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Accepted Manuscript

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Jeon and K. Han, Lab Chip, 2017, DOI: 10.1039/C7LC00925A.

Volume 16 Number 1 7 January 2016 Pages 1–218 This is an Accepted Manuscript, which has been through the
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 Page 1 of 31 Lab on a Chip
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DOI: 10.1039/C7LC00925A

A Disposable Microfluidic Device

with a Reusable Magnetophoretic Functional Substrate

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for Isolation of Circulating Tumor Cells

Lab on a Chip Accepted Manuscript

Hyungseok Cho,1 Jinho Kim,1 Chang-Wan Jeon,2 and Ki-Ho Han*,1

1
Department of Nanoscience and Engineering
Center for Nano Manufacturing, Inje University
Gimhae 50834, Republic of Korea

2
Department of Surgery
Gospel Hospital, Kosin University
Busan 49267, Republic of Korea

*Corresponding Author

Address: Department of Nanoscience and Engineering, Inje University

197, Inje-Ro, Gimhae, Gyongnam, 50834, Republic of Korea

Tel: +82-55-320-3715/ Fax: +82-55-320-3631/ E-mail: mems@inje.ac.kr

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Abstract

We describe an assembly-disposable microfluidic device based on a silicone-coated release

polymer thin film. It consists of a disposable polymeric superstrate and a reusable functional

substrate and they are assembled simply using vacuum pressure. The disposable polymeric
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superstrate is manufactured by bonding a silicone-coated release polymer thin film and a

Lab on a Chip Accepted Manuscript

microstructured polydimethylsiloxane (PDMS) replica, containing only a simple structured

microchannel. The reusable functional substrate generates an intricate energy field that can

penetrate the micrometer-thick polymer film into the microchannel and control microfluids.

This is the first report to introduce a silicone-coated release polyethylene terephthalate (PET)

thin film as a bonding layer on a microstructured PDMS replica. The bonding strength was

~600 kPa, which is the strongest among bonding methods of PDMS and PET polymer.

Additionally, accelerated tests for bond stability and leakage demonstrated that the silicone-

coated release PET film can form a very robust bond with PDMS. To demonstrate the

usefulness of the proposed assembly-disposable microfluidic device, a lateral

magnetophoretic microseparator was developed in an assembly-disposable microfluidic

device format and was evaluated for isolating circulating tumor cells (CTCs) from patients

with breast cancer.

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1. INTRODUCTION

Microfluidic devices usually include special functions for mixing, reaction, pumping, and

filtering, which are crucial for manipulating and analyzing fluids. The key functions are

usually realized by complex structures and/or implemented using complicated fabrication

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processes and expensive materials. For example, as the key functional compartments, a

Lab on a Chip Accepted Manuscript

micro-patterned interdigitated electrode array was used for dielectrophoresis1 and

acoustophoresis,2 a micrometer-sized ferromagnetic wire array for lateral magnetophoresis,3 a

tiny magnet array for magnetic cell capture,4 and a thermocycler for micro-PCR.5

Additionally, a single-crystal piezoelectric wafer was used for generating a surface acoustic

wave,6 and a semiconductor heterostructure substrate for an ultrasensitive micro-Hall sensor.7

Although such complex structures, complicated fabrication processes, and expensive

materials allow microfluidic devices to be equipped with useful functionalities, they could

also be obstacles to realizing disposable microfluidic devices.

Disposable microfluidic devices are highly desirable in medical and biological

applications, because it is essential to avoid false-positive signals caused by biological

contamination and cumbersome cleaning steps. Typically, disposable microfluidic devices

have been fabricated with polymeric materials8-10 and paper11 due to the advantages of a wide

range of mastering and prototyping methods with low-cost and high-volume manufacturing

methods.12, 13
Most polymeric microfluidic devices have been manufactured by limited

fabrication methods,14 such as embossing,15 injection molding,16 imprinting,17 laser

ablation,18 and soft lithography,19 which can only provide restricted structures and

functionalities. For this reason, a lot of efficiency and precision microfluidic devices are still

realized based on glass and semiconductor materials because of their compatibility with most

microfabrication processes.

To address these problems, a few researches20, 21

developed reusable functional

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compartments, which work together with disposable microfluidic devices. The reusable

functional compartments generated external energy fields for manipulating fluid substances

in microchannel of the disposable device. The disposable microfluidic devices were usually

fabricated by typical polydimethylsiloxane (PDMS)-glass bonding method. Then, the glass

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substrate was used as a thin layer for penetration of the external energy fields, such as

Lab on a Chip Accepted Manuscript

magnetic field20 and standing surface acoustic wave.21 Meanwhile, theoretical analyses3, 22

showed that the magnitude of the external energy field is dramatically decreased with the

distance from the energy source, thereby declining the force of the energy field transmitted

into the microchannel. Since the thickness of the used glass substrate was in a range of

several-hundred-micron, it was thick enough to obliterate the closely created force in

microscaled space. Even though the force can be effectively transferred into the microchannel

by thinning the glass substrate, it could be easily broken with only a slight impact, making it

difficult to practical use.

Here, we introduce an assembly-disposable microfluidic device that consists of a

disposable polymeric superstrate and a reusable functional substrate. The proposed

disposable polymeric superstrate only includes a simple structured microchannel and is

readily fabricated by bonding a silicone-coated release polyethylene terephthalate (PET) film

(~10-µm-thick) and a microstructured PDMS replica. The reusable functional substrate

provides the main functional compartments, which may be structured in a complex manner

and fabricated using complicated fabrication processes with expensive materials. The

disposable superstrate and the reusable substrate can be assembled and disassembled simply

using vacuum pressure. The silicone-coated release PET film used here can also form strong

bonds with PDMS, and the energy fields, such as acoustic, electric, magnetic, and thermal,

can efficiently transmit through it. Silicone-coated release polymer films are widely used in a

range of shielding and packaging products, printing, protecting adhesive films, and electronic

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and medical applications.23 They are available with various polymer materials and

thicknesses. To demonstrate the usefulness of the proposed assembly-disposable microfluidic

device, we developed an assembly-disposable lateral magnetophoretic microseparator for

isolating circulating tumor cells (the ‘assembly-disposable CTC-µChip’) and evaluated its
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performance in a clinical study of patients with breast cancer.

Lab on a Chip Accepted Manuscript

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2. MATERIALS AND METHODS

See the Supporting Information for detailed descriptions of the following methods: (a)

instrument set-up, (b) theoretical analysis, and (c) RT-PCR protocol.

2.1. Design and working principle

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The assembly-disposable CTC-µChip consists of a simple-structured disposable polymeric

Lab on a Chip Accepted Manuscript

superstrate and a reusable functional substrate (Fig. 1(a)). The disposable polymeric

superstrate is fabricated by bonding a simple-structured microchannel PDMS and a

micrometer-thick silicone-coated release PET film. It includes a microchannel having two

inlets and two outlets, and a vacuum hole leading to a vacuum trench surrounding the

microchannel. The reusable substrate includes a ferromagnetic wire array, which is inlaid into

the substrate and placed at an angle (θ = 5.7°) to the direction of flow. The disposable

superstrate and the reusable substrate can be assembled and disassembled simply using

vacuum contact, which strongly attaches them and removes the air gap between the PET film

and the ferromagnetic wire array (Fig. 1(b)).

After vacuum assembly, nucleated blood cells mixed with magnetic nanobeads and

phosphate-buffered saline (PBS) with 0.2% bovine serum albumin (BSA) were respectively

injected through the sample and buffer inlets, each 500 µm in width. To enhance the purity of

isolated CTCs, the widths of the waste and CTC outlets were designed to be 800 and 200 µm,

respectively. To generate a high-gradient magnetic field, the width and thickness of the

ferromagnetic wire were designed to be 70 and 40 µm, respectively. Detailed design of the

ferromagnetic wire array has been described previously.3

When a uniform external magnetic field is applied to the ferromagnetic wire, the

external magnetic field is deformed near the wire, generating a high-gradient magnetic field

over the whole area of the microchannel. Due to the vacuum contact between the superstrate

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and the substrate, the gradient magnetic field can effectively penetrate the micrometer-thick

PET film into the microchannel (Fig. 1(b)). Then, CTCs with bound magnetic nanobeads

passing over the wire experience magnetic force Fm and hydrodynamic drag force Fd, which

generates a lateral magnetic force, Fl, on CTCs (Fig. 1(c)). Consequently, with a uniform
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external magnetic field, CTCs with bound magnetic nanobeads are forced laterally and flow

Lab on a Chip Accepted Manuscript

into CTC outlet, while normal blood cells flow into the waste outlet.

7
 Superstrate

Inlets

Vacuum hole
Microchannel
Vacuum trench
Outlets
Vacuum Inlets Outlets
Substrate assembly

Ferromagnetic wires Disassembly

(a)

Inlet Outlet Vacuum

Vacuum
trench PDMS
Superstrate
CTC
Vacuum
SU-8 contact
Substrate
Glass

Ferromagnetic wires Polymer thin film

Drag force (Fd) Magnetic force (Fm) External magnetic field (H0)
(b)

Sample inlet Waste outlet

Magnetic field

Buffer inlet Ferromagnetic wires CTC outlet

Circulating tumor cell bound with magnetic nanobeads

(c)

Fig. 1 (a) Conception of the assembly-disposable CTC-µChip, consisting of a disposable

polymeric superstrate and a reusable substrate. (b) A cross-sectional view of the gradient

magnetic field, generated from a ferromagnetic wire array inlaid into the reusable substrate and

penetrating the micrometer-thick polymer thin film into the microchannel. (c) Working

principle of the assembly-disposable CTC-µChip for isolating CTCs from nucleated blood

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cells.

2.2. Fabrication process

The disposable polymeric superstrate was manufactured simply by bonding a 12-µm-thick

silicone-coated release PET film and a microstructured PDMS replica. An SU-8 mold on a
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glass master was first fabricated to make the PDMS replica, including the microchannel and

Lab on a Chip Accepted Manuscript

the vacuum trench. A 50-µm-thick layer of SU-8 3050 photoresist (MicroChem Corp.) was

spun and patterned for creating a mold for the microchannel on the glass master, along with

an evaporated 1000 Å Cr layer to increase the adhesion between the SU-8 and the glass

master. Then, an acrylic square bar of 2 × 2 mm2 was bonded adhesively on the glass master

to define the vacuum trench (Fig. S1(a)). A polymer mold frame, fabricated by

stereolithography (ViperSI2; 3D Systems), was used to pour the liquid PDMS. The SU-8

mold was completed by assembling the glass master and the polymer mold frame. Liquid-

phase PDMS, prepared by mixing resin and curing agent in a 10 : 1 ratio (Sylgard 184; Dow

Corning), was poured into the SU-8 mold and cured for 1 h at 75°C in an oven (Fig. S1(b)).

After peeling the PDMS replica off the SU-8 mold, the inlet and outlet reservoirs and the

vacuum hole of the PDMS replica were generated using a punch, 1.5 mm in diameter. Then,

the PDMS replica and the 12-µm-thick silicone-coated release PET film (5 g release force,

Shanghai Guangtai Adhesive Products Co.) were bonded together after oxygen plasma

treatment for 60 s at 6.8 W RF power (PDC-32G-2; Harrick Plasma) (Figs. 2(a) and S1(c)).

Figure 2(b) shows enlarged cross-sectional views of the microchannel made by the

microstructured PDMS replica and the 12-µm-thick PET film.

The fabrication process for the reusable substrate used a 0.7-mm-thick glass slide

(Borofloat33 Pyrex; Schott). A Ti/Cu/Cr seed layer was first electron beam-evaporated on the

glass slide. A SU-8 3050 photoresist was spun and patterned to create 40-µm-thick

micromolds for ferromagnetic wires (Fig. S1(d)). Ferromagnetic permalloy (Ni0.8Fe0.2) wires

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were then electroplated on the glass slide (Fig. S1(e)). The ferromagnetic wire array, 40 µm

in thickness, was subsequently formed by a chemical and mechanical polishing technique and

inlaid in the reusable substrate (Figs. 2(c) and S1(f)).

For instrument set-up of the assembly-disposable CTC-µChip, the reusable substrate

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was first placed on the center of two stacked neodymium-iron-boron (Nd-Fe-B) permanent

Lab on a Chip Accepted Manuscript

magnets. After alignment of the disposable superstrate to the reusable substrate, an air

vacuum pressure of -50 kPa was applied through the vacuum hole to tightly attach the

superstrate and substrate (Fig. 2(d)). The vacuum contact also removed the air gap between

the PET thin film and the ferromagnetic wire array, so the high-gradient magnetic field

generated from the ferromagnetic wire array can efficiently penetrate the micrometer-thick

PET film into the microchannel and finely manipulate magnetic particles in fluids.

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(a)
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Lab on a Chip Accepted Manuscript

(b)

(c)

(d)
Fig. 2 (a) A photograph of the fabricated disposable polymeric superstrate and (b) enlarged
cross-sectional views of the microchannel. (c) A photograph of the fabricated reusable
substrate, including the inlaid ferromagnetic wire array. (d) Vacuum-assembled assembly-
disposable CTC-µChip, placed on two stacked permanent magnets. (i) WBCs (red), spiked
with MCF7 breast cancer cell lines (green), mixed with magnetic nanobeads and PBS
solution are injected through sample and buffer inlets, respectively. (ii) With a uniform
external magnetic field, MCF7 cells bound with magnetic nanobeads are forced laterally and
(iii) flow into CTC outlet, while WBCs flow into the waste outlet.
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2.3. Sample preparation

Healthy human peripheral blood samples were collected in Vacutainer tubes (G-Tube, Green

Cross) containing the anticoagulant EDTA and processed within 12 h. For the analytical

evaluation, MCF7 breast cancer cell lines were first stained with a membrane-permeable
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green fluorescent nucleic acid dye (SYTO 13, Invitrogen). The fluorescently stained MCF7

Lab on a Chip Accepted Manuscript

cells were washed and carefully suspended to a concentration of ~104 cells per milliliter.

Then, 10 µl of the concentrated solution was added to 5 ml of healthy whole blood in a 50-ml

conical tube, thereby spiking ~100 MCF7 cells into the blood. Patient blood samples were

drawn from patients (n = 20) with breast cancer using a protocol approved by the institutional

review board of Kosin University Gospel Hospital and informed consent was obtained from

the patients. The characteristics of the peripheral blood donors are summarized in Supporting

Information Table S2. All blood samples from cancer patients were first filtered using a 40-

µm grid strainer (Cell Strainer, SPL Life Sciences Co.) to eliminate matted substances and

debris.

Nucleated cells in prepared 5-ml blood samples were first extracted by a density

gradient centrifugation (700×g, 30 min) using 1.119 g ml-1 Ficoll solution (Histopaque-1119,

Sigma-Aldrich) and transferred into 10 ml of ice-cold PBS with 0.2% BSA in a 50-ml conical

tube. The extracted nucleated cells were washed by centrifugation (300×g, 10 min) and

resuspended in 1 ml of ice-cold PBS with 0.2% BSA in a 1.5-ml microcentrifuge tube. The

nucleated cells were washed once again and suspended in 200 µl of ice-cold PBS with 0.2%

BSA. The nucleated cells in 200 µl of medium were then mixed with anti-EpCAM antibodies

and magnetic nanobeads in sequence and incubated on ice for 60 and 90 min, respectively,

according to the manufacturer’s instructions (STEMCELL Technologies). The sample was

finally prepared by dilution with a 4-fold volume (800 µl) of ice-cold PBS with 0.2% BSA.

CTCs isolated from blood of patients was fixed with 100 µl of 4% paraformaldehyde

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for 5 min and subsequently permeabilized with 100 µl of 0.2% Triton X-100 (AMRESCO)

for 5 min. Then, CTCs were stained using a membrane-permeable nucleic acid fluorescent

dye (DAPI, Invitrogen) for nuclei, anti-cytokeratin-Alexa 488 (eBioscience) antibodies for

epithelial cells, and anti-CD45-Alexa 647 (Biolegend) antibodies for normal blood cells for
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30 min. The fluorescently stained CTCs were identified and counted using a confocal

Lab on a Chip Accepted Manuscript

microscope (LSM510META; Carl Zeiss).

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3. RESULTS AND DISCUSSION

3.1. Bond strength

The bond strength between the silicone-coated release PET film and PDMS was measured

from normal stress using a materials testing machine (LR10K Plus, Ametek) (Fig. 3(a)).
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Because the thickness of the PET film is 12 µm, it was first attached on an acrylic round bar

Lab on a Chip Accepted Manuscript

(20 mm in diameter and 100 mm in length) and PDMS was cast in the shape of a round bar (9

mm in diameter and 100 mm in length). The non-silicone coated side of the PET film was

permanently bonded on a single end of the acrylic round bar using a superglue. Then, the

PDMS round bar and the silicone-coated release PET film on the acrylic round bar were

bonded using oxygen plasma treatment. The two round bar assembly was fixed in the

materials testing machine and pulled apart at a speed of 20 mm/min to measure the interfacial

normal bond strength.

The bond strength was defined as the force required to separate the two round bars.

After the bond strength test, PDMS residue remained on the PET film surface (Fig. 3(b)).

This result indicates that PDMS permanently bonded to the silicone-coated release PET film.

The bond strength between PDMS and the silicone-coated release PET film was measured at

617±106 kPa (Fig. 3(c)). Then, 10 sets of identical tests were conducted for statistical

evaluation of the bond strength. The average of the bond strength was higher than that

between PDMS and a PDMS linker-coated PET film (514.0±255 kPa),24 which has shown

the highest bond strength among bonding methods for PDMS and PET polymer. Even though,

a statistical analysis (Table S1) indicates that the difference between the bond strengths of the

silicone-coated PET film (617±106 kPa) and the PDMS linker-coated PET film (514.0±255

kPa) to the PDMS is not meaningful. Indeed, the bond strength of more than 600 kPa is a

very high value in comparison with the driving pressure for fluid flow of the assembly-

disposable CTC-µChip, which is typically in the range of 10–20 kPa.

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Lab on a Chip Accepted Manuscript

(a)

(b)

800

700

600
Bond strength (kPa)

500

400

300

200

100

0
Silicone PDMS–linker
(c) coated PET coated PET

Fig. 3 Photographs of (a) instrument set-up for bond strength test and (b) PDMS residue

remaining on the silicone-coated release PET film. (c) Measured bond strengths of PDMS

with the silicone-coated release PET film and PDMS-linker coated PET film. The error bars

represent one standard deviation, calculated from 10 datasets.

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3.2. Bond stability

To measure the maximum pressure the bond between PDMS and the silicone-coated release

PET thin film could withstand, air pressure from 0–700 kPa was applied to the microchannel

through a gas regulator (Fig. 4(a)). Then, a vacuum pressure of -50 kPa was used for vacuum
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assembly of the disposable superstrate and the reusable substrate. To confirm that the air

Lab on a Chip Accepted Manuscript

pressure was applied uniformly to the overall microchannel, four pressure sensors

(XGZP6847001MPG, CFSensor) were connected to monitor pressure at each inlet and outlet.

The burst test was conducted at 100-kPa increases for 30 min each, which is the operating

time of the CTC-µChip, and identical burst tests were repeated using three devices (Fig. 4(b)

and Supporting Video 1). The results demonstrated that the assembly-disposable CTC-µChip

can withstand at air pressure up to ~600 kPa. Thus, the burst pressure is much higher than the

10–20 kPa normally required to drive fluid flow in the assembly-disposable CTC-µChip. The

measured burst pressure was also similar to the value of 617 kPa obtained in the bond

strength test. Also similar to the result of the bond strength test, PDMS residue remained on

the PET film after the burst test, again indicating that PDMS permanently bonds on the

silicone-coated release PET film.

The hydrolytic stability of the bonding between the PDMS and the silicone-coated

release PET film was measured over 0–28 days, during which the disposable polymeric

superstrate was immersed in PBS solution. As shown in Fig. 4(b), bonding was maintained

until at least 28 days, although the typical intended operating time of the assembly-disposable

CTC-µChip is only ~30 min. The results also demonstrated that the proposed silicone-coated

release PET film can form strong and stable bonds with PDMS and is useful for realizing

disposable microfluidic devices. The high bond strength and hydrolytic stability will allow

disposable microfluidic devices based on the silicone-coated release polymer thin film to be

used in other applications, such as on-chip cell culture and PCR functionalities.

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Leakage testing of the assembly-disposable CTC-µChip was performed to measure

the physical bonding stability of PDMS and the silicone-coated release PET film. Red and

blue colored solutions were injected into sample and buffer inlets at several flow rates, 20, 60,

100, 200, and 300 ml h-1, for 10 min (see Supporting Video 2) so 10, 30, 50, 100, and 150
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times higher, respectively, than the ordinary injection flow rate of 2 ml h-1. The assembly-

Lab on a Chip Accepted Manuscript

disposable CTC-µChip endured with the flow rate of at least 300 ml h−1, at which two

injection syringe pumps (Legato 100, KD Scientific) were stopped due to high repulsive

forces. This result confirmed again that a strong bond was formed between PDMS and the

silicone-coated release PET film.

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Vacuum (−
−50 kPa) (mm)
2
2
Air pressure
Pressure 4 (0~700 kPa) 4 Pressure
sensor 1 0.5 0.8 sensor 3
5 1 5
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Pressure 5 22 Pressure
sensor 2 0.5 0.2 sensor 4

Lab on a Chip Accepted Manuscript

Vacuum trench
(a)

800

700
Burst pressure (kPa)

600

500

400

300

200

100

0
0 7 14 21 28
(b) Time (days)

Fig. 4 (a) Illustration of instrument set-up for burst test of the assembly-disposable CTC-

µChip. (b) Measured burst pressure of the silicone-coated release PET film and PDMS of the

disposable polymeric superstrate, immersed in PBS solution over 0–28 days after bonding the

PET film and the microstructured PDMS replica.

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3.3. Analytical evaluation using cancer cells spiked into whole blood

The performances of assembly-disposable CTC-µChips fabricated with various film

thicknesses, from 12–30 µm, were evaluated using nucleated blood cells, prepared by Ficoll

density gradient centrifugation of 5 ml whole blood spiked with ~100 MCF7 breast cancer
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cell lines. For the analytical evaluation, the CTC-µChip was placed on a stack of two

Lab on a Chip Accepted Manuscript

neodymium magnets, generating an external magnetic flux of 0.2 T, which was applied in a

horizontal direction to the microchannel. During injection of the nucleated blood cells, MCF7

cells separated into the CTC outlet were counted using a fluorescence microscope and the

recovery rate of spiked MCF7 cells was evaluated (Fig. 5(a)). The analytical evaluation

demonstrated that the assembly-disposable CTC-µChip with 12-µm-thick PET film could

recover > 90% of the MCF7 cells from spiked blood samples at a sample flow rate of 1–4 ml

h-1. The result demonstrated that the recovery rate decreased as the thickness of the PET film

increased. It also corresponds with the theoretical analysis (Fig. S2(a)) that the lateral

displacement ( ∆‫ ݕ‬ᇱ ) of a magnetically labeled cell passing over a ferromagnetic wire

decreased as the thickness of the PET film increased. As the flow rate increased, more MCF7

cells easily passed over the ferromagnetic wires, as shown in Fig. S2(b), and thus the

recovery rate decreased. Interestingly, in the condition of 12-µm-thick PET film, the recovery

rate at the flow rate of 1 ml h-1 is slightly lower than that at the flow rate of 2 ml h-1. It might

be because some MCF7 cells adhere to the ferromagnetic wires due to the strong magnetic

force in this condition. The majority of MCF7 cells were laterally forced and separated by the

first and second ferromagnetic wires. However, a few MCF7 cells were not strongly

influenced by the magnetic forces of the forward wires due to their initial levitation height in

the microchannel.

The number of contaminating WBCs ranged from 153–741 cells per 5-ml blood

sample (Fig. 5(b)). The average contaminating number of WBCs was 386.6 cells and thus the

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average WBC depletion rate could be calculated as 64,666-fold (4.81 log), on the assumption

there are 5×106 WBCs per milliliter of blood. Figure 5(b) also shows that the contaminating

WBCs were reduced as the flow rate increased. From the measured results, the purity of

MCF7 cells isolated by the assembly-disposable CTC-µChip could be calculated as the ratio
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of the number of isolated MCF7 cells to the total number of isolated nucleated cells, which

Lab on a Chip Accepted Manuscript

includes MCF7 cells and WBCs. Thus, the purities were calculated in the range of 15.6–35.0 %

(Fig. 5(c)) in the case of 20 MCF7 cells per milliliter of blood.

A previous non-disposable CTC-µChip3 was fabricated based on PDMS-PDMS

bonding and the thickness of PDMS spun-layer right on the ferromagnetic wire substrate was

~10 µm. As shown in Figs. S3(a) and S3(b), the x-directional and z-directional magnetic

forces generated from the assembly-disposable CTC-µChip with a 12-µm-thick PET film

were similar to those generated with the non-disposable CTC-µChip with a 10-µm-thick

PDMS spun-layer. Similar to the experimental results with the non-disposable CTC-µChip,

the assembly-disposable CTC-µChip with a 12-µm-thick PET film yielded a ~90% recovery

rate. This finding suggests that both PET thin film and PDMS spun-layer are favorable for the

magnetic field to penetrate into the microchannel, because PET and PDMS are non-magnetic

materials. In particular, the non-magnetic properties of the PET film are compatible with the

high-gradient magnetic separation (HGMS) method used here, allowing the realization of a

disposable microfluidic device for controlling magnetic particles in the microchannel.

To show the superiority of the assembly-disposable CTC-µChip, its CTC-isolation

performance was compared with that of EasySep Human EpCAM Positive Selection Kit

(STEMCELL Technologies) as a conventional MACS method. The recovery rate of MCF7

cells, the number of contaminating WBCs and isolation purity of MCF7 cells were

respectively measured as 60.7%, 18,756 WBCs and 0.32%, as shown in Fig. S4. Although

the CTC-isolation procedure from the prepared nucleated blood cells takes only 15 min,

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which is faster than 30 min of the assembly-disposable CTC-µChip, the results showed that

the performance of the assembly-disposable CTC-µChip is much superior to that of the

conventional MACS method due to the precise magnetophoretic isolation of target cells in

microfluidics. Especially, the purity (33.3%) of the assembly-disposable CTC-µChip is over

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100 times that (0.32%) by the conventional MACS method. Since high purity of isolated

Lab on a Chip Accepted Manuscript

CTCs is critical to obtain accurate cancer genetic information, the proposed assembly-

disposable CTC-µChip is a highly promising device for CTC-based liquid biopsy.

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 Lab on a Chip Page 22 of 31
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-1
1 ml h
100 -1
2 ml h
-1
4 ml h
80
Recovery rate (%)
60
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Lab on a Chip Accepted Manuscript

40

20

0
12 µm 19 µm 25 µm 30 µm

(a) Film thickness

-1
1 ml h
-1 5
800 2 ml h 2.0x10
The number of contaminated WBCs

-1
4 ml h
700 WBC depletion rate
5
600 1.5x10

WBC depletion rate

500

5
400 1.0x10

300

4
200 5.0x10

100

0 0.0
12 µm 19 µm 25 µm 30 µm

(b) Film thickness

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60

-1
50 1 ml h
-1
2 ml h
-1
4 ml h
40
Purity (%)
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30

Lab on a Chip Accepted Manuscript

20

10

0
12 µm 19 µm 25 µm 30 µm

(c) Film thickness

Fig. 5 (a) Recovery rates of MCF7 cells, (b) the number of contaminating WBCs, and (c) the

purities of MCF7 cells, isolated with the assembly-disposable CTC-µChips with various

thicknesses of the silicone-coated release PET film and flow rates. Breast cancer cell lines

(~100 MCF7 cells) were spiked into 5 ml of human peripheral blood and tagged by anti-

EpCAM magnetic nanobeads and isolated at sample and buffer flow rates from 1–4 ml h-1

with an external magnetic flux of 0.2 T. The error bars represent one standard deviation of

three measured datasets.

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3.4. Isolation of CTCs from patients with breast cancer

To isolate CTCs from patients with breast cancer, 5-ml samples of peripheral blood from

patients (n = 20) were collected in vacuum tubes. The thinnest film, a silicone-coated release

12-µm-thick PET film, was used to obtain the highest performance. The sample and the
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buffer flow rates were 2 ml h-1 each, with the separation process taking < 30 min. The number

Lab on a Chip Accepted Manuscript

of isolated CTCs, monitored by immunofluorescence (Figs. 6(a) and S5), ranged from 0–92

(33.6±29.4; mean ± SD) CTCs per 5 ml of blood from patients with breast cancer (n = 20), as

shown in Fig. 6(b) and Table S2. Neither of the two healthy donors had any identifiable CTCs.

The number (33.6±29.4) of CTCs isolated from 5-ml peripheral blood samples of patients can

be compared with previous experimental results3 of the number (4±2) of CTCs isolated from

200-µl peripheral blood samples of patients with breast cancer. Because the quantity of blood

used in this study (5 ml) was 25 times more than the 200 µl used previously, the previously

isolated 4 CTCs corresponds to 100 CTCs here. Thus, the average number of isolated 33.6

CTCs in this study is less than that reported in the previous study. This might have been

caused by use of the 40-µm grid strainer, RBC removal, and washing steps during blood

sample preparation. Moreover, because the present and previous results were obtained from

only 20 and 3 patients, respectively, larger clinical trials are needed to obtain more reliable

results. The number of CTCs would seem to be correlated with the stage of the cancer (Fig.

6(b)). Many studies25-28 have examined the possibility of such a relationship. Interestingly,

the numbers of CTCs isolated from two patients (nos. 19 and 20) under treatment were

markedly reduced.

The average purity of CTCs isolated from patients, defined as the ratio of

cytokeratin-positive cells (CTCs, 33.6±29.4) to CD 45-positive cells (WBCs; 350.7±122.8),

was 9.5%. This means that the WBC depletion rate of the assembly-disposable CTC-µChip

was ~71,429-fold (4.85 log) in the clinical experiments. The WBC depletion rate obtained

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using clinical samples was similar, at 64,666-fold (4.81 log), measured from the biomimetic

blood samples spiked with MCF7 breast cancer cell lines. Because the WBC depletion rate of

the assembly-disposable CTC-µChip, based on a positive separation method using only anti-

EpCAM, is almost constant, the purity of CTCs tends to increase with the stage of the cancer.
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For the sample injection flow rate of 2 ml h-1, the Reynolds number in the

Lab on a Chip Accepted Manuscript

microchannel can be calculated to be ~2, so the fluidic flow is laminar.29 Laminar flow is

important for high purity because it allows for a multi-stream fluid in which CTCs are

selectively transported across the sample and buffer stream boundary into the CTC outlet.

Additionally, the continuous separation, by lateral magnetophoresis, enables the WBC

depletion rate of the assembly-disposable CTC-µChip to be 4.85 log, which is much higher

than that of other approaches.30-32 Indeed, the high purity of 9.5% of the assembly-disposable

CTC-µChip will make it an ideal tool for high-precision genetic analyses, such as next-

generation sequencing (NGS).33

To determine whether CTCs isolated from patients were suitable for subsequent

genetic analysis, RT-PCR analyses were conducted for detecting EpCAM and keratin 19

(KRT19) transcripts as human epithelial-specific markers. Figure 7 shows EpCAM and

KRT19 transcripts amplified from CTCs isolated from patients (nos. 5, 6, 12) and a healthy

donor. EpCAM transcript was detected in 12 (63.2%) of 19 patients with breast cancer (Table

S2). However, among the 20 patients, the KRT19 transcript could be detected in 5 (25.0%),

meaning that the KRT19 transcript is not always expressed in CTCs. This result may support

the explanation that the phenotype of CTCs could be changed due to the epithelial-

mesenchymal transition (EMT).34-37

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Lab on a Chip Accepted Manuscript

(a)

100
The Number of CTCs per 5 ml Blood

80

60

40

20

0
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20

Healthy I II III IV II*

(b) donors Patients * Under treatment

Fig. 6 (a) Magnified images of fluorescently stained CTCs isolated from peripheral blood of

patients with breast cancer. (b) Frequency of CTCs per 5 ml of peripheral blood from patients

(n = 20) and healthy donors (n = 2).

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Lab on a Chip Accepted Manuscript

Fig. 7 RT-PCR amplifications of β-actin (244 bp), keratin-19 (KRT19, 211 bp), and EpCAM

(287 bp) transcripts using CTCs isolated from peripheral blood of patients (nos. 5, 6, 12) with

breast cancer. Lanes 1 and 14, 100-bp DNA size markers; lanes from 2–10, β-actin, EpCAM,

and KRT19 amplified using CTCs isolated from peripheral blood of patients (nos. 5, 6, 12)

with breast cancer; lanes from 11–13, controls using CTCs isolated from peripheral blood of

a healthy donor.

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4. CONCLUSIONS

This paper described an assembly-disposable microfluidic device based on a silicone-coated

release polymer thin film. The disposable format is an important feature for medical and

biological microfluidic devices. In this study, as the test vehicle, a lateral magnetophoretic
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microseparator for isolating CTCs (CTC-µChip) was developed in the proposed assembly-

Lab on a Chip Accepted Manuscript

disposable microfluidic device format, which consists of a disposable polymeric superstrate

and a reusable substrate. The disposable superstrate was fabricated simply by oxygen plasma

bonding of a microstructured PDMS replica and a silicone-coated release PET film and does

not include any complex structures in the microchannel. Thus, no clogging problems

occurred while nucleated blood cells flowed through the microchannel for isolating CTCs.

The analytical and clinical evaluations demonstrated that the assembly-disposable CTC-

µChip is a practical device to isolate CTCs from cancer patients with high performance.

Typically, polymers have superior mechanical and electrical properties, making them

readily compatible with microfluidic devices. Because various polymeric materials can be

used for silicone-coated release films, there are many choices depending on the application of

microfluidic devices. Thus, the proposed assembly-disposable microfluidic device can be

used for various functionalities based on energy fields, such as acoustic, electric, magnetic,

and thermal fields, which can transmit through the polymer thin film to manipulate target

substances in the microchannel. Additionally, because the assembly-disposable microfluidic

device can be realized simply with multiple functions, it could be further advanced as a

disposable microscaled platform for various applications.

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ACKNOWLEDGMENTS

This work (NRF-2016R1A2B4012077) was supported by the Midcareer Researcher Program

through an NRF grant funded by the MEST.

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