Separation Microfluidic Devices Fabricated by

Different Milling Processes

Inês M. Gonçalves

1,2

, Miguel Madureira, Inês Miranda

, Helmut Schütte

, Ana S. Moita

2,5 a

Graça Minas

, Stefan Gassmann

and Rui Lima

1,6

METRICS, University of Minho, Guimarães, Portugal

IN+, Instituto Superior Téncico, Universidade de Lisboa, Lisboa, Portugal

Center for MicroElectromechanical Systems (CMEMS-UMinho), University of Minho, Guimarães, Portugal

Jade University of Applied Science, Wilhelmshaven, Germany

CINAMIL, Departamento de Ciências Exatas e de Engenharia, Academia Militar,

Instituto Universitário Militar, Lisboa, Portugal

CEFT, Faculty of Engineering of the University of Porto, Porto, Portugal

Keywords: Separation Methods, Blood, Microfluidic Biomedical Devices, Micromilling.

Abstract: The diagnostic of several diseases can be performed by analysing the blood plasma of the patient. Despite of

the extensive research work, there is still the need to improve the current low-cost fabrication techniques and

devices for the separation of the plasma from the blood cells. Microfluidic biomedical devices have great

potential for that process. Hence, two microfluidic devices made by micromilling and sealed with or without

the solvent bonding technique were tested by means of a blood analogue fluid. A high-speed video microscopy

system was used for the visualization and acquisition of the analogue fluid flow. Then, the separation of

particles and plasma was evaluated using the software ImageJ. The device manufactured by the micromilling

process without bonding showed a significant reduction of the amount of cells between the entrance and the

exit of the microchannels. However, further analysis and optimizations of the microfluidic devices will be

conducted in future work.

1 INTRODUCTION

Several diseases can affect the components of the

blood, altering its rheological properties.

Consequently, blood plasma and cells are commonly

used for disease diagnosis and therapeutics (Yin et

al., 2013; Nam et al., 2015; Shamloo et al., 2016).

Different information can be obtained from different

components. For instance, the blood plasma is used to

evaluate the inflammatory response and in proteomic

studies, while the red blood cells are used to diagnose

certain diseases (Lee et al., 2014). Therefore, cell

separation is important in medicine and performed

millions of times per day over the world (Al-Fandi et

al., 2011; Bento et al., 2019, Catarino et al., 2019;

Pinho et al. 2020). Nevertheless, some limitations still

hinder the separation process. The most common

techniques used are filtration and centrifugation. The

former was the earliest to be used but has the

http://orcid.org/0000-0001-9801-7617

disadvantage of filter clogging that leads to a

reduction of the process efficiency (Lee et al., 2014).

Centrifugation is the most common method to

separate blood cells from plasma and makes use of

the density difference between the plasma and the

cellular components (Nam et al., 2019). One of the

problems that arise from this technique is the cell

damage due to the high shear forces which can lead

to cell lysis and contamination of the plasma (Geng et

al., 2013). Developments in the field of microfluidics

allowed the improvement of blood separation

techniques with the fabrication of smaller devices

with precise geometries, making the technology

suitable for automation, low-cost, portable, with

improved sterility and requiring less preparation time

and amount of sample and reagents (Bento et al.,

2019, Nam et al., 2019; Tsutsui and Ho, 2009;

Ookawara et al., 2010).

Gonçalves, I., Madureira, M., Miranda, I., Schütte, H., Moita, A., Minas, G., Gassmann, S. and Lima, R.

Separation Microﬂuidic Devices Fabricated by Different Milling Processes.

DOI: 10.5220/0010906500003123

In Proceedings of the 15th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2022) - Volume 1: BIODEVICES, pages 185-190

ISBN: 978-989-758-552-4; ISSN: 2184-4305

185

The design of the microfluidic devices has been

developed to better mimic the geometry of in vivo

microvascular networks. Different separation

mechanisms were also developed and can be divided

in active or passive methods (Dominical et al. 2015;

Shamloo et al., 2016). In the active methods, an

external force is applied to move the cells apart from

the plasma. Those forces can be gravitational,

acoustic, magnetic, optical, chemical, electronic or

electrical. The gravitational method is the most

popular but has some limitations in microfluidic

devices due to the interface tension, low Reynolds

number and laminar flow that occur in flows at those

dimensions (Xue et al., 2012). On the other hand,

passive techniques do not require an external force

and are preferred to the active techniques due to ease

of use and fabrication (Lee et al., 2011; Nam et al.,

2015, Catarino et al., 2019). The passive techniques

depend on hydrodynamic effects that occur in the

fluid when flowing through capillaries. If the

Reynolds number is in values of unity order, the

viscous forces balance the inertial forces and the red

blood cells have the tendency to move towards the

center of the channel, leaving a cell free layer (CFL)

near the walls (Shamloo et al., 2016). This

phenomenon leads to the reduction of the apparent

viscosity, a phenomenon called the Fåhræus-

Lindqvist effect, and to less hematocrit in small

vessels than in large ones (Yin et al., 2013, Losserand

et al., 2019; Medhi et al., 2019). The geometrical

designs used for this type of microchannels include

channel constriction, bending channel and bifurcated

channels. Bending channels make use of centrifugal

forces, constrictions in channels force particles to the

center of the channel while bifurcations make use of

the Zweifach–Fung effect. This effect consists in the

tendency of the cells to travel to the branch channel

of an asymmetric bifurcation with the higher flow rate

since the pressure is lower (Fekete, et al., 2012; Xue

et al., 2012; Meyari et al., 2020).

In this work a microfluidic device for separation

of cells and plasma was investigated. The device was

composed of a microchannel with a series of three

bifurcations and was fabricated by two different

methods. Two blood analogues were used to infer on

the efficiency of the cell clearance and the results

were analysed using ImageJ software. Though further

optimization of the design and fabrication process is

still required, a significant reduction of cells was

obtained with one of the microchannels.

2 MATERIALS AND METHODS

2.1 Microchannel Geometry

The proposed geometry was selected and optimized

by taking into account several previous flow

experiments performed by Lopes et al., 2015, Singhal

et al., 2016 and Madureira et al., 2018. Hence, the

proposed microfluidic device is composed by four

microchannels with the same geometry, but different

depths. The microchannels comprise three separation

segments connected in sequence. After the entrance

the channel width narrows to force the cells to the

center of the channel. Then, an intersection was

placed so the cells continue forward to the exit while

the plasma flows upward or downward through

branch channels. Those channels that will merge

again to form the main channel and the structure are

repeated two more times. A scheme of the

microchannel can be seen in Figure 1and the depth of

each channel is presented in Table 1.

Figure 1: Microchannel geometry with detail showing the

dimensions of the different regions of the segments.

Table 1: Depth of the different microchannels of the

microfluidic devices.

Microchannel

1 and 2

and

Manufacture 1

, W

300 μm

150

μm

100 μm

μm

Manufacture 2

, W

300 μm

150

μm

150 μm

μm

Inlet

Outlet 2

Outlet 1

Outlet 3

Outlet 4

= 260 μm

= 150 μm

= 360 μm

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186

2.2 Fabrication Methods

2.2.1 Manufacture Process 1

One of the microfluidic devices was fabricated from

poly(methyl methacrylate) (PMMA) through a

micromilling process. The geometry of the

microchannels was outlined using a CAD software

and then the device was manufactured using a

micromilling machine (Minitech Mini-Mill/GX. Two

different micromilling tools were used for the

manufacture, one with 100 μm and the other with 1

mm in diameter. The geometry was converted to NC

code using the software Visual Mill so it could be read

by the milling machine. Also, several parameters,

such as, the angle, depth and velocity of the tool, were

defined using that software. All the parameters were

set to each of the used tools considering their different

properties. Starting the milling process, the reference

for the z-axis has to be set manually since the milling

equipment has no height control. A magnifying

camera was used to observe the first touch of the

milling tool on the material. To avoid the damaging

the milling tools due to the heat resulting from the

high rotation speed, the work piece and the tools were

cooled with a mixture of water and detergent during

the milling process. More details of the micromilling

process can be found elsewhere (Lopes et al., 2015;

Singhal et al., 2016; Madureira et al., 2018).

2.2.2 Manufacture Process 2

The second microchannel device was also

manufactured by the micromilling technique above

described. However, for this device, two PMMA

plates were milled and then bonded together using the

solvent bonding technique. The microchannels were

milled in one plate and the other plate worked as the

lid. After the milling and washing, the plates were

placed together and exposed to a solvent vapor which

creates an irreversible bonding between them. In the

end of the treatment, the plates were pressed to ensure

the bonding was maintained.

On both devices, stainless steel tubes with a length of

about 1 cm and an inner diameter of about 1 mm were

used as inlets and outlets of the microchannels. The

tubes were fixed to the Plexiglass (acrylic glass,

Plexiglas XT 0A000) using epoxy glue. The

microchannel devices were then cleaned with water

and detergent for 20 minutes using ultrasonication.

Lastly, the devices were dried with pressurized air

and sealed with an adhesive foil for PCR plates

(Polyester 50 μm ultra-clear, non-sterile, heat-

resistant).

2.3 Experimental Set-up

Flow visualization was performed using an inverted

microscope (IX71, Olympus, Japan) and a high-speed

camera (Fastcam SA3, Photron, USA). The

microchannels were placed on the microscope

connected to a syringe pump (CetoniNEMESYS

Syringe Pump) to control the fluid flow. The

experimental apparatus is presented in Figure 2.

Figure 2: Experimental set-up used to perform the

experimental microfluidic high-speed video recordings.

Two blood analogue solutions were made by adding

the surfactant Brij L4 to distilled water at a

concentration of 1 wt.%, as presented in (Lima et al.,

2020). The emulsion suspended phase is composed by

spherical micelles with sizes ranging between 10 and

20m. One of the solutions (Analogue 1) was then

filtered by two filters of 10 and 20 μm average pore

size while the other solution (Analogue 2) was just

filtered with the 20 μm filter. Consequently, the fluids

present spherical micelles with size ranging from 10

to 20 μm. Initially three different fluid flows, 60

μL/min, 100 μL/min and 150 μL/min, were compared

and 100 μL/min was set for the remaining

experiments.

2.4 Image Processing

The images from the microscope were obtained using

a high-speed camera using a frame rate of 2000 and

6000 frames/second. The image sequences obtained

were analysed using the minimum intensity feature of

the Z-Project from the ImageJ software (Abràmoff et

al., 2004, Carvalho et al. 2021). The intensity of the

pixels of a selected region was then evaluated and

converted to a grey scale using the plot profile

feature. High values correspond to brighter pixels

which indicate regions with less particle density while

lower values correspond to darker pixels and,

consequently, high particle density or channel walls.

Syringe Pump

High-Speed

Camera

Inverted

Microscope

Microfluidic

Device

Separation Microﬂuidic Devices Fabricated by Different Milling Processes

187

The grey value was measured transversely to the

entrance and exit of the channel, using the Plot Profile

feature of ImageJ. The average of the values obtained

was then calculated and normalized, considering the

maximum grey value obtained using water, to a scale

of 0 to 1 for result comparison.

3 RESULTS AND DISCUSSION

The grey value was measured for the microchannel

made by the first manufacture process using the

Analogue 1 at three different fluid flows. Preliminary

tests show that a greater variation between the entry

and the exit is noted for 100 μL/min. The variation is

similar for the other two flows, being lower for 60

μL/min (Figure 3).

Figure 3: Variation of average grey value at the entrance

and exit of the microchannel for different fluid flows.

Figure 4: Variation of average grey value at the entrance

and exit of the microchannel for manufacture process 1 at a

100 μL/min fluid flow.

Subsequently, the grey value variation was

measured using both analogue fluids for the

microchannel of the manufacture process 1 (Figure

4), and of the manufacture process 2, (Figure 5). The

variation of the grey value between the entry and exit

on both the microchannels was similar between the

analogues. For the first manufacture process the

variation was higher using the analogue 2, while for

the second process the variation was higher with

Analogue 1.

The influence of the depth of the microchannel

was also investigated. The variation of the grey

intensity on the different microchannels of the

devices of manufacture 1 and 2 is presented in Figures

6 and 7, respectively. In the case of the former, the

microchannels with lower depths showed higher

clearance of cells while in the later the contrary was

verified. Consequently, the tested deepness of the

microchannels do not have a significant impact on the

cell and plasma separation.

Figure 5: Variation of average grey value at the entrance

and exit of the microchannel for manufacture process 2 at a

100 μL/min fluid flow.

Figure 6: Variation of average grey value at the entrance

and exit of the different microchannels of manufacture

process 1 at a 100 μL/min fluid flow.

Figure 7: Variation of average grey value at the entrance

and exit of the different microchannels of manufacture

process 2 at a 100 μL/min fluid flow.

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As expected, the grey value increased from the

entry to the exit, indicating a reduction of the particle

concentration throughout the microchannels.

Difference in particle size does not seem to affect the

clearance process since the results were similar using

both of the analogue fluids. On the other hand, the

fluid flow affects the efficiency of the microchannel.

For first manufacture process it was possible to obtain

high grey values corresponding to a reduction of the

particle concentration. The grey values were also near

the value obtained with water indicating a successful

separation of the particles and plasma. Nevertheless,

the detection is dependent on the resolution of the

acquired images. Consequently, the minimum value,

corresponding to the maximum amount of particles,

obtained in each measurement varied. Further

techniques and models for image acquisition and data

treatment need to be improved for a better evaluation

of the effectiveness of the device.

4 CONCLUSIONS

The present study shows the potentiality of a novel

geometry for a microfluidic device for the separation

of blood cells and plasma. The separation of the

microparticles from the base fluid was mainly due to

the successive contractions effect, where the particles

tend to migrate into the center whereas the base fluid

tend to flow towards the walls of the microchannel.

From the two manufacture processes tested, the

micromilling process without bonding showed

promising results with grey values close to the ones

obtained with water. The depth of the channels and

the size of the particles did not show a major influence

on the separation process. On the other hand, the fluid

flow affected the results obtained, being 100 μL/min

the flow that presented better results. Further image

analysis studies and optimization of the geometry to

improve the separation process will be performed in

the near future.

ACKNOWLEDGEMENTS

Authors acknowledge FCT – Fundação para a Ciência

e a Tecnologia for partially supporting this

work through projects UIDB/00319/2020,

UIDB/04077/2020, UIDB/00690/2020, UIDB/

04436/2020 and PTDC/EME-SIS/30171/2017

NORTE-01-0145-FEDER-030171, and NORTE-01-

0145-FEDER-029394 funded by COMPETE2020,

NORTE 2020, PORTUGAL 2020, Lisb@2020 and

FEDER and for supporting the PhD Fellowship

(2020.08646.BD).

REFERENCES

Faustino, V.; Rodrigues, R.O.; Pinho, D.; Costa, E.; Santos-

Silva, A.; Miranda, V.; Amaral, J.S.; Lima, R. A (2019),

Microfluidic Deformability Assessment of Pathological

Red Blood Cells Flowing in a Hyperbolic Converging

Microchannel. Micromachines, 10, 645.

Yin, X., Thomas, T., Zhang, J. (2013) Multiple red blood

cell flows through microvascular bifurcations: Cell free

layer, cell trajectory, and hematocrit separation.

Microvasc. Res., 89:47–56.

Shamloo, A., Vatankhah, P., Bijarchi, M.A. (2016)

Numerical optimization and inverse study of a

microfluidic device for blood plasma separation. Eur. J.

Mech. B/Fluids, 57: 31–39.

Nam, J., Namgung, B., Lim, C.T., Bae, J.E., Leo, H.L.,

Cho, K.S., Kim, S. (2015) Microfluidic device for

sheathless particle focusing and separation using a

viscoelastic fluid. J. Chromatogr. A, 1406:244–250.

Lee, M.G., Shin, J.H., Choi, S., Park, J.K. (2014) Enhanced

blood plasma separation by modulation of inertial lift

force. Sensors Actuators, B Chem., 190:311–317.

Al-Fandi, M., Al-Rousan, M., Jaradat, M.A.K., Al-Ebbini,

L. (2011) New design for the separation of

microorganisms using microfluidic deterministic lateral

displacement. Robot. Comput. Integr. Manuf., 27:237–

244.

Bento, D., Fernandes, C.S. Miranda, J.M., Lima, R. (2019)

In vitro blood flow visualizations and cell-free layer

(CFL) measurements in a microchannel network. Exp.

Therm. Fluid Sci., 109:109847.

Catarino, S. O., R. O. Rodrigues, D. Pinho, J. M. Miranda,

G. Minas, R. Lima. (2019) Blood Cells Separation and

Sorting Techniques of Passive Microfluidic Devices:

From Fabrication to Applications. Micromachines, 10,

593.

Pinho D, Carvalho V, Gonçalves IM, Teixeira S, Lima R.

(2020) Visualization and Measurements of Blood Cells

Flowing in Microfluidic Systems and Blood Rheology:

A Personalized Medicine Perspective Journal of

Personalized Medicine 10, 4, 249.

Nam, J., Yoon, J., Kim, J., Jang, W.S., Lim, C.S. (2019)

Continuous erythrocyte removal and leukocyte

separation from whole blood based on viscoelastic cell

focusing and the margination phenomenon. J.

Chromatogr. A, 1595:230–239.

Geng, Z., Ju, Y., Wang, W., Li, Z. (2013) Continuous blood

separation utilizing spiral filtration microchannel with

gradually varied width and micro-pillar array. Sensors

Actuators, B Chem., 180:122–129.

Tsutsui, H., Ho, C.M. (2009) Cell separation by non-inertial

force fields in microfluidic systems. Mech. Res.

Commun., 36:92–103.

Ookawara, S., Oozeki, N., Ogawa, K., Löb, P., Hessel, V.

(2010) Process intensification of particle separation by

Separation Microﬂuidic Devices Fabricated by Different Milling Processes

189

lift force in arc microchannel with bifurcation. Chem.

Eng. Process. Process Intensif., 49:697–703.

Dominical, V.M., Vital, D.M., O’Dowd, F., Saad, S.T.O.,

Costa, F.F., Conran, N. (2015) In vitro microfluidic

model for the study of vaso-occlusive processes. Exp.

Hematol., 43: 223–228.

Xue, X., Patel, M.K., Kersaudy-Kerhoas, M., Desmulliez,

M.P.Y., Bailey, C., Topham, D. (2012) Analysis of

fluid separation in microfluidic T-channels. Appl. Math.

Model., 36:743–755.

Lee, M.G., Choi, S., Park, J.K. Inertial separation in a

contraction-expansion array microchannel. J.

Chromatogr. A, 1218:4138–4143.

Losserand, S., Coupier, G., Podgorski, T. (2019) Migration

velocity of red blood cells in microchannels.

Microvasc. Res., 124:30–36.

Lima, R.; Vega, E.J.; Moita, A.S.; Miranda, J.M.; Pinho,

D.; Moreira, A.L.N. (2020) Fast, flexible and low-cost

multiphase blood analogue for biomedical and energy

applications. Experiments in Fluids, 61, 231,

doi:10.1007/s00348-020-03066-7.

Medhi, B.J., Reddy, M.M., Singh, A. (2019) Particle

migration of concentrated suspension flow in

bifurcating channels. Adv. Powder Technol., 30:1897–

1909.

Fekete, Z., Nagy, P., Huszka, G., Tolner, F., Pongrácz, A.,

Fürjes, P. (2012) Performance characterization of

micromachined particle separation system based on

Zweifach-Fung effect. Sensors Actuators, B Chem.,

162:89–94.

Meyari, M., Salehi, Z., Zarghami, R., Saeedipour, M.

(2020) Numerical investigation of particle separation in

Y-shaped bifurcating microchannels. Particuology,

Corpus ID: 228863362

Lopes R., Rodrigues R. O., Pinho D., Garcia, V., Schutte

H., Lima R., Gassmann S., (2015). Low cost

microfluidic device for partial cell separation:

micromilling approach. in Proceedings of 2015 IEEE

International Conference on Industrial Technology,

Seville, Spain.

Singhal, J., Pinho, D., Lopes, R., Sousa, P., Garcia, V.,

Schütte, H., Lima, R., Gassmann, S. (2016) Blood Flow

Visualization and Measurements in Microfluidic

Devices Fabricated by a Micromilling Technique.

Micro Nanosyst, 7:148–153.

Madureira, M., Faustino, V., Schütte, H., Gassmann, S.,

Lima, R. (2018) Blood cells separation in a T-shaped

microchannel fabricated by a micromilling technique.

J. Mech. Eng. Biomech., 2:43–49.

Abràmoff, M.D., Magalhães, P.J., Ram, S.J. (2004) Image

processing with imageJ. Biophotonics Int., 11:36–41.

Carvalho, V.; Gonçalves, I.M.; Souza, A.; Souza, M.S.;

Bento, D.; Ribeiro, J.E.; Lima, R.; Pinho, D. (2021)

Manual and Automatic Image Analysis Segmentation

Methods for Blood Flow Studies in

Microchannels. Micromachines, 12, 317.

BIODEVICES 2022 - 15th International Conference on Biomedical Electronics and Devices

190