Magnetic viruses via nano-capsid templates

Article abstract of DOI:10.1016/j.jmmm.2005.08.027

Biological templating of inorganic nanoparticles provides promising opportunities to address the grand challenge in nanoscience of realizing the full potential of self-assembled materials. We implement such biotemplating to create magnetic nanoparticles b

Full text of DOI:10.1016/j.jmmm.2005.08.027

ARTICLE IN PRESS
Journal of Magnetism and Magnetic Materials 302 (2006) 47–51
www.elsevier.com/locate/jmmm
Magnetic viruses via nano-capsid templates$
Chinmei Liua, Seok-Hwan Chunga, Qiaoling Jina, April Suttona,b, Funing Yana,c,
Ã
Ã
Axel Hoffmanna, Brian K. Kaya, Samuel D. Badera, , Lee Makowskia, Liaohai Chena,
aBiosciences Division, Materials Science Division and Center for Nanoscale Materials, Argonne National Laboratory, Argonne, IL 60439
bDept. of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824
cDepartment of Biological, Chemical and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616
Received 11 August 2005
Available online 10 October 2005
Abstract
Biological templating of inorganic nanoparticles provides promising opportunities to address the grand challenge in nanoscience of
realizing the full potential of self-assembled materials. We implement such biotemplating to create magnetic nanoparticles by utilizing
native protein capsid shells derived in high yield from the T7 bacteriophage virus. The magnetic nanoparticles are grown via bio-
mineralization reactions inside of hollowed-out capsids that retain their original chemical recognition properties. The resultant
‘‘magnetic viruses’’ are uniform in geometry, physical properties, and biochemical functionality. We first coax the DNA out of the T7
virus by means of an alkaline treatment, and then grow magnetic cobalt particles inside the remaining hollow capsid shell. Related
methods of fabricating bio-functional magnetic nanoparticles have utilized either recombinant, single-protein-type capsids, or involve
coating previously synthesized inorganic particles with bio-ligands. Given the richness of the protein types that form the native T7 capsid,
our magnetic viruses can be tailored to tune the bio-functionality and/or bio-tagging of a sample. As an example, we consider a nano-
biomagnetic sensing scheme that would utilize the T7 capsid to control the magnetic nanoparticle size distribution.
r 2005 Elsevier B.V. All rights reserved.
Keywords: Magnetic nanoparticles; Biomagnetic sensing; Brownian relaxation
Biological architectures have recently attracted much
attention as a spatially defined template for fabricating
mono-dispersed nanoparticles. It is exciting to explore the
possibilities that bio-templating approaches offer in order
to specifically create a rich variety of well-defined magnetic
nanostructures with interesting properties and functional-
ities. For example, the apo form of the ferritin protein has
been used as a template to fabricate magnetic particles at
the nanometer scale [1–2]. In general, biotemplating has
included the utilization of bacteria as a host to accom-
modate the formation of polymer material on the micro-
meter scale for drug delivery [3], and lipid assemblies,
DNA, and multicellular superstructures have been used to
direct the patterning and deposition of inorganic material
in general in the micro-to-nanometer scale [4–6]. In the
present work we utilize a virus-derived template to explore
a novel approach to the production of magnetic nanopar-
ticles.
Viruses exemplify an extraordinarily organized nano-
architecture which can be harnessed as templates for
material synthesis [7–10]. Viruses are complex molecular
biosystems in which nucleic acid strands are confined
within a nano-sized (ꢀ10–500 nm) compartment (capsid)
[11–12]. The function of the capsid is to provide a rigid and
robust container protecting the nucleic acids in the passage
from one host cell to another and to deliver the nucleic acid
to the appropriate site. Thus, capsid proteins often have the
capacity to be self-assembled into hollow cages in vitro.
$The submitted manuscript has been created by the University of
Chicago as Operator of Argonne National Laboratory (‘‘Argonne’’) under
Contract no. W-31-109-ENG-38 with the US Department of Energy. The
US Government retains for itself, and others acting on its behalf, a paid-
up, nonexclusive, irrevocable worldwide license in said article to
reproduce, prepare derivative works, distribute copies to the public, and
perform publicly and display publicly, by or on behalf of the Government.
Ã
Corresponding authors. Tel.: +1 630 252 4960; fax: +1 630 252 5219.
E-mail addresses: bader@anl.gov (S.D. Bader), lhchen@anl.gov
(L. Chen).
0304-8853/$ - see front matter r 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jmmm.2005.08.027

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48
Indeed, in vitro self-assembled protein cages derived
from plant viruses have been used as containers to host
inorganic, including magnetic, oxide mineralization in
pioneering work that highlighted the rich opportunities
that lie ahead for viral templating [13–16].
nant protein. In general, it is very difficult to produce
recombinant proteins in high yield. And even if the yields
were high, the cost associated with protein purification and
production would become prohibitive. In our case, the
capsids can be readily obtained directly from the virus. T7
phage can be massively cultured in Escherichia coli at low
cost, thus our approach offers a practical use of virus
capsids as templates for material synthesis. (ii) Because of
the establishment of T7 phage display, molecular biology
approaches can be engaged to integrate an affinity reagent
to the template against virtually any targets via phage
display, while maintaining the integration of the virus
capsid shell. More importantly, the density (or the number)
of the affinity reagent on each template can be precisely
controlled. To anchor an affinity reagent to an artificial
capsid, one has to generate the affinity reagent first, then
link the affinity reagent to the capsid either via chemical
conjugation or recombinant fusion, both of which may
affect the protein assembling ability, thus altering the
assembled capsid’s structure. In addition, precise control of
the affinity reagent density is very difficulty. Again, our
approach provides a practical way of generating a
functional virus capsid template, which can lead to uniform
functional nanoparticles.
The first goal towards the synthesis of the magnetic
viruses is to prepare ghost T7. Previously, ghost T7 capsids
have been obtained by normal release from host cells (very
low yield) or by osmotic shock [22], which had a reasonably
high yield of as high as 55%. In this work, we improved the
procedure for preparing T7 ghosts and obtained yields as
high as 498%. This was achieved by treating the purified
T7 phage with an alkali buffer: the strong alkali condition
denatures the capsid proteins and the DNA, permitting the
DNA strand to escape. Followed by PEG precipitation of
the phage, the capsid proteins are renatured, stable ghost
particles form, and ghosts are collected by ultracentrifuga-
tion.
The high yield of T7 ghost particles was confirmed by
transmission electron microscopy (TEM). As shown in
Fig. 1, ghost particles dominate the image. In contrast to
the normal T7 (left inset), which have regular icosahedral
heads (56 nm in diameter) and short tails (ꢀ8 nm), the
ghost T7 particles lose their symmetric nature, and are
slightly shrunk with an average diameter of 48 nm. Both
the normal and ghost T7 phage were negatively stained
with uranyl acetate (1%) before TEM imaging. Since
uranyl acetate can diffuse inside the capsid, ghost particles
have dark core contrast (Fig. 1, and right inset), while the
high packing density of DNA inside a normal T7 particle
blocks the uranyl acetate uptake into the virus core of the
intact viruses. Hence, the normal T7 phage particle has
brighter core contrast.
T7 bacteriophage particles are especially well suited to be
used as templates for inorganic nanoparticles. While many
viruses build their capsids and encapsulate their nucleic
acids either by co-condensation or by condensing the
nucleic acid first and subsequently building a protein shell
around it, some viruses, such as tailed T7 phage (a fast
growing and extremely stable double-strand DNA phage),
build their protein shells first and subsequently condense
the nucleic acids within them [17]. As a result, an empty T7
capsid (‘‘ghost virus’’) is stable without interior DNA. This
is confirmed by the fact that ghost T7 particles are always
present in bacterial cultures infected with T7 phage. The
outer diameter of the T7 phage is ꢀ55 nm; thus, a T7 ghost
can provide a cavity of ꢀ40 nm in the absence of its DNA,
which can serve as a template for fabrication of nano-sized
particles.
Furthermore, the biological functionality of T7 phage
can be readily tailored by generating affinity reagents
through phage display technology [18–20]. In phage
display, molecules such as antibody fragments, cDNA
encoded segments, or combinatorial peptides are expressed
as fusions to a capsid protein present on the surface of viral
particles. Libraries of millions to billions of phage particles,
each displaying a different fusion protein, are screened
(usually by affinity selection) for members displaying the
desired properties or binding affinities. Phage display offers
the following advantages: (a) the peptide or proteins which
are expressed on the surface of the viral particles are
accessible for interactions with their targets, (b) the
recombinant viral particles are highly stable, (c) the viruses
can be amplified (grown), and (d) each viral particle
contains the DNA encoding its recombinant genome,
thereby providing a physical linkage between the genotype
and phenotype. Thus, phage libraries can be methodically
screened by isolating viral particles that bind to targets,
plaque-purifying the recovered phage, and sequencing the
phage DNA inserts. Usually three rounds of affinity
selection are sufficient to isolate tightly binding phage.
Virtually any affinity reagent specific to any target can be
displayed on a T7 phage surface via phage display. In our
study, we utilized both wild type as well as S-tag peptide, a
short peptide with nanomolar affinity to a fragment of
RNAse A [21], displayed T7 phage.
Compared to previous work, the significance of using a
T7 ghost as a template for magnetic nanoparticle synthesis
is two-fold: (i) Because of the morphogenesis of T7, we can
use a native virus capsid shell, instead of a recombinant
capsid shell assembled in vitro from a single type capsid
protein. Not only is the native capsid more robust due to
the existence of all of its capsid components, but it also can
be produced in large quantities. Artificial capsid templates
reported on previously were assembled using a recombi-
The next goal is to use the ghost virus as a template to
grow metallic Co inside the particle instead of the original
DNA. This was achieved by utilizing the chemistry of
reducing cobalt ions (II) with sodium borohydride [23]. In
brief, ghost T7 particles (0.4 mg/ml) were incubated with

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49
Fig. 2. TEM image of cobalt chimeric T7 phages without negative
staining (scale bar ¼ 100 nm). The small particles (1–2 nm) in the
background come from the gold particles used for anchoring the chimeric
phage; inset: EDX spectrum of the cobalt chimeric T7 phages.
Fig. 1. TEM image of uranyl acetate negatively stained T7 ghost particles
after osmotic shock (scale bar ¼ 200 nm). Up–left inset: normal phage
particles at higher magnification (scale bar ¼ 50 nm); low–right inset:
ghost phage particles (scale bar ¼ 50 nm).
cobalt ions (II) in a degassed, buffered solution at room
temperature. After a 1-h incubation, 10 mM NaBH4
was added to the ghost T7-Co (II) mixture under N2 over
a 2-h period. A color change (pink to gray) indicated the
formation of cobalt particles. Aside from using Co (II)
ions, we conducted a control experiment with hexanitro-
cobaltate anions, which also can be reduced with NaBH4 to
form cobalt metal. However, the yield of magnetic cobalt
viruses with hexanitrocobaltate is much lower compared to
cobalt (II), which suggests that the negatively charged
capsid proteins of the T7 ghost [22] may play a role in
attracting the positive cobalt ions, and suggests that
positive metal ions are preferred for the formation of
chimeric phage.
In order to purify the cobalt particles within the T7
capsid from that formed outside the capsid, we took
advantage of the protein coating of the magnetic phage.
Thus, amino groups are exhibited outside the magnetic
phage, while the free cobalt particles lack such amino
groups. By using succinimidyl 3-(2-pyridyldithio) propio-
nate (SPDP) cross-linker, we selectively anchored the
magnetic phages onto the surface of a substrate via amide
bonds and washed away the cobalt particles formed outside
the T7 capsid. Accordingly, we used a gold-coated TEM
grid as the substrate to anchor the magnetic phage. After
washing off unbound cobalt particles, we were able to
image the magnetic phages via TEM. Such an ‘‘in vivo’’
sample preparation process is required to visualize the
magnetic phage. Otherwise, the additional cobalt particles
(outside the capsid) would induce strong background noise
that would make obtaining a clear image impossible. That
is why a signal from the gold particles, which were used to
anchor the magnetic phage, is present in Fig. 2. However,
this does not affect the image quality of the magnetic
phage, nor does it interfere with the interpretation. In the
future, we could detach the magnetic phage from the
substrate after purification. We are working on making a
photo-cleavable cross-linker in our lab. With such a cross-
linker to anchor the magnetic phage, we will be able to
release the magnetic phages from the substrate after the
washing steps.
To visualize the magnetic viruses, samples immobilized
on a gold-coated TEM grid were imaged by TEM, as
shown in Fig. 2. Uniform cobalt particles (4272 nm) were
formed inside the ghost T7. This is supported by energy-
dispersive X-ray (EDX) analysis (Fig. 2, inset), where the
dominant element inside the T7 ghost is confirmed to be
cobalt. The small particles (1–2 nm) in the background
come from the gold particles used for anchoring the
magnetic viruses.
The next challenge is to determine if the bio-recognition
capability of the capsid was preserved. To do this we
prepared Co viruses from wild-type and S-tag peptide-
displayed T7 phage particles. The S-tag peptide was
genetically fused to the capsid protein using T7 Select
vector-415-1 (from Novagen). Each S-tag peptide-dis-
played T7 phage has 415 copies of a 15 amino acid (aa)
S-tag displayed on its capsid and the S-tag peptide can
interact with 104 aa S-proteins derived from pancreatic
ribonuclease A with a 10 nanomolar dissociation constant

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50
Row for S-tag displayed
cobalt magnetic phage
Experiment
Theory
1.0
0.5
Decreasing phage
concentration
Substrate
Repeat
0.0
200
150
100
50
S-protein
Cobalt
S-tag displayed
T7 phage
-0.5
-1.0
Dye labeled
S-protein
Row for wild type
cobalt magnetic phage
0
0
100
T (K)
200
300
Fig. 3. Left: cartoon for enzyme-linked assay; right: ELISA result for the
cobalt chimeric T7 phage.
-3
-2
-1
0
1
2
3
H (kOe)
[20]. In contrast the wild-type magnetic viruses serve as a
negative control. The different binding affinity of the wild-
type and the S-tag-displayed magnetic viruses was demon-
strated using an enzyme-linked assay. As shown in Fig. 3,
when cobalt magnetic viruses bearing the S-peptide were
added to microtiter plate wells coated with the S-protein,
we observed strong binding behavior, whereas wild-type
cobalt magnetic viruses did not bind to the S-protein at all
due to the lack of ligands on their surface. This behavior is
the same as that known for the original viruses. Hence,
ligand-displayed magnetic viruses maintain their parent’s
bio-recognition functionality.
Having achieved the goal of preparation while preser-
ving the bio-recognition functionality, we turn to the
question of characterizing the magnetic properties, mea-
suring the room-temperature magnetization curve of cobalt
magnetic viruses in liquid solution shows paramagnetic
behavior, as indicated by the absence of hysteresis (see
Fig. 4). This behavior is due to cobalt phages rotating
freely in the liquid. In order to estimate the magnetic
moment of each cobalt phage, the magnetization curve can
be fitted to the classic model of paramagnetism (Langevin
function), which is given by
Fig. 4. Magnetization vs. applied magnetic field for cobalt phage in liquid
solution. The solid line shows a fit to Eq. (1). The inset shows the
temperature dependence of the coercivity for immobilized cobalt phage.
In order to ascertain that an individual cobalt phage is
truly ferromagnetic at room temperature, we also measured
the magnetic behavior of cobalt-chimeric phage that was
immobilized on a gold-coated substrate. Fig. 4 (inset)
shows the coercivity as a function of temperature for these
immobilized cobalt phages. The coercivity decreases with
increasing temperature, but remains non-zero at room
temperature indicating that the magnetic viruses are indeed
ferromagnetic. Therefore, the paramagnetic response of the
phage suspended in the liquid is indeed due to rotational
Brownian diffusion of the whole particle.
The future challenge is to utilize the magnetic viruses as a
new functional entity, for example in biosensing applica-
tions. We have considered a scenario whereby we utilize the
frequency response of the magnetic susceptibility. The
imaginary part of the magnetic susceptibility w00 has a peak
when the applied frequency is the inverse of the Brownian
relaxation time [24,25],
ꢂ
ꢀ
ꢁ
ꢃ
pZd3
2kBT
mH
kBT
kBT
M ¼ Ms coth
ꢁ
,
(1)
tB ¼
,
(2)
mH
where m is the magnetic moment of each particle. From
this fit the magnetic moment for each cobalt phage
is 4.7 ꢂ 104 mB, which is significantly smaller than the
theoretical value 5.2 ꢂ 106 mB estimated for a spherical
single-domain cobalt nanoparticle with 40-nm diameter
using the value of the bulk saturation magnetization of
cobalt (Ms ¼ 1440 emu=cm3) at room temperature. This
reduced magnetization could be due either to (i) multiple
ferromagnetic domains that would arise from the presence
of several weakly coupled randomly oriented grains, or (ii)
oxidation or hydration of the cobalt during or after the
precipitation process. Below the freezing point of the
liquid, the magnetization shows hysteresis with non-zero
coercivity, as expected for ferromagnetic particles.
where Z is the viscosity of the liquid, and d is the particle’s
effective hydrodynamic diameter. To date, we have shown
that we can detect S-protein attachment to suitable coated
magnetic nanoparticles, since the hydrodynamic diameter
and hence the relaxation time increases after binding [26].
This substrateless approach allows for easy mix-and-
measure sensing, where the analyte is mixed into the
solution of ferromagnetic particles without any need for
further sample preparation. Initial prototype applications
of this approach demonstrate a sensitivity of 0.3 pmol [27].
The magnetic virus, with its sharp size control, is expected
to yield sharp resonant peaks in w00 that make the frequency
shift analysis an even more powerful tool. Magnetic
susceptibility measurements of the cobalt magnetic viruses

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51
in aqueous solution show a peak in w00 at ꢀ5 kHz. The
Acknowledgments
estimated hydrodynamic diameter is 70 nm using the
dynamic viscosity of water at 280 K. This peak disappears
when the solution is cooled to 250 K, since the freezing of
the liquid immobilizes the nanoparticles. This implies that
the frequency peak at room temperature is due to the
rotational diffusive Brownian relaxation of the magnetiza-
tion, and thus the magnetic viruses are suitable for
biosensing applications. In contrast to the previously used
magnetic nanoparticles, the availability of almost any
affinity reagent through phage display allows a straight-
forward adaptation of this sensing approach to virtually
any target using the magnetic viruses.
This work was supported by DARPA (8C67400-110)
and the US DOE BES-MS under Contract no. W-31-109-
ENG-38. We thank Ms. Yimei Chen and Dr. Xiaozhou
Liao from University of Chicago for their help with
the TEM imaging and EDX analysis. We also thank
Dr. Zhaozhong Han for help in preparing the ghost phage.
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