Microfluidic Prototype of a Lab-on-Chip Device for Lung Cancer

Diagnostics

Dalila Vieira, Filipa Mata, Ana Moita and António Moreira

IN+, Instituto Superior Tecnico Universidade de Lisboa, Lisbon, Portugal

Keywords: Microfluidic Device, Electrowetting, Biofluid Dynamics, Wettability, Lung Cancer.

Abstract: Cell sorting for disease diagnostics is often achieved by fluorescence based identification of specific

markers. However, in lung cancer diagnostics, cytological analysis of pleural fluids is not always reliable

and immunofluorescence essays demand for specific sample preparation. Hence, this paper addresses the

development of a microfluidic device for lung cancer diagnostics which infers on the potential of a

diagnosis based on analysing the cell deformability (stiffness) that alters the rheological properties and

consequently the flow characteristics. Cell deformability will be induced by external actuation.

Electrowetting is used to transport the samples in an open configuration system using microdroplets. Effects

of the test chip configuration, sample physico-chemical properties and potential adsorption mechanisms are

discussed. Wettability plays here a vital role in the sample transport and in the diagnostic method to be

tested. Hence, an innovative approach is presented, the 3D Laser Scanning Fluorescence Confocal

Microscopy (3D-LSCFM) to provide a detailed reconstruction of the surface topology at the liquid-solid

interface region thus allowing contact angles measurement with high spatial resolution.

1 INTRODUCTION

Cell separation and sorting are critical in various

biomedical applications including diagnosis,

therapeutics and cell biology (Takahashi et al., 2004,

Gossett et al., 2010, Shields IV et al., 201,). Samples

of interest are often heterogeneous populations of

cells in a culture that comprises tissue. For instance,

the analysis of pleural fluid for lung cancer

diagnosis requires the previous separation of various

components, including blood cells (Gossett et al.,

2010). Although many standard techniques have

been developed for cell sorting, there are still several

challenges to overcome: they are often labour

intensive, require multiple additional tags or labels

to identify cells, have high costs, use large sized

equipment with low portability and require highly

skilled staff (Omori et al., 2015). Microfluidic

devices are pointed to be able to solve many of the

aforementioned problems and are actually

considered a fundamental pillar for the development

of point-of-care diagnostics (Yager et al., 2008).

Within this scope, this work aims at devising a

microfluidic chip for a lung cancer diagnosis. One

explores here the possibility to provide an earlier

diagnosis based on the deformability (stiffness) of

the cell, which alters the rheological properties and

consequently the flow characteristics. This new

approach follows the method suggested by (Gossett

et al., 2010), although these authors focused the

diagnostics on image analysis of the cells. Instead,

here, main emphasis is put on the cell deformation

inside microdroplets, as droplet spreading is

expected to be correlated to the rheological

modifications due to cells stiffness (Moita et al.,

2015). Microdroplets are also interesting regarding

sample handling in the microfluidic device. Sample

transport in continuous medium using microchannels

is the most popular approach in microfluidics, but

has problems associated with clogging, maintenance

and access to the samples. These difficulties can be

overcome with an open configuration system,

handling the samples in microdroplets, combining an

external actuation with custom made wetting

properties of the surfaces (Pollack et al., 2011, Moita

et al, 2016). However, external actuation (e.g.

electrostatic) may influence the internal droplet

flow, thus affecting droplet motion (Mugele, 2009).

Hence, a complete characterization of the flow is

required to tune the appropriate conditions for the

transport. Lab-on-a-chip open configuration systems

are still sparsely reported in the literature,

concerning the transport of biofluids. Several

Vieira D., Mata F., Moita A. and Moreira A.

Microﬂuidic Prototype of a Lab-on-Chip Device for Lung Cancer Diagnostics.

DOI: 10.5220/0006252700630068

In Proceedings of the 10th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2017), pages 63-68

ISBN: 978-989-758-216-5

authors report the effective electrowetting-induced

transport of physiological fluids, proteins and DNA

(Wheeler et al., 2005), but it is not clear which are

the most suitable electrochemical properties of the

fluids or the most important parameters governing

biofluids transport and manipulation. Adsorption of

the biocomponents on the dielectric substrate is also

a problem that is not completely understood yet and

its effects on the surface wettability are taken to a

secondary level. Yoon and Garrell (2003) showed

that protein solutions are often adsorbed by the

dielectric substrates. Recent work by Moita et al.

(2016) evidences that adsorption locally reduces the

contact angle, which aids droplet spreading but also

promotes energy dissipation at the contact line, thus

precluding droplet receding and making the droplet

transport more difficult, so this effect should not be

neglected. Finally, studies concerning EWOD

applications on microchips barely assess the

influence of design and configuration parameters. As

the droplet is transported on the chip surface, the

size and distance between electrodes must account

for the wetting properties and how they affect

droplet dynamics, which should be balanced by the

parameters affecting the electrical field. In this

context, for an initial stage of the development of the

microfluidic device, an optimization of the chip

configuration, taking into account the effect of the

liquid and surface properties is required.

Wettability plays a vital role in these flows, but

also here improved diagnostic techniques are

required to measure the micro-contact angles, which

can be significantly different form the apparent

angles typically obtained with tensiometers

(Sundberg et al., 2007, Vieira et al., 2016). In this

context, confocal fluorescence microscopy, which is

already used to infer on the potential adsorption

mechanisms is further explored here as a new

technique providing contact angle measurements

with high spatial resolution.

2 EXPERIMENTAL PROCEDURE

At this stage of the work, simple microfluidic chips

were devised to select the appropriate chip

configurations and materials and to infer on the

efficacy of the sample transport using

electrowetting. Biomimetic solutions of the pleural

fluid (as well as pleural fluid) are intended to be

used in the near future but for these preliminary tests

protein solutions and cell suspensions were used as

biofluid samples. GFP – Green Fluorescent Protein

(produced and purified in house) solution with

1.71x10

-3

mM concentration and GFP-expressing E.

coli suspensions with concentrations of 1x10

cells/ml and 2x10

cells/ml were the solutions

chosen, whose main physico-chemical properties,

namely density ρ, surface tension σ

and dynamic

viscosity μ are summarized in Table 1. Regarding

their rheology, all the fluids used here are

Newtonian.

Table 1: Physico-chemical properties of the biofluids.

Solution

Density ρ

[kg/m

]

Surface

tension σ

[mN/m]

Dynamic

viscosity μ

[Ns/m

]

GFP (1.71x10

-3

mM)

998

72.2±0.7

1x10

-3

GFP-expressing

E. coli (1x10

cells/ml)

998

73.8±0.04

1x10

-3

GFP-expressing

E. coli (2x10

cells/ml)

998

73.8±0.04

1x10

-3

For the measurements with the 3D Laser

Scanning Fluorescence Confocal Microscopy – 3D

LSCFM a fluorescent dye - Rhodamine B (Sigma

Aldrich) is used, which was chosen taken into

account its excitation and emission wavelengths, to

be compatible with the wavelengths available in the

Laser Scanning Confocal Microscope (Leica SP8),

but also due to particular characteristics of the

experimental conditions, in the present study. For

the concentrations used here

(0.0007936mg/ml<Concentration<0.496mg/ml) the

physico-chemical properties of the water-dye

solutions are very close to those of water. Detailed

description of the measurement procedures is

provided in Vieira et al. (2016).

The test chips, manufactured at INESC-MN are

printed by lithography and the patterned transferred

by wet etch. Finally, a thin film of a dielectric

material is deposited on the chip assembly. The

chips mainly comprise numerous interdigited

electrodes displaced with a fixed distance of 60μm

between them. The variable in the chips

configuration is the width of the electrodes, which

varies between 80μm and 1400μm. The length of the

electrodes is 24mm. The usable chip area is

32x22mm

. The applied voltage is varied from 0 to

250V, also to infer on its influence on the droplet

motion. The frequency was varied between 50Hz

and 450Hz. To infer on the possible adsorption of

the biocomponents on the dielectric substrates over

which the droplets of the biofluids are transported,

simple tests are performed in which droplets of the

biofluids are deposited on the surfaces. Afterwards,

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

a sequence of tests with and without electrostatic

actuation is performed and the “footprints” of the

droplets are observed on the Laser Scanning

Confocal Microscope (Leica SP8). The obtained

images are then post-processed to determine the

mean grey intensity (sum of intensities divided by

the number of pixels in the region of interest of the

droplet footprint) and the Area Integrated Intensity

(sum of intensities of pixels in the region of interest

of the droplet footprint normalized by unit of area

(μm

). Since the droplet spreads after actuation, the

integrated density is weighted with the area. To

reduce the noise, the average grey intensity levels of

the background image were also subtracted. The

final result is the herein so-called Total Corrected

Droplet Fluorescence – TCDF, as proposed by

Moita et al. (2016). Higher values of TCDF can be

associated to a larger quantity of the protein or cells

adsorbed by the substrate. The wetting properties of

the dielectric substrates play a vital role. Different

chemical and topographical characteristics are tested

to infer on the most favourable to handle the

biosamples in the various sections of the

microfluidic device. The topography is measured

using a Dektak 3 profile meter (Veeco) with a

vertical resolution of 20nm.

The wettability of the dielectric substrates is

quantified with an optical tensiometer (THETA from

Attention), by the static contact angle θ

and by

hysteresis. Given the relatively low resolution of

these measurements and considering the typical

scale of the processes governing the transport of the

samples within the droplets (micro-to-nano scale) an

alternative method is explored to provide more

accurate measurements. Detailed description of this

technique can be found in Vieira et al. (2016).

Finally, transport and deformation of the biofluid

droplets are characterized based on high-speed

visualization at 2200 fps using a Phantom v4.2 from

Vision Research Inc., with 512x512 pixels@2100fps

resolution. Post-processing home-made routines are

then used to measure deformability, spreading

diameters, velocity of droplets transport and

displacement velocity of the contact line (droplet-

dielectric substrate).

3 DEVICE CONFIGURATION

The microfluidic device under development has

three main working sections, namely, the transport

section, the diagnostic section and the

sorting/selection section. In the transport section,

which enables the transport of the biofluid droplet to

the diagnostic section, droplet motion is governed by

electrostatic actuation aided by custom made wetting

properties of the dielectric material. In this section,

hydrophobic/superhydrophobic regions are preferred

to minimize the adhesive forces, which are

proportional to hysteresis and consequently

minimize the energy dissipated at the contact line

between the droplet and the surface. Similar wetting

properties are required in the sorting/selection

section. On the other hand, in the diagnostic section

it worth promoting adhesion to constrain the sample

in the sensor area. The diagnostic methodology to

explore considers the correlation of different ratios

of cell stiffness with the degrees of the pathology,

being expected to be able to detect cell malignancy

at very early stages (Gosset et al, 2010). The

deformation of the cells inside the microdroplets is

induced. Different approaches will be explored to

impose the deformation but at this stage of the work,

electrostatic actuation is being tested. A numerical

model is being developed at two scales. At the

mesoscale, the deformability of the cell is simulated.

At this scale surface wettability effects, such as

adhesion and repulsion, are related to molecular

bonding and Van de Walls forces. The main outputs

of this model are the forces that must be exerted to

impose the various degrees of deformability of the

cell to validate the diagnostic method. On the other

hand, despite being a macroscopic flow, the

spreading of a microdroplet is known to be very

sensitive to small rheological modifications of the

fluid and can be correlated with the parameters

governing the constitutive models (Moita et al.,

2015). Hence droplet spreading is expected to be

correlated to the various levels of cell stiffness.

These levels of cell stiffness can be grouped in

classes of internal cell viscosity/water viscosity and

correlated to different degrees of the pathology. At

this scale, surface wettability can simply be

modelled based on interfacial tensions, hysteresis

and contact angle values, but still requires very

accurate contact angle measurements combined with

a detailed characterization of the droplet internal

flow, particularly near the surface.

Following the discussion in the previous

paragraphs, the selection of the materials to use as

the dielectric film, plays here a determinant role, as

different sections of the microfluidic chip demand

for opposite wetting regimes. The first approach

considered here was tailoring surface topography to

alter the wettability. Hence, photolithography was

used to define micro-patterns of regular

cavities/pillars. For an accurate measurement of the

contact angles and clearer description of the contact

Microﬂuidic Prototype of a Lab-on-Chip Device for Lung Cancer Diagnostics

line, the contact angles were with a new technique,

the 3D LSCFM, described in the next paragraphs.

3.1 Contact Angle Measurements with

3D LSCFM

To validate and explore the 3D LSCFM technique,

preliminary results were obtained by measuring the

equilibrium contact angles on smooth glass slides.

The equilibrium angles are measured for millimetric

(with diameters of 3mm) and micrometric (with

diameters ranging between tens of microns up to

1mm) droplets, to infer on their dependence on

droplet size. The obtained measures are compared

with those obtained with the optical tensiometer

(Figure 1).

Figure 1: Comparison between the equilibrium angles

measured with tensiometer and with the LSCFM

technique, measured for a smooth glass and coated Al

surfaces.

Overall the results show that both techniques

provide similar measurements, although the values

obtained from the LSCFM tend to be lower, when

compared to those given by the optical tensiometer.

This is due to the scale and resolution that are being

considered in the LSCFM. Hence, the error

associated to the worse resolution used in the

LSCFM is of 1.87µm (with negligible propagation

to the angle measurement) against 156µm obtained

with the optical tensiometer. So a more detailed

view of the contact line region can be obtained with

the 3D LSFCM, which consequently provides a

more accurate shape of the region defining the

contact angle. These observations are qualitatively in

agreement with those reported by Salim et al.

(2008).

Once the measurement technique is validated, it

can be applied to more complex surfaces, namely

those with patterned micro-structures. For these

surfaces a large difference is observed between the

measures obtained with the optical tensiometer and

those taken with the 3D LSCFM technique, as

depicted in Figure 2. Hence, there are apparent

angles of 120º, as measured by the tensiometer, thus

evidencing a hydrophobic behavior, which are in

fact up to 40º lower, when measured with the 3D

LSCFM technique.

Figure 2: Comparison between the equilibrium angles

measured with the optical tensiometer and with the

LSCFM technique measured on micro-textured silicon

wafer surfaces.

The lower angles measured with the 3D LSCFM

are in agreement with the observations of the contact

line region. So, the apparent hydrophobic regime of

some of the surfaces characterized with the

tensiometer is not accurate and often not stable, as

the liquid droplet sags in between the surface

patterns. Furthermore, the 3D reconstructions of the

droplet on both XZ and YZ planes show an evident

distortion of the contact line. These observations of

the contact line may also support our previous

results (e.g. Moita et al 2016) which showed that

surface topography in these non-stable hydrophobic

surfaces promotes energy dissipation at the contact

line, thus precluding droplet motion. In line with

these results, the safest way to alter wettability, for

the current stage of development of the work is

towards the chemical modification and/or selection

of the appropriate dielectric materials, as discussed

in the following sub-section.

3.2 Selection of the Materials as a

Function of the Influencing

Parameters

The selection of the dielectric materials to use

should be based on the contact angle measurements

but also on the hysteresis, which should be the

smallest possible, since the resistance force opposing

to droplet motion is proportional to the surface

tension σ

and to the hysteresis. In this context

Table 2 depicts the equilibrium angles, obtained for

each biofluid tested, on various dielectric coatings

which are commonly used in electrowetting chips.

Water is used as reference. The Table shows that

only the SU8 resist and Si

surfaces are

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

hydrophilic, being the others hydrophobic. The

highest contact angle of 121º is obtained for the

PDMS substrate. Despite having high contact

angles, which is desired for the transport of the

samples, PDMS and Teflon substrates depict also

high hysteresis (Figure 3).

Table 2: Equilibrium contact angles, obtained for each pair

fluid-dielectric substrate considered in the present work.

Dielectric

coating

Contact angle [°]

Water E-coli GFP

Teflon 112±5 103±6 121±6

Teflon with

Glaco

145±1 141±9 153±3

PDMS 121±1 112±1 119.5±0.4

PDMS with

Glaco

153±3 153±2 155±3

SU8 resist 67.1±0.7 65±2 71.8±0.2

SU8 with

Glaco

160±7 162±1 153±4

64.1±0.7 59±4 65±2

Glaco® is a commercial coating which is mainly

a perfluoroalkyltrichlorosilane combined with

perfluoropolyether carboxylic acid and a fluorinated

solvent (Kato et al., 2008). Its application allows

obtaining superhydrophobic surfaces with high

contact angles (>150º) and low hysteresis (<10º)

being therefore a good option to consider in the

transport section of the microfluidic device.

Concerning adsorption, Moita et al. (2016) report

that the GFP protein was adsorbed by Teflon

substrates, leading to a local increase of surface

wettability and further contributing to preclude the

receding motion, as this wettability increase is

irreversible, taking the contact angles to values near

saturation.

Figure 3: Contact angle hysteresis evaluated for GFP

solution (1.71x10

-3

mM), GFP-expressing E-coli

suspension (1x10

cells/mL) and distilled water on the

tested dielectric substrates.

In this context, this work infers on the possible

adsorption of the GFP and E-coli cells by the

substrates, evaluated by the TCDF value. The results

evidenced a minimum value of TCDF =

8.88x10

and TCDF = 1.65x10

obtained for the

adsorption of GFP (1.71x10

-3

mM) and E-Coli

suspension (1x10

cells/mL) on PDMS, respectively.

The alternative material with lower adsorption of the

biocomponents tested here was SU8, but the TCDF

values obtained were about one order of magnitude

higher than those evaluated for PDMS. Hence,

PDMS is a good choice. In addition, the application

of Glaco® coating is observed to further reduce the

adsorption of both proteins and cells, decreasing the

TCDF values in about one order of magnitude. Once

the material is selected, its effect must be further

evaluated in the dynamic response of the droplet and

its deformability, which also depends on the

properties of the biofluids. For illustrative purposes

and due to paper length constrains, Figure 4 depicts

the effect of the biofluid properties on droplet

motion on electrowetting (in the transport section),

evaluated based on its maximum spreading diameter,

made non-dimensional with the initial droplet

diameter as it is collected on the surface, d

max

The Figure evidences the better response of the

droplets of protein solutions, although the surface

tension and density only vary slightly between the

different solutions (Table 1). Since the cellular

compounds are bigger and heavier than the proteins,

they should be more difficult to carry. In fact, the

cells have a greater propensity to adhere to the

surface and tend to agglomerate, increasing the local

density and ending up creating resistance to motion.

All these aspects must be considered in the

following new configurations of the microfluidic

chip, once the materials are selected.

Figure 4: Maximum spreading dimensionless diameter of

GFP (1.71x10

-3

mM) and GFP-expressing E-coli

suspension (1x10

cells/mL), for droplets moving between

coplanar electrodes (transport section of the device).

4 SUMMARY

The present paper presents the preliminary stages of

development of a microfluidic device for lung

50 100 150 200 250 300 350 400

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

max

[-]

Fequency [Hz]

GFP

E. Coli

Microﬂuidic Prototype of a Lab-on-Chip Device for Lung Cancer Diagnostics

cancer diagnostics which infers on the potential of a

diagnosis based on analysing the cell deformability

(stiffness). The cell stiffness is expected to alter the

rheological properties and consequently the flow

characteristics in a detectable way, which is

correlated with cell malignancy. The main concepts

behind this new diagnostic method are explained

together with the global description of the

microfluidic device. Given the important role of the

wettability, a new methodology is explored here to

obtain contact angle measurements with high spatial

resolution. At this preliminary stage of the work, the

importance of the wettability is discussed in the

selection of the materials. The dynamic response of

different biofluids is also briefly discussed.

ACKNOWLEDGEMENTS

The authors are grateful to Fundação para a Ciência

e a Tecnologia (FCT) for partially financing this

research through the project UID/EEA/50009/2013,

which also supports Dalila Vieira with a fellowship.

The work was also partially financed by FCT

through the project RECI/EMS-SIS/0147/2012,

which also supported Filipa Mata with a fellowship.

A.S. Moita also acknowledges the contribution of

FCT for financing her contract through the IF 2015

recruitment program. Finally, the authors

acknowledge the contribution of Joana Pereira in the

data acquisition and post-processing of the 3D-

LSCFM data.

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