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Plasma extraction by centrifugo-pneumatically induced gating of flow

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2013 J. Micromech. Microeng. 23 035035

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IOP PUBLISHING JOURNAL OF MICROMECHANICS AND MICROENGINEERING
J. Micromech. Microeng. 23 (2013) 035035 (5pp) doi:10.1088/0960-1317/23/3/035035

Plasma extraction by
centrifugo-pneumatically induced
gating of flow
Robert Burger 1,2 , Nuno Reis 2 , João Garcia da Fonseca 2 and Jens Ducrée 1
1
Biomedical Diagnostics Institute, National Centre for Sensor Research, School of Physical Sciences,
Dublin City University, Ireland
2
Biosurfit SA, Edifı́cio ICAT, Campus da FCUL, 1749-016, Lisbon, Portugal
E-mail: robert.burger2@mail.dcu.ie and jens.ducree@dcu.ie

Received 8 August 2012, in final form 17 December 2012

Published 8 February 2013
Online at stacks.iop.org/JMM/23/035035

Abstract
We present a novel valving mechanism to implement plasma extraction from whole blood on a
centrifugal microfluidic ‘lab-on-a-disc’ platform. The new scheme is based on
pressure-induced deflection of a liquid membrane which gates the centrifugally driven flow
through a microfluidic structure. Compared to conventional concepts such as capillary burst
valves, siphons or sacrificial materials, the valving structure presented here is represented by a
compact, small-footprint design which obviates the need for (local) surface functionalization
or hybrid materials integration, thus significantly reducing the complexity (and hence cost) of
manufacture. As a pilot study of this new centrifugal flow control element, we demonstrate
here the efficient separation of metered plasma from whole blood. While the flow is stopped,
blood is separated into plasma and its cellular constituents by centrifugally induced
sedimentation. After completion, the flow resumes by elevating the spinning frequency,
providing up to 80% of the original plasma content to an overflow chamber within a short,
2 min interval. The amount of residual cells in the plasma amounts to less than 20 cells μl−1 .
(Some figures may appear in colour only in the online journal)

1. Introduction of many immunoassay protocols. The lab-on-a-disc platform

The recent decades have seen a dynamic surge of activities
in the field of centrifugal microfluidic technologies. Even at
the infancy of microfluidics in the early 1990s, companies
such as Abaxis [1], Gyros Microlabs [2] and Gamera
(later acquired by Tecan) [3] employed the robust liquid
handling of these compact ‘lab-on-a-disc’ systems to develop
pioneering bioanalytical sample-to-answer products, some
of them with still maintaining commercial success. In
particular, since the mid-2000s, a rapidly growing number
of academic and corporate groups have created a wealth
of innovative concepts for liquid handling and detection on
rotating platforms. Basically the full spectrum of common
bioanalytical assays ranging from clinical chemistry over
immunoassays to molecular and cell-based diagnostics has
been successfully implemented. Separation of plasma from
whole blood is a common preparative step at the beginning
lends itself particularly well to plasma separation since the
centrifugal force experienced by suspended cells readily
empowers density-based plasma extraction. In the past, a wide
range of centrifugal microfluidic blood separation structures
have been engineered [4–8].
A key distinction of centrifugal microfluidic platform
from conventional lab-on-a-chip systems is that all on-board
liquids simultaneously experience the centrifugal force. This
fact puts valving strategies which are capable of establishing a
time sequence representing complex liquid handling protocols
into the pivot of most application-focused research initiatives.
In order to be commercially viable, such multi-liquid valving
strategies need to be precise, robust and amenable to mass
manufacture without requiring additional backend processes
such as surface coatings and hybrid integration of sacrificial
materials.

0960-1317/13/035035+05$33.00 1 © 2013 IOP Publishing Ltd Printed in the UK & the USA
J. Micromech. Microeng. 23 (2013) 035035
These demands prove to be particularly challenging
for plasma extraction steps at the beginning of centrifugal
protocols as, by virtue of the centrifugally imposed, radially
outward direction of flow, sedimentation would have to be
run at rather elevated frequencies in reservoirs located close
to the center of rotation where real estate is scarce. Various
valving concepts have been proposed which offer different
degrees of flow control and manufacturing issues. One of
the most commonly used methods is siphoning, where two
basic actuation mechanisms have been pursued. In a capillary
siphon, a reservoir is connected to a hydrophilic siphon
channel. At high spin rates, the liquid heights in the reservoir
and the connected siphon outlet are level, since the centrifugal
force exceeds the capillary force in the siphon. Upon reduction
of the spin rate, capillary action propels the liquid across the
crest point of the channel until the advancing meniscus falls
below the liquid level in the reservoir which then empties [6,
9]. These capillary siphons thus stay closed at high frequencies
while priming below a certain threshold. In an overflow siphon,
liquid is simply added to the upstream reservoir until the
siphon overflows [10]. Siphoning is therefore triggered by an
external event, the addition of a volume to the reservoir. Lately,
centrifugo-pneumatic siphoning has been introduced which
eliminates the need for volume addition while not requiring
hydrophilization, either [11, 12]. The so-called capillary burst
valves utilize capillary stops usually defined by a combination
of channel geometry and contact angle. Popular types are
hydrophobic constrictions and hydrophilic expansions [13].
In both cases, the liquid stops at the valve and only progresses
after a threshold frequency is exceeded. In contrast to siphon
valves, capillary valves remain closed at low frequencies and
yield at high rotation speeds. While they are conceptually
simple, the practically achievable manufacturing precision and
tolerances of these capillary valves limit the maximum burst
frequencies and the definition of narrow frequency bands, thus
restricting their use for high-frequency centrifugation steps and
multi-step assay protocols, and their parallelization on a single
disc. Mark and colleagues presented a pneumatic valving
scheme for metering highly wetting liquids [14, 15] using
a counteracting air pressure to prevent liquid from passing the
valve below a certain threshold frequency.
In recent years, research has been focused on developing
sacrificial valves which offer certain advantages such as
a wider range of operational frequencies. These valves
are based on ancillary materials such as flow blocking
membranes or plugs which are opened by external stimuli
such as a light source [16], a laser [17, 18] or heat [19].
Since these physical shunts additionally act as diffusion
barriers for vapors, they also offer a route toward on-
board reagent storage. Furthermore, valving can often be
controlled independent of the rotational frequency; however,
some actuation concepts require stopping the disc for opening.
Recently, water dissolvable membranes have been employed to
allow a mere rotational control through centrifugo-pneumatic
actuation [20]. The caveat of sacrificial valving schemes is
the rather complex system assembly. Another approach has
been to form normally closed valves by a pre-stressed, elastic
membrane which exerts a pressure against a valve seat [21].
2
R Burger and N Reis
The membrane is deflected to open a passageway for the liquid
under the impact of a centrifugal field. In this case, the flow
rate is governed by the elastic modulus of the material and
complete release of the entire stored reagent volumes is hard
to achieve.
In contrast to these previous approaches, the device
presented here exploits the novel effect of self-induced
membrane deflection to control liquid flow under rotation. In
this work, we will engineer a structure for high-frequency
extraction and metering of plasma from whole blood.
Our centrifugal microfluidic structure possesses a compact
footprint in the radial direction, does not require local surface
modifications or the integration of sacrificial materials. Our
scheme is well amenable to standard injection molding
techniques, e.g. for compact discs (CDs).
This paper first outlines the novel valving principle based
on the centrifugo-pneumatic deflection of a liquid membrane.
Next, experimental details are surveyed before we present the
results on extraction of metered plasma, finally we summarize
and conclude.
2. Working principle
Figure 1 illustrates the working principle using a simplified
geometry. The system comprises two chambers displaying a
radial offset. The up- and downstream chambers are insulated
from atmospheric pressure and in fluidic communication with
each other via two independent channels. For the sake of
clarity, the channel through which liquid will initially flow
will be designated the liquid channel while the other duct
will be referred to as the air channel. At the start, liquid
is introduced into the radially inner chamber via an inlet
port distant from the channels. The chamber is designed
such that the liquid does not initially reach the channel
inlets. Subsequently, the structure is sealed from atmospheric
pressure. Rotation of the disc substrate propels a flow to
the radially outward chamber via the liquid channel. The
outer chamber is dimensioned sufficiently shallow (typically
between 50 to 500 μm) so a liquid jet forms which acts as
a continuous, gas-tight membrane. This dynamically formed,
‘elastic’ membrane asymmetrically divides the cavity into two
fluidically isolated compartments a and b.
For a simplified model of the working principle, we
assume a static and flat membrane, i.e. a pressure differential
P across the membrane will not induce deflection (figure 1
(i)). Furthermore, we set the initial gas pressure in the structure
equal to atmospheric pressure P0 .
Displacement of a liquid volume Vl from the upper
chamber will consequently result in a compression of the air
in the lower chamber, while the pressure in the upper chamber
reduces. The pressure Pb on the left side of the membrane in
the lower chamber
Vb0
Pb = P0
Vb0 − Vl
follows the ideal gas law. Similarly, the pressure in the
right side compartment can be calculated
Va0
Pa = P0
Va0 + Vl
J. Micromech. Microeng. 23 (2013) 035035 R Burger and N Reis

f1
f1 Liquid Channel f1 f2 > f 1

+
Air Channel

+
Vl Pa
Pa Va0 Pa Fc
Fcap
F
Vb0 Pb
Pb Pb Fc

i Vl ii Liquid Membrane iii Liquid Plug iv

Figure 1. Working principle of the valving scheme. The chamber is divided in two compartments a and b by the liquid membrane.
Displacement of a liquid volume Vl from the upper to the lower chamber (compartment b) results in a decreased pressure Pa in
compartment a while the pressure Pb in compartment b increases (i). The pressure difference P deflects the membrane toward the lower
pressure region in order to compensate for the pressure difference (ii). The membrane disintegrates once it reaches the air channel, leaving a
liquid plug in the channel. This plug is stabilized by the interplay of centrifugal force Fc with the counteracting force FP due to the pressure
difference and the force Fcap due to the capillary pressure (valve closed) (iii). A further increase of the rotational frequency destabilizes the
plug so the transfer of the liquid from the upper to the lower chamber (valve open) resumes until the upper reservoir is completely depleted
(iv).

with the environmental pressure P0 , while Vb0 and Va0 represent
the initial air volumes in the compartment on the left- and right-
hand sides of the membrane, respectively. Consequently, the
equation
 
Vb0 Va0
P = P0 −
Vb0 − Vl Va0 + Vl
expresses the pressure difference P across the membrane.
In the real scenario, the membrane is evidently flexible.
The system will hence seek to equilibrate this pressure
difference P by deflecting the membrane toward the air
channel (figure 1 (ii)). During the displacement phase, liquid
is continuously flowing from the upper to the lower chamber.
Once the membrane reaches the air channel, the membrane
disrupts and a liquid plug enters the air channel (figure 1 (iii)).
At this stage, the lower chamber is pneumatically sealed
by the two plugs residing within the liquid and the air channel.
Droplet formation from these plugs is suppressed by the
capillary pinning of the outer menisci along the circumferential
edges of the two orifices. In addition, the surface tension
introduces an elastic element which counteracts the protrusion
of the meniscus into the lower chamber. During this state,
the system is very similar to the centrifugo-pneumatic valving
mechanism developed by Mark and colleagues [15]. The work
by Mark et al also provides a good theoretical description of
the forces acting on a liquid plug entering a dead end chamber,
which can also be applied in the case presented here. Only
once the rotational frequency is raised beyond a significantly
higher threshold, the force equilibrium will be unbalanced by
the now dominant centrifugal field to remove the plugs from
the connecting channels. Radially outward directed flow then
resumes through the liquid channel, while air simultaneously
escapes quickly in the opposite direction via the parallel air
channel to compensate for the increasing pressure in the
outer chamber. Depending on the ratio of liquid volume to
chamber volumes, this process may occur once or several
times until liquid has been fully transferred from the inner
to the outer chamber (figure 1 (iv)). It should be noted that
our experiments showed that the deflection of the membrane
is independent of the sense of rotation, thus ruling out the
Coriolis force as the dominant effect.
3. Plasma extraction and metering
The design of the disc-based blood separation structure
(figure 2) is derived from this novel valving mechanism
(figure 1). The separation structure is constituted by an initial
blood loading chamber with a first cell retention reservoir,
a radially outer chamber compartmentalized in a second cell
collector and a plasma metering structure overflowing into a
waste. Both chambers are connected by an air and a liquid
channel to enable the above described centrifugo-pneumatic
valving. During centrifugation, cells sediment and therefore
tend to remain in the retention reservoirs of these two overflow
structures. Since the liquid membrane needs to establish before
the valve reaches the closed state (during which the plasma
separation is performed), a certain fraction of the blood will
leak into the lower, second reservoir. However, the pneumatic
layout and frequency protocol is engineered in a way that the
stopped-flow occurs prior to the filling of the second retention
reservoir. The spin rate is ramped up again (valve open) after
the cells in the chambers had sufficient time to sediment.
This way only pure, cell-free plasma will reach the final
metering unit, or further assay stages in a future, integrated
microfluidic layout. As can be seen in figure 2, several of these
plasma extraction and metering structures can be distributed
azimuthally according to the rotational symmetry of the disc,
and be used simultaneously or sequentially.
4. Materials and fabrication
All structures used in this work have been engraved and cut
by CO2 laser ablation of PMMA cast sheets (Repsol, Spain).
First, all reservoirs were engraved with a constant depth of
200 μm. Next the reservoirs were connected by engraving the
liquid and air channels, also with a depth of 200 μm. Finally,
fluidic ports were cut using the laser. Discs were then cleaned

3
J. Micromech. Microeng. 23 (2013) 035035 R Burger and N Reis

+
2 mm
ii

19
.8
21.

23.3

m
1m

m
Liquid Channel
(200x200 µm2)

mm
m

20
.7
Sample Inlet Air Channel

mm
(200x200 µm2)
1. Cell Reservoir
(V = 2.28 µl)

2. Cell Reservoir
Plasma Waste (V = 1.46 µl)
(V = 2 µl) Plasma Metering
i (V = 1 µl)

Figure 2. The design of a disc containing four identical blood separation structures (i). The inset shows a detailed view of one separation
structure, the individual components and their dimensions (ii).

Blood Sampling Blocking (Valve Closed) Plasma Separation Plasma Metering (Valve Open)

Liquid Blocked Metered

i Membrane ii Channel iii Plasma iv
f (Hz)
75

50

25

t (s)
1s 3s 0.2s 60s 9s 25s

Figure 3. Separation of plasma from 5 μl of whole blood. First blood is collected from a fingerprick and loaded into the separation chamber
(i). The disc is then spun at 50 Hz which results in formation of the liquid membrane and subsequent blocking of the air channel (ii). After
blocking the air channel, no more liquid is exchanged between the two chambers. The rotation frequency is lowered to 40 Hz and the
cellular components sediment within 60 s (iii). Subsequently the disc is accelerated to 85 Hz to remove the block from the air channel,
resulting in plasma flowing from the inner to the outer chamber. Residual cells are collected in the second cell reservoir, while only plasma
overflows to the metering chamber (iv). Scale bars are 2 mm.

by a 5 min sonication in DI water and dried using nitrogen. The
disc was then sealed using unexposed dry film resist (DFR)
(SY 330, Elga Europe, Italy). To this end, DFR was laminated
on a blank DVD halve at a role temperature of 80 ◦ C.
Subsequently, the DVD halve was bonded to the laser
machined disc using the same parameters.
Blood for all experiments was taken via fingerprick from
a healthy donor. Experiments were conducted on a custom
built test stand comprising of a computer controlled motor for
spinning the disc (Coolmuscle CM2, Myostat Motion Control
Inc., Canada) and a synchronized camera for image acquisition
(Unibrain Fire-I 501, Unibrain, Greece). A similar setup has
been presented by Grumann et al [22].
5. Experimental results
Separation was performed using the following protocol: first
a blood sample is collected with a 5 μl capillary tube
(figure 3 (i)). The blood is injected into the first sedimentation
chamber and the inlet is sealed with adhesive tape. The disc is
then accelerated to a frequency of 50 Hz at an acceleration
of 50 revolutions s−2 . At this speed, blood flows into the
second sedimentation reservoir, the liquid membrane is formed
and eventually the liquid plug emerges in the air channel
(figure 3 (ii)). After 3 s the disc is slowed down to 40 Hz with
a deceleration of 50 revolutions s−2 . This speed is maintained
for 60 s in order to complete the plasma separation while

4
J. Micromech. Microeng. 23 (2013) 035035
flow from the upper to the lower reservoir is suppressed
(figure 3 (iii)). Next the disc is moderately accelerated at
5 revolutions s−2 , since the impact of the incoming plasma
in combination with a high acceleration might lead to a
redistribution of the cell pellet in the second cell reservoir.
Next a high frequency of 85 Hz is set for 25 s in order to
remove the plug. Once the critical frequency is reached, the
block breaks and plasma with a small fraction of residual cells
advancing into the second reservoir from where the plasma
later overflows to the metering chamber. Subsequently, the
excess volume is collected in the waste reservoir (figure 3
(iv)). This results in a metered volume of 1 μl of plasma which
is available for downstream analysis. It has been observed that
the valving mechanism works reliably despite the rather low-
precision manufacturing method (CO2 laser ablation) which
has been used. This suggests that the valving mechanism is
robust and can tolerate manufacturing variations.
With this structure, up to 80% of the total plasma content
was extracted in less than 2 min. The extraction efficiency has
been calculated by first measuring the haematocrit in an aliquot
of the donor blood off-disc. Following on-disc extraction,
the separated plasma has been collected from the extraction
structure and the volume has been determined. The amount
of residual erythrocytes in the extracted plasma was then
manually counted. In all cases (n = 10), less than 20 cells per
μl of extracted plasma were determined, thus demonstrating
the high efficiency of this structure.
6. Summary and conclusions
We have developed a compact blood separation structure for
centrifugal lab-on-a-disc systems based on novel valving by
flow-induced membrane deflection. Starting with initial whole
blood volumes of typically 5 μl, our structure meters 1 μl of
highly pure plasma (<20 residual cells μl−1 ) for subsequent
assay steps within less than two min. Compared to state-of-the-
art techniques, the new mechanism does not require patterning
of hydrophobic/hydrophilic surface coatings or integration of
sacrificial materials. This lower system complexity tends to
enhance robustness of operation and facilitates monolithic
manufacture by common polymer mass replication schemes
such as injection molding. Apart from plasma extraction, the
here presented membrane deflection mechanism may be used
to control a variety of other liquid handling operations in
centrifugal microfluidic systems.
Acknowledgments
This work was supported by the Science Foundation Ireland
(grant no 10/CE/B1821).
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