologies nsed in micromachining in recent years, cell assays and experiments have become fast with high relisbility Guilhem et.al summarized the tools of microscopic ongincering to the study of biological cclls, focusing on the use of

PDMS bivvompatible mutcrials [153]

1.2 Biological cell sensing systems based on mi-

crofluidic

In order to sludy aud design the experimental chip [or cell assays, most scientivie

and research granpa now nae microfluidic teckhmology for tha design of flow channels, incubating chambers, and other mechanical structures for the construction of biological processing chips, especially with cell assays Krom a technical point of view, must cell tesls, expecially circulating Lumor cells (CTC) experiments include three basiv stepy

as follows: preparation of bioad samples and pretreatment needed to separate tumor cell; Cell staining with antibodies or gene exploration with DNA probes and CTC cell detection steps 63] (Figure 1.1) ‘l'ypically, the first step of the cell tests is the prepa ration of blood samples including red blood cells lysis and separation of white blood cells from the whole Ulved solution Thea, the separution of tumor cells can be donc

by varions methods snch as antigen-antibody interactions, aptamer - receptors or some physical properties of cancer cells including size, specific weight or electrical proper

tics, In the next step, tumor cells can be stained with antigen, aptamer or markers and

gene amplifiers using DNA probes or DNA primers [63], Iu the Gual step, oolly an be

detected and analyzed based on a mmber of different methods snch as cell analysis

in fluid flow, nsing microscopes, nsing flnorescence (aptoflnidic) method or molecular

biological methods such as polymerase chain reaction (PCI)

20

Figure 1.1: Block diagram showing three basic steps for a CTCs cell test

In reality, recent studies on living cells testing systems based on the microfluidic

platform are designed and fabricated by combining microfluidic systems with biological

interactions, physical interactions These properties and interactions are specific to the

products of various fields ranging from nanotechnology, electronics, sensors, biology,

fluid mechanics and microfabrication In addition, the separation system needs to be

designed with the requirement of high accuracy that the microfluidic systems with the

above-mentioned advantages meet these requirements

Microelectromechanical laboratories and micro-

tems can basically meet the research requirements of this topic However, the combination of biological components

and microstructures based on MEMS platform to capture and detect cells correctly is

still a problem that requires a lot of interdisciplinary experience

1.2.1 Overview of the cancer situation and the urgency of

cancer cell diagnosis

Current situation of cancer and traditional diagnostic methods

Cancer is the second leading cause of death after cardiovascular disease Cancer

is a malignant disease of cells with infinite reproductive properties, not following the

21

According to statistics in 2018 on the cancer situation in the last 10 years in the United States, the number of new canccrs in men and women are 856,370 and

‘tively Whereas, there were 323,630 deaths in men und 286,010

in lung cancer, tumors are detected when they are 2-10 cm in diameter or larger, so treauuent is often ineffective Therefore, recuut studies are trying tu find aud create molecular diagnoses to detect canoer at an early stage, before metastasis The detection of cancer in general and hing cancer in particular as soon as possible can facilitate doctors have appropriate and effective treatments

Currently, diagnosis ix bused on many different methods In the clini

cal diagnosis step, doctors’ determinations often rely ou a fawily history, personal history, patient's mediesl history and aymptoms that signal some types af cancer be- fore taking the clinical examination ‘Ihe physical examination can detect tumors (by looking, touch), tumor size, Lumor propertics (stiffness, suobility, and uleeration), lyar phadenopathy and some other phenomena, In addition, sub-clinical diagnosis may also provide more detailed information for the patient's disease situation The sub-clinical diagnosis investigation commonly inctude blood tests, such as haemogram, myelogram; use tumor markers to screen, track and evaluate the effectiveness of treatment, relapse, prognosis; Eudoscupie methods, iugiug, diugnoslivs such us rudiogtuphy, ulurasouegra- phy, computerized tornography or maguelie resonance imaging can also detect shallow tumor (breast, thyroid .) and deep tumor (liver, pancreas .) In addition, cytology

diagnosis method using smear, spread, cell block, small needle aspiration technique also

brings some advantages oompared to diagnosis of histopathology such as fast, simple diagnosis, low cost, Jess complications, can nse the template to stain imme tissue culture Besides, the diagnosis based on histopatholegical methods is considered the

"gold" standard to have a diognosis of cancer with methods of taking samples sueh os needle bivpyy, biopyy und interual biopsy examination aud surgery of ypeeimens, The application of histapathological methods allows determining the henign or malignant property of the tumor, derermining the type of cancer, histology and supporting for other tests such os immunohistochcmistry, molecular biology diagnosis

Cancer metastasis mechanism

Figure 1.2 shows a diugram of the nelasiatic process through CTOs circulating

cirenlation, a majority of CTCs die dne ta programmed death (apoptosis) and necrosis,

releasing debris, call fragments, intracellnlar substances (CTMat and CTDNA) Besides C'l'Cs in the blood stream, there is appearance of C1l'M ‘I'he presence of (1M in the bload circulation system is even rarer than CTCs C's tumor cells can be separated from CTM when subjected to shear or impuel forces in the blood Mow: they can also altach lo tumors or other blood cells when collided due tv adhesion phenomencu, The micro-environment: built into CTM is uniqne protecting the internal tumor celis from damage Therefore, CTM is considered to be more dangerous than individual CTCs when tumor cells can separate and grow in blood vessels und unay break blood vessels

Meanwhile, CTCs often have to penetrate through the blood vessels and develop to

form metastases

‘The discovery of circulating tumor cells was first reported around 1869 Ilowever, studies of C1'Cs are net common herause the circulating tumor cells are rare cells in blood stream with the ameunt of abour oue CTC in 108 - 107 white blood cells in cancer patients Therefore, this leads to the difficulties in enrichment and detection of

33

Primary tumor Micro-metastasis

Blood-vessel reupture &

secondary colony formation

Figure 1.2: Schematic showing the tumor metastasis through CTCs/CTM

CTCs cells

research field of CTCs has attracted the attention of many research groups in the world

In the study of CTCs detection and analysis equipment, the isolation and separation

of CTCs to enrich CTCs concentration plays an important role in enhancing detection

efficiency and measuring CTCs concentration in patient's samples This has a great

influence on the accuracy and effectiveness of treatment and provides a method for

determining the cancer status of patients with the lowest level of intervention In lung,

cancer, A549 cell line is a typical type of carcinoma cell of non-small cell lung cancer, which is defined as a circulatory tumor cell in the early stages of cancer [69]

With the development of micro fabrication technology, cancer detection and di-

agnosis systems based on the detection of circulatory tumor cells in blood samples are being studied by many research groups and are optimally integrated into small

sized devices (lab-on-a-chip) Usually, these devices need to be fully functional from

24

cell sereening, cell enrichment, trap and capture target calls to measure parameters, analyze and process for the purpose of retrieving necessary information of the cell

1.2.2 Current studies on the detection cancer cells

General situation

Beeause CTC cella are a rare type of cell with the appearance of about 1 CTC cell

iu the mixture of about 10*-10" while blood cells Therefore, the separation, manipula- tion and detection system shonid he designed with very high sensitivity requirements, neually to single cell levels A majority of detecting circulating timor cells assaya are im- plemented by commercial equipment systems such as flow cytometers 163], CellSearch syste [L16], high-quality Muorcsecuce scauniug microseope [LL] fiber-optic array scanning technology (FAST) 93], isnlated hy the siza of the tumor cells (isolation by size) of epithelial tumor cells - TSET) [141] and Taser scanning cet! analysis device 137]

Along with the development of micro-fabrication technology, combined with the integration of physical methods on the microfluidic technology platform, current studies

are mostly directed lowards overall teat chips Vab-ouew-chip) upplicd in point-of-care

diagnostic This integration brings benefits in reducing the size, reducing costa as well

as minimizing the chemical substances needed for the device In addition, the mi crofluidic platform also allows the integration of sub-components and proccasca such os prolreatinnt of yainpley suck as coll Isis or cell staining right ou the sicrolluidie ubip Besides, the chips used in the above studies may be dispasable which provides a disin- fection environment, avoiding the risk of cross-contamination and risk of manipntating

the biological dangerous samples [20]

Situation of domestic research

With the actual status of the early detection and diagnosis of cancers in Vietnam, many research groups have conducted research towards the field of biomedical sensors to detoct biological elements by various different mothods In which, there are orientations

to the polymer structure of biomedical applications in drug delivery system and immune sensors, electrochemical micro sensor for rapid identification of aflatoxin in milk by the

35

resaarch group of Nano Medicine Materiala Department, Institin

2 of Materials Science, Vietnam Academy of Science and Technology In addition, the research team has alsa focused on studying several biccompatible nano-systems of magnetic particle cores for cancer diagnosis and trcatment; rescarch on manufacturing and testing nanometer- sized drug matcrials capable of slow relumse, selective targeting Wo cancer culls aud study the proress of fabricating nanostrnctnred drug systems and evaluate the effects

of they on cancer cells enitnred in vitro [51, 131, 132, 170] Ar ITTMS, Manoi University

of Science and Technology, the rcscarch group of Assoc Prof Dr Mai Anh Tuan also hud the results in the study of bivciewieal seusur for detection of iuflucua virus aud Rubella nsing highly sensitive DNA sensor based on nanostrnctured porans membrane [161, 165° Besides, with the focus on stndying the system of hialogical residue detection sensors in seafood based on quartz crystal microbalance structures, [CDC team at Vietnam National University of Ho Chi Minh City achieved several initial successes and devclopineut of jutreduciug products te applicutious in (he detection of certain substances snch as narcoties, F.cali Q157-IT7 bacteria

In recent years, the Department of Animal Cell ‘lechnelogy, Institute of Liotech nology, Vietnam Academy of Science and ‘lechnology has collaborated with Institute

of Material Scicuee, Institute of Physics o conduct research ou iuterdiseipliaary co-

search directions for geuevic engineering aud nanotechnology, Oue of (ie main research

orientations is to create a targeted dmg delivery system in cancer diagnosis and treat-

ment ''he research group introduced a drug-delivery system made of natural polymers

or synthetic polyners und a tagel portion mude by recombinant antibody wolecules specific to HER2 and HERA antigens, Cyira 21-1, CD25, CD20 were purified after ex- pression in different systems (R coli, insect cells, in silleworms, in eslls CTIO, ITTIK-293)

or the antigen-specific aptamer of cancer cells [J] In addition to the above research groups, there are a nuunber of other groups that are also beginning to study and con- duct reseruch ou bioscuser sysluus However, studies on scusor dutcction of caucer ells have not been conducted much in Vieluam due to the high seusitivity rcequirernenly and related to the interdisciplinary research field such as micro-fahrication, electron-

ies, biology, and physics, etc ‘Ihe study of designing and manufacturing microtluidic

syatems oamhinsd with hiolagical srrnetnres for early detection of cancer is a highly

practical and scientifie demand

Situation of international research

In addition to conventional cancer diagnosis methods, the method of detecting the number of OTC in the whole blood also brings many benefits in supporting tu- quor growth proguusis and moniloring Weutraent cfficicney Aluhough the diuguosis aud prognosis of CTC by this method can be implemented quickly, the determination and detection of CVC cells by this method is seldom used in clinical tests due to the diffi culties in CTC isolation and identification Biological cclls cnzichment, manipulation, und detuclion are important issues in wany differont biornodioal applications, including isolation and detectian of rare CTCs, enrichment of calls in anspension, separation cells based on physical properties, captnre or locate individual celis ta determine the nature property and type of cells Despite many difficulties, the research and development of

CE cell detection in whole blood systems have attracted many research groups in the world in recent: years

Tignre 1.3 shows the block diagram af a CTC: cell separation aystem msing mag- netic nanoparticles and biological antibodies combined with microfluidic techniques

"Lhe biological chip structure consists of two parts: the first part operates to sepa- rute the while bleed cells (WBCs) from the solution using » combination of maguetic nanoparticles and WBCs-spevilic aulibodies (Antibody - CD45), The second part of this stmnetnre is the geometrically activated snrface interaction (GAST) region: the celle after being filtered in the first part are passed through the GASI region to isolate Ep CAM protein-expressing cells using, Ue auti-BpCAM antibodies immobilized on wall

of the channel In this stage, EpCAM-expressing cells are enriched in fluidic chamber According ta the anthors' report, this microfluidic magnetic activated cell sorting (j1- MACS) system allows 763 cancer celle to be separated in 5 ml of blood at a tlow rate

of 400 m/min Cancer cells have been classified with a separation efficiency of 10.19%

and 22.91% bused on EpCAM or HER2 membrane proteins respectively [74].

tt CTC(EpCAM) ÍỔ)CTC(EpCAM+) Whitebloodcell '#' ˆ Maonetc nanoparicle

Figure 1.3: Block diagram of a CTC cell separation system using magnetic nanoparticles

and biological antibodies

1.2.3 Material for microfluidic systems

Polydimethylsiloxane (PDMS) is a group of high-molecular organosilicon com- pounds commonly referred to as silicon (Figure 1.4) PDMS is transparent, inert, non- toxic, and non-flammable It is also called dimethicone and is one of many silicone oils (polime siloxane) Its application is applied from manufacturing contact lenses and medical devices to elastomers It is also present in shampoos (dimethicone makes hair shiny and smooth), food (antifoam), solder, lubricant, sand, and heat-resistant bricks Due to the biological compatibility property, PDMS material is widely used in microfluidic channel in biological equipment and devices

The PDMS material can be used in quickly creating microfluidic mold chan- nel PDMS is flexible, hydrophobic and can be welded very tightly under a uniform

pressure PDMS has the ability to heal various smooth surfaces, including glass, sili-

28

Figure 1.4: Molecular structure of Polydimethylsiloxanc (PDMS)

con, silicon nitride, polyethylene, carbon glass, polystyrene oxidation, fluorocarbon and metal Since soft lithography can be used for rapid prototyping, PDMS is often used

iu livhography as an easy cauterinl for sketching saicro-sized chunuely Compared to glass, PDMS has a lower thermal conductivity, much higher hydrophobic, and these characteristics are sufficient conditions for PDMS ta he a suitable material for making microfluidic channels On the other hand, /DMS is inflated by many types of organic solvents such as oils, but still not affected by water, nitromethane, ethylene glycol, acetonitrile, perflucrotributylamine, perfluorodecalin and propylene carbonate More- aver, PT)MS can he enhanced by PDMS surface coating with sodium silicate to resist organic solvents Llowever, hecause L-DMS shape is flexible, the metal electrode may not be patterned on the surface of this material Another advantage of POMS is ite transparcut property, it cum be cusily viewed inside the chunucl Thus, PDMS :aterial

is very suitable for use ag a fuidiv duauuel,

Glass slides are prepared ta fabricate micro electrode strnernres by depositing Chrominum and gold in tum onto the surface of the glass slide ‘I'he thickness of the two layers of Chrominumn and gold on the suelace of the glass blade afler vie evaporation prowess iy about 20 nin and 100 mu respectively Chrominua layer is used as a layer to adhere gold layer on the surface of the glass The nse af gold electrades provides certain benefits such as relatively chemically inert, difficult to oxidize during the experiment, and is capable of binding to sulfur-hased or easily functionalize the surface to bond with other organic molceules

1.3 Conclusion of the chapter and the direction of

research

From the above analysis, it is shown that the research of designing, manufacturing,

measuring and testing the operation of a microfluidic system combining aptamer and impedance sensors lo deleet cancer cells, However, when combining biologicaÏ sirue- tures based on MEMS chips to capture and detect rare cells, it shows a problem that

le

requires alot of experience in this interdisciplinary work Chirrentiy, there ïs mmill a li

in the number of rescarch and report about this rescarch ficld in Victnam Research and

development of bio-chip systuus thus play an importaul role in Vietnum’y seicutific and technological develapment However, this is a very new research direction and requires interdisciplinary as well as high technology to conduct, the experimental research This research topic proposes a novel approach in biological device studies, aptamer achieve- ments and cell detection technologies using MUMS microelectromechanical technology With the development of ueicuee and tocbnology rocenty, the ability to auanipulate, trap and derect single cell in the fluid flow can he evidenced This approach pramises ta produce a high tech product that meets the practical requirements indiseases diagnosis

in particular, in the treatment of cancer, and some other diseases requiring the need

Ww scleclively count single coll during, diagnosis and treutment,

on the reviewed methods, the author has sclected diclectrophoresis method to manip- ulate cells with many advanuges in inlegruting with microfluidic systaus Thea, ä microfinidie chip design is proposed in order to perform manipnilation and enrichment

of biological celia in the microfiudic chamber The simnlation af the proposed model hag been implemented Microfluidic chips were also successfully fabricated and experi mentally tested to confirm the ability to manipulate and enrich cells in the microfluidic chamber of the proposed siruerure

2.1 Overview of biological cell manipulation

‘The manipulation of biological particles such as biological cells plays an impor- tant role in biological studies and biomedical applications or clinical diagnostics, ie., the separation or enriching rare celts from diluted cell solution, or separating cells from the mixtnre based on special properties for each cell line, or performing trapping and positioning of separated! cells for cell classification In the field of research, the sepa-

ration of heterngeneans cell samples allows identification of the phenotypes and the subseqnent physical and biochemical analyzes In clinical diagnosis, before analyzing biochemical or cellular components, blcod samples should be separated and filtered out In some cases, cell assays require the isolation of rare cclls for discasc diagnosis, quvnitoring and Ureatmcnt such us isolation of circulating, Lumor culls froin patieul's

hlaad samples In regenerative medicine, call samples nmust he separated and refined

before transplantation to ensure the safery and effectiveness of treatment [76]

Recent advances in the ficld of lab-on-chip in combination with microfluidic plat- forms have ¢reated many lavorable conditions for the development and manufacturing

of biological applications and the expansion of capahilities of biomedical applications vesearch Tn particular, microfhridie technology has provided a, promising tool in the study of cell biology, microfluidic technology that allows us to control and manipulate precisely in the cellular scale environment Many techniques for cell manipulation in iicrofluidie systems lmve been sbudicd und developed in uccordunee wilh specific ol- jectives and applications In which, the general methods used for manipulating cells

can be classified based on the mechanism of forces acting on micro particles and cells

such as mechanical force, hydrodynamic force, optical force, magnetic force, electric

foree und some other forces 776, 195]

to the mechanical method that, affects cella and micro particles is the development of

a micro gripper system [24, 158]

Previously, in 20U5, Nikolas Chronis and Luke P Lee alsa used standard surface micromachining to successfully fabricate micro gripper systems that can capture and hold HeLa cells in solution medium |24| They used SU-8 photoresist umdtyrial and

»

a silicon wafer to hnild a micro gripper system based on the electric thermal effec: Because the microchip fabrication is based on two kind of metala ineluding gold and Chrominum, thermal expansion will affect the capture and retention of cells in beth

dry and fluidic environments In Chronis and Lcc’s cxpcriments, closing and opening

operation of micre gripper play an important role iu he whole process Iu this report,

the anthors also discussed the effect of temperature, heating rate, heating transfer,

opening and closing angles af micro-tweezers an the behavior of micro gripper Fignre

2.1 (A) shows an image of a micro clamping system and an activity experiment in

imunipulating HeLa cells in » solution of the author's group Nikolas Chrouis und Luke

P Lee In 2008, B Solana er al proposed the design of a micro gripper system for cell manipularion [158] The experiments of B Solano showed the ability to capture, hold and transport mouse egg celis (with an average diameter of about 100 ym) in the biological environment of the proposed micro pair system

Although utiero grippers show gucvess operution in capvuring and retuining single

cells, this technique is a direct mechanical contact method The mechanical action of

micro gripper when in direct contact with cells can affect, even damage the structure of the membrane layer, leading to deviations in the following measurements Furthermore,

ir order to close and open (he miervchipy in tke solution medium, this tecknique takes

time Lo heat aud expand the thermal sLructures needed, sv Lhis structure cannot capture

cells quickly and may not he snitable for fast: biological derection applications

In reality, in addition to the method of using micro gripper to manipulate bi- ological culls in aqueous incdiumn, the hydrodynumie method is aye implemented by many researchers in manipulating cells in the fluidic solution, especially in fluidic flow

environments such as microfluidic channels The technique of manipnlating cells hy

hydrodynamic methods is based on the intrinsic physical properties of the cells and the fact that these properties affect the movement of cells in laminar How ‘The bự: đrodyueunie TH force in the Dow bưlueuee ou the movement of a cell depending on the size, shape and distortion of the cell and changes Ue movement of the cell on the fluid flow in the micro-channel ‘Therefore, cells with different mechanical properties

will be influenced differently by the flow force {i.e., drag force) and will have different

field microscopy shows the operation of separating Ecoli K-12 bacteria from blood

solutions containing red blood cells

positions in the liquid channel (11, 71-73, 115] Since this is a passive mechanism, it is

easier to implement than other methods Applications of hydrodynamic methods are often used in the separation of blood cells, because of the significant differences in size

between different components of blood cells Other applications including isolating cir-

culating tumor cells and bacteria from blood solutions are also studied and improved

(46, 110, 138, 167, 186)

Albert J Mach and Dino Di Carlo firstly presented a microfluidic device capa-

ble of passively separating bacterial cells from the blood sample [110] This device is designed to continuously handle large sample volumes by employing 40 single chan-

nels placed in a radial structure with one input and many outputs are designed in a

circular way The results showed that red blood cells were separated from bacterial

3

celis contimme ta move to the middie ontput channel Tn addition, in 2013, Vahidreza Parichehreh ef al introduced a new design of a microfluidic technique combining water phase with inertial force to enrich cells in blood solutions 138] 'This technique exploits the selective relationship of RBCs with the dextran phase (DEX) to perform separation

iu chunuely with high efficiency In experiments, the rescarch group mixed RBC sane ples with representative artificial cells (MOTT-3 cells - peripheral human blood cells,

T iymphoblast, cell lines) and MCP-7 cells (human breast cancer call Tine} ata rate

of 500 : 1 (RBO ; cells) and scparation cfficioncy up to 96% of RBCs while retaining about 98% of target oly

Although hydrodynamic methads can bring high thronghput (> 10 000 celis/s), Tow-cell damage, easy fabrication and high enriching ability, it also shaws disadvantages such as slow response, low purity and requires bulky external actuators (e.g., external

check valve, syringe pump, and pneumatic pump) [2U

2.1.2 Optical method

Cul manipulation techniques use optical methods bused ou tbe balance of two types af optical forces: scattering force is the repulsion of objects in the direction of light and gradient force is the pulling force of objects along the gradient field of light intensity im space [122, 130 If the gradient force exceeds the scattering force on the particle, it ean be trapped at the ueur center of the target lens, La the opposite cust, the particle will move outward of the beam area Typically, this technique uses a force created hy a highly focused laser beam and is often set np with a high-apertnre mi- eroscope lens Since techniques for manipulating biological samples, such as viruses,

baeclcria and live mautunalian cells were firstly introduced in the mid~1980s, these techie

niques have beeu developed by mauy research groups in order to improve molecules

or biological cells manipulation efficiency Recently, cell manipulation techniques us- ing optical methods have been successfully developed and integrated in microfluidic devices for various biological applications, such as cell classification, cell trapping, and cell analysis In particular, the use of lascr beams to manipulate biological cclls in the

flow of microfluidic channels for the purpose of separation und trapping hus gamcred

3ã

mneh attention from research groups around the world Piological cells flowing in the microfinidie channel can be manipulated and separated based on the optical proper- ties of each cell type such as light absorption, refractive index of individual cells or cquivalcut-in-size micro particles Bosides, microfluidic systems using optical tweezers and [uorcsvence microscopy xyslenw were ulso developed Wo iuvestignte cell respouses ander physical and chemical stimnli [27, 38, 39, 9%, 129] In 2008, the research veam

of Masaya Murata and his team successfully established a new cell separation method based on the use of optical trapping and microfluidic trapping force [129] Murata’s report hes shows Unnt Une imorphology, size, absorption of light and relruetive index of cells are important factors affecting the aptical trapping faree acting on cells An apti- cal trap was created by a highly facnsed laser heam integrated into the fluidic channel

to change the trajectory of the moving cells in the channel to target highly efficient cell separation In their report, the team successfully performed manipulation of HeLa cervical canver cully int the Muid Dow

Typically, high-foonsing laser beams in cell manipulation techniques nsing optical methods can cause even the destruction of the structure of biological samples Although the optical effects on biological samples can be reduced by using the light of a certain wavelength tle effet of using laser bows enn still Ioad to deviations for the following measurements Iu briel, ulilizing optical fore methods van bring many benelity such a contactless manipnlation, fluidic flow-independent particle movement and high purity

‘This method also shows disadvantages such as limited throughput, extensive optical

solup incluiding, use a bulky high-power taser [20]

‘The manipulation of the biclugival cell using imagnetiv wethods is also very com mon in recent, studies Magnetically manipntating cells can be divided into two types: internal magnetism and external magnetic use Recently, the implementation of in- trinsic magnetic cell manipulation has been proposed such as utilizing hemoglobin containing iron components in red blood cells that can be used to scparate leukocytes and erythrocytes in blood solutions with external uraguctiv fields [53, 88, 118)

36