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Cite this: Chem. Soc. Rev., 2012, 41, 2912–2942
www.rsc.org/csr CRITICAL REVIEW

Magnetic nanoparticles for the manipulation of proteins and cellsw
Yue Pan, Xuewen Du, Fan Zhao and Bing Xu*
Received 20th November 2011

DOI: 10.1039/c2cs15315g
In the rapidly developing areas of nanobiotechnology, magnetic nanoparticles (MNPs) are one
type of the most well-established nanomaterials because of their biocompatibility and the
potential applications as alternative contrast enhancing agents for magnetic resonance imaging
(MRI). While the development of MNPs as alternative contrast agents for MRI application has
moved quickly to testing in animal models and clinical trials, other applications of biofunctional
MNPs have been explored extensively at the stage of qualitative or conceptual demonstration. In
this critical review, we summarize the development of two straightforward applications of
biofunctional MNPs—manipulating proteins and manipulating cells—in the last five years or so
and hope to provide a relatively comprehensive assessment that may help the future
developments. Specifically, we start with the examination of the strategy for the surface
functionalization of MNPs because the applications of MNPs essentially depend on the molecular
interactions between the functional molecules on the MNPs and the intended biological targets.
Then, we discuss the use of MNPs for manipulating proteins since protein interactions are critical
for biological functions. Afterwards, we evaluate the development of the use of MNPs to
manipulate cells because the response of MNPs to a magnetic field offers a unique way to
modulate cellular behavior in a non-contact or ‘‘remote’’ mode (i.e. the magnet exerts force on
the cells without direct contact). Finally, we provide a perspective on the future directions and
challenges in the development of MNPs for these two applications. By reviewing the examples of
the design and applications of biofunctional MNPs, we hope that this article will provide a
reference point for the future development of MNPs that address the present challenges and lead
to new opportunities in nanomedicine and nanobiotechnology (137 references).
1. Introduction
Nanobiotechnology, the integration of nanotechnology with
Department of Chemistry, Brandeis University, 415 South St,
molecular biology and medicine, is an emerging research area
Waltham, MA 02454, USA. E-mail: bxu@brandeis.edu;
Fax: +1 781-736-5201; Tel: +1 781-736-2516 that has experienced rapid growth over the past decade because
w Part of the nanomedicine themed issue. it offers exciting opportunities for discovering new science,
Yue Pan received his BS Xuewen Du obtained his BS

degree in Chemistry in 2003, degree in the Department for
followed by a MS degree Intensive Instruction (DII) of
under the supervision of Prof. Kuang Yaming Honors School
Quanxin Li from University of from Nanjing University in
Science and Technology of 2010. He is currently in his
China (USTC) in 2006. He second year as a graduate
is currently a graduate student student in Chemistry with
in Professor Bing Xu’s group Professor Bing Xu at Brandeis
at Brandeis University. His University. His current work
current work focuses on the focuses on nanomaterials.
functionalized magnetic nano-
particles as a novel kind of
biomaterials for proteins and
Yue Pan cells manipulation. Xuewen Du
2912 Chem. Soc. Rev., 2012, 41, 2912–2942 This journal is c The Royal Society of Chemistry 2012
producing novel materials, and developing efficient processes.
Particularly, the advances in the synthesis, characterization,
and applications of nanoscale materials allow scientists to
explore the interactions between nanomaterials (e.g. nano-
particles, nanowires, nanofibers, and nanotubes) and biological
entities (e.g. nucleic acid, proteins, bacteria, and mammalian cells)
at molecular or cellular levels for developing new capabilities to
address important problems. Among the variety of promising
nanoparticles developed so far, magnetic nanoparticles (MNPs),
a class of nanoparticles that exhibit superparamagnetism and
respond to a magnetic field, offer many exciting opportunities

in biology and biomedicine because of several unique features
of MNPs. First, the sizes of MNPs, ranging from a few up to
tens of nanometres, make it easy to optimize their sizes and
properties to match the interest of study or the purpose of
Fig. 1 (A) A MNP can enter the cell while a microparticle can’t (or is
application. Second, an external magnetic force is able to

difficult to do so). (B) Magnetic field induced movement of proteins or
manipulate the MNPs easily. This ‘‘action at a distance’’ or
cells.
‘‘remote control’’ provides considerable advantages for many
applications. Third, the magnetic moment of a MNP enhances
the signal of nearby protons, which allows MNPs to play an the functionalized MNPs to the targets if all the other factors
important role as MRI contrast agents. These attractive features are the same. Because of their nanometre sizes, MNPs move
of MNPs have also stimulated the development of new techniques much faster than microparticles at the same temperature.
and procedures for producing monodispersed and size-controlled Thus, the use of MNPs for applications related to cells is
MNPs (e.g. FePt, Fe3O4, Fe2O3, and CoFe2O4), albeit that the more advantageous than the use of micron sized magnetic
seminal work on the synthesis of MNPs (e.g. monodispersed beads that already have been used for separating proteins or
FePt nanoparticles1) initially aimed to provide materials for cells.3,4
the high density memory devices.2 In this critical review, we summarize the development of two
Besides responding to non-contact manipulation (e.g. the rather straightforward applications of MNPs—the uses of
change of magnetic field or the application of magnetic forces), biofunctional MNPs to manipulate proteins and cells
another important feature of MNPs is that their sizes, as small (Fig. 1B). We arrange the content of this review in the
as a few nanometres, are comparable to biologically important following way: first, we examine the strategy for the surface
objects (e.g. proteins). Compared to micrometre sized magnetic functionalization of MNPs because the stabilization of MNPs
particles, this seemingly small difference, in fact, renders significant depends on surface chemistry, and the applications of MNPs
advantages to MNPs. As shown in Fig. 1A, unlike microparticles, must rely on the molecular interactions between the functional
MNPs offer an unparalleled opportunity to manipulate subcellular molecules on the MNPs and the intended biological targets
organelles or other cellular targets inside the cells since they have outside or inside cells. Second, we discuss the use of MNPs for
nanometre sizes and are able to undergo endocytosis to enter cells. manipulating proteins since protein interactions determine or
Moreover, MNPs have much higher surface to volume ratios than closely associate with biological functions. Third, we evaluate
those of microparticles, which should increase the affinity of the use of MNPs to manipulate cells because the response of
Fan Zhao obtained his BS in Bing Xu received his BS and

Biomedical Engineering from MS from Nanjing University
Sichuan University in 2007, with in 1987 and 1990. He obtained
an emphasis on biomaterials. his PhD in 1996 from the
He is currently in his fourth University of Pennsylvania
year as a graduate student in under the supervision of
Chemistry with Professor Bing Timothy Swager. Before
Xu at Brandeis University, with starting his independent
an additional specialization in research at the Hong Kong
Quantitative Biology (HHMI- University of Science and
NIBIB Interfaces Initiative). Technology on Aug. 2000, he
His current work focuses on was an NIH postdoctoral
molecular nanomaterials for fellow with George Whitesides
immunomodulation. at Harvard University. He
Fan Zhao Bing Xu currently is a professor in the
Department of Chemistry,
Brandeis University, and his research focuses on the applications
of supramolecular chemistry to materials science, nanoscience,
and biological science.
This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 2912–2942 2913
MNPs to a magnetic field offers a unique way to modulate Table 1 Surface chemistry of common magnetic nanoparticles
cellular behavior in a non-contact mode. Fourth, we provide a
perspective on the future directions and challenges in the
development of MNPs for these two applications. By reviewing
the examples of the design and applications of biofunctional
MNPs, we expect to stimulate the future development of
biofunctional MNPs that ultimately contribute to address the
current challenges and lead to new opportunities in nanomedicine
and nanobiotechnology.
Because there are several excellent reviews on the historical
perspective of MNPs5 and the application of MNPs as MRI

contrast agents, we will not discuss MNPs applications in
MRI in this review. Readers interested in MRI applications of
MNPs may consult more specific reviews on that subject.6
2. Surface functionalization of
magnetic nanoparticles
Surface functionalization of MNPs is a necessary step for the
applications of MNPs for three reasons: first, being nanometre
sizes, the high surface to volume ratios of MNPs render them
to be highly active, which requires surface functionalization to
lower their surface energy for maintaining the proper chemical
stability. Second, the advantages of MNPs only become
available when they disperse well in solution, at least before
interacting with the targets. Thus, surface functionalization is an
effective and necessary strategy to prevent the aggregation of the to the surface of iron oxide nanoparticles (e.g. Fe3O4, Fe2O3,
MNPs. Third and most importantly, MNPs must bear a specific and CoFe2O4) turns out to be quite suitable for biological
ligand or ligands for most of the applications, including manip- applications due to the biocompatibility of dopamine and the
ulation of proteins or cells. Therefore, surface functionalization robust binding of the catechol unit (of the dopamine) to iron
of MNPs achieves two goals—‘‘soluble’’ and ‘‘target specific’’— oxides. In other words, the improved orbital overlap of the
that are essential for the biological applications of MNPs. five-membered ring on the iron oxide10 offers tight interaction
Of course, the surface functionalization of a particular between dopamine and iron oxide. After the first report by Xu
MNP depends on the type of MNPs, the intended applications, et al. on the use of a dopamine (DA) derivative as the surface
and the ability of molecules, proteins, or other targeting agents anchor for iron oxide nanoparticles,10 Sun et al. used the
to solubilize the MNPs in water. Conversely, biological applica- dopamine-derivatized ligands to functionalize the surface of
tions of MNPs demand the MNPs to have a stable surface that CoFe2O4 MNPs.11 Recently, Reimhult et al. reported that the
allows the anchor of a wide range of ligand molecules and nitration of the catechol of dopamine further enhanced the
exhibits good solvent compatibility. Generally, the freshly made stability of the dopamine-based anchors on the iron oxide
MNPs already have surface modifiers on them. Depending on nanoparticles, which provided more options for the surface
the specific methods used for making the MNPs, these surface modification of iron oxide MNPs for applications under
modifiers likely are trioctylphosphine (TOP),7 oleic acids (OA), physiologic conditions.12 In these reports, the primary amine of
or oleylamine (OM).1,8 The formation of metal carboxylate, dopamine serves as the point to connect with other functional
metal–amine, or metal–phosphine bonds at their surfaces allows groups via the formation of the amide bond. Although the
the MNPs to be surrounded with a layer of hydrocarbon, thus connection of the dopamine to the functional group prior to
making them hydrophobic and soluble in non-polar or weakly attaching the MNPs is required, this simple derivatization effec-
polar organic solvents. To make these MNPs hydrophilic for tively stabilizes the catechol unit of the dopamine, thus increasing
biological applications, the functionalization of their surfaces the long-term stability of dopamine-based anchors and rendering
should make the MNPs to be water soluble/dispersible and dopamine as a versatile anchor to attach a wide range of
stable at various pH values, ranging from 5–9, and at physio- molecules onto iron oxide MNPs for biomedical applications.
logical temperatures. Therefore, the first step of the surface Because gold nanoparticles exhibit excellent biocompatibility,
modification of MNPs for biological applications is to exchange chemical stability, and easiness for functionalization,9 several
the surface ligands and to increase the dispersity of MNPs groups have explored the use of gold as the coating or a part of
in water. MNPs.16 For example, Sun and co-workers first reported the
Table 1 shows the common MNPs and the surface anchors heterodimer of Au–Fe3O4,52 in which the Au and Fe3O4 are
used for the modification of their surfaces based on the functionalized by poly(ethyleneglycol) (PEG) attached mole-
development of self-assembled monolayers (SAMs).9 For cules, such as HS–PEG–NH2 and DA–PEG, respectively.53
example, among the methods used to modify the surface of Recently, Li et al. synthesized bifunctional Au–Fe3O4 nano-
iron oxide nanoparticles, the coordination of dopamine ligands particles by simply linking two separately-prepared nanomaterials
via chemical bonds. They first modified the surfaces of Fe3O4 using an amphiphilic polymer for transferring nanocrystals
nanoparticles with cysteine molecules by the formation of amide from organic to aqueous solutions. This simple strategy is
bonds between the surface amino groups of Fe3O4 and the suitable for a wide range of nanocrystals, including MNPs.55
carboxylic groups of cysteine. Then, by reacting these thiol- Using a similar approach, Pellegrino et al. produced multi-
modified Fe3O4 nanoparticles with Au nanoparticles, they functional nanobeads by adding a destabilizing solvent to a
obtained the Au–Fe3O4 particles.15 In this approach, the mixture of MNPs, quantum dots, and amphiphilic polymers.
average sizes of the Au particles are about 10 nm. This method Although the sizes of the nanobeads unavoidably are poly-
appears quite easy to be carried out, and it may find applications disperse, it is one of the simplest ways to obtain multifunctional
when the requirement for the reproducibility of the surface nanobeads that exhibit both magnetism and fluorescence.45
composition is less stringent. Using atom transfer radical polymerization, Vestal and Zhang
A slightly different way to put Au on iron oxide nanoparticles pioneered to coat the MNPs (MnFe2O4) with polystyrene,
is to use iron oxide as the core and Au as the shell. Fang et al. though this strategy also suffers from the problem of poly-
first prepared g-Fe2O3@Au core–shell nanoparticles through a dispersity in final particle sizes.56 Recently, Zhang et al. used
route that combines high temperature thermolysis and colloidal biocompatible polymers (e.g. poly(galacturonic acid)) to coat
microemulsion.54 Later, Zhong et al. produced gold-coated iron CoFe2O4 nanoparticles by sonicating the mixture in NaOH
solution for 5 h,44 which generated nanoparticles with negative
oxide nanoparticles by using Fe2O3 and Fe3O4 as the core and

Au as the shell, which is formed by the reductive deposition of surface charges for the adsorption of a oligopeptide containing
gold onto the pre-synthesized iron oxide nanoparticles.39 lysine. Also using biocompatible polymers (e.g. poly(lactide-
Following that, Hao et al. prepared Fe3O4/Au core/shell co-glycolide) (PLGA)), Chung et al. encapsulated a fluores-
nanoparticles that use poly(ethyleneimine) (PEI) as the linker. cence dye, disulfonated indocyanine green (ICG), and iron
First, PEI coated Fe3O4 NPs and citrate-coated gold nano- oxide nanoparticles in the polymer matrix of PLGA.41 In
particles (Au NPs) formed in aqueous phase. Second, Fe3O4 order to have sufficient ICG in the PLGA matrix, they used
nanoparticles immobilized a number of Au nanoparticles on the human serum albumin (HSA) to bind with ICG molecules in a
surface by electrostatic interaction, denoted as Fe3O4–Au seeds. non-covalent manner.
Third, Au shell-coated Fe3O4 nanoparticles, denoted as Other biocompatible polymers also are explored for coating
Fe3O4–Au coats, were produced by forming the Au shell via MNPs. Since chitosan (another kind of biocompatible polymer)
the reduction of HAuCl4 on the Au nanoparticles attached to the has characteristics of biocompatibility, biodegradability, and
Fe3O4 surface as the crystal seeds.16 While Au provides a highly low-toxicity, Minami et al. coated MNPs with chitosan by
stable coating and allows thiol-based molecules to be easily adding a sodium hydrogen carbonate solution dropwise to a
attached, the synthetic process for coating Au on iron oxide suspended mixture of MNPs and chitosan solution in dilute
remains to be improved. It would be a significant improvement if acetic acid.49 Recently, Bao et al. reported selective surface
a simpler process and better controlled coating were developed. functionality of iron oxide nanoparticles with high stability.57
Another more explored surface coating is silica (SiO2), likely While this type of mix and match methods is quite easy to be
due to the stability of SiO2 in the neutral, aqueous environ- carried out, the resulting nanoparticles tend to aggregate, thus
ment. The coating process is relatively simple and widely-used increasing polydispersity that usually diminishes the advantages
because silanes have the ability to bind to ferrite nanoparticles of nanoparticles. Another limitation of this type of approach is
(MFe2O4, M = Fe, Co).18,20 Several groups prepared the the lack of strong bonding between the polymers and the
Fe3O4 nanoparticles coated with a silica by the sol–gel process MNPs. To address this problem, Tremel et al. functionalized
that used tetraethyl orthosilicate (TEOS)17–20 as the precursor. the Fe2O3 nanoparticles using a multidentate functional copolymer
For example, Lee et al. coated the Fe3O4 nanoparticles with silica carrying catechol groups as surface binding ligands for the iron
shells by the reverse microemulsion method using a surfactant and oxide nanoparticles and free amino groups for the attachment of
TEOS as the silica precursor.23 Bentley et al. synthesized amino- the poly(IC) ligands.42 This approach offers excellent stability and
silane modified MNPs by soaking the Fe3O4 nanoparticles with surface chemistry for the polymer-coated MNPs.
N-b-aminoethyl-g-aminopropyltrimethoxysilane (AEAPT).25 The Besides the well-known Au–S bonds, the Pt–S and Fe–S
advantage of this approach is biocompatibility and stability under bonds are relatively strong and stable, which makes thiols as a
aqueous conditions, easy surface modification, and easy control of suitable anchor for the functionalization of the FePt MNPs.
interparticle interactions through variation of the shell thickness, For example, Xu et al. used thiol-modified vancomycin47 or
but the surface chemistry of silica is less defined than that of gold. nitrilotriacetic acid (NTA) to functionalize FePt MNP by
Sometimes the silica layer is too thick,43 which may reduce the replacing the hydrophobic surface monolayer on FePt with
ability of the MNPs to respond to the magnetic manipulation. hydrophilic vancomycin or NTA molecules.48 Although it
Encouraged by the successful use of dextran to coat iron requires a noble metal, this direct anchoring by thiol derivatives
oxide nanoparticles,30 MNPs functionalized by polymers are eliminates the use of polymers, thus improving reproducibility
receiving increased attention because of the versatility of and expanding the scope of the functional molecules.
polymeric coatings. The control of the composition of the Other small molecules also served as the anchors for MNPs.
polymers allows researchers to tailor the repulsive forces For example, Hyeon et al. replaced the hydrophobic surfactants
between the nanoparticles, thus developing a reliable approach with imidazoles to stabilize the Ni/NiO nanoparticles in water.3
to balance the magnetic and the van der Waals attractive Lee et al. immobilized Ni(II) on the iron oxide nanoparticle
forces acting on the MNPs. For example, Parak et al. have surface through a redox process: they formed tiny Ni(0)
reported an elegant strategy to wrap hydrophobic nanocrystals nanoparticles or deposited Ni(0) species on the surface of iron
oxide through the reduction of NaBH4, then they oxidized the
nickel into NiO or Ni(II) species in an aqueous environment.40
Of course, many other molecules have served as the candidates
of anchors or coating for MNPs.50,51,58 For the applications
discussed in this review, the most commonly used surface
chemistry is listed in Table 1.
Fig. 2 Histidine-tagged protein purification using biofunctional FePt

3. Magnetic nanoparticles for protein MNPs: (A) NTA-terminated MNPs selectively binding to histidine-
manipulation tagged proteins; (B) SDS/PAGE analysis of the fraction of proteins
washed off the MNPs by imidazole solution at 10 (lane 3), 80 (lane 4),

It is important to purify and manipulate recombinant proteins
and 500 mM (lane 5) and the fractions washed off the reused
with different tags (histidine-tagged, GST, eDHFR, etc.) in life nanoparticles using imidazole solution at 10 (lane 6), 20 (lane 7),
sciences, which has led to the development of the various and 500 mM (lane 8). Adapted with permission from ref. 48.
protocols for protein manipulation. Among the existing protocols, Copyright 2004 American Chemical Society.
magnetic separation and purification is a convenient method
for selective and reliable capture of specific proteins. Like
iron oxide based on the tight bonds between dopamine and iron
magnetic microbeads, MNPs are able to serve as an effective
oxide due to octahedral geometry of the oxygen-coordinated
tool to separate tagged proteins with high selectivity. Further-
iron. The resulting product reacts with NiCl2 to give
more, MNPs perform better than micron-sized resins or beads
MNP–DA–NTA–Ni2+, which not only exhibits high specificity
used in metal-chelate affinity chromatography (MCAC) because
for separating histidine-tagged proteins, but also shows exceptional
their high surface-to-volume ratio, fast movement, and good
stability to heat and high salt concentration. The specificity for
dispersity result in high binding rate, large protein binding
the histidine-tagged proteins is much higher than the microbead-
capacity, and simplified analytical procedures. Generally speaking,
based commercial HiTrap affinity column. This work demon-
there are two different ways to conjugate MNPs for protein
strates a useful way to link other biofunctional molecules to iron
manipulation. One is to conjugate MNPs with small ligands, and
oxide surfaces through dopamine and offers new opportunities
the other is to conjugate MNPs with antibodies. The following
for the biological applications of MNPs.10
sections describe both of them, in addition to discussing MNPs
The specificity of the MNPs exhibited in protein separation
for binding post-translational modified proteins and mentioning
suggests that MNPs, as a general and versatile system, should
a few other examples.
selectively bind with other biological targets at low concentra-
3.1 The conjugates of MNPs and small ligands for separating tions if proper anchors and ligands are used. For example, Hyeon
recombinant proteins and co-workers synthesized Ni/NiO MNPs and conjugated
imidazole to the surface of the nickel-based nanoparticles,
The use of magnetic force to separate proteins from a mixture,
which have high affinity to polyhistidine and also selectively
such as cell lysates, is a well-established procedure for the
bind to the histidine-tagged proteins (Fig. 4A).3 According to
purification of recombinant proteins.59 Since it is rather common
the authors, this approach provided a more convenient
for many research groups to use commercially available magnetic
method for efficiently purifying the histidine-tagged proteins,
microbeads for the purification of histidine-tagged proteins, it is
as compared to the other methods that need several steps to
not surprising that the early success of the manipulation of
synthesize and conjugate NTA derivatives on the MNPs. Also
proteins by MNPs is to use nickel–nitrilotriacetic acid (Ni–NTA)
using the NiO MNPs, Hyeon and co-workers designed and
modified MNPs for separating histidine-tagged proteins from cell
lysate. For example, Xu and co-workers, based on the seminal
synthesis of monodispersed FePt MNPs reported by Sun et al.,1
used a simple mercaptoalkanoic acid as the anchor to decorate
FePt MNPs with the Ni–NTA complex via the Fe–S/Pt–S
bond.48 As shown in Fig. 2, the resulting biofunctional MNPs
separate the over-expressed 6His-GFP protein from the lysate
of E. coli with exceptionally high selectivity and capacity. That
work, besides confirming the merits of MNPs, such as their high
surface-to-volume ratio, fast movement, high capacity, good
dispersity in water, and the elimination of excessive washing,
suggests that the target proteins cover the surface of the MNPs
effectively and quickly, and reduce the overall unoccupied surface
Fig. 3 (A) Structure of MNP–DA–NTA–Ni2+ and (B) SDS/PAGE
area that might be the origin of nonspecific absorption of
analysis of the purity of the proteins: the cell lysate (lane 1), the molecular
proteins, thus achieving much higher specificity than microbeads.
weight marker (lane 8), the fractions washed from the fresh-made
Using iron-oxide coated cobalt nanoparticles, Xu and MNP–DA–NTA–Ni2+ (lanes 2 and 3), the boiled MNP–DA–NTA–Ni2+
co-workers also confirmed that the high specificity conferred (lanes 4 and 5), and the commercial HiTrap affinity column (lanes 6 and 9).
by the nanoparticles for the separation of histidine-tagged The concentrations of imidazole are 10 mM (lanes 2, 4, and 6) and
proteins is a general feature of MNPs. As shown in Fig. 3, they 500 mM (lanes 3, 5, 9). Adapted with permission from ref. 10.
linked dopamine (DA) to NTA for anchoring DA–NTA onto Copyright 2004 American Chemical Society.
Fig. 5 Illustration of the surface modified silica-coated MNPs as an

agent to bind histidine-tagged proteins. The terpyridine receptor was
immobilized into the silica-coated MNPs by sol–gel reaction following
vortex-mixed in aqueous copper chloride solution to get Cu(II)-loaded
Fig. 4 (A) Imidazole-immobilized Ni/NiO for purification of histidine- terpyridine-immobilized silica-coated MNPs. Adapted with permission
tagged proteins from a solution of mixtures. (B) The schematic presenta- from ref. 17. Copyright 2010 Royal Society of Chemistry.
tion of a magnetically recyclable protein separation system using NiO
MNPs. Adapted with permission from ref. 3 and 43. Copyright 2006,
Despite that these spheres have relatively large diameters (220 nm),
American Chemical Society, and Copyright 2010 Wiley-VCH.
the polymer coating renders these spheres with excellent magnetic
response and water dispersity. Because of the poly(styrene-alt-
developed a recyclable protein separation system by combining maleic anhydride) in the shell, NTA can be attached to the
ferrimagnetic magnetite cores and NiO nanoparticles decorated polymer coating. This approach appears to be an easy way to
onto the mesoporous silica shell with a high surface area incorporate more Ni–NTA affinity sites onto the surface of the
(Fig. 4B).43 The exposed NiO nanoparticles selectively bind to magnetic spheres than the direct reaction of NTA to amine
the histidine-tagged protein from the mixed protein solution, as terminated Fe3O4@SiO2 MNPs. According to the authors,
well as the cell lysate, and the magnetic core allows the particles this approach also should provide the multivalent effect so that
to be separated from the solution by applying an external the capacity of the separation of histidine-tagged proteins by
magnetic field. Being used repeatedly, this system would be the as-prepared Fe3O4@SiO2@polymer/Ni–NTA composites
very useful when the cost of the MNPs is a concern.43 Since was four times as that by Fe3O4@SiO2/Ni–NTA.18
both methods are based on NiO, they might introduce nickel As shown in Fig. 6B, taking advantage of silica-coated
ions to the systems, which would be a concern if the targeted MNPs, Bruening and co-workers grew poly(2-hydroxyethyl
proteins are intolerant to transition metal ions. methacrylate) brushes on the SiO2 shell and subsequently used
Based on the complexes between copper ions and terpyridine, Ni–NTA to functionalize the polymer brushes. They obtained
Bae and co-workers anchored the terpyridine via silicon oxygen the MNPs that had diameters of about 100 nm and selectively
bonds on the surface of silica-coated MNPs for the separation of bound histidine-tagged proteins directly from cell extracts. These
histidine-tagged proteins (Fig. 5). Although the silica coating, in polymer brush MNPs exhibit significantly increased binding
this case, causes the aggregation of the MNPs, the authors capacities for proteins (220 mg BSA per gram of beads and
separated the histidine-tagged proteins with excellent selectivity. 245 mg histidine-tagged ubiquitin per gram of beads), which
According to the authors, compared with the current multistep are an order of magnitude greater than those of commercial
methods, this simple synthetic strategy should provide a useful magnetic beads. According to the authors, these brushes allow
method of selective detection of histidine-tagged proteins.17 As the adsorption of multilayers of proteins, thus may greatly
shown in Fig. 5, ethylenediaminetetraacetic acid (EDTA) can improve the separation of recombinant proteins.20 It would be
competitively bind copper ions from the terpyridine copper com- more significant if the capacity for other tagged proteins can
plexes, which put some restraint on the cell lysates (e.g. the lysates be increased substantially.
should be free of EDTA). In addition, the cost of terpyridine By introducing Au nanoparticles onto magnetite MNPs via
might become an issue when large scale separation is needed. chemical bonds, Li and coworkers reported the synthesis of bifunc-
Also using silica-coated iron oxide MNPs and by precipita- tional Au–Fe3O4 nanoparticles that allowed functional molecules
tion polymerization, Zheng and co-workers prepared magnetic to be easily anchored for protein separation and detection.
core–shell spheres Fe3O4@SiO2@poly(styrene-alt-maleic Through Au–S interaction, they modified these as-prepared
anhydride) enriched with Ni–NTA on their surface (Fig. 6A). Au–Fe3O4 nanoparticles by NTA molecules and used them to
It appears quite reliable for the use of MNPs to separate and
manipulate histidine-tagged proteins, and many variations
have been achieved by different groups.40,60,61 For example,
Lee and co-workers used NaBH4 to reduce Ni(NO3)2 directly
on the surface of pluronic copolymer coated magnetic nano-
particles and demonstrated that these Ni-MNPs bound to
histidine-tagged proteins and were able to separate the histidine-
tagged proteins from a multi-component solution by applying
magnetic field.40 Similarly, Lee et al. used nickel ferrite
(NiFe2O4) nanoparticle clusters (100 nm in diameters) for
the separation of histidine-tagged proteins and demonstrated

that these clusters were reusable (up to seven cycles).61 Chen
and coworkers not only demonstrated that Ni–NTA modified
Fe3O4 MNPs were able to trap histidine-tagged streptopain
from cell lysates, but also showed that either Zr(IV) or Ga(III)
ions could replace Ni(II) for selectively enriching phosphorylated

Fig. 6 (A) Scheme of Fe3O4@SiO2@polymer/Ni–NTA to separate peptides from tryptic digests of a-, b-caseins, and diluted milk.
histidine-tagged proteins. (B) The scheme of brush-modified beads and Such enrichment allows the detection limit for the tryptic digests
SDS/PAGE analysis of cell lysate containing overexpressed histidine- of a- and b-caseins to be as low as B50 fmol. This approach
tagged protein before and after purification using MNPs. Adapted is particularly attractive because it, in combination with
with permission from ref. 18, Copyright 2010 Royal Society of MALDI-MS, is able to detect multiple protein biomarkers.60
Chemistry and ref. 20, Copyright 2011 American Chemical Society. These excellent works further confirm that MNPs, as a general
and versatile system, perform better than microbeads for
separate proteins simply with the assistance of a magnet. Their manipulating histidine-tagged proteins.
result shows that these bifunctional materials separate protein Although protein purification based on 6His-tag has been
with relatively high efficiency.15 Although this approach might widely carried out using microbeads and intensively explored
have limited advantages for the separation of proteins, it is a using MNPs, the long term cytotoxicity of the metal–NTA
versatile strategy that may lead to the preparation of diverse complexes62 in this technique requires caution for in vivo
bifunctional and even multifunctional nanomaterials. applications. While glutathione (GSH) based magnetic micro-
Similar to the approach reported by Li et al.,15 Xie and beads have been commercialized for quite a number of years,
coworkers also prepared Fe3O4/Au core/shell MNPs (Fig. 7).16 there is no report on GSH modified MNPs until recently Xu
Based on the electrostatic attraction between PEI-functionalized and co-workers demonstrated a general strategy for the
Fe3O4 nanoparticles and the citrate-modified Au nanoparticles, Xie separation of GST fusion proteins from a cell lysate (Fig. 8)
et al. obtained well-defined Fe3O4/Au core/shell nanoparticles. by MNPs. The well-established ligand–target interaction
Despite that the TEM revealed aggregation among the MNPs, between GSH and GST ensures that MNP–GSH selectively
the authors reported good dispersity and strong magnetism of these binds targeted fusion proteins (e.g. GST–GFP), which forms
core/shell nanoparticles. After using 3-mercaptopropionic acid to the conjugate of MNP–GSH/GST–GFP. The use of magnetic
prepare the gold surface to be coated with carboxylic acid groups, separation yields the pure fusion protein upon elution, or
they also used NTA to react with the acid and subsequently affords GFP upon enzymatic cleavage of the TEV linker
generated Fe3O4/Au–NTA–Ni2+ nanoparticles for the separation between the two proteins.14 This work further expanded the
of histidine-tagged maltose-binding protein (MBP) directly from scope of MNPs for manipulating proteins from cell lysate to live
the mixture of lysed cells. According to their results, the enrichment cells because of the cell-compatibility of the MNP–GSH.13,14
efficiency of the MNPs is much higher than that of metal-chelate While the use of GSH as the ligand satisfies the requirement
affinity chromatography (MCAC). It would be more exciting if of biocompatibility, endogenously expressed GST may cause
that work also verified that the MBPs were still able to bind unwanted binding. To have more options for selectively bind-
maltose. ing to targeted proteins, Xu et al. also synthesized the first
example of trimethoprim (TMP) decorated MNPs (Fig. 8B) for
manipulating fusion proteins containing E. coli dihydrofolate
reductase (eDHFR).13 Based on the high affinity of TMP to
eDHFR and the orthogonality of the TMP/eDHFR ligand–
receptor pair to mammalian cells,63 this approach promises a
versatile method for validating the functions of proteins as well
as a platform for the delivery of protein drugs.
3.2 The conjugate of MNPs and antibodies for manipulating

Fig. 7 Illustration of the biofunctionalized magnetic Fe3O4/Au– proteins
NTA–Ni2+ nanoparticles and their enrichment and separation of
the proteins. Adapted with permission from ref. 16. Copyright 2010 Antibodies, as another type of biomolecules to be anchored
American Chemical Society. onto MNPs, allow the use of MNPs for immunoassay and
Fig. 9 The experimental procedure of the sandwich immunoassay

that uses the automated immunoassay system and antibody–protein
A–BacMP complexes. Adapted with permission from ref. 64. Copyright
2000 American Chemical Society.
Fig. 8 The structures of (A) GSH decorated iron oxide MNPs (1)
and (B) TMP decorated iron oxide MNPs (2). (C) The decorated in dispersion as the origin of the different intensities, they
MNPs selectively bind to target protein from a cell lysate and their suggested that the antibody–protein A–BacMP complexes
down-stream applications. (D) SDS/PAGE analysis of the binding of 1 promise a fully automated sandwich immunoassay system
to the GST in an E. coli lysate: lane 1, elution by GSH; lane 2, 1st for precise detection of human insulin in serum.
wash; lane 3, lysate after the removal of 1; lane 4, cell lysate; lane 5, Also based on an ingenious use of MNPs, Kriz and
molecular weight marker, (E) binding of 2 to the eDHFR-HA-GFP in co-workers have demonstrated a portable assay for detecting
a mammalian cell lysate: lane 1, cell lysate; lane 2, elution by 2sds the concentration of C reactive protein (CRP).29 As shown in
loading buffer at 60 1C for 4 h; lane 3, molecular weight marker. Fig. 10, they developed a two-site heterogeneous immunoassay
Adapted with permission from ref. 13 and 14. Copyright 2011 American
that employed two types of nanoparticles: silica particles coated
Chemical Society and copyright 2011 Royal Society of Chemistry.
with polyclonal anti-CRP antibodies and iron oxide MNPs
coated with monoclonal anti-CRP antibodies. The presence of
other applications that require high affinities. In 2000, Matsunaga CRP causes the MNPs to co-precipitate with silica particles,
et al. reported the use of antibody modified bacterial magnetic and the measurement of the magnetic permeability of the
particle (BacMP) for a fully automated immunoassay to sediments reveals the amounts of CRP. As a result, the two-site
determine human insulin. Based on the observation that the heterogeneous immunoassay offers a rapid (11.5 min) procedure
BacMPs have good dispersion and that BacMPs without a with a low detection limit (0.2 mg L1) and high accuracy. Based
membrane form large aggregates, they cloned a fusion gene on the principle illustrated in the work of Kriz et al., Ouyang
(proteinA–magA) into magnetobacteria (Magnetospirillum sp. et al. made hemoglobin-functionalized MNPs that were able to
AMB-1) and obtained the BacMPs that had protein A on the enrich human serum amyloid P component (SAP), vitamin
membrane surface of BacMP (i.e. protein A–BacMP complexes). D-binding protein, and serine peptidase inhibitor.65
By adding the anti-human insulin antibody to protein A–BacMP, Using the luminescent nanoparticles of rare earth metal
the authors obtained the antibody–protein A–BacMP complexes oxides, Nichkova and co-workers developed a simple and
as the nanoparticles with the diameters mainly at 80–120 nm, robust platform for quantitative multiple protein immuno-
which are used for automated detection of insulin, as outlined in analysis. They synthesized an interesting type of core/shell
Fig. 9. They found that the luminescence intensity ((kilocounts s1) MNPs that had Co:Nd:Fe2O3 as the core and luminescent
per mg of antibody) from antibody–protein A–BacMP complexes Eu:Gd2O3 as the shell. The magnetism of the nanoparticles
after immunoreaction was higher than that from BacMPs renders them to respond to the manipulation of an external
chemically conjugated to an antibody. Attributing the difference magnetic field in the separation and washing steps of the
Fig. 10 Detection of CRP using a magnetic permeability detector: Fig. 12 (top) Idealized illustration of the immobilization of the anti-
(A) polyclonal CRP antibody-conjugated silica microparticles (reagent body on the gold-coated MNPs and the subsequent recognition of the
vial), monoclonal CRP antibody-conjugated MNPs (reagent vial), and proteins and (bottom) utilization of this strategy for the separation of
whole blood from a patient (capillary), (B) immune complexes, proteins via application of a magnetic field. Adapted with permission
(C) sedimentation on the bottom of the reagent vial due to the large from ref. 39. Copyright 2007 American Chemical Society.
density of silica microparticles, and (D) a magnetic permeability
detector used to measure the concentration of C reactive protein. this work clearly demonstrates the versatility of the iron
Adapted with permission from ref. 29. Copyright 2005 American oxide@Au MNPs. However, the requirement of additional
Chemical Society. gold particles to present the proteins for enhanced SERS
signals remains to be simplified.
immunoassay (Fig. 11). The luminescence signal of the Combining the exceptional sensitivity of spin valves and the
Eu:Gd2O3 serves as an internal standard to quantify the selectivity of antibodies attached to MNPs, Reekmans and
fluorescence intensity of the reporter dye so that the target co-workers exploited an alternative magnetic bead sensing
proteins can be identified and quantified in a single step. The concept for the development of point-of-care diagnostics. As
authors demonstrated this elegant concept for three model shown in Fig. 13, based on the repositioning of the magnetic
proteins (human, rabbit and mouse IgGs) and envisioned that beads (300 nm in diameter) toward the most sensitive location
this method might find applications for disease diagnostics and on the spin valve sensors to allow for highly sensitive immuno-
for the detection of biological threats.46 sensing over a wide range of target concentrations, the authors
To meet the challenge on the fabrication of gold-coated optimized the magnetoimmuno assay. By tailoring the surface
MNPs, Zhong and co-workers reported a method to thermal chemistry (e.g. the use of mixed thiols), the blocking procedure
anneal gold nanoparticles onto iron oxide nanoparticles to (e.g. the use of bovine serum albumin (BSA) to block non-specific
generate gold-coated iron oxide (Fe2O3 and Fe3O4) core@shell protein adsorption), and the type of magnetic particles (e.g.
nanoparticles (Fe oxide@Au) that had controllable sizes ranging the use of Ademtech 300 nm), they achieved the highly
from 5 to 100 nm. The authors then immobilized antibodies on sensitive and specific detection of S100bb, a diagnostic marker
these gold-coated MNPs for the recognition of specific proteins for stroke and minor head injury, down to the concentration
that adhered to another gold nanoparticle.39 As shown in Fig. 12, of 27 pg mL1. This work not only illustrates the promise of
MNPs and spin valve sensors for diagnostic applications,66 but
also reveals that the major limitation of large MNPs (e.g. 300 nm
in diameter) is the nonspecific adsorption of proteins.
3.3 MNPs for binding post-translational modified proteins

Based on the affinity of phosphates to early transition metal
oxide and matrix-assisted laser desorption/ionization mass
spectrometry (MALDI-MS), Chen and co-workers developed
an elegant approach for detecting phosphopeptides in fluid
solutions (Fig. 14).27 Specifically, they synthesized titanium
oxide coated iron oxide (Fe3O4/TiO2) MNPs for concentrating
phosphopeptides from the products of protein digestion onto
the MNPs. After isolating the MNPs by using a magnetic field,
they treated these MNPs as the protein-bound matrix for
MALDI-MS. These Fe3O4/TiO2 core/shell MNPs, acting as
Fig. 11 Process of the multiplexed fluoroimmunoassay with magnetic
an effective MALDI matrix, achieve the detection of the
luminescent nanoparticles (MLNPs): (A) introduction of antibody-
modified MLNPs; (B) binding of the protein analytes on the surface of phosphopeptides with upper detectable mass limit at about
the nanomagneto-phosphors; (C) capture of the dye-labeled secondary 24 kDa and the sensitivity in the low-femtomole range. Their
(reporter) antibodies; (D) magnetic extraction; (E) fluorescence detection approach is straightforward because there is no need to
of reporters and nanomagneto-phosphors. Adapted with permission modify any chelating agents on the surface of the MNPs.
from ref. 46. Copyright 2007 Elsevier. Although the binding between the phosphates and TiO2 is less
Fig. 13 (A) Illustration of the magnetosandwich assay for the detection

of S100bb. (B) The 3 steps performed during the MBS measurement.
First (1) the MPs are specifically bound (i.e. randomly distributed) to the
sensor surface, followed by (2) the regeneration of the biomolecular
bonds and alignment of the MPs to the most sensitive part of the spin
valves. (3) Finally, all particles are washed away from the sensor surface
to perform a blank measurement. Adapted with permission from ref. 66.

Copyright 2007 American Chemical Society.
Fig. 15 Schematic representation of (A) APBA-functionalized Fe3O4
MNPs and (B) their application in the selective capture of glycoprotein
with the help of an external magnet. Adapted with permission from
ref. 19. Copyright 2011 Royal Society of Chemistry.
they modified the surface of silica-coated MNPs with various

alkyl groups at various pH levels and obtained MNPs with
hydrophobic pockets that had various sizes. The authors
demonstrated that this system was able to separate BSA with
Fig. 14 Immunoseparation of phosphopeptides using MNPs (Fe3O4/
high efficiency. They also found that the efficiency of separa-
TiO2) and TiO2-MALDI MS. Adapted with permission from ref. 27.
tion depended on the size of the hydrophobic pocket and
Copyright 2005 American Chemical Society.
several other factors, such as the alkyl chain length, the salt
concentration, and the pH levels.22 Despite that the orienta-
well-defined, this approach would become very useful for the
tion and activity of the proteins in hydrophobic pocket remain
analysis of the phosphorylation of proteins if the upper mass
to be evaluated, it would be more useful if the use of the
limit can be increased.
hydrophobic chains could mimic cell membranes for the
Besides recombinant proteins and phosphorylated proteins,
manipulation of membrane proteins.
glycoproteins are important targets for protein manipulation
Realizing the importance of the orientation of the proteins on
because of their essential functions in many biological processes,
the MNPs, Bentley et al. have developed a simple model for
especially to those related to post-translational modification.
predicting the spatial arrangement of proteins on MNPs. Using
Recently, Chen and co-workers used aminophenylboronic acid
the silanized copper-loaded ‘‘functionalized’’ MNPs to separate
(APBA) to functionalize MNPs for selectively binding glyco-
the histidine-tagged GFP from solution,25 the authors used a
proteins from protein mixtures. As shown in Fig. 15, the authors
Langmuir isotherm model to estimate the maximum adsorption
used silica-coated iron oxide MNPs. After functionalizing the
capacity of the GFP on the surfaces. Based on the net available
silica shell with amino groups, the authors used glycerol dial-
surface area of these nanoparticles, they suggested a vertical
dehyde to connect APBA to the surface of MNPs. Like other
alignment of 88 GFP molecules in a nearly closest packed
silica-coated MNPs, the resulting APBA-functionalized MNPs
configuration on these MNPs with 10 nm diameters. Although
exhibit considerable aggregation, probably due to the interaction
the coverage of GFP on the MNPs unlikely follows a perfect
among boronic acids itself. The authors used the APBA-coated
monolayer, this approach represents a useful way for estimating
MNPs to bind protein mixtures consisting of both glycoproteins
the assembly of other proteins with known shape onto MNPs.
(e.g. ovalbumin (OVA)) and non-glycoproteins (e.g. bovine
Recently, Nienhaus and co-workers used dual-focus fluorescence
hemoglobin (BHb), lysozyme (Lyz), and BSA) and found that
correlation spectroscopy to analyze the adsorption of three human
the decrease of the OVA in the mixture, which suggested the
blood serum proteins onto polymer-coated, fluorescently labeled
preferred binding between APBA-MNP and glycoproteins.
FePt MNPs (B12 nm diameter). They found that all three
Although it requires relatively large amount of MNPs, the authors
proteins gave a well-defined increase in the size of nanoparticles
suggested that the improvement and further investigation of the
upon binding, and the thickness of the protein corona was
MNPs should make this system useful for real samples.19
associated to a particular orientation of the protein (with known
structure). Although the evidence is indirect, they suggested that
3.4 Other examples
future studies using single-particle FRET should yield more
While most of the modification of MNPs generated hydro- detailed insights into the structural properties of the protein
philic surfaces, Park and co-workers demonstrated a different corona surrounding MNPs.67
kind of MNPs that relied on hydrophobic interaction for the A more important purpose of the use of MNPs is to manipulate
separation of proteins such as BSA. As illustrated in Fig. 16, the interaction between proteins and nucleic acid. Recently, Tremel
Fig. 17 The process of the isolation of (2–5)A synthetase from sponge

extract using dsRNA[poly(IC)] functionalized g-Fe2O3 MNPs. Adapted

with permission from ref. 42. Copyright 2007 Royal Society of Chemistry.
the manipulation of cells in vitro and in vivo, which are gaining

Fig. 16 (A) Scheme of hydrophobic partitioning modification on the increased attention and being demonstrated for various applica-
silica coated MNPs (Si-MNPs) with various alkyl groups as a hydro- tions, including photokilling bacteria, the capture and detection
phobic pocket and various amine groups as a receptor. TEM image of pathogens, cell sorting (separating/isolating), the capture of
shows the core–shell structured Si-MNPs with 40 nm of average particle stem cells, in vivo cell targeting and extraction, remote control of
size. In amine receptor at Si-MNPs, x = 0, 1 and 2 denotes monoamine,
cellular behavior or cell signaling, multicellular spheroids and cell
diamine, and triamine, respectively. Alkyl groups stand for n-propyl (C3),
sheet construction, 3D cell cultures, cell destruction, magnetically
n-octyl (C8), n-dodecyl (C12), and n-octadecyl (C18) groups as a function
of alkyl chain-lengths corresponding to n = 1, 6, 10, 16, respectively. controlling cell migration, and modulating cell focal adhesion. In
(B) Confocal microscopic images of various hydrophobic pocket sized the following part, we first discuss the manipulation of bacteria
Si-MNPs at pH 4.65. (The scale bar is 4 mm.) (C) Schematic representa- and yeast using MNPs, and then describe the use of magnetic
tion of BSA adsorption on various hydrophobic pockets on Si-MNPs. force to control mammalian cells.
Adapted with permission from ref. 22. Copyright 2010 Elsevier Inc.
4.1 MNPs for binding bacteria and yeast
and co-workers have made significant progress towards the direc-
tion. Based on the interaction of a mismatched double-stranded The enrichment of bacteria from clinical samples for detecting
RNA (with one strand being a polymer of inosinic acid, the other a bacteria at ultralow concentrations without time-consuming
polymer of cytidylic acid, and denoted as dsRNA[poly(inosinic procedures is an attractive application of MNPs. Since it is
acid):poly(cytidylic acid)] (poly(IC)) with 20 -50 -oligoadenylate unlikely that a single nanoparticle can generate enough
synthetase ((2–5)A synthetase), they developed polymer coated response to allow a cell to be manipulated by a magnetic field,
MNPs for binding an enzyme that was difficult to be purified. it is necessary to use multiple MNPs for cell manipulation by
As shown in Fig. 17, using a multidentate functional copolymer magnetic force. Because multiple MNPs may lead to aggrega-
that carried dopamine anchors for the surface binding and tion and cause unwanted non-specific binding, it becomes
1,3-butyldiamine ligands for the dsRNA[poly(IC)] binding, imperative to use highly specific ligand–receptor interactions
they immobilized the dsRNA[poly(IC)] onto the surface of for MNPs to bind cells, which not only ensures high efficiency,
iron oxide MNPs. Because dsRNA[poly(IC)] is a known but also minimizes ineffective aggregation. The examples
allosteric activator of the latent (2–5)A synthetase, these discussed in this section, indeed, validate this simple notion.
dsRNA[poly(IC)] modified MNPs are effective for separating Based on the high affinity of vancomycin (Van) to the
the Lubomirskia baicalensis (2–5)A synthetase by the incuba- receptors on the surface of Gram-positive bacteria, Xu and
tion of sponge extract with dsRNA[poly(IC)]-conjugated iron co-workers developed a simple strategy that used vancomycin-
oxide MNPs. In this case, the dsRNA[poly(IC)] is necessary conjugated FePt nanoparticles (FePt@Van conjugates) to
for preserving the activity of the (2–5)A synthetase.42 This capture and detect pathogens, such as vancomycin-resistant
result not only further confirms the versatility of MNPs for enterococci (VRE) and other Gram-positive bacteria, at
meeting a variety of needs in the manipulation of proteins, but exceptionally low concentrations.47 Fig. 18A shows the experi-
also illustrates a new direction for the applications of MNPs. mental procedure of bacterial detection using the biofunctional
MNPs. The mixing of FePt@Van conjugates with a solution
of bacteria results in a sufficient amount of MNPs binding onto
the bacteria because of their strong interactions (i.e. polyvalent
4. Magnetic nanoparticles for cell manipulation
interactions between Van and D-Ala-D-Ala on the bacterial
The concept of the use of magnetic force to manipulate cells has surface). A small magnet attracts and enriches these bacteria–
been explored before the development of MNPs.68 The develop- nanoparticle composites for the analysis. In the control experi-
ment of biofunctional MNPs, thus, offers new opportunities for ment, the use of FePt nanoparticles capped with nonspecific
groups (FePt–NH2) fails to capture the bacteria because of the The detection limit achieved using FePt@Van magnetic
lack of specific molecular recognition (Fig. 18B). Fig. 18C MNPs is comparable to that of assays based on polymerase
shows the SEM image of ‘‘magnetized’’ S. aureus aggregates chain reaction (PCR), and this protocol is faster than PCR
with FePt@Van conjugates. When FePt–NH2 is used, the when the bacterium count is low.
SEM image (Fig. 18D) shows no S. aureus. Fig. 18E shows One of the advantages of the use of biofunctional MNPs to
the TEM image of vancomycin-resistant bacteria (VanA capture bacteria is that it may serve as an alternative when
genotype) captured by the FePt@Van nanoparticles. As shown PCR is inapplicable. Xu and co-workers combined FePt@Van
in Fig. 18E and F, the nanoparticles are uniformly distributed biofunctional MNPs with vancomycin conjugated with a
and cover the entire surface of the E. faecium (VanA) cells, fluorescence dye to achieve quick, sensitive, and low-cost
which confirms that the multivalent interaction between the detection of bacteria in clinical blood samples. While the FePt
vancomycins on the surface of MNPs and the receptors on the nanoparticles provide the platform for introducing vancomycin
surface of the bacteria minimizes the aggregation of MNPs. molecules to generate multivalent interactions, the fluorescent
Surprisingly, these FePt@Van nanoparticles also capture Gram- vancomycin stains the enriched bacteria for the quick detection
negative bacteria, such as E. coli, from very diluted samples due using fluorescence microscopy. Fig. 19 illustrates the straight-
to the partial exposure of the proper receptors on the surface of forward steps for detecting bacteria in a blood sample: (i)
E. coli cells. The detection limits for bacteria achieved by these mixing, (ii) separating, (iii) staining, and (iv) washing. It is very
FePt@Van MNPs can be as low as 4 cfu mL1 for S. aureus easy to observe the captured bacteria using a fluorescence
and 15 cfu mL1 for E. coli, which remain the lowest among microscope.70 This protocol allows the detection of bacteria
all the reports on using MNPs to capture bacteria.47,69 (E. coli and coagulase-negative Staphylococcus) from the
samples within 2 h and has sensitivity as low as 10 cfu mL1.
Instead of using antibiotics, Hatton et al. coated magnetite
or cobalt MNPs with antiseptic agents for binding and killing
Gram-negative bacteria. After synthesizing magnetite MNPs,
they used the MNPs to react with PEI to generate the PEI-
modified magnetite nanoparticles, which were subsequently
added to the solution of poly(hexamethylene biguanide)
(PHMBG) to give PHMBG–PEI-MNPs (Fig. 20). The
authors found that these nanoparticles showed strong affinity
(same or higher than that of polymyxin) to lipid A, the
glycolipid component of LPS. More interestingly, they found
that the smaller MNPs (o50 nm) displayed the affinity to lipid
A several fold higher than that of larger MNPs (4100 nm),
indicating that MNPs were superior than microbeads for
capturing bacteria. In addition, they reported that these MNPs
Fig. 18 The capture of bacteria by MNPs. (A) Interaction between

FePt@Van nanoparticles and bacteria. (B) FePt–NH2 nanoparticles
as the control. SEM images of (C) aggregates of S. aureus and Fig. 19 Detecting bacteria in a clinical blood sample: (i) addition of
FePt@Van nanoparticles and (D) aggregates of FePt–NH2 nano- FePt@Van; (ii) capturing bacteria assisted by a magnet; (iii) addition
particles. (E) TEM and (F) HRTEM images of the bacteria captured of Van–FLA for staining the bacteria; and (iv) magnetically separating
by FePt@Van nanoparticles. Adapted with permission from ref. 47 stained bacteria from Van–FLA solution. Bottom, a fluorescent image
and 69. Copyright 2003 American Chemical Society and Copyright of stained bacteria after the capture. Adapted with permission from
2006 Royal Society of Chemistry. ref. 70. Copyright 2006 Wiley-VCH.
Fig. 21 Illustration of pathogen detection by magnetic glycol-nano-
particles (MGNPs). Adapted with permission from ref. 24. Copyright
2007 American Chemical Society.
strains of E. coli, which was an appealing feature of this

approach. Of course, it would be more practical if this kind
of capture had been demonstrated on a real sample with the
Fig. 20 The hypothesized interactions between a PHMBG segment
presence of other mammalian cells. It is likely that the
challenge lies on how to control the glycosides on the surface
attached to the polysiloxane shell of the core–shell paramagnetic

particle and an L-Lys-D-Ala-D-Ala segment of the peptidoglycan of the nanoparticles for more specific and selective binding to
(PG) on the surface of a Gram-positive bacterium. Color code: white the targeted cells.
(hydrogen), red (oxygen), gray (carbon), blue (nitrogen), purple Also using the interaction between mannose and E. coli,
(silicon), and pink (lone pair of electrons). Adapted with permission Chen and co-workers successfully combined microfluidic devices
from ref. 71. Copyright 2011 American Chemical Society. and mannose modified MNPs for sorting the bacteria cells. As
shown in Fig. 22, they used soft lithography73 to construct the
inhibited the growth of E. coli at a concentration of microfluidic devices consisting of two inlets and an electromagnet
10 mg mL1,72 a MIC value that remains to be lowered. or a permanent magnet.74 These devices can separate a specific
Following the work by Hatton, Alvarez-Lorenzo and strain of bacteria from a mixture solution based on the sugar
co-workers further developed the above concept (Fig. 20) by (mannose)-encapsulated MNPs. The electromagnet or the
conjugating the PHMBG to the surface of the SiO2 shell of permanent magnet is able to generate a sufficient magnetic
MNPs.71 The use of SiO2 significantly increases the stability of field to attract the bacteria bound to MNPs to cross the stream
these PHMBG–MNP/SiO2 nanoparticles, which exhibit a high boundary of the laminar flow. Although it is difficult to
saturation magnetization for more than 10 months in contact control the formed aggregates (at high bacteria and high
with air in aqueous suspensions. Similar to the results of nanoparticles concentration), which may block the microchannels,
Hatton, the authors also found that the minimum inhibitory the authors reported that the sorting efficiency was higher than
concentration (MIC) of the encapsulated particles was size- 90% and the selectivity was almost 100%. The authors also
dependent (against of eight types of bacteria). For example, compared the sorting efficiency of the nanoparticle with
the smaller MNPs (B150 nm in diameters) show the MICs microbeads and found that microbeads exhibited high sorting
from 15–120 mg mL1, but the larger MNPs (diameters in efficiency in the case of microfluidic devices. The authors
submillimetres) exhibit a MIC of 50–1000 mg mL1. Although suggested that the increase of flow rate and magnetic field
these values are generally higher than those of MNPs that should improve the sorting efficiency of the approach that
have PEI layers, these PHMBG–MNP/SiO2 nanoparticles are
nontoxic to mouse fibroblast cells, suggesting that these MNPs
may be biocompatible. However, the MIC values of these
MNPs are much larger than PHMBG alone, suggesting that
there is still room for optimizing these MNPs. It would be
interesting to see the MIC values against bacteria and IC50
values against mammalian cells if smaller MNPs are used
(e.g. diameters o 10 nm) to conjugate with PHMBG.
Recently, Huang and co-workers have reported a very
interesting type of MNPs for capturing and detecting
E. coli.24 Based on the fact that many pathogens use mamma-
lian cell surface carbohydrates as anchors for attachments, the
authors synthesized a mannose amido acid and a galactose
amido acid and used these acids to react with amino groups on
the surface of the SiO2 shell of MNPs. After decorating the
MNPs with these glycosides, they used these MNPs to detect
E. coli (Fig. 21). Although the sensitivity of this type of
magnetic glycol-nanoparticles remained to be improved (the
concentration of E. coli has to be 4103 cfu mL1), the authors
used the response patterns of different MNPs (i.e. mannose Fig. 22 The integrated microfluidic cell sorting system. Adapted with
coated MNPs and galactose coated MNPs) to differentiate two permission from ref. 74. Copyright 2008 American Institute of Physics.
combined nanoparticles and microfluidics. Nevertheless, the fragments (scFv) of the antibody (e.g. IgG), the authors used
authors demonstrated that their sorting device was able to single-domain antibodies (sdAbs, e.g., VH in Fig. 24A)78 for
separate 103 bacterial cells within 1 min, suggesting that it may decorating the MNPs. Because the readily cloning of VH
find applications in large scale and automated identification of provides small, stable, and selective targeting moieties that are
pathogens. suitable for decorating the MNPs, the nanoparticle agglutination
Instead of attaching carbohydrate molecules on the surface is not an issue with VH. Because of the single domain nature and
of MNPs, Chen and co-workers used glycoproteins to decorate a low number of reactive sites on VH, the conjugation-induced
MNPs as the affinity probes for binding bacteria.75 Because aggregation of MNPs is minimized. In addition, the smaller size
P-fimbriated E. coli had specificity toward Gal(a1–4)Galb units, of VH allows higher binding density on the surface of MNPs
the authors, thus, used pigeon ovalbumin (POA),76 a phospho- (Fig. 24B). Thus, these single-domain-antibody MNPs not only
protein that contains high level of terminal Gal(a1–4)Galb exhibit high affinity toward their target S. aureus, but also display
units, to modify the surface of magnetite nanoparticles coated long shelf life. This approach minimizes cross-reactivity that
with alumina (Fe3O4@Al2O3). The phosphates of POA allow inherently associates with whole antibodies, thus offering a
the formation of phosphate–alumina complexes within 30 s general and useful strategy for developing other biofunctional
under microwave heating. The POA–Fe3O4@Al2O3 MNPs nanoparticles besides MNPs.77
(Fig. 23) are able to capture the P-fimbriated E. coli for the Because of antimicrobial drug resistance, it always needs new
characterization by MALDI-MS. The authors reported that the ways for killing or inhibiting bacteria. Chen and co-workers
detection limit toward uropathogenic bacteria was about 105 demonstrated a quite innovative approach for photokilling
cfu mL1. Based on the same principle, the authors also used pathogens (Fig. 25). After adding a titanium oxide (TiO2) layer
these POA–Fe3O4@Al2O3 MNPs as the affinity probes for on the SiO2 encapsulated magnetite nanoparticles, the authors
P. aeruginosa, functioning via the recognition of galactophilic used dopamine to immobilize succinic anhydride on the surface
PA-IL towards Gal(a1–4)Galb oligosaccharides. Similarly, of TiO2. The succinic anhydride molecules react with IgG to
they used MALDI-MS to profile the bacteria. Although the form IgG–Fe3O4@TiO2 MNPs. When these MNPs are being
need of MALDI-MS excludes this approach to be bedside irradiated by low power UV light, they not only capture patho-
diagnostics, it allows rapid profiling of bacteria without the genic bacteria, but also inhibit the growth of several strains of
culture steps if the counts of bacteria are relatively sufficient. bacteria (e.g. S. pyogenes, multiantibiotic-resistant S. pyogenes,
In addition, the use of glycoproteins should preserve the native
conformation of the oligosaccharide units, which is a very
useful strategy for the applications that demand proper
orientation of the carbohydrate molecules. Based on the
TEM analysis, this approach may be optimized to achieve
higher sensitivity if the aggregation of MNPs can be reduced
before binding with the bacteria.
Using only a part of an antibody, Tanha et al. developed
single-domain-antibody nanoparticles and demonstrated that
these MNPs captured a few dozens of S. aureus from mixed cells
with almost 100% efficiency and specificity. As shown in Fig. 24,
recognizing the cross-reactivity associated with the constant
region (Fc) and the instability of the smaller, single-chain variable
Fig. 24 (A) Illustration of an antibody and antibody fragments. The

antibody domains are shown as ovals. An antibody, e.g., an IgG,
comprises two identical heavy chains (yellow and blue) and two
identical light chains (red and green). The Fc region of the IgG can
interact indiscriminately with several different bacterial targets,
decreasing specificity. Alternatives to whole antibodies include scFv
(a light chain binding domain (VL) plus a heavy chain binding domain
(VH) covalently joined by a linker (orange)) and, most simply, VH
domain. (B) The MNPs covalently modified with single-domain
Fig. 23 Schematic demonstration of surface modification of antibodies-HVHP428 VH with average 4 VH per nanoparticle and
Fe3O4@Al2O3 NPs by pigeon ovalbumin for Gal(a1–4)Galb presenta- (C) the representative TEM images of the HVHP428 VH modified
tion. Adapted with permission from ref. 75. Copyright 2009 Royal MNPs interacts with S. aureus. Adapted with permission from ref. 77.
Society of Chemistry. Copyright 2009 American Chemical Society.
Fig. 25 Illustration to show the steps in the use of MNPs for photo-
killing bacteria. Adapted with permission from ref. 26. Copyright 2008
Wiley-VCH.
and methicillin-resistant S. aureus (MRSA)). Because it is

unlikely for the bacteria to develop resistance against light,
this approach certainly is worthy of further exploration to
improve the efficiency of bacteria killing and to use, ideally,
Fig. 27 Transmission electron microscopy images of thin sections of:
longer wavelength light or visible light.26
(A) native yeast cells; (B) yeast cells coated with PAH/PSS/PAH/PSS/
Instead of using MNPs for the detection of bacteria, Ansari PAH-stabilised MNPs/PSS. Adapted with permission from ref. 81.
and co-workers proposed a novel application that used MNPs Copyright 2010 Royal Society of Chemistry.
to bind bacteria for industrial biotransformation. In the
processes that use freely dispersed microorganisms as the advantages of the use of the interactions between MNPs and
catalysts for biotransformation, it is usually necessary to microorganisms for the development of bioreactors.
separate the microorganisms from the products after the Derived from layer-by-layer (LBL) surface chemistry,82,83
reactions complete. Based on that some biodesulfurization Paunov and coworkers reported an interesting procedure for
was done by using immobilized R. erythropolis on calcium coating yeast cells by magnetite MNPs via electrostatic inter-
alginate,80 Ansari et al. used magnetite nanoparticles with an actions.81 They first coated poly(allylamine hydrochloride)
average size of 45–50 nm to decorate the R. erythropolis IGST8 (PAH) onto the hydrated yeast cells whose surfaces were
(Fig. 26) for the biodesulfurization of dibenzothiophene (DBT) negatively charged in water, then the cells were coated with
and for the post-reaction separation of the bacteria from the poly(sodium polystyrene sulfonate) (PSS). After repeating the
reaction mixture. Using scanning electron microscopy (SEM), procedure to build PAH/PSS/PAH coatings on the cells, they
they found that the MNPs substantially coated the surfaces of deposited magnetite MNPs on the cells before the deposition
the bacteria. Notably, they also found that the decorated cells of two additional polyelectrolyte layers. The final products
had 56% higher DBT desulfurization compared to the undeco- have the layer structures of PAH/PSS/PAH/MNPs/PAH/PSS,
rated cells. Based on that the nanoparticles indeed enhance which preserve the viability of the yeast cells. TEM shows that
membrane permeability of black lipid membranes (BLM), the the MNPs form a multilayered coating on the outer side of the
authors proposed that MNPs increased the permeability of yeast cell walls (Fig. 27). Besides using these magnetically
the bacterial membrane, thus facilitating the mass transport functionalized yeast cells to express GFP and demonstrating
of the reactant and product.79 This result illustrates the that the presence of MNP–polyelectrolyte composite coating
affected little the fluorescence emission, the authors demonstrated
the successful manipulation of individual cells by an external
magnetic field. These results indicate the promises of the use of
MNPs to manipulate eukaryotic cells, which is the focus of the
following section.
4.2 MNPs for mammalian cell manipulation and organization

In order to use MNPs to manipulate mammalian cells, one
major concern is the biocompatibility of the MNPs if the
Fig. 26 SEM images of two different Rhodococcus erythropolis desired outcome is other than the cell death. This requirement
IGTS8 bacteria decorated with MNPs. The bacterium shown on the more or less limits the types of MNPs to be used. Thus, most
right-hand panel is about to undergo division. Adapted with permission of the MNPs used for the manipulation of mammalian cells
from ref. 79. Copyright 2009 Wiley Periodicals, Inc. are iron oxide nanoparticles. For the same reason, the surface
modifier or anchors should be biocompatible as well. Despite
these restrictions, many exciting and promising developments
have emerged in recent years. Generally, these developments
of the use of MNPs for the manipulation of mammalian cells
fall into several categories: (i) to capture cells for diagnosis; (ii)
to label cells for in vivo monitoring or targeting; (ii) to attract
or arrange cells in patterns or three-dimensional cell culture.
Thus, we arrange this section into four subsections, the first
three corresponding to the above three categories, and the
fourth collects those applications of MNPs outside the scopes
of the first three.
4.2.1 MNPs capture cells for diagnosis. Similar to the use

of MNPs for the capture of bacteria from the background of a
large amount of other cells,47,70 one useful application of
MNPs is to separate and purify hematopoietic stem cells from

blood. Xu and co-workers used N-(2-aminoethyl)-3-amino-
propyltrimethoxysilane (AEAPS) to modify the silica-coated
iron oxide nanoparticles that had a diameter of about 60 nm
for attaching anti-CD34+ monoclonal antibodies.84 Using the
process illustrated in Fig. 28 and the MNPs coated with anti-
CD34+ antibodies, they were able to increase the percentage
of CD34+ cells from approximately 1% before the separation
to 75% after the separation of the cells from human umbilical
cord blood. Their success indicates that the proper immuno- Fig. 29 (A) Illustration of magnetic active SERS tagging material
(SERS-MNPs) for cancer-cell targeting and separation. (B) Illustra-
magnetic nanoparticles are able to separate desired stem cells
tion of cancer-cell targeting. (C) Optical microscopy and Raman
for further analysis or differentiation. This work would be
mapping images of SERS-MNPs-targeted cancer cells. Adapted with
more illustrative on the efficiency and selectivity of these MNPs permission from ref. 23. Copyright 2010 Wiley-VCH.
if the authors had performed the comparison experiments using
the anti-CD34+ microbeads.
a 1000-times-stronger SERS intensity due to aggregation of
In biomedicine, there is an increased need for tracking cells,
the SERS-MNPs.
which usually requires highly specific and sensitive spectro-
The most attractive feature of MNPs is the high efficiency of
scopic modality for imaging the cells. Based on the fact that
the use of MNPs to bind and capture cells, which leads to the
surface-enhanced Raman spectroscopy (SERS) easily distinguish
exploration of identification and isolation of cancer stem cells
the Raman-labelled compound from the background, Lee and
(CSCs), a challenge in cancer research for decades. Jeong and
co-workers prepared SERS-encoded MNPs as a multifunctional
co-workers demonstrated the potential of using SERS-MNPs
tagging material for cancer-cell targeting, separation, and
for isolating and identifying bronchioalveolar stem cells
imaging.23 As shown in Fig. 29, these magnetic active SERS
(BASC, a type of lung stem cells). As shown in Fig. 30, after
tagging materials (SERS-MNPs) consist of several components:
the extraction of lung tissues, the authors mixed the cell sample
magnetite (ca. 18 nm in diameter), silver nanoparticles (embedded),
with the SERS-MNPs that targeted to the CD34 marker. After
silica shell (16 nm thick), Raman labels (e.g. 2-chlorobenzenethiol),
applying a magnetic force to separate the CD34+ cells, the
a silica layer, and antibodies. To demonstrate their application, the
authors used fluorescence-activated cell sorting (FACS)85
authors used these SERS-MNPs for targeting breast-cancer cells
analysis to further identify the BASCs based on a series of other
(SKBR3) and floating leukemia cells (SP2/O) and showed the
cell markers. According to the authors, this approach enabled the
separation of the targeted cancer cells from the untargeted cells by
BASCs to be enriched to an approximately 4- to 5-fold higher
using an external magnetic field. According to the authors, this
level than that in the case without pre-separation.86 Ideally, the
system proves to be an efficient tool for separating and imaging the
next step is to develop a more efficient approach to identify and
targeted cells because the magnetic-field-induced hot spots provide
quantify BASCs without the use of FACS.87
Using a top-down physical technique called laser ablation
synthesis in solution (LASiS) to obtain iron oxide MNPs that does
not require chemicals or stabilizers, Amendola and co-workers
investigated the resulting MNPs for macrophage labeling and
manipulation. The authors reported that 80 000 laser pulses
(1064 nm, 9 ns, 10 J cm2) irradiated at an iron plate in
distilled water to generate 1 mg of magnetite nanoparticles
(5–30 nm in diameters) directly, which was incubated with
Fig. 28 Immunomagnetic separation of CD34+ hematopoietic stem proper ligands (e.g. fluorescein–bovine serum albumin
cells. Adapted with permission from ref. 84. Copyright 2006 Elsevier. (F–BSA), amine terminated fluorescein–poly(ethyleneglycol)
Fig. 32 Illustration of the recognition of specific cancer cells by

MNPs. (A) Two types of nanobioprobes coated with anti-CD3 or

anti-PSMA mAb recognize Jurkat T cells or LNCaP cells, respectively.
(B) Magnetic isolation of cancer cells bound by nanobioprobes.
(C) Fluorescent imaging of target cancer cells under a fluorescence
Fig. 30 Isolation of BASCs using SERS-MNPs. The total lung cells microscope. When a mixture of the two types of cancer cells in A was
were extracted from lung tissues and treated with SERS-MNPs. The bound by their respective nanobioprobes, they can be distinguished
targeted cells, CD34+ cells, moved in the direction of the magnetic under UV due to different colors of the attached nanobioprobes.
force and, in this way, it was possible to remove the other unnecessary Adapted with permission from ref. 89. Copyright 2011 American
cells. Then, the collected cells (CD34+ cells) were applied to FACS Chemical Society.
analysis in order to isolate the BASCs. Another marker, Sca-1, was
conjugated with FITC. Finally, BASCs (CD34+Sca1+CD45CD31)
were extracted from the CD34+ cells. Adapted with permission from anti-prostate-specific membrane antigen (anti-PSMA), onto
ref. 86. Copyright 2009 Elsevier Inc. fluorescent-magnetic bifunctional nanoparticles, Pang and
co-workers made two types of multifunctional MNPs for
(F–PEG–NH2), and fluorescein or tetramethylrhodamine isothio- detecting and isolating multiple tumor markers or tumor cells
cyanate (FITC or TRITC) to produce multifunctional MNPs. from complex samples.89 Using a magnet and an ordinary
According to the authors, these iron oxide MNPs show excellent fluorescence microscope, they were able to demonstrate the
biocompatibility so that they can be up-taken by macrophage detection and collection of leukemia cells and prostate cancer
cells (U937), thus a magnet is able to sort and manipulate the cells from mixed samples within 25 minutes. They also found
macrophages (Fig. 31).88 Moreover, the authors suggested that that the capture efficiency of mAb-coupled MNPs for the
these MNPs were able to serve as vehicles for autonomously cancer cells was as high as 96%. Furthermore, without any
transporting theranostic agents into tissues or lesions that were sample pre-treatment before cell analysis, they were able to
hard to reach by the hematic flux, which was an exciting idea detect and to separate leukemia cells and prostate cancer cells
that deserved further exploration. in a large population of cultured normal cells (about 0.01%
Because of their applications in biomedical research, clinical were tumor cells). These encouraging results further confirm
diagnosis, and biomedicine, multifunctional MNPs (i.e. fluorescent, the promises of mAb-coupled multifunctional MNPs for very
biotargeting, and magnetic) have received increased attentions sensitive detection and rapid isolation of multiple cancer cells.
in recent years.90 Not surprisingly, they also have been explored It, however, remains to be seen if this approach can compete
for manipulation of cells. As shown in Fig. 32, by attaching with established protocols. Moreover, this procedure remains
monoclonal antibodies (mAb), such as mAbs of anti-CD3 or to be improved to exclude false positive. One solution would
be to perform magnetic capture and fluorophore staining in
two separated steps.70
One of the most attractive features of MNPs for cell capture
is that they capture cells at ultralow concentration.47 While
most of the works on cell captures give qualitative results, few
reported quantitative assessment. Larson and co-workers
recently showed an excellent example on the quantification
of the magnetic capture of leukemia cells.91 They used MNPs
conjugated to anti-CD34 antibodies to target three cell lines
that expressed different amounts of CD34 sites. They used
anti-CD34 antibody-tagged MNPs and unlabeled MNPs to
Fig. 31 Macrophages, after incubation with FITC-labeled MNPs incubate with U937, GA-10, Jurkat cell lines that expressed
and staining with a CD13-APC, were separated by a magnet. Adapted 4.6 104, 6.2 103, and 2.3 103 CD34 sites, respectively. Using
with permission from ref. 88. Copyright 2011 Royal Society of three separate approaches, light microscopy, superconducting
Chemistry. quantum interference device magnetometry, and in vitro magnetic
Fig. 33 Analysis of nanoparticle binding using light microscopy and

SQUID. (A) Human leukemic cell lines U937, GA-10, and Jurkat.
Photomicrographs show 20 imaging of Prussian blue staining for the
Fig. 34 Characterization of the extent of biotinylation using the
presence of iron-oxide nanoparticles on cells incubated with either CD34
HABA–avidin assay and scanning electron microscopy images of
antibody-conjugated or unconjugated nanoparticles. Scale bar, 20 mm.
magnetically labeled HeLa cells. (A) Biotinamidohexanoic acid
(B) Nanoparticle (NP) binding was confirmed by SQUID magnetometry.
N-hydroxysuccinimide ester (BiotinSE) concentration-dependent
Nanoparticles conjugated with CD34 (+) or unconjugated nanoparticles
studies. Adherent HeLa cells were biotinylated using different BiotinSE
() are indicated. (C) The schematic of the magnetic needle covered with
concentrations at room temperature for 30 min. (B) Labeled HeLa cells.
a polyimide sheath prepared for insertion into a bone marrow sample
Arrows point to individual streptavidin paramagnetic particles, with
(left), as well as the magnetic needle inserted into an experimental sample
scale bar 5 mm. (C) Labeled HeLa cells with a higher concentration of
(right). Adapted with permission from ref. 91. Copyright 2009 American
particles on the cell surface, with scale bar 10 mm. Adapted with
Association for Cancer Research.
permission from ref. 92. Copyright 2009 Elsevier Inc.
needle extraction to quantify the binding of the anti-CD34 (Fig. 34A). By adjusting the concentration of MNPs added to
conjugated MNPs on the cells, they found that these anti- the cells, they were able to control the degree of magnetic
CD34-conjugated MNPs preferentially bound to high CD34- labeling (Fig. 34B and C). The authors also verified that the
expressing cell lines. As shown in Fig. 33A, light microscopy density of biotins on the cell surfaces affected little the binding
shows more anti-CD34 conjugated MNPs on U937 cells than of MNPs onto the cell surface. At the applied field strength of
on other two cell lines, while the unconjugated MNPs exhibit 1 T, the authors measured the dependence of magnetic moment
little preference to any one of the cell lines. This observation of labeled HeLa cells on the concentration of the paramagnetic
correlates well with SQUID magnetometry (Fig. 33B). More particles added, and they found that the average magnetic
importantly, they demonstrated that the use of the magnetic moment per cell increased almost linearly with the concentration
needle (Fig. 33C) allowed the identification of leukemia cells of paramagnetic particles. The cell viability test (for 72 h)
at concentrations below those normally found in remission indicated that the labeling by the commercial magnetic beads
marrow samples. The authors also reported that the magnetic exerted little cytotoxicity. The authors also extended their work
needle enhanced the percentage of lymphoblasts detectable by on magnetic cell patterning94 and used magnetic manipulation
light microscopy by 10-fold. These results not only indicate to form three dimensional multicellular structures made by
that the use of antigen-targeted MNPs improves the sensitivity biotinylated HeLa and TE671 cells.
of detection in the marrow,91 but also serve as an excellent Bouix et al. also evaluated the technology development that
illustration on the quantitative assessment of the application used commercial MNPs to magnetize erythrocytes for automatic
of MNPs. phenotyping, including ABO grouping, RH-K phenotyping, and
Prior to the intensive development of MNPs, there is the antibody screening, in the automated system.93,95 As shown in
use of immunoselective magnetic microbeads. Using those Fig. 35, the materials for screening assemble in the following
commercially available magnetic beads, some important concepts way: a layer of anti-IgG antibody coated on the microplate, a
have been explored and parameter mapped. Since the key step high density solution (NanoLys) to separate the plasma and
in the use of MNPs for cell manipulation is to label the cells by coated anti-IgG, a diluent, patient’s plasma, pre-magnetized red
MNPs, considerable efforts have been spent on the relevant blood cells (RBC)s for reverse ABO grouping.96 After the plate is
development. Recently, Slater and co-workers have demon- incubated and then placed on the magnetic shaker, sensitized
strated a quick way to label cells in a two-step method cells migrate through the NanoLys layer to react with coated
composed of biotinylating the proteins on the cell membrane anti-IgG at the bottom of the wells. Thus, positive reaction gives
and binding streptavidin modified MNP onto the biotinylated a cellular layer, and negative reaction results in a dot at the
proteins.92 The authors found that a substantial surface biotin bottom of the well. This technique relies on the adsorption of
density (B108 biotin per cell) could be achieved within 30 min MNPs on the membrane of the erythrocytes. In contrast to other
when the concentration of the biotinylation agent was 0.75 mM magnetic bead technologies, this method eliminates the washing
Fig. 35 Principle of erythrocyte-magnetized technology (EMT) anti-

body detection. Adapted with permission from ref. 93. Copyright
2008, 2009 John Wiley & Sons, Inc.
steps for removing free IgG because these antibodies remain in

the upper layer. The authors reported that the success of this
MNP based automated system relied on the balance of
sensitivity with specificity, a design principle that might be
useful in other assays.
In order to monitor the immune status of HIV-infected
patients, routine count of CD4+ and CD8+ T lymphocytes is
necessary. Terstappen and co-workers used commercially avail-
able MNPs to develop a low-cost platform to analyze the CD4/
CD8 ratio for HIV monitoring.97 Since CD3+ T lymphocytes
comprise CD4+ and CD8+ T lymphocytes, the authors combined
the use of commercial anti-CD3 ferrofluid with anti-CD4–
phycoerythrin (PE) and anti-CD8–peridinin–chlorophyll–protein
complex (PerCP) labeling. After using anti-CD3 ferrofluids to
separate CD3 cells from whole blood, the authors used Fig. 37 Schematic diagram of production of protein A–BacMPs with
a reconstructed magnetosome membrane (A) AMB-1 transformant
fluorescence microscopy to count CD4+ and CD8+ T lym-
harboring pUM13ZZ and extracted protein A–BacMPs. (B) Procedure
phocytes based on the fluorescent image (Fig. 36). The authors
for reconstruction of the magnetosome membrane of protein A–BacMPs.
reported that the use of a simple single platform image (C) Magnetic separation system of target cells from PBMCs. Adapted
cytometer showed promising results and was in good correla- with permission from ref. 98. Copyright 2008 Wiley Periodicals, Inc.
tion with the high cost flow cytometry. This work indicates
that it is feasible to conduct sophisticated diagnostic protocols
Along this direction, Matsunaga and co-workers reported an
at low cost if the use of MNPs is properly combined with the
elegant approach that employed MNPs produced by magne-
use of fluorescence.70
totactic bacterium, bacterial magnetic particles (BacMPs), for
Besides the use and modification of the MNPs made by chemical
separating target cells.98 As shown in Fig. 37, the authors
reactions, it also feasible to use the MNPs made in nature.
collected the magnetite nanoparticles (Fe3O4, 50–100 nm in
diameter) made by the magnetotactic bacteria after disrupting
the bacteria cells that expressed the fusion proteins of Mms13
and protein A (Mms13 serving as the anchor protein and
protein A for binding with IgG antibodies). After using
detergents to remove other proteins and lipid bilayers on the
BacMPs, the authors reconstructed the magnetosome membrane
of BacMP by adding phosphatidylcholine (PC). Then, they used
the reconstructed magnetosome to immobilize IgG antibodies
and achieved magnetic separation of monocytes and B-lympho-
cytes from the peripheral blood. According to the authors, this
reconstruction process, indeed, reduces the amount of membrane
surface proteins and endotoxins, which are observed on untreated
protein A–BacMPs. One advantage of these bacterial mag-
netic particles (BacMPs) is that they have a single magnetic
domain of magnetite and exhibit strong ferrimagnetism.99,100
Fig. 36 Color merge of CD4–PE (green) and CD8–PerCP (red) The disadvantages of this method are that these BacMPs still
images. Cells in yellow circle indicate CD4–PE labeled cells and cells aggregate together, and it is quite tedious for the reconstruc-
in blank circle indicate CD81dim T cells. Adapted with permission tion of magnetosome, and the presence of endotoxin still
from ref. 97. Copyright 2009 John Wiley & Sons, Inc. might be a concern.100 Also using the interaction of protein
Fig. 38 The presentation of major histocompatibility complex (MHC)/
MAGE-1 A24 peptide complex on MNPs via biotin–streptavidin inter-

actions. Adapted with permission from ref. 102. Copyright 2009 American
Chemical Society.
A and IgG, Zhang et al. immobilized protein A on the surface

of Fe3O4@Au MNPs for the depletion of CD3+ T cell from
mouse splenocytes with high efficiency (98.4%).101

Based on their pioneering works on BacMP, Matsunaga and
co-workers used MHC/peptide complex-conjugated BacMP for
separating melanoma-specific cytotoxic T lymphocytes (CTLs).102
As shown in Fig. 38, they decorated the magnetic nano-
particle with major histocompatibility complex (MHC)/peptide
complexes via the biotin–streptavidin complex. Despite this
rather elaborated surface modification, the authors reported
that the melanoma-reactive cells and melanoma-specific cyto-
toxic T lymphocytes were successfully separated from stimu-
lated peripheral blood mononuclear cells derived from a
vaccinated melanoma patient with 93.1% and 87.7% purity,
respectively. Compared to the previous two-step process for
magnetic separation of antigen-specific CTLs, this approach is
single-step separation of antigen-specific CTLs. These results Fig. 39 (A) An image of targeted EPCs at a specific location within
validate the potential of MHC/peptide complex-conjugated the microfluidic system. (B) Quantification of the targeted cells as
MNPs for efficient separation of antigen-specific CTLs in a function of flow rate. Adapted with permission from ref. 104.
cancer immunotherapy and fundamental research. The principle Copyright 2009 Springer.
established by this work likely will help the emergence of a
simpler process in the future. 4.2.2 MNPs for cell tracking and targeting in vivo. By
Similar to the previous example, Park and co-workers used connecting ephrin mimetic peptides on magnetic CoFe2O4
bacterial magnetic nanoparticles (BacMPs) to target endo- nanoparticles, Zhang and co-workers reported a promising
thelial progenitor cells (EPCs) to a specific location within a result on the use of MNPs for targeting ovarian cancer cells
microfluidic channel, and did a very useful quantitative analysis in vitro and in vivo.44 Based on the work of Pasquale et al.,105
for this approach. The authors incubated the MNPs with the the authors attached a YSAYPDSVPMMS (YSA) peptide to
EPCs at a concentration ranging from 2 mg per 104 cells to the polygalacturonic acid-coated CoFe2O4 nanoparticles and
20 mg per 104 cells and found that the up-take of the MNPs demonstrated that these YAS-conjugated MNPs were able to
into the EPCs was dose-dependent in 24 h incubation. Generally, selectively bind to the ovarian cancer cells overexpressing
one-third of the MNPs were up-taken by the EPCs. As shown EphA2 receptors. Despite that the orientations of the peptides
in Fig. 39, when higher amount of MNPs was used for on the MNPs were unknown, the authors were able to use
incorporation (10 mg MNP per 104 cells), higher number of these peptide-modified MNPs for capturing cancer cells from
cells were attracted toward the magnet with a local magnetic the peritoneal cavity of mice in vivo (Fig. 40). Although the
field of about 400 mT. About 40% of EPCs remained at the authors suggested that this approach could be useful for
targeted site with a flow rate of 5 ml min1 and an incubation capturing other cancer cells, Bosma et al. recently reported
concentration of 10 mg MNP per 104 cells. Although higher that EphA2 receptor targeting does not increase adenoviral
flow rate decreased the amount of EPCs adhered at the pancreatic cancer transduction in vivo.106 These results indi-
targeted site, the authors reported that many cells still adhered cate that there is an unmet need for optimizing or maintaining
to the channel wall at the flow rate of 10 ml min1 (equivalent the interaction of the peptides and the receptors in vivo.
to the linear velocity of 7 mm s1 and a significantly fast flow One of the important objectives in the use of MNPs
rate corresponding to the blood flow rate in arteriole). This for manipulating cells is to control and monitor the position
result helps understand the use of MNPs for labeling cells and of targeted cells in vivo, Lythgoe and co-workers recently
tracking cell in vivo and provides useful insights for the use of demonstrated this approach by magnetically guiding endo-
MNPs in cell therapy.103 thelial progenitor cells (EPCs) to a site of arterial injury.107
Fig. 40 In vivo peritoneal targeting of Hey and BG-1 cells with
MNP–YSA peptide conjugates. (A) Green fluorescence of FDA

loaded Hey cells in the center of illumination through the abdominal
skin of an anesthetized mouse. The cells were pulled to the cavity
surface by the magnet via the MNP conjugates. (B) No visible
fluorescence of FDA-loaded BG-1 cells through the abdominal skin
of an anesthetized mouse under illumination after the magnet was
removed. Adapted with permission from ref. 44. Copyright 2008
American Chemical Society.
Because EPCs are involved in vascular re-endothelialization

and post-ischemic neovascularization, the authors labeled
human EPCs with FDA-approved iron oxide nanoparticles
(Feridex). As shown in Fig. 41A and B, these MNP labeled
cells migrate to the site of magnet 15 s after the exposure to the
magnet. It is likely that the cells transiently acquire a magnetic
dipole moment and experience intercell attraction to form
aggregates in vitro. By placing the magnet outside the body
of rats, the authors successfully enhanced the localization of
EPCs at the site of common carotid artery injury. This promising
result indicates the potential of the use of a magnetic device Fig. 41 (A) Count of cells in field of view (20 objective) with the
positioned outside the body for homing cells to the targeted video frames for t = 0 (application of magnet), 5, 10, and 15 s after
organs and may lead to a useful tool for the systemic injection of exposure to the magnet. Relative position of the magnet is displayed.
cell therapies.107,108 (B) High power magnification of specimen showing CellTracker-
Taking advantage of the ability of MNPs for targeting a labeled cells (green) adhering to the injured vascular intima. Cell nuclei
specific site, Pfeifer and co-workers combined gene delivery are labeled with 4,6-diamino-2-phenylindole (blue). Inset: the reflectance
confocal image of a CD133+ cell showing SPIO-nanoparticles (yellow).
and magnetic positioning of cells. In their rather comprehensive
Adapted with permission from ref. 107. Copyright 2009 American
study, they incubated lentiviral (LV) particles with positively
College of Cardiology Foundation.
charged MNPs or negatively charged MNPs and found that both
types of MNPs exhibited a similar virus-binding capacity with a
maximum capacity of more than 75% within 20 min of incuba- HUVECs at the injured vessel walls (Fig. 42C, left). The
tion. More importantly, they found that the use of the MNPs control experiments, performed under the same conditions
enhanced lentiviral transduction under non-permissive condi- without placing a magnet to the CCA, show little retention of
tions (e.g. like hydrodynamic flow forces and low temperature). the LV/MNP transduced HUVECs (Fig. 42C, right). According
As shown in Fig. 42A, when the LV/MNP mixtures were to the authors, these results suggest that MNP-labeled cells can
applied to the cells followed by 30 min (at room temperature) be positioned to the vessel wall in vivo by magnetic force at sites
shaking, the presence of a magnetic gradient field led to higher of arterial injury. This exciting result demonstrates the use of
level of transduction (indicated by the strong green fluores- MNPs for intravascular gene- and cell-based therapies.109
cence) in human umbilical vein endothelial cells (HUVECs) Because it allows gene transfection in vitro before the injection
than the case of the absence of MNPs. These LV/MNP- of the cells, it may hold great promises for both gene therapy
transduced cells can be positioned to walls of blood vessels. and cell therapy.
As shown in Fig. 42B, under induced hydrodynamic forces, the For detecting circulating tumor cells (CTCs), Zharov and
aortic strips incubated with LV/MNP-transduced HUVECs co-workers combined magnetic capture of circulating tumor
show strong EGFP fluorescence (Fig. 42B, left); aortas cultured cells with photoacoustic detection. As shown in Fig. 43, the
without a magnet exhibit only marginal EGFP expression authors used an amino-terminal fragment (ATF) of the
(Fig. 42B, right). By positioning of endothelial cells to injured urokinase plasminogen activator and polyethylene glycol to
vessels, the authors were able to demonstrate the utility of the functionalize MNPs for capturing the cancer cells and folate
technology in vivo. As shown in Fig. 42C, with small magnets at functionalized gold carbon nanotubes (GNT) for enhancing
the injured common carotid artery (CCA), the infusion of photoacoustic imaging. Using the human breast cancer cells
40 000 LV/MNP transduced HUVECs via the external carotid (MDA-MB-231), which were positive for the urokinase plasmino-
artery results in the retention of MNP-labeled, transgenic gen activator and folate receptors, the authors mimicked the
Fig. 43 Using antibody modified MNPs and folic acid-conjugated
gold-plated carbon nanotubes to detect circulating tumour cells. (A)

The scheme of the detection setup. The laser beam is delivered either
close to the external magnet or through a hole in the magnet using a
fibre-based delivery system. (B) Schematic (left) and transmission
TEM image (right) of MNPs, each with a 10 nm core, a thin (2 nm)
layer of amphiphilic triblock copolymers modified with short PEG
chains and the amino-terminal fragment (ATF) of the urokinase

plasminogen activator. Scale bar, 10 nm. (C) Schematic (left) and
Fig. 42 Transduction efficiency after MNP-assisted lentiviral infec- topographic atomic force microscopy image (right) of a GNT (12 nm
tion and ex vivo and in vivo positioning of HUVECs to the vessel walls 98 nm) coated with PEG and folic acid. Adapted with permission
in the presence of hydrodynamic forces or blood flow. (A) Transduc- from ref. 110. Copyright 2009 Nature Publishing Group.
tion of HUVECs under hydrodynamic flow stress (cells were shaken in
the presence of a magnetic gradient field). Note that the area of EGFP multifunctional nanoprobes not only serve as the contrast for
expression is close to the site of the magnetic gradient field after LV/ electron microscopy, magnetic resonance imaging, scattering-
TM (virus + TM 30 0 , center) and LV/CM (virus + CM 30 0 , right) based imaging, but also lead to a new imaging mode, magneto-
transduction, but not after transduction without MNPs (virus 30 0 ,
motive photoacoustic imaging,111 which promises remarkable
left). (B) LV/TM-transduced HUVECs were transferred to aortic
contrast enhancement compared with conventional photoacoustic
strips and cultured for 24 h with (+magnet) and without (magnet)
magnets below the strips while being shaken. Shown are brightfield
imaging.
(upper) and fluorescence pictures (lower) taken using a stereomicro-
scope (4-fold magnification). (C) In vivo positioning of LV/ 4.2.3 MNPs for 3D cell culture and 2D cell patterning. To
TM-transduced HUVECs to the intima of injured common carotid develop a new way for in vitro three-dimensional (3D) cell culture
arteries by magnetic forces (+magnet). Control experiments were that is critical for tissue engineering, Honda and co-workers used
performed under the same conditions, but without placing a magnet MNPs to develop an innovative tissue engineering strategy,
(magnet). Images were taken 10 min after application of the cells and termed magnetic force-based tissue engineering (Mag-TE), which
restoration of the blood flow. Yellow arrows indicate retention of eliminated the use of conventional scaffolds. As shown in Fig. 44,
MNP-labeled HUVECs in the area of the magnetic field (left), but not
they used magnetite cationic liposomes (MCLs) to label human
in the control animal (right). Shown are bright-field (upper) and
mesenchymal stem cells (MSCs). By placing a magnet (4000 G)
fluorescence images (lower) taken using a stereomicroscope (1.8-fold
magnification). Adapted with permission from ref. 109. Copyright on the reverse side of the surface that had ultralow attachment
2009 National Academy of Sciences. points, they were able to seed these MNP labeled MSCs onto
the surface. After a 24 h incubation, these MSCs formed
occurrence of natural CTCs by intravenously injecting 1 105 multilayered sheet-like structures (300 mm in thickness). More
unlabeled cancer cells in 50 mL PBS solution followed by importantly, the MSCs in the sheets were able to differentiate
injection of the GNT/MNP cocktail. Based on the gradual into osteoblasts, adipocytes, or chondrocytes after a 21-day
increase in flash photoacoustic signals within 8–10 min after culture period using proper induction medium. Using an
injection of nanoparticles to the approximate level of the electromagnet, the authors harvested and transplanted the
photoacoustic signals observed from cells labeled in vitro MSC sheets constructed by Mag-TE into the bone defect in
and a similar clearance behavior, the authors concluded that the crania of nude rats for bone regeneration. The authors also
they successfully targeted CTCs in vivo. More importantly, they reported that MCLs did not inhibit proliferation or differentia-
were able to demonstrate the detection of CTCs originated from tion of MSCs at a magnetite concentration of 100 pg-magnetite/
a primary tumor when the tumor was quite visible on nude cell and below. These results, undoubtedly, demonstrate the
mice. These results suggest that duplex molecular targeting of exciting opportunities presented by MNPs and have stimulated
CTCs by the use of MNPs and functionalized nanoparticles for the development of Mag-TE for tissue engineering.112,113
magnetic-photoacoustic flow cytometry should be feasible for Despite its importance in tissue engineering, it is not easy to
the detection of CTCs in the bloodstream in vivo and in real achieve three-dimensional (3D) cell culture because the superficial
time,110 though the potential of this approach for the early seeding of cells on the surface of scaffolds causes tissue necrosis in
diagnosis of cancer remains to be demonstrated. the central part of 3D scaffolds. To solve this issue, Iwasaki et al.
Recently, Gao and coworkers reported multifunctional pioneered the exploration of alternative cell seeding procedures
probes for photoacoustic imaging by using iron oxide and based on chitosan modified MNPs.49 Taking advantage of the
gold-coupled core–shell nanoparticles (NPs) with well-defined biocompatibility, biodegradability, and low-toxicity of chitosan,
structural characteristics and physical properties. The resulting they developed MNPs coated with chitosan for enhancing cellular
Fig. 44 Procedure for construction of MSC sheets by Mag-TE. Fig. 45 Fabrication and characterization of C2C12/VEGF cell
(A) Magnetite cationic liposome (MCL) for magnetic labeling of sheets. (A) Illustration of fabrication procedure for cell sheets:
MSCs; MCLs were added to MSCs grown in tissue culture plates. MCL-labeled C2C12/VEGF cells are seeded inside a silicone rubber
(B) Construction of MSC sheets by Mag-TE: MSCs labeled with tube frame placed in the dish. The cells are attracted and accumulated
MCLs were seeded onto an ultra-low-attachment plate, and a cylindrical in 3-D on the bottom of dish by a magnet placed beneath the dish,
magnet was then placed under the plate. Cells attracted to the culture and then cultured to form a multilayered cell sheet. (B and C) The
surface by magnetic force were cultured to construct a cell sheet. Upon overall view of the C2C12/VEGF cell sheets. Micrographs were taken
removal of the magnet, cell sheets detached from the culture surface, under the bright-field (B) and fluorescent microscope (C), respectively.
and could be harvested without enzymatic treatment. Adapted with (D) The fluorescence image of cross-section of the C2C12/VEGF
permission from ref. 34. Copyright 2007 Wiley Periodicals, Inc. cell sheet. Adapted with permission from ref. 35. Copyright 2010
Elsevier.
invasion upon the application of a magnetic force. They found
that the cell-invasion efficiency depended on the strength of tissue culture based on magnetic levitation of cells.114 As
magnetic force. The introduction of these MNPs into cells and shown in Fig. 46A, the authors produced a hydrogel consisting
the presence of magnetic force increase the invasion efficiency, of gold nanoparticles, iron oxide MNPs, and filamentous
likely due to up-regulation of matrix metalloproteinases bacteriophage. After the incubation of the hydrogels with
(MMPs) and adhesion molecules that play a crucial role in cells, the fractions of the phage, the gold nanoparticles, and
improving the ability of cell invasion. Overall, the authors the MNPs enter the cells or bind to cell membranes. Thus, by
demonstrated that these chitosan modified MNPs efficiently spatially controlling the magnetic field, the authors were able
enhanced cell seeding into a deep scaffold, increased cell–cell to levitate the cells, to control the geometry of the cell mass,
interactions, and shortened the period of cell proliferation. and to produce multicellular clustering of different cell types in
To address the problem of the lack of blood vessels in co-culture. More encouragingly, the authors reported that the
implanted tissues, the Kamihira group reported the fabrica- magnetically levitated human glioblastoma cells exhibited
tion of angiogenic cell sheets using MCLs for both retroviral similar protein expression profiles to those observed in human
transfection and cell accumulation assisted by magnetic tumor xenografts. These results indicate that magnetic levita-
forces.35,36 After labeling a retroviral vector encoding an tion of cells might be a useful method for recapitulating in vivo
expression cassette of vascular endothelial growth factor protein expression of cancer cells and may be more feasible for
(VEGF) and magnetically attracting the MCL labeled retroviral long-term multicellular studies (Fig. 46B–D).117
vector particles onto a monolayer of mouse myoblast C2C12 Similarly, Slater and co-workers also generated magnetic
cells for gene delivery, they found that MCL-mediated infection multicellular spheroids using the simple hanging drop
achieved increased efficiency by 6.7-fold compared with the method.118 To enable a simple manipulation of the cell
conventional method. As shown in Fig. 45, the authors used spheroids, the authors labeled the membrane of HeLa cells
these MCL labeled, VEGF gene-engineered C2C12 (C2C12/ with biotin molecules, and then used streptavidin decorated
VEGF) cells to form a multilayered cell sheet (300 mm in MNPs to bind the HeLa cells. As shown in Fig. 47A, the
thickness) in the presence of a magnet. After the construction spheroid formation was completed 48 h after the hanging drop
of the cell sheets using both magnetic force-based techniques cultures. Assay of the F-actin distribution within the spheroids
(magnetofection115 and magnetic cell accumulation116), the indicated a three dimensional reorganization of the cellular
authors also transplanted the cells subcutaneously into nude cytoskeleton compared to monolayer cultures. The most
mice and reported that the C2C12/VEGF cell sheet grafts had useful feature of these magnetic multicellular spheroids is that
produced thick tissues, with a high-cell density and promoted they allow easy and quick magnetic separation without the
vascularization, on day 14. This result further demonstrates the need for centrifugation. After the formation of multicellular
versatility and promises of MNPs for cell manipulation that spheroids of different sizes by the change of the concentrations
may achieve sophisticated biological functions. of the seeding cell within the hanging drops, the authors also
Because cellular activities in two-dimensional cell culture demonstrated that magnetic field induced the formation of cell
differ from those found in vivo and 3D cell culture is believed patterns (Fig. 47C). More interestingly, it was possible to use
to be clinically relevant, many efforts have focused on developing magnetic fields to pattern the magnetic spheroids within a few
3D cell culture. Pasqualini and co-workers reported an innova- seconds, which might provide a way to fuse the patterned
tive approach that used MNPs to achieve a three-dimensional spheroids together to form a larger tissue construct.
Fig. 46 MNP-containing hydrogels. (A) Vial of a MNP containing Fig. 48 Magnetophoresis for label-free cell assembly. (A) Schematic
hydrogel (arrow) in water. (B) Scheme of electrostatic interactions of
of BSA-passivated nanoparticle. (B) Suspension of cells in ferrofluid

MNP (brown spheres) and gold (yellow spheres) nanoparticles with assumes a random orientation in the absence of a magnetic field.
phage (elongated structures; pIII and pVIII indicate surface capsid (C) Suspended cells form linear arrangements in ferrofluid in the
proteins). (C) MRI image (T2*-weighted) of purified hydrogel in presence of magnetic field (arrows), where the ferrofluid particles
solution: MNP-free hydrogel control (top panel), average T2* = 46.2 shepherd the cells into chains due to their induced magnetic dipoles.
ms; MNP-containing hydrogel (bottom panel), average T2* = 16 ms. (D) Linearly arranged cells adherent to cell-adhesive surface survive
Scale bar, 2 mm. (D) Three-dimensional cell culture with magnetic- and grow upon removal of ferrofluid and magnetic field. Adapted with
based levitation. Adapted with permission from ref. 114. Copyright permission from ref. 119 Copyright 2009 American Chemical Society.
2010 Nature Publishing Group.
The dimensions of the ferrofluid particles have to be fairly

narrow (B10–20 nm). If the cells or other factors cause the
aggregation of the MNPs in ferrofluid, it would lead to
difficulties. Besides it requires fairly high concentration of
Fe3O4–BSA (30 mg mL1), the whole procedure is still quite
complicated and restrictive for the purpose of the creation of a
fairly simple linear cell organization. Nevertheless, this
Fig. 47 (A) Formation of magnetic multicellular HeLa spheroids. method may lead to complicated cell patterns (e.g. arrays of
48 h after start of spheroid culture. Spheroid formation is completed. squares). The principle demonstrated in this work was also
(B) Random distribution of magnetic HeLa spheroids without any used for label-free cell sorting.119
applied magnetic field. 3-day-old spheroids were used. Scale bar
represents 500 mm. (C) Magnetic HeLa spheroids were patterned with
4.2.4 Other examples of the manipulation of cells by MNPs.
an applied magnetic field within a few seconds. Adapted with permission
from ref. 118 Copyright 2010 Elsevier Inc. Gedanken and co-workers developed a one-step process for
making poly(vinyl alcohol) (PVA) coated magnetite MNPs for
While most works relied on the binding between functionalized loading the MNPs into sperm cells.123 Using high-intensity
MNPs and the target cells for magnetic cell manipulation, Alsberg ultrasound, the authors reacted PVA with iron acetate at
and co-workers proposed a quite novel approach to create ordered
cellular structures based on label-free negative magnetophoresis.119
Although the details on how to produce Fe3O4–BSA ferrofluid
was absent in that report, according to Fig. 48A, the authors
passivated the magnetite MNPs with BSA for generating a
magnetic continuum. Because cells, as the nonmagnetic spherical
particles in a magnetized ferrofluid, behave like a nonmagnetic
cavity inside a magnetized medium (Fig. 48B) and exhibit the field
characteristics of a point dipole,122 these cells, thus, experience
dipole–dipole interactions that result in the formation of linear
chains oriented along the external field direction (Fig. 48C). After
the cells form the linear pattern and adhere to the surface, the
ferrofluid can be removed magnetically (Fig. 48D). This interesting
strategy may find applications in regenerative medicine because
the MNPs need not bind to or be up-taken by cells and the need of
magnetic fluid is temporary.119 However, it also has several Fig. 49 TEM images of bovine sperm cells loaded with PVA–SPION
drawbacks. The generated structures are still limited to linear for the given times: (A) 0 min, (B) 30 min, (C) 60 min, and (D) 80 min.
structures; it would be more attractive if more sophisticated or Adapted with permission from ref. 123. Copyright 2006 American
even three-dimensional structures can be made by this method. Chemical Society.
diluted solution and obtained the PVA coated MNPs with
5 nm diameter Fe3O4. At pH 7.4 and in glucose-free modified
Tyrode solution (m-TALP), the MNPs entered the sperm cells
within 3 h (Fig. 49). The authors found that a relatively high
concentration of the MNPs (7.35 mM) was internalized into
bovine sperm cells (108 cells per mL) and the internalization of
MNPs exhibited little negative effects on the motility and the
ability to undergo the acrosome reaction of the sperm cells.
Later, the same group also used a similar approach to load
PVA or poly(vinylpyrrolidone) (PVP) coated Eu(OH)3 nano-
particles into the bovine sperm cells, and found the loaded
MNPs exhibiting little spermiotoxicity,124 though the concen-
trations of Eu3+ inside the cells reached 4.8–27.8 mg L1. In a
more extensive study, Kadar and co-workers also examined
the effects of the loading of MNPs inside sperm cells,125
including the effects on the development of the embryos after

the fertilization. They found that zero-valent nano irons
(nZVI), with or without polyacrylic stabilizers, caused 30%
mortality of the sperms of Mytilus galloprovincialis, 20%
decline in fertilization success, and 50% of the larvae to have
Fig. 52 Magnetism-induced aggregation of Ab–Zn-MNPs on the

293-hTie2 cell surface. (A) Tie2 receptor-bound nanoparticles before
and after application of the magnetic field. (B) TEM images of the
nanoparticles. Nanoparticles are indicated by arrows. (C) N-MACS
on tubulogenesis of HUVECs after treatment with Ab–Zn-MNPs and
application of a magnetic field. Adapted with permission from ref. 128.
Copyright 2010 Wiley-VCH.
Fig. 50 Optical microscopy images of macrophage cells (A) without delay of embryo development. They also detected significant
MNP for 24 h, and (B) with He-MNP for 24 h. Scale bar = 25 mm. DNA damage in sperm exposed to the high concentrations
Adapted with permission from ref. 126. Copyright 2006 Wiley-VCH. (10 mg L1) of the nZVIs. These results suggest that surface
chemistry of MNPs and the valence of the metals are major
factors of the cellular impacts of the MNPs on the use of
MNPs for manipulating mammalian cells.
In order to minimize or delay the phagocytosis of MNPs,
Neoh and co-workers have developed heparin-immobilized
MNP (He-MNP) and studied the uptake of these MNPs by
macrophage cells.126 Using a surface-initiated atom-transfer
radical polymerization (ATRP), they grafted poly(N-isopropyl-
acrylamide) (poly(NIPAAM)) on the surface of MNPs with
average diameters of 6–8 nm. After immobilizing heparin on
the functionalized MNPs, they incubated these heparinized
MNPs (He-MNPs) with mouse macrophages. Using optical
microscopy to visualize and inductively coupled plasma
spectroscopy to quantify the internalization of the MNPs into
the macrophages (Fig. 50), they concluded that the He-MNPs
were able to delay phagocytosis of macrophages. This result
illustrates a useful strategy for enabling the applications of
MNPs in vivo.
While most of the works demonstrated binding of MNPs to
the targeted cells for capture and separation, few works explored
the arrangements of the magnets for precise control of the
location of the magnetized ‘‘cells’’, Ge and co-workers designed
Fig. 51 (A) Magnetic apparatus. (B) Sectional drawing. (C) Image of a very intriguing magnetic pole for magnetic attenuation of
sample at the same average velocity of 0.8 mm s1, at a magnetic flux cells.127 By arranging the two cylindrical magnetic poles that had
density of 640 mT. Adapted with permission from ref. 127. Copyright holes in the middle (Fig. 51A), the authors generated the
2010 Elsevier. magnetic flux as shown in Fig. 51B, which was a magnetic field
Fig. 54 The concept of targeted magnetomechanical cancer-cell destruc-

tion using disc-shaped magnetic particles possessing a spin-vortex ground
state. The microdiscs are biofunctionalized with antibody, specifically
Fig. 53 (A) Possible binding of target protein on the decorated mag- targeting human glioblastoma cells. When an alternating magnetic field
netic iron oxide nanoparticles; and relative size of magnetic iron oxide is applied, the magnetic discs oscillate, compromising membrane integrity
nanoparticles, a ribosome, and a mammalian cell. (B) Fluorescence and initiating spin-vortex-mediated programmed cell death. Adapted with
images and (C) scheme of magnetically modulated focal adhesion of permission from ref. 129. Copyright 2010 Nature Publishing Group.
COS-1 cell for 4 hours: t = 0 h (elongate forming a diagonal (451) axis
running upwards from right to left), t = 1.5 h (shift down slightly and the of the magnetic flux depended on the flow velocity and the
axis move to 201), t = 3 h (twist and run vertically, overall a 451 rotation), magnetic flux density. If this spatially-focused feature can be
t = 4 h (detached, no longer visible). Adapted with permission from scaled up, it promises a new strategy to promote the use of
ref. 13. Copyright 2011 American Chemical Society. magnetic field for homing and engraftment in cell therapy.
Taking advantage of the magnetically-induced aggregation
with z-axial symmetry in the cylindrical space between the poles. of MNPs, Cheon and co-workers have demonstrated an
Using this apparatus and flowing the cells slowly through a tube elegant example of the control of cell activities by a magnetic force
inserted in the hole, the authors enriched mesenchymal stem cells (termed by the authors as N-MACS‘).128 As shown in Fig. 52A,
(MSCs) loaded with MNPs at controlled distance from the they used a TiMo214 monoclonal antibody (mAb)-conjugated
magnetic pole (Fig. 51C). The amount of cells at the focal point Zn2+-doped ferrite magnetic nanoparticle (Ab–Zn-MNP) to
Table 2 Biofunctional magnetic nanoparticles for the manipulation of proteins
target and to manipulate Tie2 receptors magnetically. By oxide MNPs to manipulate cellular components and cells via a
creating horizontal magnetic field lines (from two permanent magnetic force.13 To use MNPs that have comparable size of
NdFeB magnets) for inducing strong attraction between the proteins certainly provided more precise control of biological
dipoles of neighboring nanoparticles, they were able to control processes by a magnetic force. In addition, the use of smaller
the aggregation of Ab–Zn-MNPs. Since these MNPs were MNPs may lead to single cell manipulation. As shown in
selectively linked to cell surface receptors, aggregation of Fig. 53B, under the magnetic attraction for four hours, the
MNPs induced downstream cell signaling (Fig. 52B) and initiated iron oxide MNPs in the cells exerted a mechanical force on
tubulogenesis in the pre-angiogenesis stage of endothelial cells the cell to cause initially cell shape distortion and then the
(Fig. 52C). This work demonstrates a useful approach to use detachment from the surface after 4 hours. This result suggests
magnetic force for controlling a specific cellular property or that iron oxide MNPs permit magnetically modulated focal
behavior. adhesion of a single live cell.13

While most of the works utilize MNPs with the diameter It is also important to kill cancer cells selectively. Kim et al.
larger than 10 nm, the Xu group further developed biofunc- reported a novel way to induce apoptosis of several cancer
tional, small molecules (GSH or TMP) decorated ultra-small cells by the mechanical forces generated by the spin-vortex at
MNPs (o10 nm) for live cell manipulation.13,14 As shown in very low frequency (tens of Hz) for a short time (10 min). As
Fig. 53, the comparison of the diameters of iron oxide MNPs shown in Fig. 54, they attached anti-IL13a2R antibody to a
(6 nm), a ribosome (20–30 nm), and a cell (e.g. COS-1 cell is magnetic microdisc made from lithography. These antibody-
about 20–30 mm) suggested that it was possible to use iron functionalized magnetic microdiscs selectively bound to the
Table 3 Biofunctional magnetic nanoparticles for the manipulation of cells
cancer cells. When a low frequency alternating magnetic field As shown in Fig. 55, the author depicted several intriguing
caused the microdisc vortices shift, the created oscillation applications, such as nanomagnetic actuation for perturbing
transmitted a mechanical force to the cell, which resulted in actin filament, mechanosensitive ion-channel activation,
membrane destruction, DNA damage, and apoptosis. Amazingly, targeted ion-channel activation, and receptor clustering.
a low-frequency field of a few tens of hertz applied for only ten Although the use of MNPs to activate cells offers means to
minutes was sufficient to achieve B90% cancer-cell destruction isolate cellular mechanics and ion channel activation to gain
in vitro. Although this demonstration uses magnetic microdiscs, better understanding of these cellular processes, these applica-
it is possible to create a similar effect using MNPs based on the tions likely will demand more precise control/measurement on
principle of selective induction of apoptosis, which is of great the number of MNPs at the targeted location, and some
importance for developing the anticancer therapeutic strategies. application may require the use of single MNP. How to use
a magnetic field to control a single MNP for exploring a

biological process remains a challenge to be met.
5. Perspectives and challenges
Obviously, the successful binding of the MNPs to either
The above browse of the recent literature, though incomplete, proteins or receptors on cell surfaces is impossible without the
highlights the potential of MNPs in the manipulation of attachment of proper ligands or antibodies on the MNPs.
proteins or cells (also see Tables 2 and 3). The past successes Despite their effectiveness, the ligands or antibodies rarely are
in the capture of cells or separation of proteins mainly developed for being used on MNPs. A more attractive approach
benefited from the response of the aggregates or multiple is to use MNPs as the platform to screen new ligands that bind
MNPs to the applied magnetic field. These successful examples to the target of interest, which has been recently demonstrated
also inspired highly innovative use of the unique feature of by Weissleder and co-workers.132 As shown in Fig. 56, they
MNPs—‘‘remote control’’ (or action without contact). For investigated the multivalent effect of the small molecules
example, Dobson et al. recently described the concept of nano- attached on the dextran coating of MNPs and found that
magnetic actuation, a possible way to regulate cell functions the multivalency of the small molecules increased specific
via the magnetic control of MNPs on the surface of cells.130 binding affinity and revealed new biological properties of such
nanomaterials. They synthesized a library of nanoparticles
decorated with different synthetic small molecules for rapidly
screening the library against different cell lines. Using fluorescent
MNPs, the authors reported to discover a series of nano-
particles with high specificity towards pancreatic cancer cells.
Although these results confirm that it is feasible to use MNPs
to explore suitable ligands for the modification of MNPs, the
dextran coating on those MNPs is less-defined and may pose
Fig. 55 Illustration of different types of nanomagnetic actuation.

(A) Magnetic twisting cytometry: large (micrometre-sized) magnetic
particles attach to receptors on the cell membrane. The receptors are
linked to actin filaments (black lines). A magnetizing pulse (left) gives
the particle a remanent magnetization (B = magnetic field vector) to
allow responses to a ‘twisting field’ (right). The force required to twist
the particle is related to the mechanical properties of the actin
filaments. (B) Mechanosensitive ion-channel activation: magnetic
particles, again generally larger than 1 mm, are bound to the integrin
receptors (left) and, upon the application of a high-gradient magnetic
field (right), the particles are pulled towards the field, deforming the
cell membrane and activating adjacent mechanosensitive ion channels.
(C) Targeted ion-channel activation: magnetic nanoparticles Fig. 56 Nanoparticle and derived-nanoparticle library. (A and B)
(100 nm–2.7 mm) are attached to an ion channel via an antibody (left). Laser light scattering (A) and atomic force microscopy (B) were used
Upon activation of a high-gradient magnetic field source (right), the to reveal nanoparticles (38 nm mean diameter) comprising a magnetic
ion channel is forced open. (D) Receptor clustering: magnetic nano- core and surface-bound, crosslinked dextran. (C) Model of the crosslinked
particles are bound to receptor complexes. In the absence of a dextran coating modified with small molecules. (D) The nanoparticles are
magnetic field (left), the receptors are spaced along the membrane fully soluble in water, are fluorescent and superparamagnetic, that is,
surface. When a high-gradient field is applied with a magnetic needle detectable by magnetic resonance imaging (MRI). (E) Different classes of
(right), the receptors are pulled towards the field source, initiating small molecules with amino, sulfhydryl, carboxyl or anhydride function-
receptor clustering that can, for example, trigger intracellular signal- alities were anchored onto the nanoparticles and stored in multiwell plates
ling. Adapted with permission from ref. 131. Copyright 2008 Nature for testing. Adapted with permission from ref. 132. Copyright 2005
Publishing Group. Nature Publishing Group.
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View Online / Journal Homepage / Table of Contents for this issue

This article was published as part of the

Nanomedicine themed issue

Guest editors Frank Caruso, Taeghwan Hyeon and Vincent Rotello

Please take a look at the issue 7 2012 table of contents to

Cite this: Chem. Soc. Rev., 2012, 41, 2912–2942

www.rsc.org/csr CRITICAL REVIEW

Received 20th November 2011

Yue Pan received his BS Xuewen Du obtained his BS

respond to a magnetic ﬁeld, oﬀer many exciting opportunities

application. Second, an external magnetic force is able to

Fan Zhao obtained his BS in Bing Xu received his BS and

perspective of MNPs5 and the application of MNPs as MRI

oxide nanoparticles by using Fe2O3 and Fe3O4 as the core and

Fig. 2 Histidine-tagged protein puriﬁcation using biofunctional FePt

washed oﬀ the MNPs by imidazole solution at 10 (lane 3), 80 (lane 4),

Fig. 5 Illustration of the surface modiﬁed silica-coated MNPs as an

the separation of histidine-tagged proteins and demonstrated

ions could replace Ni(II) for selectively enriching phosphorylated

3.2 The conjugate of MNPs and antibodies for manipulating

Fig. 9 The experimental procedure of the sandwich immunoassay

3.3 MNPs for binding post-translational modiﬁed proteins

Fig. 13 (A) Illustration of the magnetosandwich assay for the detection

to perform a blank measurement. Adapted with permission from ref. 66.

they modiﬁed the surface of silica-coated MNPs with various

Fig. 17 The process of the isolation of (2–5)A synthetase from sponge

extract using dsRNA[poly(IC)] functionalized g-Fe2O3 MNPs. Adapted

the manipulation of cells in vitro and in vivo, which are gaining

Fig. 18 The capture of bacteria by MNPs. (A) Interaction between

strains of E. coli, which was an appealing feature of this

attached to the polysiloxane shell of the core–shell paramagnetic

Fig. 24 (A) Illustration of an antibody and antibody fragments. The

and methicillin-resistant S. aureus (MRSA)). Because it is

4.2 MNPs for mammalian cell manipulation and organization

of the ﬁrst three.

4.2.1 MNPs capture cells for diagnosis. Similar to the use

MNPs is to separate and purify hematopoietic stem cells from

Fig. 32 Illustration of the recognition of speciﬁc cancer cells by

MNPs. (A) Two types of nanobioprobes coated with anti-CD3 or

Fig. 33 Analysis of nanoparticle binding using light microscopy and

Fig. 35 Principle of erythrocyte-magnetized technology (EMT) anti-

steps for removing free IgG because these antibodies remain in

MAGE-1 A24 peptide complex on MNPs via biotin–streptavidin inter-

A and IgG, Zhang et al. immobilized protein A on the surface

mouse splenocytes with high eﬃciency (98.4%).101

MNP–YSA peptide conjugates. (A) Green ﬂuorescence of FDA

American Chemical Society.

Because EPCs are involved in vascular re-endothelialization

gold-plated carbon nanotubes to detect circulating tumour cells. (A)

chains and the amino-terminal fragment (ATF) of the urokinase

of BSA-passivated nanoparticle. (B) Suspension of cells in ferroﬂuid

The dimensions of the ferroﬂuid particles have to be fairly

including the eﬀects on the development of the embryos after

Fig. 52 Magnetism-induced aggregation of Ab–Zn-MNPs on the

Fig. 54 The concept of targeted magnetomechanical cancer-cell destruc-

Table 2 Biofunctional magnetic nanoparticles for the manipulation of proteins

behavior. adhesion of a single live cell.13