Trends in Analytical Chemistry 114 (2019) 89e97

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Trends in analytical separations of magnetic (nano)particles

b, Michael C. Breadmore a, Mirek Macka a, c, d, *

nica N. Alves a, Manuel Miro
Mo
a
School of Natural Sciences and Australian Centre for Research on Separation Science (ACROSS), University of Tasmania, Hobart, 7001, Australia
b
FI-TRACE Group, Department of Chemistry, University of the Balearic Islands, Carretera de Valldemossa, km. 7.5, E-07122, Palma de Mallorca, Spain
c
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czech Republic
d
Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00, Brno, Czech Republic

a r t i c l e i n f o a b s t r a c t

Article history: Magnetic particles (MPs) and magnetic nanoparticles (MNPs) are appealing candidates for biomedical
Available online 2 March 2019 and analytical applications due to their unique physical and chemical properties. Given that magnetic
fields can be readily used to control the motion and properties of M(N)Ps, their integration in analytical
Keywords: methods opens new avenues for sensing and quantitative analysis. There is a large body of literature
Capillary electrophoresis related to their synthesis, with a relatively small number of methods reporting the analysis of M(N)Ps
Field flow fractionation
using separation methods, which provide information on their purity and monodispersity. This review
Magnetic (nano)particles
discusses analytical separation methods of M(N)Ps published between 2013 and June 2018. The analytical
Magnetophoresis
Microfluidic chip
separation methods evaluated in this work include (i) field flow fractionation, (ii) capillary electropho-
Separation resis, (iii) macroscale magnetophoresis and (iv) microchip magnetophoresis. Among the trends in
analytical separations of M(N)Ps an inclination towards miniaturization is moving from conventional
benchtop methods to rapid and low-cost methods based on microfluidic devices.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction magnetisation due to the rapid reversal of their magnetic moment.

It makes superparamagnetic nanoparticles especially suitable
Magnetic (nano)particles (M(N)Ps) offer the unique advantage when looking for fast responses to external magnetic fields without
of being manipulated (moved or held in place) using permanent agglomeration effects [6]. For this reason, superparamagnetic
magnets or electromagnets, a significant reason behind their nanoparticles have been extensively pursued for a vast variety of
popularity, which grew rapidly in the past decades [1e3]. As an biomedical applications, including biosensing [7], bioanalysis [8],
example, one of the most routinely used methods exploiting MPs is drug delivery [9], magnetic resonance imaging (MRI) [10], and
magnetic sorting of cell populations from biological suspensions hyperthermia treatment of tumours [11].
[4]. This method is now standardized for tissue engineering and The unique behavior and increasing applications of M(N)Ps have
medical analysis. stimulated the advancement of new synthesis methods. The core of
Magnetic nanoparticles (MNPs) exhibit physical properties that M(N)Ps can be made of a wide range of magnetic materials such as
differ remarkably from those of the bulk ferromagnetic material nickel, cobalt, iron and iron oxides. Iron oxides and their corre-
due to finite size effects such as high surface-to-volume ratio, and a sponding ferrites are the most commonly used due to their high
special magnetic property at diameters typically lower than 20 nm magnetic moments, biological compatibility, simple synthesis and
called superparamagnetism [5]. At such small size, MNPs do not low cost of production. However, bare iron oxide nanoparticles are
exhibit multiple magnetic domains like ferromagnetic particles, but only stable in low ionic strength solutions at pH values above (pH
instead a single domain. Therefore, under an external magnetic 9e12) or below (pH 2e5) their point zero charge. To prevent ag-
field, the magnetic moment of single domain nanoparticles quickly gregation and increase selectivity, the magnetic cores are usually
aligns with the applied field, but in its absence, they exhibit no net coated with inorganic materials [12], polymers [13e16] and/or
functionalised with organic and biological molecules [1,17]. There is
a large quantity of literature exploiting the synthesis and surface
* Corresponding author. Private Bag 75, School of Natural Sciences and Australian engineering of M(N)Ps for many purposes. Compared to this large
Centre for Research on Separation Science, University of Tasmania, Hobart, 7001, output, the body of reports on separation approaches used for the
Australia. Fax: þ61 362262858. analysis of M(N)Ps is considerably smaller, but still very significant.
E-mail address: Mirek.Macka@utas.edu.au (M. Macka).

https://doi.org/10.1016/j.trac.2019.02.026
0165-9936/© 2019 Elsevier B.V. All rights reserved.
90 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97

A search in Elsevier's database Scopus shows that only ca. 12% of the of polydisperse M(N)Ps by simply controlling the flow rate. How-
total publications on M(N)Ps address analytical separation tech- ever, broad size distributions were obtained. Thus, further optimi-
niques. Yet, the separation and analysis of M(N)Ps is critically sation of the system is crucial to allow for specific applications such
important to obtain information on their size, shape and chemistry as in biomedicine.
surface, enabling their practical use for many applications.
In 2012, Stephens et al. [18] reviewed 58 papers describing 2.1.1.1. Magnetic quadrupole field flow fractionation. The first pro-
separation of M(N)Ps by means of applied magnetic fields and field totype of a magnetic quadrupole field flow fractionation (MQFFF)
gradients for improved purification and analysis [18]. The main goal was developed and evaluated by Zborowski et al. [26] for contin-
of this review is to pinpoint separation techniques of M(N)Ps for the uous separation of human peripherical lymphocytes labeled with
period between 2013 and June 2018, by employing three distinct magnetic colloids. It consisted of a quadrupole electromagnet as-
types of field-flow fractionation (FFF) (magnetic FFF, asymmetrical sembly of four steel pole tips with two of them opposed the
FFF and cyclical electrical FFF), capillary electrophoresis (CE), magnetic north poles and the other two opposed the magnetic
macroscale magnetophoresis (high/low gradient magnetic separa- south poles. The electromagnet assembly was radially symmetric.
tion), and microchip magnetophoresis. Analytical separation and The steel was magnetized by an electric current in the coils wrap-
sample preparation approaches using M(N)Ps are deemed outside ped around the poles. This configuration creates a magnetic field
the scope of our review, for which there is a large body of literature, whose magnitude increases linearly with the radial distance from
including recent comprehensive reviews [1e3,19e23]. All the the axis. This methodology is well described by Carpino et al. [27].
studies discussed and critically analyzed are summarized in Table 1 The results showed that the separation process was close to the
in terms of particle composition and size, magnetic field applied, predicted behavior of an ideal quadrupole magnetic field [26]. Later
separation principle, separation time and complexity of the in- on, Orita et al. [28] developed a simple on-off field MQFFF to
frastructures used. separate and quantify two distinct sub micrometer commercial
M(N)Ps (90 and 200 nm) at specific magnetic field and flow con-
2. Separations of magnetic (nano)particles ditions [28]. This on-off field MQFFF system was inspired by the
system previously developed by Zborowski et al. [26]. It consisted
2.1. Field flow fractionation of a separation channel volume of 0.94 mL fitted into a stainless-
steel cylinder that was implemented in a flow injection setup
FFF is a separation technique that uses an external field applied with downstream optical detection (Fig. 1). The fractograms
perpendicular to the direction of flow causing differential migra- exhibited improved retention (98.6% vs. 53.3%) for the larger M(N)
tion of M(N)Ps. Typically, the flow profile in a FFF channel is Ps (200 nm vs.90 nm) at higher flow rates (0.05 mL/min vs. 0.01 mL/
laminar, so particles which interact more strongly with the field are min) [28]. Thus, for given field and flow conditions, the on-off field
found closer to the channel walls and will move more slowly due to MQFFF system can be used for the quantification of retained and
slower flow streams. Analytes can be separated by different unretained fractions. This is useful for the separation of unwanted
mechanisms of FFF according to the type of the field applied. weakly magnetic particulate contaminants from M(N)P suspen-
Typical fields include centrifugal and gravitational forces, cross flow sions. Compared to the magnetic coil setup previously described,
of solvent, and thermal, electrical and magnetic gradients [24]. the on-off field MQFFF system requires less handling.
Moore et al. [29] used MQFFF for red blood cell (RBC, with mean
2.1.1. Magnetic field flow fractionation diameter of 8 mm) separation as an alternative to centrifugal sepa-
Magnetic field flow fractionation (MFFF) has been shown to be ration. A quadrupole field was designed having a maximum field of
an effective method for the separation of polydisperse suspensions 1.68 T at the magnet pole tips, zero field at the aperture axis, and a
of M(N)Ps when an external magnetic field is applied along a nearly constant radial field gradient of 1.75 T mm1 inside a cylin-
flowing channel. Rogers et al. [25] used MATLAB to simulate the drical aperture. A light-scattering detector downstream of the mag-
separation of fluidMAG-D (starch-coated magnetite (Fe3O4)) M(N) net measured light attenuation caused by the cells eluting from the
Ps of sizes between 50 and 400 nm by simulating particle trajec- magnet as a function of time. The cell samples were composed of
tories and magnetic forces. The results obtained from the simula- high spin RBC (obtained by chemical conversion of hemoglobin to
tion showed that M(N)Ps within the size range of interest could be methemoglobin - met RBC - or exposure to anoxic conditions - deoxy
separated and collected in a size dependent manner in fraction 1 RBC), low spin RBC (obtained by exposure to ambient air e oxy RBC),
(smaller sized) and fraction 2 (larger sized). To validate this model, and mixtures of deoxy RBC and white blood cells (WBC). Cell tracking
the simulated conditions were replicated experimentally. However, velocimetry was used to measure the magnetophoretic mobility of
the theoretical and experimental results did not agree. This the RBC and the results showed that the mobility depended on the
inconsistency could be due to the fact that particle-particle in- presence of high-spin hemoglobin. Only high spin RBC were attrac-
teractions are not taken into consideration into the model. Due to ted by the magnet, while low-spin RBC demonstrated magnetic
the failure of the initial experiments, the same authors changed the susceptibility comparable to WBC. It was also found that RBC did not
approach to a simple magnetic coil setup composed of a tubing elute within 15 min from the channel at flow rate 0.05 mL/min but
wrapped around a Grade N42 diametrically magnetized neodymi- as would be expected rapidly eluted at a higher flow rate of 2.0 mL/
um cylinder [25]. An inlet for both the M(N)Ps suspension and the min. These results agreed with earlier studies on the magnetic
mobile phase was inserted at the top of the magnetic coil and a properties of hemoglobin using other techniques [30,31]. The frac-
single outlet at the bottom was used for the collection of magnetic tionation experiments of RBC and a RBC/WBC mixture showed that a
particle fractions. The tubing was filled with M(N)Ps suspension. As 5  107/mL cell suspension pumped at 0.1 mL/min through a mag-
soon as a steady state level of accumulation of M(N)Ps across the netic field of 1.5T and gradient of 1000 T m1 is depleted to less than
inner wall of the tubing was achieved, an initial flow rate of 5% of the initial RBC number concentration. The 5% residual
0.25 mL/min was applied to wash out the M(N)Ps remaining sus- contamination was comparable to that typically seen in WBC ob-
pended. Then, the flow rate was increased up to 50 mL/min. DLS tained by blood centrifugation. One advantage over blood centrifu-
measurements and TEM showed that the particles collected at gation, is that MQFFF RBC separation can be scaled to microliter
lower flow rates were smaller than particles collected at higher devices for RBC debulking, which can be portable and operated
flow rates. This approach was low cost and allowed the separation immediately after donation with minimal human labor.
 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97 91

Table 1
Particle composition and size, magnetic field applied, separation principle, separation time and complexity of the infrastructure used for M(N)Ps and magnetically susceptible
RBCs between 2013 and 2018.

Particle composition Particle size (nm) Magnetic field applied Separation Separation Complexity of the infrastructure Ref
principle time

Starch-coated magnetite (nano) 50e400 51 mm length Grade N42 MFFF <1 h Basic laboratory equipment [25]
particles diametrically magnetized
NdFeB cylinder
Dextran-coated magnetite 90 and 200 Quadrupole electromagnet MQFFF 50 min Stainless-steel cylinder within a [28]
nano(particles) quadrupole electromagnet
implemented into a flow injection setup
with downstream optical detection
RBCs 8000 Quadrupole magnet MQFFF 25 min Cylindrical flow channel centred inside [29]
of a quadrupole magnet with
downstream light scattering detection
Carboxydextran-coated 6e60 n.a. AF4 15 min AF4 instrument connected to MALLS [32]
maghemite nanoparticles detection
Hybrid polymer magnetic 54 n.a. AF4 15 min AF4 instrument connected to UV and [33]
micelles MALLS detection
Lipid and polystyrene 50 and 100 n.a. CyEFFF 30 min HPLC pump connected to an EFFF [34]
sulfonate-coated magnetite channel with downstream UV
nanoparticles detection; ac and dc voltages induced
by a signal generator and a dc power
supply
Polyvinylpyrrolidone (PVP)- 127 0.56 T permanent magnetic HGMS 1h HGMS instrument [45]
coated magnetic particles assembly consisting of two 2
4  0.5 inch NdFeB blocks with
a minimum gap of 5/8 inch
Poly (diallyl 50 NdFeB permanent magnet LGMS 6 min Basic laboratory equipment [46]
dimethylammonium
chloride) (PDDA) and
chitosan (Chi)-coated
magnetic nanoparticles
Poly(diallyldimethylamonium 50 nm (spherical) Cylindrical shaped N50-graded LGMS 6h Basic laboratory equipment [48]
chloride) (PDDA)-coated iron and 20  300 nm NdBFe (1.20 T) and Alnico
oxide nanoparticles (rod-like) permanent magnet (1.45 T)
with 14 mm in diameter and
15 mm in length
Bare and carboxylated iron 7 - 13 (bare iron n.a. CE 12 min CE instrument [39]
oxide nanoparticles oxide
nanoparticles)
and 10
(carboxylated
iron oxide
nanoparticles)
Carboxylated iron oxide 75 n.a. CE 5 min CE instrument [41]
nanoparticles
Polydisperse magnetic particles 150 and 500 Permanent NbFeB magnet Microchip 15 s Microchip fabricated in PDMS with a [49]
(6 mm length, 4 mm width, and magnetophoresis side permanent magnet, a Gaussmeter,
3 mm thickness) and DIC detection
Iron oxide magnetic beads 5000 Soft magnetic microstructures Microchip <2 s Microchip fabricated in PDMS mounted [50]
made of a mixture of iron magnetophoresis in an inverted microscope connected to
powder and PDMS into a a high-speed camera, placed in the
prefabricated channel center of parallel permanent magnets
Polystyrene (5000 and 5000, 11000, and Permanent NbFeB N42 grade Microchip 15 min Microchip fabricated in PDMS with a [51]
11000 nm) and polyethylene 35000 cuboid magnet magnetophoresis side permanent magnet mounted in an
(35000 nm) magnetic beads inverted microscope connected to a
high-speed camera
Microspheres composed of 2000, 6000 and Octupolar array of permanent Microchip 10 s Microchip fabricated in PDMS with a [52]
styrene-maleic acid 12000 magnets magnetophoresis spiral channel centred with respect to
copolymer matrix the octupolar magnetic array
encapsulating 50% by mass
magnetite cores
Magnetite nanoparticles 10 Permanent NbFeB magnet Microchip >1 h Microchip fabricated in PDMS with [53]
magnetophoresis permanent magnet mounted in an
inverted microscope connected to a
high-speed camera
Magnetite-doped and uncross- 7000 Halbach array Microchip <2 s Microchip fabricated in PDMS placed in [54]
linked polystyrene particles magnetophoresis the center of the Halbach array
with spherical and elliptical mounted in an inverted microscope
shapes connected to a high-speed camera

AF4: Asymmetrical field flow fractionation; CE: Capillary electrophoresis; CyEFFF: Cyclical electrical field flow fractionation; HGMS: High gradient magnetic separation; LGMS:
Low gradient magnetic separation; MALLS: Multi-angle laser light scattering; MFFF: Magnetic field flow fractionation; MQFFF: Magnetic quadrupole field flow fractionation;
RBCs: Red blood cells.
92 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97

Fig. 1. Schematic of the MQFFF system. The mobile phase was driven by a pump. The sample is introduced through a separate port. The flow of the mobile phase pushes the sample
from the injector into the separation channel fitted into a quadrupole electromagnet connected to a UVevisible detector. Reprinted from Ref. [28] with permission.

2.1.2. Asymmetrical flow field-flow fractionation electrophoretic analysis of lipid and polystyrene sulfonate-coated
Asymmetrical flow field flow fractionation (AF4) is a separation MNPs [34].
technique based on the theory of FFF. The cross flow is induced by
flowing liquid constantly exiting through a semi-permeable wall on 2.2. Capillary electrophoresis
the bottom of the channel. The lower size M(N)Ps, which can be
fractioned, are restricted by the molecular weight cut off mem- CE is a powerful separation method which has the advantages of
brane. The suitability of AF4 has been shown for the fractionation of minimal requirement of sample and buffer volumes, and lack of
magneto polyplexes (with mean diameter of 54 nm) [32] and generation of organic waste. Further, the use of narrow capillaries
carboxydextran-coated maghemite dispersions (with diameter of in CE with high electrical resistance, allows the application of high
6e60 nm) [33] by connecting the AF4 instrument to UV [33] and electrical fields with minimal heat generation. The use of high
multi-angle laser light scattering (MALLS) [32,33] detectors. AF4 electrical fields together with the conventional plug-type flow from
can be a simple, fast and reliable tool for quality control in com- electrically driven systems results in short analysis times and high
mercial production of M(N)Ps, while providing complementary efficiency and resolution for almost any type of ionic analytes. Yet, a
information related to nonmagnetic sample components. remarkable limitation of bare iron oxide M(N)P separations by CE
that has been previously reported [35e38] is their high tendency to
2.1.3. Cyclical electrical field flow fractionation spontaneously agglomerate to minimize surface energies, which is
Cyclical electrical field flow fractionation (CyEFFF) consists of an observed in electropherograms as spurious spikes that in turn
oscillating square voltage applied between a top and bottom elec- prevent the accurate determination of the electrophoretic mobil-
trode inside the channel. As result, M(N)Ps move back and forth ities of M(N)Ps. This limitation has been recently overcome by Alves
between the electrodes in agreement with their sizes and electro- et al. [39], who achieved symmetrical and smooth peaks of bare
phoretic mobilities. M(N)Ps with high electrophoretic mobilities iron oxide nanoparticles. This was accomplished through electro-
move further into the center of the channel and they spend more static stabilisation using complexing electrolyte anions such as
time at the faster fluid regions thus eluting earlier than the lower citrate and phosphate, and the additive tetramethylammonium
mobility M(N)Ps. One of the limitations of this technique is the hydroxide (TMAOH) within the background electrolyte (BGE), an
band broadening of the resulting UV fractograms and low resolu- ionic solution of desired concentration, co-ion and counter-ion
tion. To address and solve the diffusion issue, Tasci et al. [34] re- mobilities, and usually also providing pH-buffering capacity.
ported the separation of MNPs by CyEFFF applying square wave TMAOH is a peptizing agent (an electrolyte that converts aggre-
voltages with higher duty cycles instead of DC offset voltages gated particles into a colloidal sol) [40] used for more than two
(Fig. 2). Thus, particle diffusion was suppressed, which allowed decades in the synthesis of well-dispersed iron oxide M(N)P solu-
separations of MNPs with mean diameter of 50 and 100 nm. This tions, however never utilised for effective CE separations of M(N)Ps.
study demonstrated the capability of CyEFFF for size and The same study also showed the successful separation of bare (with
diameter between 7 and 13 nm) and carboxylated (10 nm) iron
oxide nanoparticles in 12 min using Tris-nitrate containing 20 mM
TMAOH as BGE. The electrophoretic mobilities for bare and
carboxylated iron oxide nanoparticles were 3.3E-08 m2 V1 s1 (0.9
%RSD) and 4.1E-08 m2 V1 s1 (0.4 %RSD), respectively. These
findings demonstrate that simple and rapid CE experiments are
excellent tools to characterise and monitor properties and in-
teractions of iron oxide nanoparticles with other molecules for
potential surface modification purposes [39]. Baron et al. [41]
studied the online stacking of carboxylated core-shell magnetite
Fig. 2. Schematic of the CyEFFF system. The dashed line shows the particle trajectory nanoparticles in CE. By monitoring the ionic strength of the BGE
that results from the cyclical electrical field. Reprinted from Ref. [34] with permission. and the sample zone, it was observed that stacking occurred
 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97 93

optimally when MNPs were dispersed in 10 mM borate/NaOH (pH were eliminated. The continuous use of this HGMS system over 7 h
9.5) and injected to the BGE composed of 100 mM borate/NaOH (pH allowed the treatment of 17 L oil water mixture with no reduction
9.5). The decrease of the electric double layer thickness with of the oil and MPs removal capacity [45]. Although this study in-
increasing ionic strength could induce MNP aggregation and led to troduces a new application of HGMS for oil remediation, it has the
the restructuring of the MNPs zone due to the decrease of distance disadvantages of being tedious and time-consuming.
between nanoparticles [41]. The Derjaguin-Landau-Verwey-
Overbeek (DLVO) theory, which describes van-der-Waals and 2.3.2. Low gradient magnetic separation
electrostatic interactions between charged surfaces within a liquid In contrast to conventional practice where HGMS is normally
medium [42], was suggested as a cause of peak sharpening in CE. employed, LGMS is still poorly explored and understood. Toh et al.
[46,47] showed the reliability of LGMS for magnetophoretic sepa-
2.3. Macroscale magnetophoresis ration of microalgal biomass that interacted electrostatically with
cationic polymer functionalised MNPs [46,47]. Poly (diallyl dime-
Magnetophoresis refers to the motion of magnetic particles or thylammonium chloride) (PDDA) and chitosan (Chi) (with mean
magnetizable material through a fluid under the influence of a diameter of 50 nm) worked as binding agents to promote rapid
magnetic field [43]. For almost all the M(N)Ps applications, separation of the negatively charged Chlorella sp. through LGMS at
manipulation, recovery, and collection rates using external mag- field gradient lower than 80 T m1. The obtained results indicated
nets should be done quickly. Rapid magnetophoretic separation can cell separation efficiency of about 98% for PDDA and 99% for Chi.
⃗
be attained under both high gradient (HGMS, VB/ > 1000 T m1) Though, from a practical point of view, PDDA was preferable as
⃗
and low gradient magnetic separation (LGMS, VB < 100 T m1).
/
polymer binder since the attachment mechanism involved was pH
independent [47]. Because almost all the magnetophoretic studies
2.3.1. High gradient magnetic separation have been dedicated to the behavior of spherical MNPs, and poor
HGMS is commonly employed in conventional industry practice attention has been paid to rod-like MNPs, Lim et al. [48] compared
to separate magnetic materials from non-magnetic aqueous solu- the magnetophoretic behavior of spherical and rod-like iron oxide
tions, such as for wastewater treatment of bacteria and solids. nanoparticles under LGMS. Both effects of particle concentration
Typically, HGMS is used to separate microscale or bigger particles, and magnetic field gradient on the separation kinetics were eval-
or microscale aggregates of nanoparticles, or nanoparticles uated. It was shown that at low particle concentration, the mag-
encapsulated in larger polymer beads. However, the application of netophoresis of MNPs at low magnetic gradient is significantly
HGMS to suspensions of individually dispersed M(N)Ps has been enhanced by particle anisotropy (non-spherical shape), with rod-
poorly explored so far. HGMS systems generally consist of a column like MNPs taking significantly less time than spherical MNPs to
packed with magnetically susceptible wires placed inside an elec- be separated [48]. New approaches for the separation of M(N)Ps
tromagnet. Through application of a magnetic field across the col- according to their shape, size, and coatings, along with their unique
umn, the wires dehomogenise the magnetic field in the column magnetic properties will open new opportunities for M(N)Ps.
producing high field gradients around the wires to enable the
capture of M(N)Ps onto their surfaces. The attraction of M(N)Ps 2.4. Microchip magnetophoresis
depends on the magnetic field gradients generated, particle size
and magnetic properties [44]. A study conducted by Mir- The field of microfluidics is continuously evolving as miniaturized
shahghassemi et al. [45] describes the application of HGMS for oil platforms provide quick analysis with high resolution at low cost,
remediation using polyvinylpyrrolidone (PVP)-coated MPs (mean foster portability and make use of exceptionally minute amounts of
size diameter of 127 nm) in a continuous and large volume flow reagents. Zhang et al. [49] developed a microchip based on magne-
system. This technique was analyzed as a function of magnetic field tophoresis with differential interference contrast (DIC) detection
strength, mixing time and stainless-steel wool content. Fluores- (Fig. 3A, B). The real-time moving trajectories and velocities of the
cence and inductively coupled plasma-optical emission spectrom- MPs at different magnetic field strengths (depending on the distance
eter (ICP-OES) data indicated that ca. 85% of oil and 95% of MNP to the permanent magnet) were measured based on consecutive DIC

Fig. 3. Schematic diagram (A) and photography (B) of the experimental setup of the microchip magnetophoresis. (C) Representative magnetopherograms of the MPs by microchip
magnetophoresis with the DIC detection system. (D) Magnetophoretic velocities of the MPs at different permanent magnet distances obtained using a DIC microscope. Reprinted
from Ref. [49] with permission.
94 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97

Fig. 4. Fabrication process of the microfluidic device for efficient separation of magnetic particles based on deflection in flowing streams. Reprinted from Ref. [50] with permission.

images. The results indicated that shorter distances to the magnet MPs that deflected them perpendicularly to the pressure-driven
caused higher magnetophoretic velocities of MPs, and enhanced flow. Thus, the separation depended on the magnetic forces. In
magnetophoretic velocity differences between dissimilar particle turn, magnetic forces are affected by the shape of the iron-PDMS
sizes (Fig. 3D) [49]. This study allowed the successful separation and microstructures and the mass ratio of the iron-PDMS composite.
detection of a polydisperse mixture of MPs (500 nm and 160 nm) at a Also, the flow rate in the fluid channel affects the time that MPs are
single-particle level in only about 15 s (Fig. 3C). subjected to the magnetic field, and consequently their vertical
The microfluidic separation of iron oxide beads (5 mm diameter) deflection. Numerical simulations were developed to predict the
with soft magnetic microstructures was demonstrated by Zhou particle trajectories showing good agreement with experimental
et al. [50]. The fabrication process of the microfluidic device is data. Finally, systematic experiments and simulations were con-
represented in Fig. 4. This microfluidic device consisted of two ducted to study the effect of several relevant factors on the sepa-
channels - fluidic and structural e made in polydimethylsiloxane ration of MPs: microstructure shape, mass ratio of the ironePDMS,
(PDMS). The fluidic channel contained two inlets and two outlets. A microfluidic channel width and average flow velocity. The results
mixture of iron powder and PDMS was injected into the structural demonstrated that (i) half circular ironePDMS microstructure
channel located between two external permanent magnets. Three caused greater deflections, (ii) larger mass ratio of the ironePDMS
microstructure shapes were studied: (i) half circle, (ii) 60 isosceles composite provided higher magnetic forces, and (iii) wider chan-
triangle and (iii) 120 isosceles triangle. The soft magnetic micro- nels separate MPs less efficiently than narrow microfluidic chan-
structures provided localized and strong magnetic forces on the nels when operating at the same flow rate [50]. Based on the

Fig. 5. MIMF scheme of the particle separation. The device (scale bar 25 mm) consisted of an inertio-magnetic zone (IMZ) with a side permanent magnet and an expansion zone
(EZ). The schematic representation of MIMF device shows three red-colored magnetic particles (MP) of varied sizes and a black-colored non-magnetic particle (NMP). Reprinted
from Ref. [51] with permission.
 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97 95

12 mm circulating in a spiral microfluidic channel (Fig. 6). The fluid

was injected via an input port located near the center of the spiral
and exits through a symmetric flow splitter and two outlet ports.
The exit ports were referred to as inner outlet and outer outlet,
which collected the inner and outer halves of the fluid stream,
respectively. For that purpose, an array of permanent magnets was
arranged and accurately centred beneath the spiral to produce a
magnetic field with octupolar symmetry. At low flow rates (5 mL/
min) it was observed that 6 mm MPs clustered along a streamline
near the outer wall of the spiral. At intermediate flow rates (30 mL/
min), 6 mm MPs were homogeneously distributed across the width
of the channel. At high flow rates (60 mL/min), 6 mm MPs were
focused in clusters within the inner half of the spiral. The phe-
nomenon observed at high flow rates was caused by hydrodynamic
drag forces that induced secondary (Dean) flow in the spiral
microfluidic channel. A model incorporating key forces involved in
the spiral microchip was described and used to extract quantitative
in situ information about the magnitude of local Dean drag forces
Fig. 6. Geometry of the microfluidic spiral. The fluid was injected via the input port
from experimental data. The experimental results also showed that
(in) and exits through the exit ports referred as inner outlet (io) and outer outlet (oo).
Reprinted from Ref. [52] with permission. at low flow rates (5 mL/min) all the 12 mm MPs and one third of the
2 mm MPs were drawn toward the outer wall of the spiral and
extracted from the outer outlet, whereas the remaining two thirds
obtained results, enhanced separations of MPs can be achieved in a of the 2 mm MPs were extracted from the inner outlet. Gradually
compact, simple and low-cost microfluidic device. more 6 and 12 mm MPs were extracted from the inner outlet at
Kumar and Rezai [51] introduced a novel hybrid technique higher flow rates. For example, all the 12 mm MPs were found to exit
called multiplex inertio-magnetic fractionation (MIMF) to simul- the inner outlet at 40 mL/min. The behavior of the smallest MPs
taneously fractionate up to four magnetic and non-magnetic par- (2 mm) was opposite of the larger particles, with less and less being
ticles in water at a throughput of 106e109 particles per hour. The extracted from the inner outlet as the flow rate increases [52].
MIMF device was based on interactions between flow-induced in- The effective application of MNPs is highly dependent of appro-
ertial forces and magnetic forces in an expansion microchannel priate cleaning after synthesis and/or before their use to remove
containing an external permanent magnet (Fig. 5). The particle solvents, excess of surfactants, byproducts and undesired impurities.
fractionation performance was first optimized in terms of flow rate Because manual cleaning is time consuming and inefficient, Cardoso
and aspect ratio of the channel and particle size on duplex MIMF to et al. [53] designed, fabricated and tested a microfluidic system for
understand the behavior of the particles. The obtained knowledge the continuous cleaning and separation of MNPs (average diameter
was then applied to demonstrate fourplex MIMF with three mag- of 10 nm) synthesized by coprecipitation using NH4OH as catalyst.
netic monodisperse particles (5, 11 and 35 mm) and nonmagnetic First, a theoretical study was performed to optimize the geometrical
particles (15 mm). The non-magnetic particles inertially focus at the configuration of the microfluidic device and the experimental con-
center of the channel, while magnetic particles get fractionated ditions. The optimized microfluidic system was composed of two
based on interaction between inertial and magnetic forces and inlets and two outlets (Fig. 7). The cleaning solution (water, fluid A)
positioned in a size related manner in the device with smaller was introduced through the inlet A and the synthesis solution with
particles located closer to the external magnet. The exit position for MNP (fluid B) through the inlet B. The waste fluid (fluid C) exited
each particle type and size was measured with respect to the through the outlet C, whereas the cleaned MNPs solution (fluid D)
expansion region baseline since the focus of this study was only to exited though the outlet D. A permanent magnet was located near
investigate the concept of MIMF and not M(N)Ps sorting. In the the diffusion channel to deflect the MNPs by magnetic forces from
future, outlets can be implemented based on the exit positions the synthesis solution to the cleaning solution (Fig. 7). Gas chroma-
calculated. This MIMF device addresses several disadvantages of tography was performed to indirectly calculate the cleaning effi-
currently available magnetic fractionation devices such as low ciency by measuring the decrease of the peak area of NH4OH. The
throughput, requirement of sheath flow and inability to fractionate results demonstrated a cleaning efficiency of about 99.7% by con-
multiple targets simultaneously [51]. trolling the fluid flows in the microfluidic system, whereas manual
Dutz et al. [52] studied the consequences of applying an external cleaning achieved a value of about 94.3% after cleaning six consec-
magnetic force to a suspension of MPs with diameters of 2, 6 and utive times. Both processes are time-consuming, however the

Fig. 7. Schematic of the optimized microfluidic system. Fluid A: cleaning solution; Fluid B: synthesis solution with MNPs; Fluid C: waste; Fluid D: cleaned MNPs. Channel widths (a)
600 mm; (b) 400 mm; (c) 600 mm; (d) 400 mm; diffusion channel length (e) 10 mm. Reprinted from Ref. [53] with permission.
96 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97

microfluidic system offers negligible loss of MNPs and the process is [4] S. Miltenyi, W. Müller, W. Weichel, A. Radbruch, High gradient magnetic cell
separation with MACS, Cytometry 11 (1990) 231e238.
performed autonomously [53].
[5] S.A. Wahajuddin, Superparamagnetic iron oxide nanoparticles: magnetic
The separation of magnetic particles with spherical (mean nanoplatforms as drug carriers, Int. J. Nanomed. 7 (2012) 3445.
diameter of 7 mm) and elliptical shapes was demonstrated by Zhou [6] S.R. Dave, X. Gao, Monodisperse magnetic nanoparticles for biodetection,
et al. [54] in a simple and effective manner. The microfluidic chip imaging, and drug delivery: a versatile and evolving technology, Wiley
Interdiscip. Rev. Nanomed. Nanobiotechnol. 1 (2009) 583e609.
consisted of two inlets and one outlet. The inlet 1 was injected with [7] J.M. Perez, T. O'Loughin, F.J. Simeone, R. Weissleder, L. Josephson, DNA-based
aqueous-glycerol solution that worked as buffer flow, while the magnetic nanoparticle assembly acts as a magnetic relaxation nanoswitch
inlet 2 was injected with sample particles suspended in aqueous- allowing screening of DNA-cleaving agents, J. Am. Chem. Soc. 124 (2002)
2856e2857.
glycerol solution. The microfluidic device was placed in the cen- [8] J.E. Smith, L. Wang, W. Tan, Bioconjugated silica-coated nanoparticles for
ter of a uniform magnetic field and mounted on an inverted mi- bioseparation and bioanalysis, TrAC, Trends Anal. Chem. 25 (2006) 848e855.
croscope to record the trajectories of the magnetic particles. A [9] N. Kohler, C. Sun, J. Wang, M. Zhang, Methotrexate-modified super-
paramagnetic nanoparticles and their intracellular uptake into human cancer
pressure-driven flow was combined with the magnetic field cells, Langmuir 21 (2005) 8858e8864.
applied perpendicularly to the flow direction. The results showed [10] B. Chertok, B.A. Moffat, A.E. David, F. Yu, C. Bergemann, B.D. Ross, V.C. Yang,
that the asymmetrical rotation of the ellipsoidal MPs, together with Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored mag-
netic targeting of brain tumors, Biomaterials 29 (2008) 487e496.
the particle-wall hydrodynamic interactions, resulted in a net lift [11] K. Maier-Hauff, F. Ulrich, D. Nestler, H. Niehoff, P. Wust, B. Thiesen, H. Orawa,
force towards the channel center. Differently, spherical MPs V. Budach, A. Jordan, Efficacy and safety of intratumoral thermotherapy using
remained closer to the channel wall. This uniform magnetic field magnetic iron-oxide nanoparticles combined with external beam radio-
therapy on patients with recurrent glioblastoma multiforme, J. Neuro Oncol.
technique can be applied to multiple microfluidic channels facili-
103 (2011) 317e324.
tating high throughput parallelization for biological and biomedical [12] V. Salgueirin ~ o-Maceira, M.A. Correa-Duarte, M. Spasova, L.M. Liz-Marz
an,
applications that require separation of shaped MPs [54]. M. Farle, Composite silica spheres with magnetic and luminescent function-
alities, Adv. Funct. Mater. 16 (2006) 509e514.
[13] G.S. Demirer, A.C. Okur, S. Kizilel, Synthesis and design of biologically inspired
3. Conclusion and outlook biocompatible iron oxide nanoparticles for biomedical applications, J. Mater.
Chem. B 3 (2015) 7831e7849.
[14] J. Chomoucka, J. Drbohlavova, D. Huska, V. Adam, R. Kizek, J. Hubalek, Magnetic
The improvement of the existing approaches of synthetizing
nanoparticles and targeted drug delivering, Pharmacol. Res. 62 (2010) 144e149.
uniform and more monodisperse M(N)Ps has been notorious in [15] A.K. Gupta, M. Gupta, Synthesis and surface engineering of iron oxide nano-
recent years. However, even the most efficient and highly opti- particles for biomedical applications, Biomaterials 26 (2005) 3995e4021.
mized protocols yield samples relatively polydisperse. This is an [16] D. Singh, J.M. McMillan, X.-M. Liu, H.M. Vishwasrao, A.V. Kabanov,
M. Sokolsky-Papkov, H.E. Gendelman, Formulation design facilitates magnetic
obstacle for some emerging applications of M(N)Ps with some nanoparticle delivery to diseased cells and tissues, Nanomedicine 9 (2014)
specific functions, particularly in biomedical areas, as their prop- 469e485.
erties are dependent. Analytical tools for M(N)Ps separation are [17] W. Wu, Q. He, C. Jiang, Magnetic iron oxide nanoparticles: synthesis and
surface functionalization strategies, Nanoscale Res. Lett. 3 (2008) 397.
fundamental to understand the behavior of M(N)Ps according to [18] J.R. Stephens, J.S. Beveridge, M.E. Williams, Analytical methods for separating
their size, shape and surface chemistry. This knowledge can give and isolating magnetic nanoparticles, Phys. Chem. Chem. Phys. 14 (2012)
information on the monodispersity and purity of M(N)Ps for their 3280e3289.
[19] V. Tolmacheva, V. Apyari, E. Kochuk, S. Dmitrienko, Magnetic adsorbents
ultimate practical use. Over the last 5 years, novel strategies for based on iron oxide nanoparticles for the extraction and preconcentration of
M(N)P separations have been reported based on FFF, macroscale organic compounds, J. Anal. Chem. 71 (2016) 321e338.
magnetophoresis and CE, but the trend is toward integrating [20] Y. Wang, Q. Chen, C. Gan, B. Yan, Y. Han, J. Lin, A review on magnetophoretic
immunoseparation, J. Nanosci. Nanotechnol. 16 (2016) 2152e2163.
external magnetic fields onto single microfluidic structures. From
[21] A.A. Hernandez-Hernandez, G.A. Alvarez-Romero, E. Contreras-Lopez,
about 33 journal articles that have been published in the last 5 K. Aguilar-Arteaga, A. Castaneda-Ovando, Food analysis by microextraction
years, 54% of them are microfluidic based. These can be easily methods based on the use of magnetic nanoparticles as supports: recent
advances, Food Anal. Methods 10 (2017) 2974e2993.
fabricated and are inexpensive, not requiring any extra power
[22] S. Piovesana, A.L. Capriotti, Magnetic materials for the selective analysis of
source. Moreover, separations in microchip magnetophoresis can peptide and protein biomarkers, Curr. Med. Chem. 24 (2017) 438e453.
be easily achieved with high efficiency and throughput in the order [23] T. Tangchaikeeree, D. Polpanich, A. Elaissari, K. Jangpatarapongsa, Magnetic
of seconds. Real-time moving trajectories and velocities of M(N)Ps particles for in vitro molecular diagnosis: from sample preparation to inte-
gration into microsystems, Colloids Surf., B 158 (2017) 1e8.
can also be monitored by means of microscope imaging that is seen [24] J.C. Giddings, F. Yang, M.N. Myers, Flow-field-flow fractionation: a versatile
as the new trend in microfluidic separation and identification of new separation method, Science 193 (1976) 1244e1245.
M(N)Ps. [25] H.B. Rogers, T. Anani, Y.S. Choi, R.J. Beyers, A.E. David, Exploiting size-
dependent drag and magnetic forces for size-specific separation of magnetic
nanoparticles, Int. J. Mol. Sci. 16 (2015) 20001e20019.
Acknowledgements [26] M. Zborowski, L. Sun, L.R. Moore, P.S. Williams, J.J. Chalmers, Continuous cell
separation using novel magnetic quadrupole flow sorter, J. Magn. Magn.
Mater. 194 (1999) 224e230.
Mirek Macka gratefully acknowledges the Australian Research [27] F. Carpino, L.R. Moore, J.J. Chalmers, M. Zborowski, P.S. Williams, Quadrupole
Council Future Fellowship (FT120100559). Manuel Miro
acknowl- Magnetic Field-Flow Fractionation for the Analysis of Magnetic Nanoparticles,
edges financial support from the Spanish State Research Agency IOP Publishing., 2005, p. 174.
[28] T. Orita, L.R. Moore, P. Joshi, M. Tomita, T. Horiuchi, M. Zborowski,
through project CTM2017-84763-C3-3-R (MINECO/AEI/FEDER, EU). A quantitative determination of magnetic nanoparticle separation using on-
Michael Breadmore acknowledges an Australian Research Council off field operation of quadrupole magnetic field-flow fractionation
Future Fellowship Award (FT130100101). (QMgFFF), Anal. Sci. 29 (2013) 761e764.
[29] L.R. Moore, P.S. Williams, F. Nehl, K. Abe, J.J. Chalmers, M. Zborowski, Feasi-
bility study of red blood cell debulking by magnetic field-flow fractionation
References with step-programmed flow, Anal. Bioanal. Chem. 406 (2014) 1661e1670.
[30] L. Pauling, C.D. Coryell, The magnetic properties and structure of hemoglobin,

Ríos, M. Zougagh, Recent advances in magnetic nanomaterials for
[1] A. oxyhemoglobin and carbonmonoxyhemoglobin, Proc. Natl. Acad. Sci. U.S.A. 22
improving analytical processes, TrAC, Trends Anal. Chem. 84 (2016) 72e83. (1936) 210e216.
[2] T. Jamshaid, E.T.T. Neto, M.M. Eissa, N. Zine, M.H. Kunita, A.E. El-Salhi, [31] C.D. Coryell, F. Stitt, L. Pauling, The magnetic properties and structure of fer-
A. Elaissari, Magnetic particles: from preparation to lab-on-a-chip, biosensors, rihemoglobin (methemoglobin) and some of its compounds, J. Am. Chem. Soc.
microsystems and microfluidics applications, TrAC, Trends Anal. Chem. 79 59 (1937) 633e642.
(2016) 344e362. [32] E. Wetterskog, A. Castro, L. Zeng, S. Petronis, D. Heinke, E. Olsson, L. Nilsson,
[3] I. Vasconcelos, C. Fernandes, Magnetic solid phase extraction for determina- N. Gehrke, P. Svedlindh, Size and property bimodality in magnetic nano-
tion of drugs in biological matrices, TrAC, Trends Anal. Chem. 89 (2017) particle dispersions: single domain particles vs. strongly coupled nano-
41e52. clusters, Nanoscale 9 (2017) 4227e4235.
 M.N. Alves et al. / Trends in Analytical Chemistry 114 (2019) 89e97 97

[33] E. Haladjova, S. Rangelov, M. Geisler, S. Boye, A. Lederer, G. Mountrichas, [44] G.D. Moeser, K.A. Roach, W.H. Green, T. Alan Hatton, P.E. Laibinis, High-
S. Pispas, Asymmetric flow field-flow fractionation investigation of magne- gradient magnetic separation of coated magnetic nanoparticles, AlChE J. 50
topolyplexes, Macromol. Chem. Phys. 216 (2015) 1862e1867. (2004) 2835e2848.
[34] T. Tasci, E. Manangon, D. Fernandez, W. Johnson, B. Gale, Separation of [45] S. Mirshahghassemi, A.D. Ebner, B. Cai, J.R. Lead, Application of high gradient
magnetic nanoparticles by cyclical electrical field flow fractionation, IEEE magnetic separation for oil remediation using polymer-coated magnetic
Trans. Magn. 49 (2013) 331e335. nanoparticles, Separ. Purif. Technol. 179 (2017) 328e334.
[35] G.R. Ducatte, N.E. Ballou, C. Quang, S.L. Petersen, Separation and character- [46] P.Y. Toh, B.W. Ng, C.H. Chong, A.L. Ahmad, J.-W. Yang, C.J.C. Derek, J. Lim,
ization of oxide particles by capillary electrophoresis, J. Microcolumn Sep. 8 Magnetophoretic separation of microalgae: the role of nanoparticles and
(1996) 403e412. polymer binder in harvesting biofuel, RSC Adv. 4 (2014) 4114e4121.
[36] C. Quang, S. Petersen, G. Ducatte, N. Ballou, Characterization and separation of [47] P.Y. Toh, B.W. Ng, A.L. Ahmad, D.C.J. Chieh, J. Lim, Magnetophoretic separation
inorganic fine particles by capillary electrophoresis with an indifferent elec- of Chlorella sp.: role of cationic polymer binder, Process Saf. Environ. Protect.
trolyte system, J. Chromatogr. A 732 (1996) 377e384. 92 (2014) 515e521.
[37] S.L. Petersen, N.E. Ballou, Separation of micrometer-size oxide particles by [48] J. Lim, S.P. Yeap, C.H. Leow, P.Y. Toh, S.C. Low, Magnetophoresis of iron oxide
capillary zone electrophoresis, J. Chromatogr. A 834 (1999) 445e452. nanoparticles at low field gradient: the role of shape anisotropy, J. Colloid
[38] N.G. Vanifatova, B.Y. Spivakov, J. Mattusch, U. Franck, R. Wennrich, Investi- Interface Sci. 421 (2014) 170e177.
gation of iron oxide nanoparticles by capillary zone electrophoresis, Talanta [49] P. Zhang, S. Park, S.H. Kang, Size-dependent magnetophoresis of native single
66 (2005) 605e610. super-paramagnetic nanoparticles in a microchip, Chem. Commun. 49 (2013)
[39] M.N. Alves, P.N. Nesterenko, B. Paull, P.R. Haddad, M. Macka, Separation of 7298e7300.
superparamagnetic magnetite nanoparticles by capillary zone electrophoresis [50] R. Zhou, C. Wang, Microfluidic separation of magnetic particles with soft
using non-complexing and complexing electrolyte anions and tetramethy- magnetic microstructures, Microfluid. Nanofluidics 20 (2016) 48.
lammonium as dispersing additive, Electrophoresis 39 (2018) 1429e1436. [51] V. Kumar, P. Rezai, Multiplex Inertio-Magnetic Fractionation (MIMF) of
[40] F. Me
rida, A. Chiu-Lam, A.C. Boho
rquez, L. Maldonado-Camargo, M.-E. Pe
rez, magnetic and non-magnetic microparticles in a microfluidic device, Micro-
L. Pericchi, M. Torres-Lugo, C. Rinaldi, Optimization of synthesis and pepti- fluid. Nanofluidics 21 (2017) 83.
zation steps to obtain iron oxide nanoparticles with high energy dissipation [52] S. Dutz, M. Hayden, U. Ha €feli, Fractionation of magnetic microspheres in a
rates, J. Magn. Magn. Mater. 394 (2015) 361e371. microfluidic spiral: interplay between magnetic and hydrodynamic forces,
[41] D. Baron, P. Dolansk
a, Z. Medríkov
a, R. Zboril, J. Petr, On-line stacking of PLoS One 12 (2017), e0169919.
carboxylated magnetite coreeshell nanoparticles in capillary electrophoresis, [53] V. Cardoso, D. Miranda, G. Botelho, G. Minas, S. Lanceros-Me
ndez, Highly
J. Sep. Sci. 40 (2017) 2482e2487. effective clean-up of magnetic nanoparticles using microfluidic technology,
[42] M. Bostro €m, V. Deniz, G. Franks, B. Ninham, Extended DLVO theory: electro- Sensor. Actuator. B Chem. 255 (2018) 2384e2391.
static and non-electrostatic forces in oxide suspensions, Adv. Colloid Interface [54] R. Zhou, F. Bai, C. Wang, Magnetic separation of microparticles by shape, Lab
Sci. 123 (2006) 5e15. Chip 17 (2017) 401e406.
[43] M. Zborowski, J.J. Chalmers, Magnetophoresis: Fundamentals and Applications,
Wiley Encyclopedia of Electrical and Electronics Engineering, 1999, pp. 1e23.