Dopamine as a robust anchor to immobilize functional molecules on the iron oxide shell of magnetic nanoparticles

Article abstract of DOI:10.1021/ja0464802

We report on the use of dopamine (DA) as a robust molecular anchor to link functional molecules to the iron oxide shell of magnetic nanoparticles. Using nitrilotriacetic acid (NTA) as the functional molecule, we created a system with an M/Fe₂O<

Full text of DOI:10.1021/ja0464802

Published on Web 07/27/2004
Dopamine as A Robust Anchor to Immobilize Functional Molecules on the
Iron Oxide Shell of Magnetic Nanoparticles
Chenjie Xu,† Keming Xu,†,§ Hongwei Gu,† Rongkun Zheng,‡ Hui Liu,‡ Xixiang Zhang,‡
Zhihong Guo,† and Bing Xu*,†,§
Department of Chemistry, Department of Physics, and Bioengineering Program, The Hong Kong UniVersity of
Science and Technology, Clear Water Bay, Hong Kong, China
Received June 14, 2004; E-mail: chbingxu@ust.hk
Scheme 1 a
This communication describes a general strategy that uses
dopamine (DA) as a stable anchor to present functional molecules
on the surface of iron oxide nanostructures. The successful synthesis
of monodispersed magnetic nanoparticles,1 particularly iron oxide
nanoparticles2 or core/shell nanostructures comprising iron oxides
shells,3 promises broad applications of magnetic nanoparticles in
biomedicine and biology due to the inherent biocompatibility of
iron oxide.4 These applications, however, usually require designated
molecules to be immobilized on the surfaces of the magnetic
nanoparticles. Although thiolated organic molecules serve as
excellent anchors to link the functional molecules on the magnetic
nanoparticles whose surfaces consist of noble metals,5 no demon-
stration shows that a small molecule acts as a versatile and robust
anchor on a nanoparticle whose surface comprises iron oxide.
Therefore, several alternatives have been developed to modify
magnetic nanoparticles that consist of iron oxidesfor example,
coating iron oxide with Au, polymers, and silica.6 These approaches,
however, still have drawbacks such as low magnetic moments, low
specificity, or poor tunability. Here we report an easy method that
employs DA as a robust anchor to immobilize the functional
molecules on the iron oxide shells of magnetic nanoparticles and
the use of nitrilotriacetic acid (NTA) as the functional molecule
for protein separation to demonstrate robustness and specificity of
nanostructures created by this method.
We choose DA as the anchor to connect NTA to the iron oxide
shell of the magnetic nanoparticles because the spectroscopic study
by Rajh7 suggested that bidentate enediol ligands such as DA
convert the under-coordinated Fe surface sites back to a bulk-like
lattice structure with an octahedral geometry for oxygen-coordinated
iron, which may result in tight binding of DA to iron oxide. In
addition, Langmuir isotherms indicate that the desorption of DA
from metal oxide nanoparticles (e.g., TiO2) is less favorable than
adsorption.8 Thus, we created a system with an M/Fe2O3-DA-
NTA (M ) Co or SmCo5.2) nanostructure (5) and tested its stability
and specificity. Upon chelation to Ni2+, this M/Fe2O3-DA-NTA-
Ni2+ system (6) separates histidine-tagged proteins from a cell lysate
with high efficiency and capacity. In addition, the specificity and
efficiency of 6b remain unaffected even after 6b is boiled in a buffer
solution. The simple synthetic route provided in this work also
allows a large pool of functional molecules to be connected to iron
oxide. Because of the increasing usage of iron oxide nanoparticles
in biological research, the ease of linking other biomolecules to
iron oxide surfaces through a versatile anchor, such as DA, should
lead to useful applications of iron oxide-containing nanostructures
in several areas, e.g., cell biology, biotechnology, and environment
monitoring.
a Conditions: (a) NaOH, tert-butyl dicarbonate, dioxane, H2O, 24 hrs;
(b) BnBr, K2CO3, DMF, rt, 24 h; (c) 10% CF3COOH, CH2Cl2, rt, 5 h; (d)
succinic anhydride, pyridine, rt, 3 h; (e) NHS, DCC, DMAP, CHCl3, rt, 3
h; (f) NaHCO3, NTA, H2O, CH3CH2OOH, CHCl3, rt, 24 h; HCl; (g) Pd/C,
CH3OH, H2; (h) hexane/water, sonication for 20 min; and (i) NiCl2‚6H2O.
Scheme 1 illustrates the synthetic route for making the M/Fe2O3-
DA-NTA agents. After protecting the hydroxyl groups of dopa-
mine (1) with benzyl bromide, succinic acid anhydride reacts with
2 to yield 3. NR,NR-bis(caboxymethyl)lysine reacts with 3 to give
4 after deprotecting the hydroxyl group. Co (or SmCo5.2)/Fe2O3
nanoparticles9 (in hexane phase) react with 4 (in aqueous solution)
under vigorous stirring overnight or under sonication for 20 min
to form Fe-O bonds that link 4 to the Fe2O3 shell. After the
reaction, the resulting product, 5, becomes water soluble and is
easily separated from the organic phase. Then, excess amounts of
NiCl2‚6H2O react with 5 in the aqueous phase. The removal of the
unreacted Ni2+ solution followed by washing the nanoparticles with
Tris buffer solution yields 6.
We used UV-vis, infrared, and time-of-flight second ion mass
spectra (ToF-SIMS) to characterize the DA-NTA moieties on the
surface of Fe2O3.10 In addition to the absorptions originating from
the nanoparticles, the presence of a small peak at 280 nm (originated
from 4) indicates that DA-NTA resides on the nanoparticle. The
IR spectra of 5a and 5b both exhibit broad peaks around 3440 cm-1
and strong sharp peaks centered at 1640 cm-1, which arise for
carboxylic acid groups and amide groups, indicating the presence
of DA-NTA on 5. The ToF-SIMS spectrum of 5 not only displays
the fragments of 4 (m/e ) 497, 478), but also the fragment of iron-
coordinated dihydroxylbenzyl moiety (FeO2C7H5+, m/e ) 177),
further proving that DA-NTA forms covalent bonds with the Fe2O3
shell. We also estimated the numbers of DA-NTA conjugate linked
to the core-shell magnetic nanoparticles by weight analysis. We
found that on each Co/Fe2O3 and SmCo5.2/Fe2O3 nanoparticle, there
average 27 and 50 DA-NTA molecules, respectively.10 TEM
(Figure 1) indicates that 5a or 5b exhibits well-defined core-shell
† Department of Chemistry.
‡ Department of Physics.
§ Bioengineering Program.
nanostructures (similar to the as-prepared Co/Fe2O3 or SmCo5.2
/
Fe2O3 nanoparticles).
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J. AM. CHEM. SOC. 2004, 126, 9938-9939
10.1021/ja0464802 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
We tested the thermal stability of 6b and compared its specificity
with a commonly used commercial product for separating histidine-
tagged proteins. As shown in Figure 2B, after being boiled in Tris
buffer (pH ) 7.9) for 20 min, the specificity and efficiency of 6b
remain unaffectedselectrophoresis traces show that the fractions
washed from the boiled 6b contain only the histidine-tagged protein,
the same as the fractions washed from the freshly made 6b.
Moreover, the specificity of 6b for the histidine-tagged proteins is
much higher than the commercial HiTrap affinity column that is
based on microbeads: after the affinity column and 6b were bathed
with cell lysate and washed under the same conditions, the fraction
washed from the affinity column contains many other proteins,
while the fractions from 6b contain only the histidine-tagged protein.
In conclusion, we have demonstrated for the first time that dopamine
serves as a robust anchor on the surface of the iron oxide shell of
a magnetic nanoparticle, and the resulting nanostructure exhibits
high specificity for protein separation and exceptional stability to
heating and high salt concentration. Our simple synthetic strategy
should provide a useful way to link other biofunctional molecules
to other metal oxide surfaces (e.g., TiO212 and Fe3O4) that also have
high affinity to DA7 and offer new opportunities for the biological
applications of metal oxide nanoparticles.
Figure 1. Transmission electron micrograph (TEM) images of (A) Co/
Fe2O3, (B) 5a, (C) SmCo5.2/Fe2O3, and (D) 5b. (E) Magnetism of 6a, 6a-
GFP, 6b, and 6b-GFP at the ambient conditions.10
Acknowledgment. This work was partially supported by RGC
(Hong Kong), HIA (HKUST), and a DuPont Young Faculty Grant
(to B.X.).
Figure 2. SDS/PAGE analysis of the purity of the proteins. (A) Cell lysate
(lane 1), GFP standard (lane 2), molecular weight marker (lane 3), the
fractions from freshly made SmCo5.2/Fe2O3-NTA by washing with
imidazole elutions (10 mM, lane 4; 500 mM, lane 5), fractions from the
freshly made Co/Fe2O3-NTA using imidazole elutions (10 mM, lane 6;
500 mM, lane 7). (B) Cell lysate (lane 1), GFP standard (lane 7), the
molecular weight marker (lane 8), the fractions washed from the freshly
made 6b (lanes 2 and 3), boiled 6b (lanes 4 and 5), and the microbeads of
the commercial HiTrap affinity column (lanes 6 and 9). The concentrations
of imidazole are 10 mM (lanes 2, 4, and 6) and 500 mM (lanes 3, 5, 9). All
imidazole elutions also contain 20 mM Na2HPO4 and 0.5 M NaCl.
Supporting Information Available: The details of the synthesis
and characterization of 6 and the separation of histidine-tagged proteins
of reused 6. This material is available free of charge via the Internet at
http://pub.acs.org.
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The procedure and condition of using 6 to isolate proteins is
similar to our previously reported process.11 After washing away
the physically absorbed proteins on 6 or the residual protein
solutions by deionized water, we used 10 and 500 mM of imidazole
elutions to sequentially wash the protein-bound nanoparticles. Figure
2A shows the imidazole elutions contain only the target protein
(6xHis-GFP, lanes 4, 5, and 7). Since there is no other protein being
washed off even by the low concentration (10 mM) imidazole
elution (Figure 2A, lanes 4 and 6), 6 indeed enhances the specificity
of NTA-Ni2+ to bind with histidine-tagged proteins. Control
experiment shows that the amount of GFP absorbed on 5 is almost
negligible when there is no chelation of Ni2+ ions with the NTA
groups.10 Both 6a and 6b can be regenerated and reused,10
suggesting that the bonding between DA and iron oxide is stable
at high salt concentration and the presence of sodium dodecyl
sulfonate (the cell lysate contains 0.5 M NaCl, 20 mM Tris-Cl,
and 2% SDS).
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