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Review
Cite this article: Leong SS, Yeap SP, Lim JK.
2016 Working principle and application of
magnetic separation for biomedical diagnostic
at high- and low-field gradients. Interface
Focus 6: 20160048.
http://dx.doi.org/10.1098/rsfs.2016.0048
One contribution of 12 to a theme issue
‘Multifunctional nanostructures for diagnosis
and therapy of diseases’.
Subject Areas:
biomedical engineering, chemical engineering,
nanotechnology
Keywords:
low gradient magnetic separation, high
gradient magnetic separation,
magnetophoresis, magnetic particles,
biomedical diagnostic, disease detection
Authors for correspondence:
Swee Pin Yeap
e-mail: yeapsw@ucsiuniversity.edu.my
JitKang Lim
e-mail: chjitkangl@usm.my
Electronic supplementary material is available
online at http://dx.doi.org/10.6084/m9.fig-
share.c.3493029.
Working principle and application of
magnetic separation for biomedical
diagnostic at high- and low-field
gradients
Sim Siong Leong1, Swee Pin Yeap1,2 and JitKang Lim1,3
1
School of Chemical Engineering, Universiti Sains Malaysia, Nibong Tebal, Penang 14300, Malaysia
2
Faculty of Engineering, Technology and Built Environment, UCSI University, 56000 Cheras Kuala Lumpur,
Malaysia
3
Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA
SSL, 0000-0001-8247-0169
Magnetic separation is a versatile technique used in sample preparation for
diagnostic purpose. For such application, an external magnetic field is applied
to drive the separation of target entity (e.g. bacteria, viruses, parasites and
cancer cells) from a complex raw sample in order to ease the subsequent
task(s) for disease diagnosis. This separation process not only can be achieved
via the utilization of high magnetic field gradient, but also, in most cases, low
magnetic field gradient with magnitude less than 100 T m21 is equally feas-
ible. It is the aim of this review paper to summarize the usage of both high
gradient magnetic separation and low gradient magnetic separation (LGMS)
techniques in this area of research. It is noteworthy that effectiveness of the
magnetic separation process not only determines the outcome of a diagnosis
but also directly influences its accuracy as well as sensing time involved. There-
fore, understanding the factors that simultaneously influence the efficiency of
both magnetic separation process and target detection is necessary. Moreover,
for LGMS, there are several important considerations that should be taken into
account in order to ensure its successful implementation. Hence, this review
paper aims to provide an overview to relate all this crucial information by
linking the magnetic separation theory to biomedical diagnostic applications.
1. Introduction
Fast, selective and accurate detection of diseases is critical in clinical diagnosis
as it allows physicians to provide more precise and preliminary medical assess-
ments to patients. However, biological samples are usually exceptionally
complex due to the presence of multiple components. In order to perform dis-
ease diagnosis, it is essential to isolate the specific target entity (which is the
infectious agent to be detected) from the complex raw samples. This sample
preparation step is needed prior to analysis in order to speed up the screening
process [1] as well as to enhance the detection limit [2].
Separation of targeted entity for diagnostic purpose is possible through the
application of magnetophoretic force. The idea implemented here is to magnet-
ically isolate the target entity, either those with or without intrinsic magnetic
responsive characteristic (target entity without magnetic property must be
magnetically labelled with magnetic particles) [3], from a complex mixture by
the application of an external magnetic field (figure 1). Owing to the presence
of magnetic field gradient, magnetic materials will be magnetically aligned and
driven to the region with the highest magnetic field strength by magneto-
phoretic force [4]. This phenomenon is known as magnetophoresis which
involves the motion of magnetic particles relative to their non-magnetic
surrounding medium under a non-homogeneous magnetic field [5]. The mag-
netically trapped and concentrated samples can then be used for the subsequent
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
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(a) (b) (c)
magnet
target non-target non-target
Figure 1. (a) Sample with a mixture of various components containing both
target and non-target entities, (b) isolation of target entities using magnetic
field, (c) concentrated target after removal of the non-targets. Note that the tar-
gets must possess intrinsic magnetic responsiveness, or have been pre-labelled
with magnetic responsive particles.
routine analysis of target identification. This technique, com-
monly known as ‘magnetic separation’ (MS), has gained
popularity in the preparation of samples for diagnostic pur-
pose. There are many advantages offered by the utilization of
this technique. First and foremost, physical contact between
the magnetic source and the biological samples can be omitted
throughout the entire separation process which in turn makes
MS to be biologically non-invasive and does not exert a detri-
mental effect on the biological components [6]. In addition,
MS technique is also known to be high-throughput, low-cost
[7] and less energy intensive when a permanent magnet is
employed as magnetic source [8].
Owing to the benefits offered by MS technique, a great deal
of effort has been allocated in the design of different types of
magnetic separator, which allow the MS process to occur
either in a batch or continuous manner [9]. In addition, MS
technique is feasible in wide-ranging length scales as it is
not only workable in a laboratory test tube but also displays
excellent performance in miniaturized devices [10,11]. Nam
and co-workers employed microfluidic device with two outlets
to separate late stage malaria-infected red blood cells (RBCs)
which possessed relatively higher magnetic susceptibility in
comparison with healthy RBCs [12]. Pamme & Wilhelm [13]
demonstrated continuous MS of magnetically labelled cancer
cells in a microfluidic separator with multiple outlets leading
to the fractionation of the cancer cell–magnetic parti-
cle complexes according to their magnetic dipole moment.
In addition, Chen and co-workers filled up their magnetic
separation chamber with irregular-shaped iron particles with
diameter ranging from 25 to 75 mm, in order to generate
highly localized and strong magnetic field gradient upon
exposure to an external magnetic field to allow the effective
separation of HIV virions that had been labelled with magnetic
particles [14].
In spite of all the sophisticated applications mentioned
earlier, MS itself is a subject of research with many open-
ended questions yet to be answered. Particularly, MS technique
can be further classified into high gradient magnetic separa-
tion (HGMS) and low gradient magnetic separation (LGMS)
according to the magnitude of magnetic field gradient
employed in the separation process. In comparison with
HGMS, the design rules for LGMS are ill defined [15], and
hence, more theoretical and experimental works must be
dedicated to its development in order to realize the true poten- 2
tial of LGMS technique for biomedical diagnostic as well as
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in other applications. Moreover, the underlying mechanism
of magnetophoresis also determines the LGMS rate and the
effectiveness of this separation method [4]. Concurrently, the
time consumed to separate target entity from a complex
sample is one of the key factors that decides the duration of
the diagnostic process. Therefore, a thorough understanding
of the theoretical background of the LGMS process is impera-
tive in the design of magnetic separator used in a biomedical
diagnostic application. Other factors, such as magnetic particle
size, magnetic particle concentration and magnetization, also
Interface Focus 6: 20160048
play inevitable roles in determining the effectiveness of
LGMS for target detection. In this regard, it is expected that
there will be several trade-offs originating from the complex
interplay of all these factors and they are yet to be revealed
and explored in depth.
Hence, in this paper, it is our goal (i) to review the utiliz-
ation of MS technique in sample preparation for diagnosis of
diseases, (ii) to highlight the feasibility of LGMS (less than
100 T m21) in biomedical diagnostic application, (iii) to discuss
important factors that simultaneously affect the effectiveness of
LGMS and target detection, and (iv) to discuss some criteria
that are worth considering for future implementation of
LGMS technique in biomedical diagnostic application.
2. Magnetic separation in isolation of target
entity for diagnostic purpose
As mentioned in the Introduction, MS technique has been
used in sample preparation for disease diagnosis. One of the
active researches in this area is the magnetic isolation of
malaria-infected red blood cells (i-RBCs) for clinical diagnosis
of malaria. Malaria, a life-threatening disease, is triggered by
Plasmodium parasites [16]. The malaria parasites convert hae-
moglobin in healthy cells to haemozoin [17] which contains
iron(III) (Fe3þ) that exhibits a relatively stronger magnetic sus-
ceptibility compared with iron(II) (Fe2þ) in haemoglobin [12].
Owing to the presence of iron(III), i-RBCs display stronger
paramagnetic effect than healthy red blood cells (h-RBCs)
[12]. Thus, by taking advantage offered by the difference of
their intrinsic magnetic susceptible property, fractionation of
i-RBCs and h-RBCs can be achieved through the application
of external magnetic field (figure 2a) [12]. This technique
serves as an alternative to the conventional isolation method
that relies on the density of i-RBCs [19]. By applying a similar
concept, the separation of deoxyhemoglobin RBCs (para-
magnetic) from white blood cells (diamagnetic) also can be
conducted by MS technique [20]. In this regard, the blood
cell sorting can be achieved solely depending on the difference
in intrinsic magnetic properties. For instance, the absence of
bound oxygen to Fe atom causes deoxygenated RBCs to pos-
sess relatively higher magnetic susceptibility in comparison
with that of oxygenated RBCs [3]. In fact, the feasibility of
MS technique in the isolation of RBCs from whole blood
samples has been discussed way back in the 1970s by Melville
et al. [21] in their work published in Nature.
However, it should be highlighted that most biological
compounds lack inherent magnetic properties in most situ-
ations. Thus, it is necessary to artificially enhance the
magnetic property of the target entity so that it can be separ-
ated from the other non-magnetic components under an
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(a) sample flow 3

(h-RBC and i-RBC) sheath flow h-RBC sample

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(b)
i-RBC sample
(i) (ii)
i iii
sheath flow
ii iv
permanent
h-RBC
magnet
i-RBC
(i) (ii) (iii) (iv)
Ni Ni Ni Ni 10 mm
injection of sample hydrodynamic separation process collection of
(h-RBC + i-RBC) focusing by magnetic force separated sample

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(c)
magnetic field
(i) S (ii) no magnet 500 mm (iii) with magnet 500 mm
N
M(b)

M(a) buffer

sample
non-M y mixture
non-M, M(a), M(b)
x

Figure 2. (a) Schematic diagram showing the principle of i-RBC separation from a blood sample using a microfluidic device containing ferromagnetic wire. An
external magnetic field was applied normal to the ferromagnetic nickel (Ni) wire in order to create high magnetic field gradient. Paramagnetic i-RBCs were drawn
towards the Ni wire and isolated out from the h-RBCs, reprinted with permission from [12], copyright & 2013 American Chemical Society. (b) SK-BR3 cells incu-
bated (i) with IO that had been conjugated with Ab (dark blue), and (ii) with carboxyl-functionalized IO that had not been conjugated with Ab (less blue) (note:
Prussian blue staining was implemented to confirm the presence of Fe ions on the cell surface), reprinted with permission from [18], copyright 2011 Elsevier. (c)(i)
Schematic diagram illustrating a free-flow MS technique at which laminar flow was applied in x-direction, whereas magnetic field was applied in y-direction. Note
that the non-magnetic material moved along the laminar flow, while those with magnetic responsiveness were deflected from the direction of laminar flow. This
separation technique was used to separate HeLa cells in which HeLa cells that had been incubated with magnetic particles (ii) flowing straight through the channel
when there was no applied magnetic field and (iii) deflected in their flow direction upon application of magnetic field, reprinted with permission from [13],
copyright & 2006 Royal Society of Chemistry.

externally applied magnetic field. This intention can be
accomplished by labelling the target entity with magnetic
particles before subjecting to MS. Magnetite (Fe3O4), maghe-
mite (g-Fe2O3) and commercially available Dynabeads are
some of the commonly used magnetic particles for this
purpose. This approach has been used in the detection of
various biological components such as bacteria, viruses
and parasites [10,14,22– 25]. In addition, early diagnosis of
cancer can also be achieved by magnetic separation of circu-
lating tumour cells (CTCs) that had been specifically labelled
with magnetic particles [18].
Owing to the reason stated above, surface modification of
the magnetic particles with specific molecules that only bind
to the target entity, but not on other non-target components,
is the main prerequisite. For instance, Xu and co-workers [18]
surface modified their iron oxide nanoparticles with a water-
soluble polymer which was then followed by bio-conjugation
with anti-HER2 antibody. The as-prepared iron oxide-
antibody conjugates (IO-Ab) were then incubated in a fresh
human whole blood that was spiked with SK-BR3 (a human
breast cancer cell line that is HER2-positive). The cells labelled
with IO-Ab were then magnetically isolated by using a Super-
MagTM separator before analysis on the captured cells was
conducted. It was reported that IO-Ab were preferentially
labelled to SK-BR3 cells in comparison with normal cells
found in the blood sample. As illustrated in figure 2b, it can
be seen that the amount of IO-Ab attached onto the cell is over-
whelming (figure 2b(i)) in comparison to that of IO without Ab
functionalization (figure 2b(ii)). This result has directly con-
firmed that the antibody (attached on magnetic particles)
plays an essential role in the specific binding of magnetic par-
ticles onto the targets. Likewise, Chen and co-workers have
also shown the high selectivity of their antibody-conjugated
magnetic beads towards Salmonella enterica over other species
of bacteria (Escherichia coli, Shigella spp., Staphylococcus aureus
and spirillumcholera) due to the specific antibody–antigen rec-
ognition feature [22]. In other words, magnetic particles that
serve as magnetic carriers in the MS process can be made
more versatile to label different targets based on the different
strategies/compounds used to functionalize their surface.
Besides attaching onto the target cell surface, magnetic
particles might be further internalized into the cell body.
For instance, Pamme & Wilhelm [13] found the internaliz-
ation of g-Fe2O3 nanoparticles with diameter less than
10 nm into the target cells (i.e. mouse macrophages and
human ovarian cancer (HeLa) cells) in large quantity can
later be used to facilitate the cell sorting via MS technique.
Here, the MS process was performed in continuous mode
within a customized microfluidic chip where the magnetic
particle-labelled cells were deflected from their original path-
way and driven towards the region with the strongest
magnetic field gradient (figure 2c).
More interestingly, diagnosis can also be performed by
measuring the amount of magnetic particles that are still
remained in the sample solution after the MS process. This
approach has been demonstrated in a recent work published
by Chen and co-workers in the detection of Salmonella enterica,
which is a food pathogen that affect human gastrointestinal
tract. In this work, antibody-conjugated magnetic particles of
size 250 nm (MP250) and 30 nm (MP30) were first dispersed
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free 4
(a) magnetic (b)
inlet

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particle

magnetic
particle

magnetic
particle
captured aggregate

N
S

magnetic
particle
circulating

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flow
magnetically
susceptible
wires
outlet magnet
magnetic
layer of captured
particle
magnetic particles
magnet
Figure 3. Illustration of HGMS and LGMS processes. (a) The HGMS column is packed with randomly entangled magnetizable wires, which act as magnetic field
‘dehomogenizer’ to produce strong and localized magnetic field gradient near to it. Magnetic particles which are flowing through the column will experience an
exceptionally strong magnetophoretic force in the vicinity of the magnetizable wires and be captured on them. (b) Illustration of LGMS process. Permanent magnet
is used to create magnetic field gradient across the magnetic particle solution in which magnetic particles are going to be separated. The magnetic particles migrate
towards the region with highest magnetic field gradient (which is usually the region adjacent to the magnet) and are separated from the solution. Particle aggrega-
tion (due to the magnetic dipole – dipole interaction) and continuous circulating flow (as a consequence of hydrodynamic effect) occur throughout the entire process
and contribute to the distortion of the magnetophoretic pathway of magnetic particles.

into the biological sample in order to promote the formation
of MP30-target-MP250 complex. Upon the application of an
external magnetic field, the magnetophoretic separation rates
of MP250 and MP30-target-MP250 complex are considerably
higher compared with that of MP30 owing to their greater
magnetic dipole moment. Accordingly, the amount of the
target entity in the initial biological sample can be inferred
from the transverse relaxation time of the water molecules
(T2 value) surrounding the MP30 which remained within
the biological sample after the MS process [22]. The posi-
tive outcomes obtained from this novel approach definitely
encourage more exciting exploration in this research area in
the future.
The MS process can also be conducted in a negative selec-
tion manner. In this regard, the undesired entities are labelled
with magnetic particles and thus being separated during the
MS process. On the contrary, the target entity, which is
unlabelled and magnetically irresponsive, is still suspended
in the biological sample after the MS process [26]. This
approach is appealing as the target entity remains unaltered
throughout the MS process. However, it might suffer low
target yield due to non-specific loss of the target entity or
inadequate removal of undesired biological components [27].
One of the factors that determines the successful implemen-
tation of all aforementioned MS applications is the magnetic
separator design. Hence, in the next section, design and
principle of MS technique are thoroughly discussed.
3. Strategies of magnetic separator design
Basically, MS technique can be classified into two distinct
classes, namely HGMS and LGMS, according to the magnitude
of magnetic field gradient employed in the separation process.
In fact, HGMS has widely been implemented in the isolation of
magnetically responsive particles from their suspension [28].
To date, HGMS has been proved to be feasible in mineral
processing [29–31], waste removal [32–34], biotechnology
[35], as well as drug delivery [36,37]. In general, the operation
of HGMS involves a column which is filled with randomly
entangled magnetically susceptible wires and surrounded
by an electromagnet (figure 3a) [38]. By turning on the
electromagnet around/besides the column, the magnetically
susceptible wires tend to dehomogenize the magnetic field
such that high magnetic field gradient (more than 100 T m21)
is produced in the vicinity of the magnetizable wires. As the
suspension of magnetic particles is channelled through
the column, the magnetic particles are captured on the magnet-
ically susceptible wires and hence being isolated from the initial
solution. In contrast, LGMS involves only a simple set-up in
which the separation of magnetic materials from the solution
is performed typically by a hand held permanent magnet with-
out any kind of packing. Under a non-homogeneous magnetic
field produced by the permanent magnet, magnetic particles
are driven to migrate towards the magnetic source by magneto-
phoretic force and therefore being isolated out of the solution
(figure 3b). Because the magnitude of magnetic field gradient
involved in this set-up is generally less than 100 T m21, this
type of MS technique is denoted as LGMS.
3.1. Working principle of high gradient magnetic
separation
Owing to the remarkable difference between magnetic suscep-
tibility of magnetizable wires and that of suspending medium,
the magnetic field within close range of the wires is signifi-
cantly distorted which sequentially induces an intense
magnetic field gradient in that region [28,39]. Magnetic field
gradient as high as 1260 T m21 can be created near the wires
within the HGMS column when the electromagnet is switched
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on [29]. As magnetophoretic attraction force experienced
by a magnetic particle is directly proportional to the magnetic
field gradient (see equation (3.1)), the magnetic particle experi-
ence a huge magnetophoretic force as it passes through the
region adjacent to the magnetizable wire [40]:
Fm ¼ 43pr3 mo MrH: ð3:1Þ
Here, Fm is magnetophoretic force exerted on single magnetic
particle, r is radius of magnetic particle, mo is the permeability
of free space, M is volumetric magnetization of magnetic particle
and rH is magnetic field gradient. Such a gigantic magneto-
phoretic force is able to overcome viscous drag and Brownian
fluctuation which oppose the magnetophoretic motion of mag-
netic particles. Finally, the magnetic particles will be captured
on the wires and isolated from the solution.
Even though HGMS has been well established and widely
implemented in various applications, there are several draw-
backs associated with the utilization of HGMS. First and
foremost, the installation and operation cost of the HGMS is
extremely high as huge amount of energy is required for the elec-
tromagnet to generate a magnetic field that is sufficiently intense
to induce a successful collection of magnetic particles [32]. Apart
from that, the highly complex and inhomogeneous magnetic
field within the HGMS column prevents the further develop-
ment of analytical model to accurately describe the HGMS
process which in turn renders theoretical study of the given pro-
cess exceptionally strenuous [28]. Last but not least, magnetic
particles which have been attached on the wires are difficult to
be removed due to the complicated set-up of the column. The
retention of magnetic particles on the wires will greatly reduce
the separation efficiency of the HGMS column [41].
3.2. Working principle of low gradient magnetic
separation
In order to overcome the disadvantages of HGMS described
above, another MS scheme was introduced to induce the
magnetophoretic collection of magnetic particles from sus-
pension. This separation technique is able to perform the
magnetic particle collection without the intricate set-up of
magnetizable wires within the HGMS column. On the con-
trary, the only part required is a permanent magnet which
produces non-uniform magnetic field all over the magnetic
particle solution subjected to MS [42]. Owing to the non-
homogeneity of the magnetic field throughout the magnetic
particle solution, magnetophoretic motion is initiated in
which the magnetic particles migrate towards the region
where the magnetic field gradient is the highest (figure 3b)
[43]. In such a way, magnetic particles are removed from
the suspending medium and the separation is accomplished.
Because magnetic field strength declines rapidly with the dis-
placement from the magnet, magnetic field gradient across
the entire volume of magnetic particle solution subjected to
separation is usually lower than 100 T m21 even though a
strong neodymium ferrite boron magnet (abbreviated as
NdFeB magnet and is a permanent magnet with extremely
strong remanent magnetization) is employed [44,45].
Yet, one of the most pronounced disadvantages of LGMS
is originated from the weak magnetophoretic force (as a con-
sequence of low magnetic field gradient) encountered by the
magnetic particles to be separated [15]. There are two factors
which oppose the deterministic magnetophoretic migration
of magnetic particles, namely viscous drag and thermal 5
motion [43]. Viscous drag is the resistance encountered by
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an object that performs a relative motion with respect to
the surrounding fluid. The magnitude of viscous drag can
be evaluated by the Stokes equation [46]:
Fd ¼ 6phrv, ð3:2Þ
where Fd is viscous drag experienced by magnetic particle, h
is viscosity of the suspending fluid and n is magnetophoretic
velocity of magnetic particle relative to fluid. Besides, thermal
motion is random fluctuation of magnetic particles that dis-
rupts the deterministic pathway of magnetic particles along
Interface Focus 6: 20160048
the magnetic field gradient. The intensity of thermal fluctu-
ation can be reflected by the magnitude of diffusion
coefficient D of the given particle in the suspension as
demonstrated in the Stokes–Einstein equation [47]:
kT
D¼ , ð3:3Þ
6phr
where k is Boltzmann constant and T is absolute temperature.
According to equations (3.1) and (3.2), magnetophoretic
force Fm varies linearly with respect to r 3 while the opposing
viscous drag Fd is directly proportional to r. Hence, as the
magnetic particle (and hence r) is small, viscous drag
dominates the magnetophoretic force and thus the magnetic
particles migrate slowly towards the magnet pole. Conse-
quently, more time is required to execute a successful
separation. Apart from that, the thermal fluctuation of magnetic
particle is more intense as its size is small (see equation (3.3)).
This motion randomizes the magnetophoretic pathway of mag-
netic particle and this has caused the difficulty in controlling the
motion of magnetic particles. The relevant dimensionless num-
bers include Reynolds number (Re) and Péclet number (Pe)
which characterize the ratio of inertia force (magnetophoretic
force in this case) to viscous drag force and diffusive force,
respectively [15]. According to this dimensionless analysis,
magnetophoretic force is more dominant than viscous drag
and diffusion in defining the motion of magnetic nanoparticle
providing that Re and Pe are larger than unity. Consequently,
these criteria must be fulfilled prior to the more comprehensive
design of the given LGMS process.
Despite the arguments mentioned above, LGMS is still a
feasible separation technique in the removal of magnetic
particles from their suspension. Yavuz et al. [42] reported that
magnetic nanoparticles as tiny as 12 nm can be collected
within a reasonable timescale by applying low magnetic field
gradient. This phenomenon is mainly due to the self-aggregation
of magnetic particles under an external magnetic field [48]. For
instance, when a magnetic particle is exposed to an external
magnetic field, it will be magnetized and acquire net magnetic
dipole moment. In order to lower the magnetic interaction
potential energy, magnetic particles with net magnetic dipole
moment tend to align themselves in the direction of the magnetic
field lines and being driven towards the regions with higher
magnetic field [49]. In the course of the magnetophoretic
migration of magnetic particles, some particles will come close
to one another. Because each magnetic particle with net mag-
netic dipole moment is behaving as a ‘small’ magnet, magnetic
particles which are sufficiently near will experience magnetic
attraction among each other and finally ‘stick’ together [50].
Accordingly, magnetic particles tend to collapse with one
another forming larger aggregate [51]. Owing to their higher
magnetic volume, magnetic particle aggregate acquires much
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larger magnetophoretic force which is able to overcome the
aforementioned opposing forces (i.e. viscous drag force and ther-
mal fluctuation) and move collectively towards the magnetic
source with much magnetophoretic velocity [42]. Hence, by
the aid of aggregation, magnetic particles can be separated
by low magnetic field gradient generated by a hand-held
permanent magnet within a reasonable timescale.
3.3. Application of high and low gradient magnetic
separations in biomedical diagnostic
Application of both HGMS and LGMS in biomedical diagnos-
tic has been proved to be feasible. Several published works in
this area of research are summarized in table 1. The concept ori-
ginated from HGMS has been used to separate E. coli bacteria
from whole blood sample [10] as well as to concentrate and
purify HIV viral product in human plasma [14]. In order to
generate localized and strong magnetic field gradient which
enables rapid and efficient separation of magnetically labelled
E. coli bacteria, magnetizable substance is employed to deho-
mogenize the magnetic field created by the magnetic source.
In the work reported by Xia and co-workers, magnetic field
gradient as high as 290 T m21 can be generated by NiFe micro-
comb that is acting as magnetic field dehomogenizer (table 1)
[10]. This recorded magnetic field gradient value is much
higher than the one that can be achieved within a microfluidic
channel (approx. 20 T m21) in the absence of NiFe microcomb.
In the purification of human immunodeficiency virus type 1
virions, irregular iron oxide spheres were placed within the
microfluidic separator to act as magnetic field gradient con-
centrator. Under this configuration, the solution containing
viral product can be concentrated within 20 min and yields a
solution that is 40–80 times more concentrated in comparison
with the initial solution [14]. On the other hand, Pivetal and co-
workers designed a micro-magnet array which can develop
magnetic field gradient as high as 2.5  105 T m21 to position
and immobilize magnetic particle-labelled E. coli (row 11 in
table 1) [57].
Meanwhile, there are plenty of biomedical diagnostic
applications which have employed the concept of LGMS in
their operation unwittingly and resulted in positive outcomes.
For instance, in the continuous sorting of monocytes and
macrophages by magnetic nanoparticles, permanent magnet
(NdFeB) was used to create the magnetic field throughout
the microfluidic chip which generates magnetic field gradient
within the range of 30–80 T m21 (row 7 in table 1) [55].
Despite the low magnetic field gradient (less than
100 T m21), excellent purification (more than 88%) and high-
throughput (10–100 cells/s) could be accomplished in this
magnetophoresis set-up. Apart from that, in the separation
of cancer cells from fresh whole blood as reported by Xu
and co-workers, SuperMagTM separator (Ocean NanoTech)
was used to trigger the magnetophoretic motion of the magne-
tically labelled tumour cells (row 5 in table 1) [18]. Enrichment
factor of cancer cell to normal cell as high as 1 : 10 000 000
has been achieved under the low magnetic field gradient
(100 T m21) created by permanent magnets within the mag-
netic separator. Furthermore, in the work done by Song and
co-workers, magnetic scaffold was used to create magnetic
field gradient below 100 T m21 for the isolation and detection
of particular target cell from analytical samples (see table 1
for detail) [54]. For instance, the magnetophoretic capture effi-
ciencies of leukaemia cells and prostate cancer cells under the
given experimental condition were reported as 96% and 97%, 6
respectively. It should be noted that the magnetic field gradient
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can be high at the location near to the surface of the magnetic
source and decay with the distance away from the magnetic
source. As an example, Lin and co-workers designed a mag-
netic bioseparator by using two NdFeB magnet columns [1].
A small space was left between the two magnet columns and
being employed as the MS zone. Even though the magnetic
field gradient in the separation zone can be as high as approxi-
mately 300 T m21 at the surface of the magnet columns, it
decays to 0 T m21 at the middle of the separation zone. This
phenomenon has caused the average magnetic field gradient
Interface Focus 6: 20160048
within the separation zone to be approximately 90 T m21
(table 1). By using this set-up, it only took around 2 min to sep-
arate the E. coli that had been labelled with 180 nm diameter
magnetic particles. Hence, LGMS appears as a beneficial and
effective tool in separation of particular cells, bacteria or viruses
for diagnostic purpose.
Nevertheless, in order to design and optimize the diagno-
sis process by incorporating LGMS technique, it is extremely
vital to understand some of the control parameters that influ-
ence the performance of the process in the first place. This
will be discussed in the next section.
4. Effectiveness of low gradient magnetic
separation in biomedical diagnostic
application
4.1. Factors that influence low gradient magnetic
separation rate
Size of magnetic particles plays a very important role in con-
trolling the LGMS kinetics. As depicted in equation (3.1), the
magnitude of Fm experienced by a magnetic particle is directly
proportional to r 3. Here, force balance between Fm and Fd
reveals that the magnetophoretic velocity, which determines
the separation time, varies linearly with respect to r 2. Thus,
in an LGMS process, particles with larger size are separated
first before the small ones when they are subjected to the
same magnetic field [58].
There exists plenty of literature in relating the MS effi-
ciency to the magnetic particle size. Lately, Lin and co-
workers showed that E. coli O157:H7 which had been labelled
with 180 nm magnetic particles can be successfully isolated in
less than 2 min within a magnetic bioseparator of mean mag-
netic field gradient approximately 90 T m21 [1]. Meanwhile,
when the same experiment was conducted by using magnetic
particles of size 30 nm, the optimal MS duration was notably
increased to 60 min, which is about 30 times slower in
comparison with the MS process that employed 180 nm
magnetic particles. Thus, magnetic particles of larger size
are preferential in fast detection/screening applications. By
reversing this concept, Chen and co-workers used a mixture
of magnetic particles of two different sizes (i.e. 30 nm (MP30)
and 250 nm (MP250)) in bacteria and virus detection [22].
Their preliminary study showed that MP250 was successfully
precipitated in 1 min upon subjecting to a magnetic field of
0.01 T; while MP30 remains suspended even after prolonging
the MS time to 1 h under the same magnetic field (figure 4a).
By employing the advantage offered by such a huge distinc-
tion in MS time, the concentration of the target entity within a
Table 1. Details of magnetic particles (MPs) or cells with intrinsic magnetic property used in biomedical diagnosis based on magnetic separation concept.

types of
a
magnetic particles , magnetic
or cells with intrinsic separator
magnetic size of MPs (batch/ magnetic field magnetic field application in
responsiveness (diameter) magnetic properties functionalizing layer concentration of MPs continuous) strength gradient biomedical diagnostic ref.
b 26 21
deoxygenated RBCs 7.7 mm Dx ¼ 0.265  10 — not counted batch mean ¼ 1.40 T mean ¼ 131 T m determine [52]
methemoglobin RBCs Dx b ¼ 0.301  1026 0.5  106 cells ml21 magnetophoretic
mobility of the cells
g-Fe2O3 8.4 nm 3.1  105A m21c carboxyl groups 1015 particles ml21 continuous 0.4 T 50 T m21 sorting of mouse [13]
macrophages and
human ovarian
cancer cells (HeLa
cells)
superparamagnetic green 1.6 mm n.a. streptavidin 3.1  109 beads ml21 continuous 0.048 Td 290 T m21d separation of living [10]
fluorescent bead E. coli bacteria
(43% iron oxide)
parasite-infected n.a. n.a. — n.a. continuous 1.426 T 804 T m21 concentrate erythrocytes [53]
erythrocytes parasitized by
human malaria
species
Fe3O4 27 + 5 nm n.a. oleic acid þ amphiphilic 2 mg Fe ml21 batch n.a. 100 T m – 1 separation of tumour [18]
polymer þ antibodies against human cells from fresh
epithelial growth factor receptor 2 whole blood
(anti-HER2 or anti-HER2/neu)
fluorescent–magnetic 211 nm 8.18 emu g21c monoclonal antibody (mAb) 10  1010 nanobioprobes ml21 batch 0.4 T 0– 100 T m21e detection and extraction [54]
bifunctional of leukaemia cells
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nanoparticles and prostate cancer

cells
g-Fe2O3 8.7 nm 6.6  10214 A m2f (for carboxylate functional groupsh 1 mM (extracellular iron continuous 0.15– 0.26 T (from 30– 80 T m21 sorting of monocytes [55]
1 pg of irong) concentration) middle of the and macrophages
separation chamber based on amount of
to surface of the MPs internalize into
magnet) the cells
deoxygenated RBCs 6– 8 mm n.a. — 5  107 cells ml21 semi 0– 1.68 T 1750 T m21 RBC debulking [56]
methemoglobin RBCs n.a.

(Continued.)
7

Interface Focus 6: 20160048 rsfs.royalsocietypublishing.org

Table 1. (Continued.)

types of
a
magnetic particles , magnetic
or cells with intrinsic separator
magnetic size of MPs (batch/ magnetic field magnetic field application in
responsiveness (diameter) magnetic properties functionalizing layer concentration of MPs continuous) strength gradient biomedical diagnostic ref.

Fe3O4 30 nm n.a. streptavidin þ antibodies against 40 mg ml21 for 30 nm MPs batch 1.15– 1.65 T 0– 300 T m21 isolate E. coli O157:H7 [1]
E. coli (mean ¼ 1.35 T) (mean ¼ 90 T m21) cells
180 nm carboxyl functional 100 mg ml21 for 180 nm MPs
groups þ streptavidin þ antibodies
against E. coli
magnetic beads 30 nm, n.a. capture antibody (Ab1), detection 0.2 g l21i batch 0.01 T n.a. detection of S. enterica [22]
250 nm antibody (Ab2)
microbeads 50 nm n.a. streptavidinj n.a. batch  0.12 to0.29 T  2.89  104 – selective arraying of E. [57]
2.5  10 T m21 coli bacteria by
micro-magnet
a
Magnetic particles that were used to label on target entity which is originally non-magnetically responsive.
b
Dx ¼ net magnetic susceptibility of the RBC in aqueous suspension¼ xRBC – xH2 O .
c
Saturation magnetization.
d
The values of magnetic field strength and magnetic field gradient provided were the maximum (which occurs at the location closest to the magnet within the whole domain of flowing channel) under the influence of NiFe microcomb.
e
Simulation based on the geometry configuration provided by using COMSOL Multiphysics (see electronic supplementary material for the simulation result).
f
Magnetic moment.
g
1 pg ¼ 8  105 nanoparticles.
h
The carboxylate functional groups here are to stabilize the magnetic particles in aqueous medium.
i
The concentration of magnetic beads is estimated in the solution where magnetic separation occurs.
j
During immunomagnetic labelling, the streptavidin-coated microbeads were mixed with bacterial cells that already pre-treated with biotinylated anti E. coli antibodies.
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(a) (b) 9
(i) (iii)

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50 mm

(ii) (iv)

MP250 MP30
(1 min) (60 min)

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(c) (i) (ii)
(d)
normalized absorbance (arb. units)

1.5 1.0 magnet

50 mT mm–1
0.9
0.025 g l–1
0.8
normalized opacity

0.05 0 g l–1
10 g l–1 exit
1.0 0.7 0.075 g l–1
1 g 1–1 0.25 g l–1
6
0.6 0.50 g l–1
0.1 g 1–1 0.70 g l–1
0.5 5
0.01 g 1–1 1 g l–1
4
0.4 2.5 g l–1
0.5 0.3
5 g l–1
3
10 g l–1
0.2 2
0.1 1
1 mm
0 0 12 mm
0 100 200 0 200 400 600 800 diameter cells
time (s) time (s)

Figure 4. (a) MP250 was separated in 1 min by using magnetic field of 0.01 T. Meanwhile, MP30 remains well dispersed even after 60 min subjected to the same
magnetic field, reprinted with permission from [22], copyright 2015 American Chemical Society. (b) Optical micrographs of solution with 1 g ml21 of 0.41 mm
superparamagnetic microsphere (Estaporw M1 – 030/40) are demonstrated in (i) and (ii). Figure (i) was observed under the absence of magnetic field, while figure
(ii) is the image taken after 120 s of magnetic field exposure. The alignment of superparamagnetic microsphere along the magnetic field was observed in (ii) and
this is an indication of magnetic particle aggregation under low gradient magnetic field, reprinted with permission from [48], copyright & 2008 American Chemical
Society. Optical micrograph of 425 nm magnetic nanoparticles in deionized water (iii) before the application of magnetic field and (iv) a few seconds after the
exposure to magnetic field generated by NdFeB magnet, reprinted with permission from [45], copyright & 2008 American Institute of Physics Publishing LLC. Chain
formation ( particle aggregation) was observed in (iv) and this phenomenon has proven the occurrence of cooperative magnetophoresis. (c) The magnetophoresis
profile for (i) Estaporw M1 – 030/40 particles, reprinted with permission from [48], copyright & 2008 American Chemical Society, and (ii) 30 nm bare iron oxide
magnetic nanoparticles, reprinted with permission from [58], copyright & 2014 American Chemical Society. Both (i) and (ii) show that the magnetophoresis process
is accelerated as magnetic particle solution with higher concentration is employed. The variation of magnetophoresis rate with the concentration of the magnetic
particle solution indicates the significance of particle aggregation throughout the magnetophoresis. (d ) Illustration of magnetic separation chamber used for the
fractionation of mouse macrophages or human ovarian cancer cells (HeLa cells) according to the cell magnetic moment, reprinted with permission from [13],
copyright & 2006 Royal Society of Chemistry.

biological sample can be measured by dispersing the magnetic [50,51]. Particle aggregation is initiated by the magnetic inter-
particle mixture into the given solution. Under this scenario, the action between magnetic dipole moment possessed by each
interaction between antibody (from magnetic particles) and magnetic particle. As suggested by De Las Cuevas and co-
antigen (from targets) would cause the formation of large workers, the significance of magnetic interaction between
MP250-target-MP30 complex. The target entities (bacteria or two magnetic particles can be estimated by magnetic Bjerrum
virus) which were embedded in MP30-target-MP250 complex length lB which is formulated as [48]
can then be rapidly removed upon exposure to a weak magnetic
 1=3
field due to the gigantic size of MP250-target-MP30 complex in 8pm0 M2
lB ¼ r2 , ð4:1Þ
comparison with MP30. The unreacted MP30 which were still 9kT
suspended in the solution after the MS will change the T2
value of the surrounding water molecules. The change of T2 where m0 is the permeability of free space. Particularly,
values was then correlated to the concentration of the target magnetic interaction is dominant over the thermal random
(bacteria or virus) in the initial biological sample. Results from motion and particle aggregation is initiated as the separation
the studies mentioned above have highlighted the importance distance between both magnetic particles is smaller than
of selecting magnetic particles of suitable size for successful the magnetic Bjerrum length. Because the average distance
employment of MS technique in biomedical diagnosing. between magnetic particles suspended in the solution
In addition to particle size, the separation kinetic of MS is decreases with the particle concentration of the solution,
also strongly dependent on the concentration of the magnetic magnetic interaction between particles (which induces parti-
particles. This phenomenon is attributed to the cooperative cle aggregation) is exceptionally substantial in the LGMS of
nature of the magnetic particle system in which magnetic par- highly concentrated magnetic particle solutions. In addition,
ticles tend to aggregate and move collectively towards the according to thermodynamic self-assembly theory as proposed
magnetic source upon exposure to an external magnetic field by Andreu et al. [59], the onset of particle aggregation in
 Downloaded from http://rsfs.royalsocietypublishing.org/ on July 7, 2018
magnetic particle solution subjected to magnetophoresis can
be predicted by aggregation parameter N* which is defined as
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
N  ¼ fo eG1 , ð4:2Þ
where fo is the volume fraction of magnetic particles in the
given solution and G is the magnetic coupling parameter
which is given by
m0 m2s
G¼ : ð4:3Þ
2pd3 kT
Here, ms is the total magnetic dipole moment possessed by
one magnetic particle at saturation and d is the diameter of mag-
netic particle. As N* is larger than unity, particle aggregation is
initiated by the magnetic interaction between neighbouring
magnetic particles [60]. Besides, the magnitude of N* is also
representing the average number of particles within a particle
aggregate in the cooperative regime. Both magnetic Bjerrum
length lB [48] and aggregation parameter N* [59] analysis
suggests that the particle aggregation is more significant at
higher particle concentrations. The direct consequence of particle
aggregation is the formation of larger-sized magnetic particle
clusters with much stronger magnetic response in comparison
to individual magnetic particles. In the work reported by De
Las Cuevas et al. [48], they observed instantaneous align-
ment of Estapor M1–030/40 particles (the diameter of which is
0.41 mm) upon the application of an external magnetic field
(figure 4b(i)(ii)). Meanwhile, Schaller et al. [45] also reported the
similar result in their attempt to investigate the motion of mag-
netic particles under an external magnetic field (figure 4b(iii)(iv)).
Formation of particle aggregates, which possess larger
size and more intense magnetic response, leads to accelerated
MS such that magnetic particles can be segregated from the
solution within a shorter timescale. Under this condition,
magnetic particles interact along their magnetophoretic
migration pathway; thus, this phenomenon is designated as
cooperative magnetophoresis. In line with the same coopera-
tive concept, MS is faster as the concentration of magnetic
particles increased (figure 4c) [4,48,58]. Therefore, this prin-
ciple can be used in biomedical diagnostic applications
which employ MS technique to isolate a target entity. Even
though the separation of the target entity by external mag-
netic field can be accelerated by labelling the target entity
with larger magnetic particles, the utilization of larger mag-
netic particles offers some drawbacks. First and foremost,
due to the fact that the ratio of surface area to volume is redu-
cing with the increase in magnetic particle size, the available
surface sites for labelling become fewer. Furthermore, the
relative size between magnetic particle and target entity also
has a significant effect on the sensitivity in certain biomedical
detection which exerts extra constraint on the selection of the
magnetic particle size (will be discussed in more detail in the
next section) [61]. Henceforth, cooperative nature of magnetic
particles throughout the LGMS process provides an alternative
route to optimize the separation rate of magnetically labelled
target entity in biomedical diagnostic application. By increas-
ing the concentration of magnetic particles supplied to the
sample filled with the target entity, the magnetophoretic separ-
ation rate of magnetically labelled target entity can be greatly
enhanced upon cooperative movement of the aggregated
magnetically responsive species.
In addition to the size and concentration effects, magnetiza-
tion (which is the total magnetic dipole moment per unit mass
or volume [62]) of magnetic particles is also an important cri- 10
terion which affects the magnetophoretic separation rate of
rsfs.royalsocietypublishing.org
magnetic particles in any related biomedical diagnostic appli-
cation. The linear correlation between magnetophoretic force
experienced by magnetic particles and their magnetization
has been demonstrated in equation (3.1). Magnetic particles
with stronger magnetization (and hence more intense magnetic
dipole moment) experience larger magnetophoretic force
under the same external magnetic field and undergo more
rapid MS. One related example which employs this principle
is demonstrated in the work reported by Pamme & Wilhelm
[13], in which target cells were fractionated according to their
Interface Focus 6: 20160048
magnetic loading. The target cells were firstly incubated with
nano-sized magnetic particles in order to allow the internaliz-
ation of magnetic particles into the cells. Cells that were loaded
with more magnetic particles possessed higher magnetic
moment; while those loaded with fewer magnetic particles
exhibited lower magnetic moment. When the mixture of cells
(with different magnetic moments) was driven to flow through
a microfluidic chamber where the magnetic field gradient was
applied in the direction perpendicularly to the flow velocity,
the cells start to deflect from their original flow direction
under the influence of magnetophoretic force. Cells with
higher magnetic moment were deflected to a greater extent
towards the location where the external magnet was applied
and vice versa (figure 4d). By this way, mixture of cells is fractio-
nated according to their magnetic loading due to their different
MS responsiveness. Similarly, this principle has also been
successfully applied in the sorting of monocytes and macro-
phages based on the amount of magnetic particles that have
been internalized into the cells [55].
4.2. Factors that simultaneously affect the performance
of target detection
Apart from affecting the separation rate, particle size is also
important in determining the efficiency of target detection.
Generally, micrometre-sized (1 –10 mm) and nanometre-
sized (50 –100 nm) magnetic particles are popular for the
isolation of target entities from complex biological samples
for diagnostic purpose [14]. Even though magnetic particles
of larger size are preferable in terms of their shorter LGMS
duration (as discussed in §4.1), they pose several shortcom-
ings as compared with the nano-sized counterparts. First,
the surface area to volume ratio of micrometre-sized particles
is lower compared with nano-sized particles which in turn
causes the reduction in the binding capacity [18] and capture
efficiency [63] for magnetic particles with larger size. Second,
gravitational sedimentation is the common issue encountered
by large and dense particles [14,27], hence, continuous agita-
tion is needed to keep them dispersed. On the contrary, small
particles with nano-sized dimension are usually dispersed
more uniformly within the suspension [1] and exhibit faster
reaction kinetics [1,63]. Additionally, due to their less steric
exclusion property, higher amount of nano-sized particles
can be bound onto the surface of a target cell in comparison to
that of micrometre-sized particles (as illustrated in figure 5a)
[18]. A clear comparison between target cells that were
attached with magnetic particles of different sizes is given in
figure 5b. Figure 5b(i) shows immobilization of nano-sized
magnetic particles on the surface of E. coli [64]; while
figure 5b(ii) shows E. coli O157:H7 cells which were bound to
antibody-coated magnetic beads (Dynabeads). In addition,
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(a) (b) 11
(i)

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membrane 1 mm (ii)
of target
cell

100 nm magnetic
particles

Interface Focus 6: 20160048

30 nm 200 nm

(c) (d) (e) 10 mg ml–1

1 mg ml–1
600 0.5 mg ml–1
105 0.1 mg ml–1

probe excess
100 equivalence

DT2 (ms)
400
95
T2 (ms)

target excess
90 200
85
high T2 80 0
low T2
75 1 10 102 103 104 105 106 107
0.001 0.01 0.1 1 concentration of S. enterica (cfu ml–1)
no. target analytes/ no. probes

Figure 5. (a) A pictorial drawing (not drawn to scale) showing membrane of a target cell labelled with magnetic particles of various sizes (more nano-sized
magnetic particles can be attached on the cell surface than the micrometre-sized counterparts due to less steric exclusion) [18]. (b)(i) Transmission electron
microscopy image showing E. coli immobilized with 3-mercaptophenylboronic acid/1-decanethiol-modified magnetic nanoparticles, reprinted with permission
from [64], copyright & 2013 Multidisciplinary Digital Publishing Institute; (ii) electron micrograph (scale bar, 5.14 mm) of E. coli O157:H7 cells captured by Dyna-
beadsw M-280 Streptavidin (Dynal, Lake Success, NY, USA) coated with biotinylated antibodies, reprinted with permission from [65], copyright & 2002 John Wiley
and Sons. (c) Solution with well-dispersed magnetic particles provides high T2 value. Once the target cell was mixed with the magnetic particle solution, interaction
between antibody (from the functionalization layer of magnetic particles) and antigen (from the target cells) would induce the aggregation between magnetic
particles and cells which results in large magnetic particles– cells complex. Owing to the presence of the larger-sized complex, T2 value of the solution becomes
lower, reprinted with permission from [61], copyright & 2009 American Chemical Society. (d) Variation of T2 value with the number ratio of target cells to
magnetic particle probes, reprinted with permission from [61], copyright & 2009 American Chemical Society. (e) The effect of magnetic particle (MP30) concen-
tration (0.1, 0.5, 1 and 10 mg ml21) on the sensitivity of the detection process. It can be observed that the sensitivity (change of T2 value) of the detection method
is decaying with the concentration of MP30, reprinted with permission from [22], copyright & 2015 American Chemical Society.

Pamme & Wilhelm [13] predicted that the number of magne-
tic particles bound to the surface of a target cell can be
significantly reduced from 2  106 to 102 and 100 when the
diameter of the magnetic particles is enlarged from 9 nm to
400 nm and 2.8 mm, respectively. This prediction implies that
smaller magnetic particles are more appropriate for use in
the analysis of high-density cell surface markers; while
for detection of rare cell surface markers, larger magnetic par-
ticles are more appropriate. Moreover, using smaller particles
approaching the size of surface markers, the resolution of
diagnostic process could be improved significantly.
Apart from acting as one of the non-trivial parameters
which govern the dynamical behaviour of cooperative LGMS
(separation rate), concentration of magnetic particle solution
also affects the performance of LGMS-aided biomedical diag-
nostic application in term of other aspects. After dispersing
antibody-conjugated magnetic particles into the biological
sample containing the target entity, the binding between anti-
body (from magnetic particles) and antigen (from the target
entity) fosters the aggregation of magnetic particles in the sol-
ution which in turn changes the spin–spin relaxation time (T2)
of the surrounding water protons. In general case, T2 value
declines with the degree of magnetic particle aggregation as
particle aggregates (with larger size in comparison to
individual magnetic particles) tend to cause magnetic field dis-
tortion around them and diphase the spins of water protons
which are diffusing through the region more significantly
(figure 5c) [61]. Thereby, the concentration of the target entity
in the initial solution can be inferred from the percentage
change of T2 value measured by a relaxometer [66]. However,
the degree of magnetic particle aggregation (and hence change
of T2 value) is highly dependent on the number ratio of the
target entity to magnetic particles. For instance, when magnetic
particles with a diameter of 70 nm were employed as the probe
to analyse the concentration of 8 nm entity (BSA-(Tag)6) which
has a smaller size than the magnetic particle probe, T2 value
tends to decrease with increasing concentration of target
entity until a minimum (denoted as equivalence point) is
reached (as shown in figure 5d). However, beyond the equival-
ence point, T2 value increases with target entity concentration
as the target entities which are present in excess have saturated
the binding sites of magnetic particles and prevent the further
aggregation of magnetic particles [61]. This phenomenon is
known as prozone effect. The intervention of prozone effect
has distorted the anti-correlation between concentration of
the target entity and T2 value when the number ratio of
target entity to magnetic particles exceeds the equivalence
point [66]. Thereby, in order to obtain reliable measurements
 Downloaded from http://rsfs.royalsocietypublishing.org/ on July 7, 2018
on concentration of the target entity, it is necessary to disperse
sufficient amount of magnetic particles into the given solution
so that the number ratio of target entity to magnetic particles is
well below the equivalence point.
Additionally, concentration of magnetic particles also
affects the sensitivity of target detection by MS. This phenom-
enon is observed in a bacteria and virus detection method
proposed which involved strong magnetic responsive
MP250-target-MP30 complex and poor magnetic responsive
unreacted MP30 [22]. Here, unreacted MP30 will still remain
suspended after being subjected to an external magnetic
field. Because concentration of MP30 dispersed in the solution
has a strong effect on the T2 value, the percentage change of
T2 value of the solution after the MS process can be used to
infer the concentration of the target entity in the initial sol-
ution. However, the sensitivity of this bacteria or virus
detection method is strongly dependent on the concentration
of MP30 supplied in the initial solution (figure 5e). As MP30
have been oversupplied, the fraction of MP30 involved in
the formation of MP250-target-MP30 complex is too low to
provide noticeable change in T2 value of the solution after
undergoing MS. On the contrary, when the concentration of
MP30 in the initial solution is too low, the magnetic signal
obtained from T2 value measurement is not sufficiently
intense to provide reliable result which accurately reflects
the concentration of the target in the initial solution. Further-
more, the linear relationship between the change of T2 value
and the target concentration can only be observed in a par-
ticular range of target concentration [22]. Therefore, in
order to have an accurate measurement of the target concen-
tration by using MS method, concentration of the magnetic
particles and the target must be carefully tuned to certain
range which provide reliable feedback from the analysis.
Furthermore, for MS-based in vivo diagnosis, the size and
concentration of magnetic particles employed must be con-
sidered carefully as their nanotoxicity is unclear. In this
regard, Nel et al. [67] demonstrated that nano-sized magnetic
particles are biologically toxic due to their large exposed sur-
face area that facilitates harmful interaction with biological
systems. For instance, Kunzmann and co-workers showed
the decay of cell viability of human monocyte derived
dendritic cells upon exposure to CSNPs (silica coated iron
oxide nanoparticles) with size of 30 and 50 nm. On the con-
trary, this phenomenon is not observed throughout their
exposure to larger CSNPs (70 and 120 nm) [68]. In parallel,
the same authors also observed that micro-sized titanium
oxide (TiO2) caused relatively higher DNA damage com-
pared with their nano-sized counterpart due to their higher
surface reactivity [69]. Hence, the toxicity dependency on
magnetic particle size cannot be solely coming from the
trade-off between total exposure surface area and surface
reactivity of the given magnetic particles. In addition, con-
centration is also one of the major factors that influences
bio-compatibility of a magnetic particle solution. This is
because magnetic particle solution which is highly concen-
trated (beyond certain threshold concentration) was found
to be toxic to living organism [70]. The toxicity is mainly
due to the induction of oxidative stress and generation of
reactive oxygen species which will subsequently injure or
kill the cells. For a cell –particle mixture, cell viability
decreases with increasing concentration of magnetic particles
[71]. For instance, it was reported that the viability of PC12
cells diminishes as they are exposed to a higher concentration
of iron oxide (Fe2O3) nanoparticles [72]. Hence, even though 12
the utilization of highly concentrated magnetic particle sol-
rsfs.royalsocietypublishing.org
ution is capable of increasing the MS rate in biomedical
diagnostic application, there exists certain constraint for us to
select magnetic particle solution with a suitable concentration
in the design of biomedical diagnostic process. Apart from opti-
mizing the MS rate by increasing the concentration of magnetic
particles, it is also necessary to ensure the concentration falls
within the range that is characterized by excellent sensitivity
and biological harmlessness.
To summarize all the above discussion, figure 6 is presented
to highlight the complex interplay between the particle size,
Interface Focus 6: 20160048
particle concentration and magnetization, which determine
the design of LGMS for biomedical diagnostic application.
5. Concluding remarks and future considerations
Over the past few decades, tremendous efforts have been
dedicated to the development of MS technique for sample
purification prior to the diagnosis of diseases. In this regard,
understanding the underlying principles that govern the
dynamical behaviour of MS is critical in the optimization of
separation efficiency. Concurrently, along with the progressive
growth in research works related to magnetophoresis of mag-
netic particles under low magnetic field gradient, more
findings directly related to its working principle have been dis-
covered and disclosed. In the near future, those findings
definitely should be taken into consideration for any design
of LGMS technique for biomedical diagnostic application.
First and foremost, magnetic particles employed to magne-
tically label the specified target entity must possess good
colloidal stability. This prerequisite is needed to ensure the par-
ticles present in well-dispersed condition so that more effective
mixing and tagging with target entity can be promoted. Apart
from imparting good colloidal stability to the functionalized
magnetic particles in the initial suspending medium, it is para-
mount to ensure their colloidal stability is not disrupted by
different biological matrices (e.g. DPBS, RPMI-1640, human
plasma) throughout the magnetic labelling process [18]. Gener-
ally, stability of magnetic particles can be strengthened by
imparting electrostatic repulsion and steric hindrance to the
given particles. However, it was reported that enhanced col-
loidal stability compromised the MS rate of magnetic
particles under low magnetic field gradient [73]. Hence, the
conflicting effect between colloidal stability and MS efficiency
must be carefully assessed in the design of LGMS technique for
biomedical diagnostic application.
As discussed in §3.2, several limitations exist in the isolation
of tiny size magnetic particles by employing LGMS technique.
In order to perform a successful LGMS, magnetophoretic
force exerted on magnetic particles must be sufficiently intense
to overcome viscous drag, thermal motion and gravitational
pulling. To accomplish these requirements, there are several
well-established rules of thumb which can provide a fast screen-
ing of the suitability of any selected magnetic particle system in a
particular LGMS process. Here, dimensionless number analysis
(i.e. Reynolds number, Péclet number and Froude number)
serves as a beneficial tool in determining whether the LGMS
of a particular system can be successfully conducted [15].
Another particle size-related criterion that also must
be carefully addressed is the impact of particle-size
distribution (PSD). As demonstrated in our previous work,
 Downloaded from http://rsfs.royalsocietypublishing.org/ on July 7, 2018

magnetization 13

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magnet

50 mT mm–1
exit
6
5
4
3
2
1
1 mm
12 mm
diameter cells

selectivity

Interface Focus 6: 20160048

10 mg ml–1
1 mg ml–1
600 0.5 mg ml–1
0.1 mg ml–1

S
DT2 (ms)

400

200

N
0
1 10 102 103 104 105 106 107
concentration of S. enterica (cfu ml–1) LGMS in biomedical MP250 MP30
diagnosis (1 min) (60 min)

sensitivity magnetic
separation rate
1.5
normalized opacity

10 g l–1 magnetic
1.0 1 g 1–1 particles
0.1 g 1–1
0.01 g 1–1 1 mm
0.5
100 nm 30 nm
0
0 100
time (s)
200
membrane of target cell
particle cooperative magnetophoresis magnetic loading particle
concentration size
Figure 6. Schematic diagram demonstrating the relationship between the three major factors ( particle size, particle concentration and magnetization) that influence
the performance of LGMS-aided biomedical diagnosis. Image components reprinted with permission from [13], Copyright & 2006 Royal Society of Chemistry, [22]
Copyright & 2015 American Chemical Society, [45] Copyright & 2008 American Institute of Physics and [48] Copyright & 2008 American Chemical Society.

magnetophoresis profile of an electrosterically stabilized internalize into) a unit target entity [13,18,55]. Hence, the magne-
magnetic nanoparticle suspension under LGMS is greatly tophoresis of this magnetic particle-bound target is a result of
influenced by PSD of the given particle system [58]. Prep- synergistic movement of a large number of magnetic particles.
aration of highly monodisperse magnetic particle system This process operated under the cooperative magnetophoresis
with narrow PSD is essential in order to have a high-quality mode in which magnetic particles move collectively towards
control of the LGMS process. Yet, the synthesis of mono- magnetic source. Additionally, the magnetic particle-bound tar-
disperse magnetic particles is technically challenging as gets might clump to each other during magnetophoretic
aggregation of the monodisperse particles, which results in migration which in turn further disturbs the dynamical behav-
polydisperse magnetic particles, is possible [74]. In addition, iour of the LGMS process. For this case, examining the
the aggregation of magnetic particles is further promoted by magnetophoretic migration of these magnetic particle-bound
the magnetic attraction due to the induced magnetic dipole targets at a microscopic level is highly recommended.
moment possessed by each magnetic particle in the presence Finally, our recent work has revealed that hydrodynamic
of an external magnetic field. For ferro- and ferri-magnetic effect is also playing a vital role in governing the dynamical
particles, magnetic dipole moment exists even without the behaviour of LGMS process. Owing to the momentum trans-
external magnetic field. Also, particle size is exceptionally fer between moving magnetic particles and the surrounding
vital in determining the bio-compatibility of the magne- fluid, continuous sweeping flow is created within the entire
tic particle system (as discussed in §4.2), and tight control of magnetic particle solution that is subjected to LGMS [44].
self-aggregation throughout the diagnosis process is indis- The convective flow generated due to the particle motion is
pensable. Thus, it is not only essential to synthesize highly directed towards the area where the magnetic field gradient
monodispersed magnetic particles, the PSD of the magnetic is lower. Simultaneously, this circulating flow drives mag-
particles throughout the diagnosis process must be closely netic particles located within the same area towards the
monitored and controlled as well. magnet within a relatively shorter timescale in comparison
Timescale involved for disease diagnosis is influenced by the with the motion which is purely driven by magnetophoretic
rate at which MS occurs. Therefore, it is crucial to optimize the force. In such a way, hydrodynamics engenders the fast mag-
separation rate of magnetic particles in any biomedical diagnos- netophoretic collection of magnetic particles from their
tic application. In particular, self-aggregation of magnetic suspension and makes LGMS more applicable for biomedical
particles leading to cooperative magnetophoresis is able to applications in which fast separation kinetics is needed. By
accelerate the LGMS rate. In fact, there were high amount taking advantage of the hydrodynamic interaction, it is
of magnetic particles in the cluster form, which bind to (or believed that the efficiency of diagnosis process could be
 Downloaded from http://rsfs.royalsocietypublishing.org/ on July 7, 2018

further enhanced. However, for LGMS that is conducted in framework in this aspect, such that the incorporation of 14
continuous mode, the magnetophoretic pathway of particles LGMS into a biomedical diagnostic application can be

might be considerably disrupted by the convective flow
initiated from hydrodynamic effect. In this regard, the inter-
ference of hydrodynamically driven convection has caused
difficulty in the theoretical prediction of deterministic magne-
tophoretic pathway of magnetic particles subjected to LGMS.
To date, we are only able to calculate the macroscopic
quantities of the LGMS system by solving the Navier –
Stokes equation (which is a nonlinear and fourth-order
partial differential equation in three-dimensional space [75])
numerically with the aid of computational simulation tools.
rsfs.royalsocietypublishing.org
facilitated in the upcoming future.
Authors’ contributions. The manuscript was written through contributions
of all authors. All authors have given approval to the final version of
the manuscript.
Competing interests. We declare we have no competing interests.
Funding. This project is financially supported by (i) International Foun-
dation for Sciences (IFS) and co-financed by the Organisation for the
Prohibition of Chemical Weapons (OPCW) (grant no. 6050324/I100)

Interface Focus 6: 20160048

and (ii) Fundamental Research Grant Scheme (FRGS) grant from
By contrast, theoretical description of the microscopic picture Ministry of Higher Education (MOHE) (grant no. 6071269).
of LGMS incorporating the hydrodynamic effect is still Acknowledgement. Sim Siong Leong acknowledges the support from the
unexplored. Thus, it is imperative to develop a theoretical MOHE Malaysia through the MyPhD Scholarship.

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