Annals of Biomedical Engineering, Vol. 42, No. 11, November 2014 (Ó 2014) pp. 2289–2302
DOI: 10.1007/s10439-014-1060-2
Magnetic Droplet Manipulation Platforms for Nucleic Acid Detection
at the Point of Care
DONG JIN SHIN1 and TZA-HUEI WANG1,2,3,4,5
1
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA; 2Department of Mechanical
Engineering, Johns Hopkins University, Baltimore, MD, USA; 3Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins
University, Baltimore, MD, USA; 4Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA; and
5
108 Latrobe, 3400 N Charles St, Baltimore, MD 21218, USA
(Received 28 February 2014; accepted 19 June 2014; published online 10 July 2014)
Associate Editor Tingrui Pan oversaw the review of this article.
based microfluidic devices,61 capillary pump-driven
microfluidic devices16,19 and droplet-based platforms.16,32 In addition to the traditional benefits of
microfluidics like reduced sample consumption and
integration, point-of-care diagnostic platforms should
also consider factors such as sample preparation,
simple end-user operation and portable instrumentation. Among these approaches, droplet-based techniques are of particular interest since aqueous reagents
are readily scalable in a droplet format without radical
modification to biochemical processes, which makes
them easily adaptable to a wide range of current and
future benchtop assays.
Droplet microfluidic platforms have emerged under
various formats, and can be broadly categorized under
open-surface and channel-based formats. Furthermore, platforms may be categorized based on the mode
of droplet or particle manipulation, some of which
include
magnetic,2,7,13,27,29,32,34,36,37,39,43,44,52,63–65,67
dielectrophoretic (DEP),15,22,53 electrowetting-ondielectric (EWOD)8,14,20,30,38,47,48,50,55 and optical12,35
approaches. This review focuses specifically on opensurface magnetic droplet platforms that utilize magnetic forces on particles as a means of both droplet and
particle manipulation. This is in contrast to other
droplet platforms utilizing EWOD and DEP as the
primary mechanism for droplet handling, where the
incorporation of magnetic particles requires an auxiliary mode of actuation for magnetic manipulation.
Magnetic particles are regularly utilized in biochemical
assays as solid phase substrates for analyte capture and
purification, which highlights the importance of magnetic manipulation in these platforms. Magnetic
droplet platforms are particularly well-suited to nucleic
acid-based assays for two reasons. Firstly, nucleic acid
Abstract—This review summarizes recent developments in
the use of magnetically actuated droplets in point-of-care
molecular diagnostic platforms. We discuss the fundamentals
of magnetic droplet manipulation and the various modes of
actuation. The balance of forces acting on a droplet during
transport and particle extraction, as well as the devices and
instrumentation developed to perform these operations will
be presented and discussed. Furthermore, we review some of
the recent advances on the diagnostic applications of
platforms utilizing magnetic manipulation for genetic assessment of biological samples.
Keywords—Magnetofluidics, Droplet manipulation, Electromagnetic actuation, Surface patterning, DNA extraction,
Real-time PCR.
INTRODUCTION
Recent advances in bioanalytical techniques have
enabled researchers to develop assays that are both
simple and robust. These developments have paved the
path towards integrated platforms designed to perform
all aspects of molecular diagnostic assays, from sample
preparation to detection, on a single device. Taking
advantage of these advancements, commercial development of plug-and-play benchtop platforms has
already made significant progress in recent years, as
demonstrated in products such as Cepheid GeneXpert
platform62 and BioFire FilmArray platform.5 Meanwhile, efforts to further miniaturize assays for mobile
medicine have also led to shifting paradigms in academic research towards approaches such as paperAddress correspondence to Tza-Huei Wang, 108 Latrobe, 3400 N
Charles St, Baltimore, MD 21218, USA. Electronic mail: biomems123@gmail.com, thwang@jhu.edu
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Ó 2014 Biomedical Engineering Society
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purification involves multiple steps of buffer aspiration
and addition, which is greatly simplified on a magnetic
droplet manipulation platform as a series of particle
extraction steps across various buffer droplets.63 Secondly, the issue of contamination with amplificationbased assays such as PCR makes disposable cartridges
an essential aspect of device design,32 and the simple
layout of a magnetically actuated droplet device confers an advantage over other devices that require
components such as electrodes to be fabricated directly
on the cartridge.
In this review article, the theoretical basis of opensurface magnetic droplet platforms will be discussed in
order to highlight the physical basis for magnetofluidic
droplet manipulation. Fundamentals of droplet
manipulation operations are described as a function of
parameters such as droplet volume and particle size,
which are important topics in the context of on-chip
process design. Principles of device fabrication and
various modes of magnetic actuation will be discussed,
followed by examples of their application in assessment of genetic biomarkers.
PRINCIPLES OF MAGNETOFLUIDIC DROPLET
MANIPULATION
Magnetically actuated droplet microfluidic platforms have an interdisciplinary origin, as they are the
product of recent progress in magnetofluidics, microfabrication and macromolecule purification techniques. From a magnetofluidics perspective, early
studies of manipulating liquid containing magnetic
particles by using a permanent magnet44 or an electromagnet39 paved the way for other investigators to
build on the fundamental mechanisms. Additional
progress in device fabrication techniques via surface
patterning23 and micro-elevations defined by soft
lithography63 enabled integration of multiple reagents
in order to recreate the entire workflow of complex
bioassays on a single device.
A typical magnetically actuated droplet platform as
shown in Fig. 1 may include a hydrophobic substrate,
on which aliquots of reagents utilized at various stages
of a multi-step reaction are primed as sessile droplets.
These droplets are separated by passive elements that
function as valves that facilitate extraction of magnetic
particles from a droplet. The surface-modified magnetic particle serves as a solid phase substrate on which
analytes can be purified and transported across
reagents at various stages of sample processing.
Droplet transport, fusion and magnetic particle
extraction are essential operations in any biomolecular
assay that involves the addition of reagents and aspiration of buffer solutions from magnetic particles.
AND
WANG
FIGURE 1. Sketch of a typical magnetically-actuated droplet
device. Droplets are arranged serially on a hydrophobic substrate and sealed with oil. Passive valves for particle extraction from buffer are realized in this example using
topographical barriers. In this example, extraction of nucleic
acids and DNA amplification are combined into a singlestream process to integrate all sample processing steps
involved in a PCR-based assay on a single device.
While the specifics of their implementation may vary
across platforms, droplet manipulation on an open
surface can be generalized as the interaction of three
forces. The balance of these components determines
the type of droplet handling operation being performed. The first component is the magnetic force,
which is a consequence of paramagnetic bead cluster
being attracted by an external magnetic field. For
magnetic particles clustered in a nonmagnetic medium,
the force acting on the particle cluster can be obtained
as follows42:
M
B
r B:
FM ¼
v
q
l0
Here, M and q represent the mass and density of
magnetic particle cluster, x is the magnetic susceptibility of the particles, B is the applied magnetic field,
and l0 is the permeability of free space. In magnetbased droplet manipulation, this component is the only
parameter that is actively modulated on the device. As
such, all operations are facilitated either by modulating
travel velocity or by introducing passive structural
components that can modify the other two force
components on the droplet. Magnetic force is directly
proportional to the mass of the magnetic bead cluster,
which highlights an important design parameter for
magnetic droplet-based platforms. Because magnetic
particles also serve a functional role as the solid substrate on which biochemical analytes are captured,
large particle load also increases the functional surface
area of the system. However, increase in particle load is
accompanied by a concomitant increase in the volume
of liquid carried by the magnetic particle cluster, which
may result in contamination of subsequent steps in
complex bioassays.13
The second component is the drag or frictional
force, which describes the friction imposed on the
droplet by physical impediments such as solid surfaces
Magnetic Droplet Manipulation Platforms
and barriers in contact with the droplet. This force is
described by the following equation29:
Ff ffi Kf Rb l U:
Here, Kf is the frictional constant, Rb is the radius of
contact area between the droplet and the substrate, l is
the viscosity of surrounding oil medium and U is the
velocity of the droplet. From a design perspective,
frictional force can become a hindrance to efficient
droplet transport, especially when the magnetic force is
smaller than frictional force. This is the case with
rugged surfaces as well as substrates with high surface
energies, which results in an increase in the frictional
constant and the contact area. Open-surface droplet
microfluidic platforms address this issue by using planar surfaces coated with commercial fluoropolymers
such as amorphous Teflon13,27,36,47,63,67 and Cytop30,55
or chemical vapor deposition with chemicals such as
parylene14 and organosiloxane (SiOC).50 Coating the
substrate with low surface energy chemicals has additional functional roles as a means of preventing contamination via fouling, as well as preventing loss of
biological molecules via adsorption to the surface.
A third component is the capillary force, which
arises at deformities on the surface of the droplet. This
force is particularly relevant for its involvement in
droplet fission, which generally takes place after a
process of deformation and necking. The maximum
capillary force that can be sustained before the droplet
of volume V undergoes fission can be followed from
derivation by Shikida et al.44:
1
Fc;max ffi c 6p 2 V 3 :
Here, c describes the interfacial tension between
droplet and the surrounding oil medium. From a
design perspective, reducing this component allows the
magnetic particles to dissociate from the parent droplet
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more easily. This can be facilitated by the addition of
surfactants in the oil medium, which may be a necessary condition for fission of magnetic particles from
fluids with a high interfacial tension. Optimizing the
concentration of surfactants and particle volume is an
important process in developing a robust droplet
manipulation platform. Chiou et al.13 characterized the
effect of varying bead volume and surfactant concentration on the robustness of their droplet fission
operation and observed that an increased surfactant
concentration is also accompanied by droplet instability, where reagent priming eventually becomes
challenging.
Designing a droplet microfluidic platform capable of
performing multiple fluidic handling operations
requires an intuitive understanding of the interplay
between the three forces as shown in Fig. 2. These
interactions have been investigated by Long et al.29 for
open-surface droplet manipulation on a planar hydrophobic substrate, in the form of an ‘operation diagram’
where the relative magnitudes of forces as a function of
magnet velocity and particle load result in a particular
droplet operation. In this classification scheme, the
interaction between the droplet containing magnetic
particles and a permanent magnet are categorized under
three regimes. The first is particle extraction, which
occurs when the magnetic force exceeds the maximum
capillary force holding the magnetic plug and the parent
droplet together. The second is droplet transport, where
the magnetic force is insufficient to overcome the capillary force but is sufficient enough to overcome the
friction force acting between the droplet and the device
surface. The last regime is droplet disengagement, where
the magnetic force is less than both the friction and
capillary forces and no further manipulation is achieved
by the magnet.
In a later work, Zhang and Wang67 described an
expanded operation diagram as shown in Fig. 3 to
FIGURE 2. (a) Operating diagram developed by Long et al. as a function of particle loading and magnet velocity. OB1, OB2 and
OB3 are the operating boundaries separating each operation regions. (b) Top-down schematic of three forces acting on a droplet
containing magnetic particles. In general, droplet kinematics involves the relative balance between magnetic force on the particle
(FM), friction force (Ff) and capillary force (Fc). (adopted from Ref. 29).
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FIGURE 3. Operating diagram developed by Zhang et al. as a function of particle loading, droplet volume and SET dimension.
With a fixed travel velocity, droplet manipulation operations depend only on the static parameters including droplet volume,
particle amount and SET size (from Ref. 67).
include surface patterns that can dramatically enhance
frictional forces in order to facilitate a full range of
droplet handling operations. The selective patterning
of hydrophobic substrate with hydrophilic surface
energy traps (SETs) result in an additional force component attributed to surface tension arising from the
wetting of the droplet on the SET. In this arrangement,
uniform droplet dispensing in a series of SETs becomes
possible for applications including serial dilution and
aliquoting.65,67 Because surface tension is not a function of velocity but rather the perimeter length of SET,
magnet travel velocity can be decoupled from operations such as particle extraction and the operation
diagram becomes a function of three static parameters
including droplet volume, SET dimension and particle
load. This is a significant advantage from a platform
design perspective since highly precise instrumentation
is no longer essential to achieve particle extraction and
droplet transport in a robust manner.
DEVICE AND INSTRUMENTATION
Designing and realizing an integrated bioassay platform from the basic principles highlighted in the preceding section requires several engineering problems to
be addressed. Firstly, the device must have a mechanism
to facilitate extraction and transport of magnetic solid
substrates across multiple reagents in a robust manner.
Secondly, the magnetic particles must be actuated by a
mechanism that is robust and operator-independent.
Lastly, appropriate supporting instrumentation for
thermal incubation and signal acquisition must be in
place. In this section, the discussion will focus on the
modes of magnetic actuation, device fabrication, thermal control and signal acquisition.
Magnetic Actuation Mechanism
The most intuitive method of magnetic particle
actuation is by using a strong permanent magnet such
as neodymium alloy (Nd2Fe14B) to actuate the magnetic particles from the reverse side of the device. In
this approach, magnetic particles that are initially
suspended in a droplet are concentrated by the applied
field into a tight magnetic plug. Once formed, the
motion of the magnetic plug along the device surface
can be controlled by sliding the magnet on the reverse
side. From a theoretical standpoint, the field gradient
is essentially varying slowly in time due to the motion
of the magnet, such that the force on the magnetic
particles is directed towards the motion of the magnet.
While manual handling of permanent magnet is feasible in some designs, other designs which depend on the
steady control of velocity and position would require
motorized instrument with feedback control. Moreover, automation of magnet handling has the added
benefit of reducing variability due to the operator.
Several modes of automated permanent magnet
actuation have been proposed. Translational stages are
employed in quantitative investigation of droplet
kinematics due to their ability to precisely control the
position and velocity of the magnet.29,34,52 Alternatively, a stepper motor with a permanent magnet
mounted on the rotor may be employed to facilitate
magnetic particle actuation in a circular motion37,43 as
shown in Fig. 4.
While actuation via a permanent magnet is the most
intuitive method, there are some limitations. Firstly,
the permanent magnet itself is not capable of switching
polarity in a manner that can allow the particles to
disperse. Although mixing of constituents in a single
droplet may be achieved through internal flow introduced by oscillation of the magnetic particle or by
Magnetic Droplet Manipulation Platforms
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FIGURE 4. Rotary actuation of magnetic particles using a stepper motor. (a) Device proposed by Shikida et al. incorporates
permanent magnets alongside a coil electromagnet to enable particle agitation via magnetic repulsion. (b) Device proposed by
Pipper et al. utilizes rotary actuation for thermal cycling between temperature zones H1, H2, H3 and H4 on a micro PCR platform.
The length of the scale bar is 5 mm. (from Refs. 43 and 37).
droplet transport,30,67 efficient sampling of the entire
droplet volume by the magnetic particles would require
an active mechanism for agitation. To address this,
additional actuation mechanisms may be used in tandem with a single permanent magnet. For example, the
instrument proposed by Shikida et al.43 utilizes a
stepper motor, with two spokes attached with a permanent magnet and a coil electromagnet respectively,
where the electromagnet is able to induce particle
agitation and supplement the limitations of the platform solely based on permanent magnet actuation.
Zhang and Wang66 developed a magnetic micro
gyromixer that spins and balances itself on a droplet
surface via a rotating magnetic field to mix the
enclosed reagents by stretching and folding internal
fluid streamlines in the droplet. In the method proposed by Tsuchiya et al.,52 two linearly actuated
magnets on opposite sides of the droplet are used to
facilitate agitation of magnetic particles as shown in
Fig. 5. Because the magnetic particles can be gathered
in two opposing directions inside the droplet, the
droplet volume can be sampled more thoroughly by
the particles.
Another approach to magnetic actuation is by using
an array of electromagnets to control the magnetic
field across the device surface in an addressable manner. The lack of mechanical components and the ease
of access to customized printed circuit boards make
this an attractive approach from both miniaturization
and commercial standpoints. This mode of magnetic
particle actuation is realized using planar coil-induced
magnetic field gradients in the presence of a uniform
static field as shown in Fig. 6.39 Firstly, a uniform
magnetic field is placed perpendicular to the plane of
the device, causing the magnetic particles to become
strongly polarized perpendicular to the device. Because
the magnetic force is scaled by the dot product of
magnetic moment and the applied field gradient, force
is generated in the direction where the field gradient in
alignment with the magnetic particle is maximized. The
coils are capable of generating a magnetic field perpendicular to the device in two polarities, and the
position of the maximum field gradient can be controlled by changing the polarities of current through
each coil element. While a typical neodymium alloy
magnet generates strong fields on the order of
102–103 mT, an electromagnet-based platform operates in the presence of approximately 50 mT static field
with variations in local field around ±10 mT due to the
coil electromagnet.13
Several groups have demonstrated the feasibility of
electromagnetic droplet actuation. Among the earlier
work, Rida et al.39 demonstrated the use of a coil-based
microsystem for the transport of magnetic particles over
a distance of millimeters, demonstrating the feasibility
of electromagnetic actuation on the order of microliters.
Subsequently, Lehmann et al.27 demonstrated transport
of magnetic particles through multiple aqueous buffers
by performing DNA capture with silica-coated magnetic particles using patterned surfaces combined with
coil-based manipulation. Most recently, Chiou et al.13
demonstrated the use of topographical barriers in
combination with coil-based magnetic transport in
order to recreate the entire workflow for a real-time
PCR assay from a biological sample.
While electromagnetic manipulation provides great
potential in miniaturizing the actuation mechanism,
several technical limitations remain. The requirement
for generating a field gradient of sufficient magnitude
in order to overcome the other two force components
acting on the droplet implies the use of high current to
drive the coils. This gives rise to two technical bottlenecks that may be applicable to all mobile devices,
namely power and thermal management. While a
detailed discussion of these topics is beyond the scope
of this review, it should be noted that thermal management is strongly linked to reducing the system
power requirement since additional components such
as thermoelectric cooling must be employed in order to
offset the effects of resistive heating.
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(a)
(b)
Forward primer
Magnet
Reverse primer
Magnet handling channel
30mm
45 mm
3mm 3mm 3mm
Reaction chamber
7 mm
Mineral oil
Magnetic beads
4 mm
0.5mm 0.5mm
Template DNA or RNA PCR or RT-PCR mixture
Magnet
Magnetic Beads
FIGURE 5. Mixing strategies on a permanent magnet-based platform. (a) In the example demonstrated by Zhang et al., a particle
plug is oscillated back and forth in order to generate internal flow. However, the particle is confined to the volume proximal to the
substrate and thorough sampling of the reagent by the particles may not be achieved. (b) In the droplet PCR system described by
Tsuchiya et al., two magnets are used to sample the entire volume of the droplet more efficiently. (adopted from Refs. 52 and 67).
FIGURE 6. Schematic view of electromagnetic manipulation for droplets containing magnetic particles. (a) Uniform magnetic field
B generated by placing permanent magnets (3) on a highly field-permeable soft iron plate (4) strongly polarizes magnetic particle
(1) perpendicular to the device plane (2). (b) Two layers of overlapping electromagnetic coil array generates magnetic field in the
component perpendicular to the device, such that magnetic particle can be actuated along the field gradient. (from Ref. 39).
Device Elements and Fabrication
Magnetic droplet manipulation can achieve three
operations including particle extraction, magnet disengagement and droplet transport as described by
Long et al.29 However, velocity is a difficult parameter
to control precisely, and a more robust mode of particle extraction is desirable for platforms designed to
perform biomolecular assays. In order to aid this
process, passive elements can be placed on the device
substrate. Passive elements on these platforms are
analogous to valves in the sense that reagent transport
is controlled such that only magnetic particles can be
extracted and transferred to subsequent reagents.
Here, two forms of passive elements explored in the
literature and their fabrication process are discussed.
Topographical patterns are capable of serving dual
roles as storage wells for the aqueous reagents and
physical barriers to restrain the droplets during particle
Magnetic Droplet Manipulation Platforms
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FIGURE 7. Demonstration of (a) topography assisted particle extraction and (b) SET-assisted particle extraction. (adopted from
Refs. 63 and 67).
extraction.63 During particle extraction, the narrow
sieve structures formed between topographical barriers
provide an outlet port for magnetic particles to be
transferred across, as shown in Fig. 7. Because the
maximum capillary force at the sieve neck is insufficient to maintain cohesion of the entire droplet past
the barrier, droplet fission takes place and particle is
extracted. In the regions that are not compartmentalized by topographical barriers, droplet transport can
be performed as usual.
Fabrication of topographical patterns can be
achieved using various techniques. In an earlier work
demonstrating the use of physical obstruction for
particle extraction, Shikida et al.44 constructed devices
by bonding layers of glass plates together with phenol
resin, followed by hydrophobic surface treatment.
However, glass devices are generally difficult to manufacture and prototype on a regular basis. Using an
alternative approach, a more convenient soft lithography-based fabrication58 has been proposed by Zhang
et al.63 and also utilized in a similar fashion by Berry
et al.7 In this process, master molds with depth on the
order of 1024 m are fabricated using SU-8 negative
photoresist with planar features defined by a conventional photolithography process. The mold is subsequently cast with poly-dimethylsiloxane (PDMS),
which is bonded via oxygen plasma activation to a
glass substrate and dip-coated in amorphous Teflon in
order to render the device surface hydrophobic.
Alternatively, Zhang et al.63 demonstrated the use of a
computer numeric controlled (CNC) machined PTFE
mold for fabrication of pillar features on the order of
1023 m to be utilized as topographical barriers. More
recently, Chiou et al.13 utilized rapid prototyping
instrument to generate a casting mold for fabricating
an extruded layer of PDMS. These fabrication
approaches can significantly reduce the difficulty of
generating surface topography and make it easier to
prototype new designs.
Platforms utilizing magnetically actuated droplets
can also incorporate substrates with differential wetting
properties in place of topographical barriers. Instead of
confining aqueous droplets using structural barriers,
hydrophilic spots are used to define regions where the
droplets can wet selectively. One key advantage of this
approach over the topographical features is the ability
to aliquot reagents, since liquid is retained in the
hydrophilic spot once wet. This feature is particularly
useful in several applications, including spatial multiplexing of reactions and generating dilution series.
Moreover, because the processing steps in which patterning takes place is subtractive rather than additive,
this process is particularly attractive for fabrication in
large batches. In general, substrates from which devices
are constructed assume a hydrophilic nature due to the
wide usage of glass. In an approach described by Inagaki et al. and utilized by Lehmann et al.,23,27 coating of
hydrophilic substrate with hydrophobic film is followed
by selective oxygen plasma etching to generate hydrophilic spots. More recently, Zhang and Wang67 developed an improved version of this technique by using
lithographically defined SU-8 membrane as a shadow
mask. In this work, full-range magnetic manipulation
including sample aliquoting was demonstrated for
simultaneously performing multiple PCR reactions on a
single sample. In another instance, serial dilution was
performed by using different sizes of hydrophilic spots
followed by merging in diluent droplets.65 In a techniques explored by Hong and Pan, superhydrophobic
surfaces have been formed using a mixture of SU-8
photoresist embedded with polytetrafluoroethylene
(PTFE) nanoparticles, where large-scale features can be
produced using standard soft lithography while taking
advantage of extremely low surface energy afforded by
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FIGURE 8. Thermal control on droplet platforms. (a) Schematic of a microheater designed for integration with a micro-PCR
device, as proposed by by Neuzil et al. (b) A heat map showing temperature profiles on the surface of the micro-PCR device. The
device utilizes three microheaters which maintain steady-state temperature at annealing, denaturing and extension temperatures.
(c) Schematic of a static droplet micro-PCR device proposed by Angione et al. utilizes a single heating element underneath the
reagent which cycles through three temperature zones over time. (d) Temperature profile of the static micro-PCR device during
thermal cycling. Black line represents the controlled surface temperature and the red line is the calibrated droplet temperature.
(from Refs. 32 and 3).
PTFE.18 Alternatively, Schutzius et al.41 demonstrated
hydrophilic patterning of a superhydrophobic substrate
coated with fluoroacrylic-carbon nanofiber composite
using wettable paraffin wax to generate virtual channels
on a planar substrate. More recently, a highly facile
technique was described by Xing et al.60 utilizing direct
laser patterning of PDMS structure to generate superhydrophobic surfaces with contact angle up to 165°. A
more comprehensive review of surface preparation for
droplet manipulation is available in Ref. 31
Thermal Control
Thermal control is an important parameter in
assays which require incubation at elevated temperatures for enzymatic reactions. In particular, PCR
requires cycling of reagent temperature between
approximately 60 and 95 °C for primer annealing and
duplex DNA denaturation. Thermal cycling for microdevices has been achieved using various mechanisms
including contact heating elements3,9,17,25,28,59 and
non-contact modes such as infrared heating21,26 and
microwave heating.24 In the context of open-surface
droplet platforms, two directions have been explored
as shown in Fig. 8. In the first instance, thermal cycling
is achieved in the manner of zone heating, where spatially separated zones are maintained at different
temperatures required for thermal cycling. This method is more commonly found in channel-based microfluidic platforms17,25,28 since flow-based transport of
reagents lends itself to thermal cycling via zone heating, although systems using a static heating chamber
have also been reported.9,26 The liquid reagent is
transported repeatedly across different temperature
zones to alter the temperature. Implementation of this
technique on a magnetic droplet platform requires the
Magnetic Droplet Manipulation Platforms
system to operate under droplet transport mode to
ensure that transport of the droplets across temperature zones can be performed without accidental particle extraction. Neuzil et al.32 demonstrated a circularly
actuated device with temperature zones maintained by
microfabricated heating elements, while Ohashi et al.34
demonstrated a linearly actuated droplet device for
thermal cycling reactions with a temperature gradient
established by heating one edge of an aluminum plate.
In the second instance, thermal cycling is achieved
by a single heating element placed in contact with
static incubation chamber which transitions through
target temperatures in a manner that is analogous to a
conventional thermal cycling instrument.3,59 This
approach consumes smaller device footprint due to the
static nature of the incubation zone and is also suitable
in applications where an array of samples must be
thermally controlled simultaneously.4 In a micro-PCR
platform developed by Angione et al.,3 thermal cycling
is achieved directly on a Teflon-coated indium tin
oxide substrate via resistive heating. While this type of
arrangement requires an integrated heater for each
PCR device, the scheme adapted by Xiang et al.59
utilizes a glass-PDMS chamber cartridge that is loaded
onto an external thin-film heater for thermal cycling.
Similar approaches have been adopted by Zhang
et al.63 and Chiou et al.13 for thermal cycling of
genomic DNA extracted on their cartridges.
There are several considerations in implementing
temperature control elements in a magnetic droplet
platform. In order to facilitate rapid transition of the
reagent between temperatures, it is imperative that the
thermal mass of the incubation region is minimized. In
contrast to thermal cycling of the entire device, integrated
microheaters placed directly in contact with the reagent
can minimize the thermal mass involved to the reagent
and its immediate vicinity. However, the integration of a
microfabricated heater and temperature sensor may
contribute significantly to the cost of each assay cartridge,
and it may be prudent to incorporate heating elements on
the instrument instead. When heating elements are placed
outside the microfluidic device, care must be taken to
thermally insulate the incubation zone and ensure proper
thermal contact between the heating element and the
device. Examples demonstrated by Pipper et al.36 and
Angione et al.3 both utilize a small droplet of oil encapsulating the PCR reagent such that the reagent is protected from evaporation while maintaining a small
thermal mass insulated by surrounding air.
Signal Acquisition
While open-surface fluidic manipulation can be
made compatible with various biosensor technologies,
optical modes are conveniently integrated into these
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platforms without affecting the droplet kinematics due
to their non-contact nature. Furthermore, the prevalence of fluorescent probes and indicator dyes in bioassays has encouraged development of either
luminescence or fluorescence-based approaches for
signal acquisition. A typical acquisition system
involves detectors such as photomultiplier tubes,3 silicon photodiodes63 or charge-coupled devices,59 while
illumination sources may range from light-emitting
diodes (LED)63 to halogen lamps.3 Optical setups for
collection of fluorescence from a single collection spot
are configured in an epifluorescence microscope configuration as shown in Fig. 9, where a dichroic
beamsplitter is used to route excitation and emission
light along the same objective lens. This arrangement
lends itself to compact detector design suitable for
portable instrument as demonstrated by Novak et al.18
POTENTIAL APPLICATIONS IN MOLECULAR
DIAGNOSTICS
The fluidic operations handled by magnetically actuated droplet platforms are well-suited to processing of
magnetic particles through a sequence of aqueous
reagents. In particular, magnetic particles with surfaces
coated with glass or modified with silane have been utilized
for extraction of nucleic acids.1,6 Solid phase extraction
involving the adsorption of duplex DNA to silica is a
convenient technique for purifying DNA from biological
samples. Initially demonstrated by Vogelstein and Gillespie54 using glass beads, the incorporation of magnetic
particles for the ease of handling has given rise to various
formulations that are commercially available. In this section, droplet platforms demonstrating nucleic acid based
assays will be discussed. For a more comprehensive review
of magnetic particle technologies for nucleic acid isolation,
readers are encouraged to refer to the following Ref. 6
Nucleic Acid Detection Via Polymerase-Based Signal
Amplification
Nucleic acid detection via amplification is an effective technique in identifying pathogens, and provides
some key advantages over culture-based techniques
including shorter turnaround time, assay sensitivity
and the ability to assess fastidious samples.49 While
DNA amplification-based analysis techniques have
traditionally depended on time-consuming processes
such as gel electrophoresis and Southern blotting,
advancements in fluorescent probes have simplified the
workflow such that post-amplification processes could
be substituted by optical detection.
Earlier designs by Ohashi et al.34 and Neuzil et al.32
demonstrated the feasibility of performing PCR in
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FIGURE 9. (a) Epifluorescence microscope configuration. A collimated illumination source is reflected at a right angle by a
dichroic beamsplitter and directed towards the objective lens, which focuses directly on the sample droplet. The fluorescence
emitted by the droplet is collected by the objective lens and directed through the dichroic beamsplitter, which allows the emitted
light to pass into the focusing lens and to be collected at the photodetector and converted into electrical signal. (b) Photograph
and (c) schematic of a miniaturized fluorescence detector in epifluorescence microscope configuration as proposed by Novak et al.
(adopted from Ref. 33).
magnetically actuated droplets. Later platforms demonstrated by Pipper et al.36 and Zhang et al.63 utilize
an open-surface design to combine solid phase DNA
extraction and PCR amplification on a single device
as shown in Fig. 10. Isolation of pathogenic nucleic
acid targets and subsequent detection via real-time
PCR demonstrates the potential of such platforms in
mobile biosurveillance, including pathogen identification and viral load quantification. In addition, these
platforms could be extended to perform multiplexed
reactions in a single mixture with molecular beacon
probes in order to detect a panel of pathogens
simultaneously.10
An alternative method of detecting multiple gene
targets is by performing amplification reactions from
the same DNA isolate in separate reaction wells. To this
end, Zhang and Wang67 developed a SET-based integrated platform capable of extracting and aliquoting
genomic DNA before performing multiple PCR reactions simultaneously using unique primer sets.
Genetic Mutation Detection Via Melting Curve Analysis
Alongside pathogen identification, genetic mutation
detection is also an important aspect of genetic diagnostics. While sequencing techniques provide the most
Magnetic Droplet Manipulation Platforms
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FIGURE 10. Real-time PCR droplet platforms for integrated sample processing and detection. Zhang et al.63 (top panel) demonstrates amplification of the 16S ribosomal RNA gene from E. coli using a TaqMan real-time PCR assay. Chiou et al. (bottom
panel) demonstrates real-time amplification of the KRAS oncogene target from genomic DNA that was extracted on chip directly
from human whole blood13.
comprehensive information regarding an unknown
nucleic acid target, the high level of fidelity and time
required to process sequencing samples preclude their
use in point-of-care settings. In light of this, melting
curve analysis has recently emerged as a potential
alternative to sequencing in rapid diagnostic applications.51 This technique monitors the dissociation of
double stranded DNA during heating by measuring
the fluorescence emitted by DNA intercalating dyes as
shown in Fig. 11.56 By monitoring the fraction of
remaining double stranded DNA as the temperature is
gradually increased, thermodynamic properties associated with the sequence are resolved as a function of
temperature. Since the dissociation characteristics of a
DNA strand are a function of base composition and
length, samples containing mutations generate melting
profiles that are different from their wild-type counterparts. Melting curve analysis is directly compatible
with conventional real-time PCR workflow since it
requires only a single universal primer pair for product
amplification, in contrast with other PCR-based
genotyping assays such as allele-specific PCR57 and
allele-specific oligonucleotide probes40 which require
additional probes for mutation identification.
Several droplet platforms utilizing melting curve
analysis for detecting variations in DNA sequence
have been reported. In a recent study, Shin et al.45
demonstrated identification of a single nucleotide
polymorphism in the KRAS oncogene using a melting
curve based approach. In this work, open-surface
magnetic droplet manipulation was used to combine
DNA extraction and amplification via PCR into a
single streamlined process. Another study by Athamanolap et al.4 demonstrated the utility of melting
curve analysis in a droplet format by obtaining melt
signatures from an array of droplets containing PCR
amplicons with variations in DNA sequences.
CHALLENGES AND PERSPECTIVES
The main hurdles in bringing open-surface droplet
platforms to commercialization are twofold. The first is
reagent storage and transport, which is a challenge
shared by many other technologies developed for pointof-care diagnostics. Although many of the buffers containing salts and other soluble, inorganic substances
may be kept at room temperature without degradation,
fragile organic components such as enzymes may expire
when stored outside a freezer. Enzymatic processes are
indispensable as they are involved in both the sample
preparation stage as well as signal amplification and
detection stage. To overcome this challenge, various
preservation strategies have been investigated to enhance the shelf life of enzymes.46 One example is desiccation of enzymes for long-term storage, combined with
liquid pouches for rehydration of reagents into liquid
form prior to using the device.11
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FIGURE 11. (a) Principles of melting curve analysis. As the temperature is increased, duplex DNA is dissociated along with
double-stranded DNA binding dyes, resulting in a decrease in fluorescence. (b) An example of melting curve analysis for identification of a single-nucleotide polymorphism within a 544-bp fragment of the HTR2A gene. (c) Layout of magnetically actuated
droplet cartridge for performing genomic DNA isolation, PCR and melting curve analysis from biological sample. (d) Temperature
profile achieved at the incubation chamber, including the initial PCR amplification and thermal ramp for melting curve acquisition.
(adopted from Refs. 56 and 45).
The second challenge is miniaturization and power
management of supporting instrumentation. While
microfluidic cartridges themselves are generally small
by design, the supporting instrumentation for operating the device including power management and control systems are typically designed for laboratory
setting and not suited for portable operation. To this
end, miniaturized components for various aspects of
device operation have been reported. A miniaturized
optical detector for fluorescence detection with sensitivity of 1029 M fluorescein has been proposed by
Novak et al.33 using LEDs as an illumination source
and silicon photodiode as a detector. Similarly, miniaturization of actuation instruments has also been
demonstrated by Shikida et al.,43 utilizing a stepper
motor to design a magnetic actuator in a palmtopsized format. Planar electromagnetic coil actuation
also lends itself to small form factor, although power
requirements remain to be resolved.
A key strength of magnetic droplet-based techniques comes from the ability to translate workflow
developed for tube-based benchtop assays in a minia-
turized format without significant alterations to the
assay mechanism. As the technology reaches maturity,
magnetic droplet platforms may function as a portable
replica of the benchtop environment that can dramatically shorten the development time required to
translate a benchtop assay into a point-of-care format.
As such, the utility of this technology is expected to
expand in parallel with the growing repository of
benchtop molecular diagnostic assays.
ACKNOWLEDGMENTS
The authors thank the funding support from NIH
(R01CA155305, U54CA151838 and R21CA173390)
and NSF (1159771 and 0967375).
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